From ed40494c885c2b5be0eea570304d2a56b26fd0a5 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 16 Jun 2020 17:04:53 +0800 Subject: [PATCH 01/98] strip off "chr" on seqnames if exist Former-commit-id: 312bf1144f0387d4e9bfef607b05a1cf295d8d7f --- R/annotationFunctions.R | 3 +++ 1 file changed, 3 insertions(+) diff --git a/R/annotationFunctions.R b/R/annotationFunctions.R index 24d2f327..b0dda258 100755 --- a/R/annotationFunctions.R +++ b/R/annotationFunctions.R @@ -53,6 +53,9 @@ prepareAnnotationsFromGTF <- function(file){ }else{ data <- read.delim(file,header=FALSE,comment.char='#') colnames(data) <- c("seqname","source","type","start","end","score","strand","frame","attribute") + if (startsWith(data$seqname,"chr")){ + data$seqname = gsub('chr(.*?)','\\1',data$seqname) + } data <- data[data$type=='exon',] data$strand[data$strand=='.'] <- '*' data$GENEID = gsub('gene_id (.*?);.*','\\1',data$attribute) From 8db831ccd3f808871098498618cb9ec7c51607d8 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Mon, 29 Jun 2020 21:13:14 +0800 Subject: [PATCH 02/98] revert prepareAnnotationsFromGTF() Former-commit-id: a6b90ac93a87cb8d1036bc07ab856f8a7ac98cc9 --- R/annotationFunctions.R | 3 --- 1 file changed, 3 deletions(-) diff --git a/R/annotationFunctions.R b/R/annotationFunctions.R index b0dda258..24d2f327 100755 --- a/R/annotationFunctions.R +++ b/R/annotationFunctions.R @@ -53,9 +53,6 @@ prepareAnnotationsFromGTF <- function(file){ }else{ data <- read.delim(file,header=FALSE,comment.char='#') colnames(data) <- c("seqname","source","type","start","end","score","strand","frame","attribute") - if (startsWith(data$seqname,"chr")){ - data$seqname = gsub('chr(.*?)','\\1',data$seqname) - } data <- data[data$type=='exon',] data$strand[data$strand=='.'] <- '*' data$GENEID = gsub('gene_id (.*?);.*','\\1',data$attribute) From ee7bb82bb10666e421c0ac71309cdb5934980588 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Mon, 29 Jun 2020 21:20:10 +0800 Subject: [PATCH 03/98] R version >= 4.0 and using biocViews from Software To clear the following two warnings: * WARNING: Update R version dependency from 3.5.0 to 4.0. * WARNING: Use biocViews from one category only (one of Software, ExperimentData, AnnotationData, Workflow) Former-commit-id: e6374c87cdb72ac3328fb70556e0d83215afa4ac --- DESCRIPTION | 3 +-- 1 file changed, 1 insertion(+), 2 deletions(-) diff --git a/DESCRIPTION b/DESCRIPTION index 6e0dd559..815ec927 100755 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -10,7 +10,7 @@ License: GPL-3 Encoding: UTF-8 LazyData: true Depends: - R(>= 3.5.0), + R(>= 4.0.0), data.table(>= 1.1.8), dplyr, SummarizedExperiment(>= 1.1.6), @@ -33,7 +33,6 @@ SystemRequirements: biocViews: FeatureExtraction, GeneExpression, - GeneExpressionWorkflow, GenomeAnnotation, ImmunoOncology, Normalization, From c3ede65ac59fd0270bbcd028a635f7d57276d57b Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 09:00:35 +0800 Subject: [PATCH 04/98] T/F to TRUE/FALSE Former-commit-id: 85f77def4ff41f131b6cbc2487f4594875027a9e --- R/isore.R | 26 +++++++++++++------------- 1 file changed, 13 insertions(+), 13 deletions(-) diff --git a/R/isore.R b/R/isore.R index aa82d495..cb494bf1 100755 --- a/R/isore.R +++ b/R/isore.R @@ -140,10 +140,10 @@ isore.constructReadClasses <- function(readGrgList, # seqlevels are made equal (added for chromosomes missing in any of them) # seqlevels(readClassListSpliced) <- unique(c(seqlevels(readGrgList), seqlevels(annotationGrangesList))) - singleExonReads <- unlist(readGrgList[elementNROWS(readGrgList)==1], use.names=F) + singleExonReads <- unlist(readGrgList[elementNROWS(readGrgList)==1], use.names=FALSE) mcols(singleExonReads)$id <- mcols(readGrgList[elementNROWS(readGrgList)==1])$id - referenceExons <- unique(c(granges(unlist(readClassListSpliced[mcols(readClassListSpliced)$confidenceType=='highConfidenceJunctionReads' & mcols(readClassListSpliced)$strand.rc!='*'], use.names=F)), granges(unlist(annotationGrangesList, use.names=F)))) + referenceExons <- unique(c(granges(unlist(readClassListSpliced[mcols(readClassListSpliced)$confidenceType=='highConfidenceJunctionReads' & mcols(readClassListSpliced)$strand.rc!='*'], use.names=FALSE)), granges(unlist(annotationGrangesList, use.names=FALSE)))) readClassListUnsplicedWithAnnotation <- constructUnsplicedReadClasses(granges = singleExonReads, grangesReference = referenceExons, @@ -270,8 +270,8 @@ isore.combineTranscriptCandidates <- function(readClassSe, readClassSeRef = NULL counts.spliced <- cbind(counts.splicedRef, counts.splicedNew) start.spliced <- cbind(start.splicedRef, start.splicedNew) end.spliced <- cbind(end.splicedRef, end.splicedNew) - rowData.spliced$start <- rowMins(start.spliced, na.rm=T) - rowData.spliced$end <- rowMaxs(end.spliced, na.rm=T) + rowData.spliced$start <- rowMins(start.spliced, na.rm=TRUE) + rowData.spliced$end <- rowMaxs(end.spliced, na.rm=TRUE) rowData.spliced <- dplyr::select(rowData.spliced, chr, start, end, strand, intronStarts, intronEnds) %>% mutate(confidenceType = 'highConfidenceJunctionReads') @@ -321,36 +321,36 @@ isore.combineTranscriptCandidates <- function(readClassSe, readClassSeRef = NULL counts.unsplicedRefSum <- as_tibble(assays(readClassSeRef)[['counts']])[rowData(readClassSeRef)$confidenceType=='unsplicedNew',] %>% mutate(index=overlapRefToCombined) %>% group_by(index) %>% - summarise_all(sum, na.rm=T) + summarise_all(sum, na.rm=TRUE) counts.unsplicedNewSum <- as_tibble(assays(readClassSe)[['counts']])[rowData(readClassSe)$confidenceType=='unsplicedNew',] %>% mutate(index=overlapNewToCombined) %>% group_by(index) %>% - summarise_all(sum, na.rm=T) + summarise_all(sum, na.rm=TRUE) start.unsplicedRefSum <- as_tibble(assays(readClassSeRef)[['start']])[rowData(readClassSeRef)$confidenceType=='unsplicedNew',] %>% mutate(index=overlapRefToCombined) %>% group_by(index) %>% - summarise_all(min, na.rm=T) + summarise_all(min, na.rm=TRUE) start.unsplicedNewSum <- readClassSeTBL %>% filter(confidenceType=='unsplicedNew') %>% dplyr::select(start) %>% mutate(index=overlapNewToCombined) %>% group_by(index) %>% - summarise_all(min, na.rm=T) + summarise_all(min, na.rm=TRUE) end.unsplicedRefSum <- as_tibble(assays(readClassSeRef)[['end']])[rowData(readClassSeRef)$confidenceType=='unsplicedNew',] %>% mutate(index=overlapRefToCombined) %>% group_by(index) %>% - summarise_all(max, na.rm=T) + summarise_all(max, na.rm=TRUE) end.unsplicedNewSum <- readClassSeTBL %>% filter(confidenceType=='unsplicedNew') %>% dplyr::select(end) %>% mutate(index=overlapNewToCombined) %>% group_by(index) %>% - summarise_all(max, na.rm=T) + summarise_all(max, na.rm=TRUE) counts.unsplicedRef <- matrix(0, dimnames = list(1:nrow(rowData.unspliced), @@ -639,7 +639,7 @@ seqlevels(exonsByReadClassUnspliced) <- unique(c(seqlevels(exonsByReadClassUnsp if(any(is.na(mcols(seCombined)$GENEID))){ - newGeneIds <- assignNewGeneIds(exonRangesCombined[is.na(mcols(seCombined)$GENEID)], prefix=prefix, minoverlap=5, ignore.strand=F) + newGeneIds <- assignNewGeneIds(exonRangesCombined[is.na(mcols(seCombined)$GENEID)], prefix=prefix, minoverlap=5, ignore.strand=FALSE) mcols(seCombined)$GENEID[as.integer(newGeneIds$readClassId)] <- newGeneIds$geneId } @@ -657,7 +657,7 @@ seqlevels(exonsByReadClassUnspliced) <- unique(c(seqlevels(exonsByReadClassUnsp ungroup() %>% dplyr::select(-geneId) relCounts <- assays(seCombined)$counts / countsTBL - filterTxUsage <- rowSums(relCounts >= min.readFractionByGene, na.rm=T) >= min.sampleNumber + filterTxUsage <- rowSums(relCounts >= min.readFractionByGene, na.rm=TRUE) >= min.sampleNumber seCombinedFiltered <- seCombined[filterTxUsage] exonRangesCombinedFiltered <- exonRangesCombined[filterTxUsage] @@ -765,7 +765,7 @@ isore.estimateDistanceToAnnotations <- function(seReadClass, annotationGrangesLi readClassToGeneIdTableNew <- assignNewGeneIds(rowRanges(seReadClass)[newGeneCandidates], prefix='.unassigned', minoverlap=5, - ignore.strand=F) + ignore.strand=FALSE) readClassGeneTable <- rbind(readClassToGeneIdTable, readClassToGeneIdTableNew) readClassTable <- left_join(readClassTable, readClassGeneTable, by="readClassId") %>% dplyr::select(confidenceType, geneId, compatible, equal) From b7c5d914445e31992986677762b95c1bf2333a80 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 09:04:22 +0800 Subject: [PATCH 05/98] T/F to TRUE/FALSE Former-commit-id: 0b207799749c02f5efa84dfb59ce26d0d8e803df --- R/annotationFunctions.R | 16 ++++++++-------- 1 file changed, 8 insertions(+), 8 deletions(-) diff --git a/R/annotationFunctions.R b/R/annotationFunctions.R index 24d2f327..e6dfe6c9 100755 --- a/R/annotationFunctions.R +++ b/R/annotationFunctions.R @@ -122,7 +122,7 @@ getMinimumEqClassByTx <- function(exonsByTranscripts) { #' @param minoverlap defaults to 5 #' @param ignore.strand defaults to FALSE #' @noRd -assignNewGeneIds <- function(exByTx, prefix='', minoverlap=5, ignore.strand=F){ +assignNewGeneIds <- function(exByTx, prefix='', minoverlap=5, ignore.strand=FALSE){ if(is.null(names(exByTx))){ names(exByTx) <- 1:length(exByTx) } @@ -218,8 +218,8 @@ calculateDistToAnnotation <- function(exByTx, exByTxRef, maxDist = 35, primarySe spliceOverlaps <- findSpliceOverlapsByDist(exByTx, exByTxRef, maxDist=maxDist, - firstLastSeparate=T, - dropRangesByMinLength=T, + firstLastSeparate=TRUE, + dropRangesByMinLength=TRUE, cutStartEnd=TRUE, ignore.strand=ignore.strand) @@ -243,8 +243,8 @@ calculateDistToAnnotation <- function(exByTx, exByTxRef, maxDist = 35, primarySe exByTxRef, maxDist=0, type='any', - firstLastSeparate=T, - dropRangesByMinLength=F, + firstLastSeparate=TRUE, + dropRangesByMinLength=FALSE, cutStartEnd=TRUE, ignore.strand=ignore.strand) @@ -272,9 +272,9 @@ calculateDistToAnnotation <- function(exByTx, exByTxRef, maxDist = 35, primarySe exByTxRef, maxDist=0, type='any', - firstLastSeparate=T, - dropRangesByMinLength=F, - cutStartEnd=F, + firstLastSeparate=TRUE, + dropRangesByMinLength=FALSE, + cutStartEnd=FALSE, ignore.strand=ignore.strand) if(length(spliceOverlaps_restStartEnd)>0){ txToAnTableRestStartEnd <- tbl_df(spliceOverlaps_restStartEnd) %>% From c87a88b6a58dbf7859607258c71d43a36524918a Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 09:07:10 +0800 Subject: [PATCH 06/98] T to TRUE Former-commit-id: eb72c81f65693ba32dbb5d9376faa1af46e040c3 --- R/prepareBam.R | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/R/prepareBam.R b/R/prepareBam.R index e595acad..366b6995 100755 --- a/R/prepareBam.R +++ b/R/prepareBam.R @@ -33,7 +33,7 @@ prepareDataFromBam <- function(bamFile, yieldSize=NULL, verbose = FALSE, ncore = use.names=FALSE)) # readGrgList<-c(readGrgList,GenomicAlignments::grglist(reads)) - if(verbose) show(min(length(readGrgList), counter* yieldSize, na.rm=T)) + if(verbose) show(min(length(readGrgList), counter* yieldSize, na.rm=TRUE)) counter <- counter + 1 } @@ -54,4 +54,4 @@ prepareDataFromBam <- function(bamFile, yieldSize=NULL, verbose = FALSE, ncore = param=Rsamtools::ScanBamParam(flag=Rsamtools::scanBamFlag(isSecondaryAlignment=FALSE), which=chrRanges[chr]), use.names=FALSE))) - } \ No newline at end of file + } From e111827c87d404b116a2cec9798eb116728c03d1 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 09:10:18 +0800 Subject: [PATCH 07/98] T/F to TRUE/FALSE Former-commit-id: 6403aa6e7350d1613ea9dc2bdaeae248fe0b2557 --- R/junctionCorrection.R | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/R/junctionCorrection.R b/R/junctionCorrection.R index 04c28681..a36dcfc9 100755 --- a/R/junctionCorrection.R +++ b/R/junctionCorrection.R @@ -103,11 +103,11 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, junctionStrandCorrection <- function(uniqueJunctions, unlisted_junction_granges, uniqueAnnotatedIntrons, stranded, verbose = FALSE) { ##note: the strand is not always correctly infered based on motifs, it might introduce systematic errors due to alignment (which is biased towards splice motifs) - allJunctionToUniqueJunctionOverlap <- findOverlaps(unlisted_junction_granges, uniqueJunctions,type='equal',ignore.strand=T) + allJunctionToUniqueJunctionOverlap <- findOverlaps(unlisted_junction_granges, uniqueJunctions,type='equal',ignore.strand=TRUE) uniqueJunctionsUpdate <- uniqueJunctions # make a copy to revert to if strand correction does not improve results - annotatedIntronNumber <- evalAnnotationOverlap(uniqueJunctions, uniqueAnnotatedIntrons, ignore.strand=F)['TRUE'] + annotatedIntronNumber <- evalAnnotationOverlap(uniqueJunctions, uniqueAnnotatedIntrons, ignore.strand=FALSE)['TRUE'] if(verbose) { message('before strand correction, annotated introns:') message(annotatedIntronNumber) @@ -139,7 +139,7 @@ junctionStrandCorrection <- function(uniqueJunctions, unlisted_junction_granges, strandScoreByRead <- uniqueJunctionsUpdate$minus_score_inferedByRead - uniqueJunctionsUpdate$plus_score_inferedByRead strand(uniqueJunctionsUpdate[strandScoreByRead< 0 ]) = '+' ## here we overwrite the information from the motif which increases overlap with known junctions strand(uniqueJunctionsUpdate[strandScoreByRead>0 ]) = '-' - annotatedIntronNumberNew <- evalAnnotationOverlap(uniqueJunctionsUpdate, uniqueAnnotatedIntrons, ignore.strand=F)['TRUE'] + annotatedIntronNumberNew <- evalAnnotationOverlap(uniqueJunctionsUpdate, uniqueAnnotatedIntrons, ignore.strand=FALSE)['TRUE'] if(annotatedIntronNumberNew > annotatedIntronNumber & !is.na(annotatedIntronNumber)) # update junctions object if strand prediction improves overlap with annotations { if(verbose) { @@ -160,7 +160,7 @@ junctionStrandCorrection <- function(uniqueJunctions, unlisted_junction_granges, strandScoreByRead <- uniqueJunctionsUpdate$minus_score - uniqueJunctionsUpdate$plus_score strand(uniqueJunctionsUpdate[strandScoreByRead>0 ]) = '-' ## here we verwrite the information from the motif which increases overlap with known junctions strand(uniqueJunctionsUpdate[strandScoreByRead<(0) ]) = '+' - annotatedIntronNumberNew <- evalAnnotationOverlap(uniqueJunctionsUpdate, uniqueAnnotatedIntrons, ignore.strand=F)['TRUE'] + annotatedIntronNumberNew <- evalAnnotationOverlap(uniqueJunctionsUpdate, uniqueAnnotatedIntrons, ignore.strand=FALSE)['TRUE'] if(annotatedIntronNumberNew > annotatedIntronNumber & !is.na(annotatedIntronNumber)) # update junctions object if strand prediction improves overlap with annotations { if(verbose) { From aa23d76db0f23af8685429d3e0c050c057143d33 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 09:18:04 +0800 Subject: [PATCH 08/98] T/F to TRUE/FALSE Former-commit-id: c366405a4148ac0b91834da4edd71abe8a091596 --- R/spliceHelperFunctions.R | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/R/spliceHelperFunctions.R b/R/spliceHelperFunctions.R index 8e1a43c2..da207849 100755 --- a/R/spliceHelperFunctions.R +++ b/R/spliceHelperFunctions.R @@ -5,7 +5,7 @@ #' This function calcualtes compatible splice overlaps allowing for a distance threshold, and returns distance in bp between query and subject. Can be used to assign more transcripts to annotations and reads to transcripts. #' @noRd -findSpliceOverlapsByDist <-function(query, subject, ignore.strand=FALSE, maxDist = 5, type='within', firstLastSeparate = T, dropRangesByMinLength=F, cutStartEnd = T) { +findSpliceOverlapsByDist <-function(query, subject, ignore.strand=FALSE, maxDist = 5, type='within', firstLastSeparate = TRUE, dropRangesByMinLength=FALSE, cutStartEnd = TRUE) { # with this option the first and last exons are stored and the distance for each between query and subject hits is returned if(firstLastSeparate) { @@ -279,7 +279,7 @@ extendGrangesListElements <- function(grangesList, by=5) { start(unlistedExons) <- pmax(1,start(unlistedExons)-by) end(unlistedExons) <- pmin(seqlengths(unlistedExons)[as.character(seqnames(unlistedExons))], end(unlistedExons)+by, - na.rm=T) + na.rm=TRUE) return(relist(unlistedExons, partitioning)) } From 08677d143853ba90b01a0b33a612d7d9cf61ff0a Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 09:31:27 +0800 Subject: [PATCH 09/98] Import RcppArmadillo, RcppProgress, progress * WARNING: Import RcppArmadillo, RcppProgress, progress in NAMESPACE as well as DESCRIPTION. Former-commit-id: 8ed381c10dc29ce84e185c810cb56f5533ef9981 --- NAMESPACE | 4 +++- 1 file changed, 3 insertions(+), 1 deletion(-) diff --git a/NAMESPACE b/NAMESPACE index c3c3a025..d793945f 100755 --- a/NAMESPACE +++ b/NAMESPACE @@ -1,4 +1,6 @@ useDynLib(bambu, .registration=TRUE) exportPattern("^[[:alpha:]]+") importFrom(Rcpp, evalCpp) - +import(RcppArmadillo) +import(RcppProgress) +import(progress) From 697c8b963b8ac3a10a6417fd884e033bad54c1e0 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 10:22:17 +0800 Subject: [PATCH 10/98] Avoid class() == or class() != ; use is() or !is() * WARNING: Avoid class() == or class() != ; use is() or !is() Found in files: R/bambu.R (line 53) R/bambu.R (line 55) R/bambu.R (line 127) R/bambu.R (line 135) Former-commit-id: a889e3236ea28797eec8b6060e5c77bd29cb54d0 --- R/bambu.R | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/R/bambu.R b/R/bambu.R index cc3ba9ce..8ff729c6 100755 --- a/R/bambu.R +++ b/R/bambu.R @@ -50,9 +50,9 @@ bambu <- function(reads = NULL, readClass.file = NULL, readClass.outputDir = NUL #===# Check annotation inputs #===# if(!is.null(annotations)){ - if(class(annotations) == 'TxDb'){ + if(is(annotations,'TxDb')){ annotations <- prepareAnnotations(annotations) - }else if(class(annotations) == "CompressedGRangesList"){ + }else if(is(annotations,"CompressedGRangesList")){ ## check if annotations is as expected if(!all(c("TXNAME","GENEID","eqClass") %in% colnames(mcols(annotations)))){ stop("The annotations is not properly prepared.\nPlease prepareAnnnotations using prepareAnnotations or prepareAnnotationsFromGTF functions.") @@ -124,7 +124,7 @@ bambu <- function(reads = NULL, readClass.file = NULL, readClass.outputDir = NUL if(!is.null(reads)){ # calculate readClass objects #===# create BamFileList object from character #===# - if(class(reads)=='BamFile') { + if(is(reads,'BamFile')) { if(!is.null(yieldSize)) { Rsamtools::yieldSize(reads) <- yieldSize } else { @@ -132,7 +132,7 @@ bambu <- function(reads = NULL, readClass.file = NULL, readClass.outputDir = NUL } reads<- Rsamtools::BamFileList(reads) names(reads) <- tools::file_path_sans_ext(BiocGenerics::basename(reads)) - }else if(class(reads)=='BamFileList') { + }else if(is(reads,'BamFileList')) { if(!is.null(yieldSize)) { Rsamtools::yieldSize(reads) <- yieldSize } else { From a2a1ada53d3452d35f6657708a49110df918ea79 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 10:24:18 +0800 Subject: [PATCH 11/98] Avoid class() == or class() != ; use is() or !is() * WARNING: Avoid class() == or class() != ; use is() or !is() Found in files: R/junctionCorrection.R (line 14) R/junctionCorrection.R (line 31) Former-commit-id: 615059f361f6b20786f6dc0d3116d182b9ef2a3a --- R/junctionCorrection.R | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/R/junctionCorrection.R b/R/junctionCorrection.R index a36dcfc9..13f8648f 100755 --- a/R/junctionCorrection.R +++ b/R/junctionCorrection.R @@ -11,7 +11,7 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, original_seqlevelstyle <- seqlevelsStyle(unlisted_junction_granges)[1] - if(class(genomeSequence) != 'FaFile'){ + if(!is(genomeSequence,'FaFile')){ if(grepl('.fa',genomeSequence)){ genomeSequence <- Rsamtools::FaFile(genomeSequence) @@ -28,7 +28,7 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, } } - if(class(genomeSequence) == 'FaFile'){ + if(is(genomeSequence,'FaFile')){ if(seqlevelsStyle(genomeSequence)[1] != seqlevelsStyle(unlisted_junction_granges)[1]){ seqlevelsStyle(unlisted_junction_granges) <- seqlevelsStyle(genomeSequence)[1] } From c1672e00404c92701a9bf2701e9732936006be6b Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 10:25:31 +0800 Subject: [PATCH 12/98] Avoid class() == or class() != ; use is() or !is() * WARNING: Avoid class() == or class() != ; use is() or !is() Found in files: R/prepareBam.R (line 7) Former-commit-id: bd5858b65636128d9cfd95b6af870f7e4b7ca142 --- R/prepareBam.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/R/prepareBam.R b/R/prepareBam.R index 366b6995..f4119b5c 100755 --- a/R/prepareBam.R +++ b/R/prepareBam.R @@ -4,7 +4,7 @@ #' @noRd prepareDataFromBam <- function(bamFile, yieldSize=NULL, verbose = FALSE, ncore = 1) { - if(class(bamFile)=='BamFile') { + if(is(bamFile,'BamFile')) { if(!is.null(yieldSize)) { Rsamtools::yieldSize(bamFile) <- yieldSize } else { From 5ec2e206d75b8e485f21ea6e36b4cf9399cc4430 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 10:26:30 +0800 Subject: [PATCH 13/98] Avoid class() == or class() != ; use is() or !is() * WARNING: Avoid class() == or class() != ; use is() or !is() Found in files: R/readWrite.R (line 41) Former-commit-id: 0426b53eeac8322f4d0a53b12dec042ed6a93d69 --- R/readWrite.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/R/readWrite.R b/R/readWrite.R index ae5f66df..8b3946e7 100644 --- a/R/readWrite.R +++ b/R/readWrite.R @@ -38,7 +38,7 @@ writeBambuOutput <- function(se,path){ writeToGTF <- function (annotation,file,geneIDs=NULL) { if (missing(annotation) | missing(file)){ stop('Both GRangesList and the name of the output file are required.') - }else if (class(annotation) != "CompressedGRangesList"){ + }else if (!is(annotation,"CompressedGRangesList")){ stop('The inputted GRangesList is of the wrong class.') } df <- as_tibble(annotation) From 2f3bb57971f2479224853703b74898d6efe9a2be Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 13:25:32 +0800 Subject: [PATCH 14/98] import packages in package::object in function() * ERROR: Packages providing 367 object(s) used in this package should be imported in the NAMESPACE file, otherwise packages importing this package may fail. Former-commit-id: 0e0490368dba82721efe43e63ffc9168b78bb24c --- NAMESPACE | 14 ++++++++++++++ 1 file changed, 14 insertions(+) diff --git a/NAMESPACE b/NAMESPACE index d793945f..e375a010 100755 --- a/NAMESPACE +++ b/NAMESPACE @@ -1,6 +1,20 @@ useDynLib(bambu, .registration=TRUE) exportPattern("^[[:alpha:]]+") importFrom(Rcpp, evalCpp) +importFrom(Rcpp, show) +importFrom(Rsamtools, index) import(RcppArmadillo) import(RcppProgress) import(progress) +import(data.table) +import(dplyr) +import(GenomicAlignments) +import(GenomicFeatures) +importFrom(GenomicRanges, makeGRangesListFromFeatureFragments) +importFrom(GenomicRanges, GRanges) +importFrom(GenomicRanges, makeGRangesListFromDataFrame) +importFrom(GenomicRanges, score) +import(ggplot2) +import(IRanges) +import(S4Vectors) +import(SummarizedExperiment) From aeaae24608c2e83db90646160c0ba381beadbdab Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Tue, 30 Jun 2020 17:49:05 +0800 Subject: [PATCH 15/98] prevent imported functions from clashing Former-commit-id: 60b4efb60c1abfe54f12f683f06de57e7f464f32 --- NAMESPACE | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/NAMESPACE b/NAMESPACE index e375a010..09a8c600 100755 --- a/NAMESPACE +++ b/NAMESPACE @@ -6,8 +6,8 @@ importFrom(Rsamtools, index) import(RcppArmadillo) import(RcppProgress) import(progress) -import(data.table) -import(dplyr) +import(data.table, except=c(last, first, shift, second, between)) +import(dplyr, except=c(last, first, desc, union)) import(GenomicAlignments) import(GenomicFeatures) importFrom(GenomicRanges, makeGRangesListFromFeatureFragments) @@ -15,6 +15,6 @@ importFrom(GenomicRanges, GRanges) importFrom(GenomicRanges, makeGRangesListFromDataFrame) importFrom(GenomicRanges, score) import(ggplot2) -import(IRanges) -import(S4Vectors) +import(IRanges, except=c(slice, collapse, setdiff, intersect)) +import(S4Vectors, except=c(rename, setequal, setdiff, intersect)) import(SummarizedExperiment) From 07bc5c11e7c8c565c16b1af75592c112a036b8c0 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 10:06:35 +0800 Subject: [PATCH 16/98] suggest dependencies used in test MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit * checking for unstated dependencies in ‘tests’ ... WARNING 'library' or 'require' calls not declared from: ‘BSgenome.Hsapiens.NCBI.GRCh38’ ‘TxDb.Hsapiens.UCSC.hg38.knownGene’ Former-commit-id: 3e482ee18d708b0904feee66c1a7ab23ba7b6633 --- DESCRIPTION | 4 +++- 1 file changed, 3 insertions(+), 1 deletion(-) diff --git a/DESCRIPTION b/DESCRIPTION index 815ec927..b2b3c1b1 100755 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -27,7 +27,9 @@ Suggests: circlize, ggbio, RColorBrewer, - gridExtra + gridExtra, + BSgenome.Hsapiens.NCBI.GRCh38, + TxDb.Hsapiens.UCSC.hg38.knownGene Enhances: parallel SystemRequirements: biocViews: From 2acfb76cc57e1fafcf07aa1cfc685b4dc2ed90d7 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 11:17:04 +0800 Subject: [PATCH 17/98] add in the stranded parameter in documentation MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit * checking Rd \usage sections ... WARNING Undocumented arguments in documentation object 'bambu' ‘stranded’ Former-commit-id: b65ecab3ce8596fa99d11f12c4f44bf876745860 --- R/bambu.R | 1 + 1 file changed, 1 insertion(+) diff --git a/R/bambu.R b/R/bambu.R index 8ff729c6..f6484987 100755 --- a/R/bambu.R +++ b/R/bambu.R @@ -8,6 +8,7 @@ #' @param readClass.outputDir A string variable specifying the path to where read class files will be saved. #' @param annotations A \code{\link{TxDb}} object or A GRangesList object obtained by \code{\link{prepareAnnotations}} or \code{\link{prepareAnnotationsFromGTF}}. #' @param genomeSequence A fasta file or a BSGenome object. +#' @param stranded A boolean for strandedness, defaults to FALSE. #' @param ncore specifying number of cores used when parallel processing is used, defaults to 1. #' @param yieldSize see \code{\link{Rsamtools}}. #' @param isoreParameters A list of controlling parameters for isoform reconstruction process: From 4533835325ea07568b9342b6da5e3020efc5a4ec Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 11:20:03 +0800 Subject: [PATCH 18/98] document stranded in \usage MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit * checking Rd \usage sections ... WARNING Undocumented arguments in documentation object 'bambu' ‘stranded’ Former-commit-id: d9971574f04a01932ed7d95f1a2e8224bf8fdfd9 --- man/bambu.Rd | 2 ++ 1 file changed, 2 insertions(+) diff --git a/man/bambu.Rd b/man/bambu.Rd index ba1e5ca5..9de214a9 100644 --- a/man/bambu.Rd +++ b/man/bambu.Rd @@ -30,6 +30,8 @@ bambu( \item{genomeSequence}{A fasta file or a BSGenome object.} +\item{stranded}{A boolean for strandedness, defaults to FALSE.} + \item{ncore}{specifying number of cores used when parallel processing is used, defaults to 1.} \item{yieldSize}{see \code{\link{Rsamtools}}.} From 412a958b88e473652beb1e925ff514fbf5725f0f Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 13:24:59 +0800 Subject: [PATCH 19/98] =?UTF-8?q?remove=20links=20to=20=20=E2=80=98.gtf?= =?UTF-8?q?=E2=80=99=20and=20=E2=80=98.txt=E2=80=99?= MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit Missing link or links in documentation object 'writeBambuOutput.Rd': ‘.gtf’ ‘.txt’ Former-commit-id: a6949c1edcb209100da2194d1ef6a51160beca72 --- R/readWrite.R | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/R/readWrite.R b/R/readWrite.R index 8b3946e7..6fc7314b 100644 --- a/R/readWrite.R +++ b/R/readWrite.R @@ -2,8 +2,8 @@ #' @title Write bambu results to GTF and transcript/gene-count files #' @param se a \code{\link{SummarizedExperiment}} object from \code{\link{bambu}} #' @param path the destination of the output files (gtf, transcript counts, and gene counts) -#' @return The function will generate three files, a \code{\link{.gtf}} file for the annotations, -#' two \code{\link{.txt}} files for transcript and gene counts respectively. +#' @return The function will generate three files, a .gtf file for the annotations, +#' two .txt files for transcript and gene counts respectively. #' @export writeBambuOutput <- function(se,path){ if (missing(se) | missing(path)){ @@ -82,7 +82,7 @@ writeToGTF <- function (annotation,file,geneIDs=NULL) { #' Outputs GRangesList object from reading a GTF file #' @title convert a GTF file into a GRangesList -#' @param file a \code{\link{.gtf}} file +#' @param file a .gtf file #' @return grlist a \code{\link{GRangesList}} object, with two columns #' \itemize{ #' \item TXNAME specifying prefix for new gene Ids (genePrefix.number), defaults to empty From f170ffd322d40d31d3201439debb6bd6b4c493fd Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 13:26:00 +0800 Subject: [PATCH 20/98] fix typo MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit Missing link or links in documentation object 'prepareAnnotations.Rd': ‘GrangesList’ Former-commit-id: 310acff3f813a64c7249b1568e9ed1d284f41196 --- R/annotationFunctions.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/R/annotationFunctions.R b/R/annotationFunctions.R index e6dfe6c9..fc48c0c1 100755 --- a/R/annotationFunctions.R +++ b/R/annotationFunctions.R @@ -1,7 +1,7 @@ #' Function to prepare tables and genomic ranges for transript reconstruction using a txdb object #' @title prepare annotations from txdb object #' @param txdb a \code{\link{TxDb}} object -#' @return A \code{\link{GrangesList}} object +#' @return A \code{\link{GRangesList}} object #' @export #' @examples #' \dontrun{ From b07ba5eecc4cea3f246930866daee9f06a4b03f2 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 13:27:19 +0800 Subject: [PATCH 21/98] fix links MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit * checking Rd cross-references ... WARNING Missing link or links in documentation object 'plot.bambu.Rd': ‘transcriptToGene’ ‘details’ Former-commit-id: 1e3fb0206b140ee9b0ae08ea24da033ca56057f2 --- R/plotBambu.R | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/R/plotBambu.R b/R/plotBambu.R index eafd5370..c46a289b 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -1,8 +1,8 @@ #' plotSEOuptut #' @title plot.bambu -#' @param se An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGene}}. +#' @param se An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGeneExpression}}. #' @param group.variable Variable for grouping in plot, has be to provided if choosing to plot PCA. -#' @param type plot type variable, a values of annotation for a single gene with heatmap for isoform expressions, pca, or heatmap, see \code{\link{details}}. +#' @param type plot type variable, a values of annotation for a single gene with heatmap for isoform expressions, pca, or heatmap, see details. #' @param gene_id specifying the gene_id for plotting gene annotation, either gene_id or transcript_id has to be provided when type = "annotation". #' @param transcript_id specifying the transcript_id for plotting transcript annotation, either gene_id or transcript_id has to be provided when type = "annotation" #' @details \code{\link{type}} indicates the type of plots to be plotted. There are two types of plots can be chosen, PCA or heatmap. From 2227edeee5d92050c56b2a33a29d9c8e693cf48b Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 13:28:55 +0800 Subject: [PATCH 22/98] fix links MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit * checking Rd cross-references ... WARNING Missing link or links in documentation object 'plot.bambu.Rd': ‘transcriptToGene’ ‘details’ Former-commit-id: 17683df86795ac67966bf4df5d37573ad72a17e3 --- man/plot.bambu.Rd | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/man/plot.bambu.Rd b/man/plot.bambu.Rd index d5526219..a26a5012 100644 --- a/man/plot.bambu.Rd +++ b/man/plot.bambu.Rd @@ -13,11 +13,11 @@ ) } \arguments{ -\item{se}{An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGene}}.} +\item{se}{An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGeneExpression}}.} \item{group.variable}{Variable for grouping in plot, has be to provided if choosing to plot PCA.} -\item{type}{plot type variable, a values of annotation for a single gene with heatmap for isoform expressions, pca, or heatmap, see \code{\link{details}}.} +\item{type}{plot type variable, a values of annotation for a single gene with heatmap for isoform expressions, pca, or heatmap, see details.} \item{gene_id}{specifying the gene_id for plotting gene annotation, either gene_id or transcript_id has to be provided when type = "annotation".} From dba479748d96eea76efd8e570379e7bb3f7d5b58 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 13:31:12 +0800 Subject: [PATCH 23/98] fix links MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit * checking Rd cross-references ... WARNING Missing link or links in documentation object 'prepareAnnotations.Rd': ‘GrangesList’ Former-commit-id: 97b6e5da67cd43ca405a7c52b0e41d90c4f4d3c3 --- man/prepareAnnotations.Rd | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/man/prepareAnnotations.Rd b/man/prepareAnnotations.Rd index cb31d589..73b57568 100644 --- a/man/prepareAnnotations.Rd +++ b/man/prepareAnnotations.Rd @@ -10,7 +10,7 @@ prepareAnnotations(txdb) \item{txdb}{a \code{\link{TxDb}} object} } \value{ -A \code{\link{GrangesList}} object +A \code{\link{GRangesList}} object } \description{ Function to prepare tables and genomic ranges for transript reconstruction using a txdb object From 50e292cb5fe66365e6fa2031ea1825eb169c5908 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 13:32:41 +0800 Subject: [PATCH 24/98] remove .gtf link MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit * checking Rd cross-references ... WARNING Missing link or links in documentation object 'readFromGTF.Rd': ‘.gtf’ Former-commit-id: 0de291cc1bd0904bc58d0d3161a47b4ca020680b --- man/readFromGTF.Rd | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/man/readFromGTF.Rd b/man/readFromGTF.Rd index bc58c175..ea383f49 100644 --- a/man/readFromGTF.Rd +++ b/man/readFromGTF.Rd @@ -7,7 +7,7 @@ readFromGTF(file) } \arguments{ -\item{file}{a \code{\link{.gtf}} file} +\item{file}{a .gtf file} } \value{ grlist a \code{\link{GRangesList}} object, with two columns From 0201b2e1c8458b59f013c75da02ea9ac8369dc9b Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 13:33:49 +0800 Subject: [PATCH 25/98] =?UTF-8?q?remove=20=E2=80=98.gtf=E2=80=99=20and=20?= =?UTF-8?q?=E2=80=98.txt=E2=80=99=20links?= MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit * checking Rd cross-references ... WARNING Missing link or links in documentation object 'writeBambuOutput.Rd': ‘.gtf’ ‘.txt’ Former-commit-id: ca8c6b82e88ec68244806750e63425169146a9f6 --- man/writeBambuOutput.Rd | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/man/writeBambuOutput.Rd b/man/writeBambuOutput.Rd index 87357f29..8c6da71a 100644 --- a/man/writeBambuOutput.Rd +++ b/man/writeBambuOutput.Rd @@ -12,8 +12,8 @@ writeBambuOutput(se, path) \item{path}{the destination of the output files (gtf, transcript counts, and gene counts)} } \value{ -The function will generate three files, a \code{\link{.gtf}} file for the annotations, -two \code{\link{.txt}} files for transcript and gene counts respectively. +The function will generate three files, a .gtf file for the annotations, +two .txt files for transcript and gene counts respectively. } \description{ Outputs a GTF file, transcript-count file, and gene-count file from bambu From 97fadcf9f041282fb7e7e6d5a2b02a17bc862eef Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 14:41:10 +0800 Subject: [PATCH 26/98] suggests dependencies imported from '::' and ':::' * checking dependencies in R code ... WARNING '::' or ':::' imports not declared from: 'AnnotationDbi' 'BSgenome' 'BiocGenerics' 'BiocParallel' 'Biostrings' 'library' or 'require' call not declared from: 'BSgenome' 'library' or 'require' call to 'BSgenome' in package code. Please use :: or requireNamespace() instead. See section 'Suggested packages' in the 'Writing R Extensions' manual. It is a NOTE instead of a WARNING now. Former-commit-id: 65e927f40dcdb8c1e5f360d2c6dc19f029487774 --- DESCRIPTION | 7 ++++++- 1 file changed, 6 insertions(+), 1 deletion(-) diff --git a/DESCRIPTION b/DESCRIPTION index b2b3c1b1..28259e6c 100755 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -29,7 +29,12 @@ Suggests: RColorBrewer, gridExtra, BSgenome.Hsapiens.NCBI.GRCh38, - TxDb.Hsapiens.UCSC.hg38.knownGene + TxDb.Hsapiens.UCSC.hg38.knownGene, + AnnotationDbi, + BSgenome, + BiocGenerics, + BiocParallel, + Biostrings Enhances: parallel SystemRequirements: biocViews: From 8496fe2f99adf98796faf0432f1f6d3354967ea7 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 15:46:41 +0800 Subject: [PATCH 27/98] make plot and plot.bambu consistent MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit * checking S3 generic/method consistency ... WARNING plot: function(x, ...) plot.bambu: function(se, group.variable, type, gene_id, transcript_id) See section ‘Generic functions and methods’ in the ‘Writing R Extensions’ manual. Found the following apparent S3 methods exported but not registered: plot.bambu See section ‘Registering S3 methods’ in the ‘Writing R Extensions’ manual. It is now a NOTE instead of a WARNING. Former-commit-id: fbd9108f8f7f6212fbc51c828b6ecd104109c4fa --- R/plotBambu.R | 6 +++++- 1 file changed, 5 insertions(+), 1 deletion(-) diff --git a/R/plotBambu.R b/R/plotBambu.R index c46a289b..06f94805 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -15,7 +15,11 @@ #' @importFrom ggbio autoplot #' @importFrom gridExtra grid.arrange #' @export -plot.bambu <- function(se, group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ +plot <- function(se, ..., group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ + UseMethod("plot") + } +#' @export +plot.bambu <- function(se,group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ if(type == "annotation"){ if(is.null(gene_id)&(is.null(transcript_id))){ From 9705c4efea473e83f3af9e511c4a0900062b551f Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 16:55:23 +0800 Subject: [PATCH 28/98] Update plotBambu.R Former-commit-id: f192e1301466149f06c21baec99c435739ee9783 --- R/plotBambu.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/R/plotBambu.R b/R/plotBambu.R index 06f94805..f5b2ac39 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -14,7 +14,7 @@ #' @importFrom RColorBrewer brewer.pal #' @importFrom ggbio autoplot #' @importFrom gridExtra grid.arrange -#' @export +#' @noRd plot <- function(se, ..., group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ UseMethod("plot") } From 6f597af64c7ff82057b9bc1c3d53ad09b8bb50aa Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 16:57:01 +0800 Subject: [PATCH 29/98] add in return object * WARNING: Add non-empty \value sections to the following man man/transcriptToGeneExpression.Rd Former-commit-id: 9bedbc60f92a0c1f5a0062194d7747ade0d3931e --- R/transcriptToGeneExpression.R | 1 + 1 file changed, 1 insertion(+) diff --git a/R/transcriptToGeneExpression.R b/R/transcriptToGeneExpression.R index 0dc08522..388d9358 100644 --- a/R/transcriptToGeneExpression.R +++ b/R/transcriptToGeneExpression.R @@ -1,6 +1,7 @@ #' Reduce transcript expression to gene expression #' @title transcript to gene expression #' @param se a summarizedExperiment object from \code{\link{bambu}} +#' @return A SummarizedExperiment object #' @export transcriptToGeneExpression<- function(se){ counts <- as.data.table(assays(se)$counts,keep.rownames = TRUE) From ffd5c368e8788330bd5f3d71332d47bfcb83c9c6 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 16:59:42 +0800 Subject: [PATCH 30/98] return value object * WARNING: Add non-empty \value sections to the following man pages: man/prepareAnnotationsFromGTF.Rd Former-commit-id: 695726ce010dcae2c097d90011c87cdf3dec5ff5 --- R/annotationFunctions.R | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/R/annotationFunctions.R b/R/annotationFunctions.R index fc48c0c1..da0a90b9 100755 --- a/R/annotationFunctions.R +++ b/R/annotationFunctions.R @@ -37,8 +37,8 @@ prepareAnnotations <- function(txdb) { #' Prepare annotation granges object from GTF file #' @title Prepare annotation granges object from GTF file into a GRangesList object #' @param file a GTF file -#' @return grlist a \code{\link{GRangesList}} object, unlike \code\link{readFromGTF}}, -#' this function finds out the equivalence classes between the transcripts, +#' @return A \code{\link{GRangesList}} object +#' @details Unlike \code\link{readFromGTF}}, this function finds out the equivalence classes between the transcripts, #' with \code{\link{mcols}} data having three columns: #' \itemize{ #' \item TXNAME specifying prefix for new gene Ids (genePrefix.number), defaults to empty From 6cf1f5ee7e56068ff3af2024abda5655d2d18281 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 17:00:39 +0800 Subject: [PATCH 31/98] add in value object * WARNING: Add non-empty \value sections to the following man pages: man/prepareAnnotationsFromGTF.Rd, man/transcriptToGeneExpression.Rd Former-commit-id: 2fc8d49ada0ec03c9ad5bf04a1ef24ed19cfc524 --- man/prepareAnnotationsFromGTF.Rd | 5 ++++- 1 file changed, 4 insertions(+), 1 deletion(-) diff --git a/man/prepareAnnotationsFromGTF.Rd b/man/prepareAnnotationsFromGTF.Rd index 56506ce3..a0f2ee65 100644 --- a/man/prepareAnnotationsFromGTF.Rd +++ b/man/prepareAnnotationsFromGTF.Rd @@ -10,8 +10,11 @@ prepareAnnotationsFromGTF(file) \item{file}{a GTF file} } \value{ - +A \code{\link{GRangesList}} object } \description{ Prepare annotation granges object from GTF file } +\details{ + +} From d0009cca094e3c66c0633f7c8d51ea74dc409333 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 1 Jul 2020 17:01:29 +0800 Subject: [PATCH 32/98] add in value object * WARNING: Add non-empty \value sections to the following man pages: man/prepareAnnotationsFromGTF.Rd, man/transcriptToGeneExpression.Rd Former-commit-id: 3205c4dcf9d0af243c39e620f1b7efd319805805 --- man/transcriptToGeneExpression.Rd | 3 +++ 1 file changed, 3 insertions(+) diff --git a/man/transcriptToGeneExpression.Rd b/man/transcriptToGeneExpression.Rd index 354594d6..efcc1a4a 100644 --- a/man/transcriptToGeneExpression.Rd +++ b/man/transcriptToGeneExpression.Rd @@ -9,6 +9,9 @@ transcriptToGeneExpression(se) \arguments{ \item{se}{a summarizedExperiment object from \code{\link{bambu}}} } +\value{ +A SummarizedExperiment object +} \description{ Reduce transcript expression to gene expression } From f46895178a52c97db05e928386ddc825596928ec Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:01:50 +0000 Subject: [PATCH 33/98] Added seoutput for BSgenome Former-commit-id: b297d6f0c21cf23692df73dfc5df363e1ccaca15 --- 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z>c56(`>8`ytH6C5G?mb#@MbnG)k|xDoE{*|e(ELxe9#H>&@h7x Date: Thu, 2 Jul 2020 08:02:26 +0000 Subject: [PATCH 34/98] slightly modified code when testing for bsgenome to fix test failure Former-commit-id: bd01e7024972337f860a353995c6222ffce64d64 --- tests/testthat/test_isore.R | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/tests/testthat/test_isore.R b/tests/testthat/test_isore.R index bed9d309..555f1419 100644 --- a/tests/testthat/test_isore.R +++ b/tests/testthat/test_isore.R @@ -10,7 +10,7 @@ test_that("isore.constructReadClasses completes successfully", { seReadClassUnstrandedExpected <- readRDS(system.file("extdata", "seReadClassUnstranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) seReadClassStrandedExpected <- readRDS(system.file("extdata", "seReadClassStranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) - + seReadClassFromBsgenomeExpected <- readRDS(system.file("extdata", "seReadClassBsgenomeStranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) seReadClassUnstranded <- isore.constructReadClasses(readGrgList = readGrgList, runName ='SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000_unstranded', @@ -39,7 +39,7 @@ test_that("isore.constructReadClasses completes successfully", { ncore = 1, verbose = FALSE) - expect_equal(seReadClassFromBsgenome,seReadClassStrandedExpected) + expect_equal(seReadClassFromBsgenome,seReadClassFromBsgenomeExpected) }) From fcb823b34f2c3176d48cb2a75007df558ac5c121 Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:03:01 +0000 Subject: [PATCH 35/98] Added run examples for main functions Former-commit-id: 5612f7d8aad119f5135184e6b4395038e9342b54 --- R/annotationFunctions.R | 5 +++-- R/bambu.R | 13 +++++++------ R/plotBambu.R | 6 ++++++ R/readWrite.R | 10 ++++++++++ R/transcriptToGeneExpression.R | 3 +++ 5 files changed, 29 insertions(+), 8 deletions(-) diff --git a/R/annotationFunctions.R b/R/annotationFunctions.R index da0a90b9..7a48f2c7 100755 --- a/R/annotationFunctions.R +++ b/R/annotationFunctions.R @@ -4,11 +4,9 @@ #' @return A \code{\link{GRangesList}} object #' @export #' @examples -#' \dontrun{ #' library(TxDb.Hsapiens.UCSC.hg38.knownGene) #' txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene #' prepareAnnotations(txdb) -#' } prepareAnnotations <- function(txdb) { exonsByTx = exonsBy(txdb,by='tx', use.names=TRUE) if(any(duplicated(names(exonsByTx)))) { @@ -47,6 +45,9 @@ prepareAnnotations <- function(txdb) { #' } #' #' @export +#' @examples +#' gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") +#' gr <- prepareAnnotationsFromGTF(gtf.file) prepareAnnotationsFromGTF <- function(file){ if (missing(file)){ stop('A GTF file is required.') diff --git a/R/bambu.R b/R/bambu.R index f6484987..0b537b3a 100755 --- a/R/bambu.R +++ b/R/bambu.R @@ -32,14 +32,15 @@ #' @details #' @return A list of two SummarizedExperiment object for transcript expression and gene expression. #' @examples -#' \dontrun{ +#' #' ## ===================== -#' ## More stringent new gene/isoform discovery: new isoforms are identified with at least 5 read count in 1 sample +#' ## Minimum read support 5 #' ## Increase EM convergence threshold to 10^(-6) -#' seOutput <- bambu(reads, annotationGrangesList, -#' genomeSequence, isoreParameters = list(min.readCount=5), -#' emParameters = list(conv = 10^(-6)) -#' } +#' test.bam <- system.file("extdata", "SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.bam", package = "bambu") +#' gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) +#' fa.file <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", package = "bambu") +#' se = bambu(reads = test.bam, annotations = gr, genomeSequence = fa.file, extendAnnotations = FALSE) +#' #' @export bambu <- function(reads = NULL, readClass.file = NULL, readClass.outputDir = NULL, annotations = NULL, genomeSequence = NULL, diff --git a/R/plotBambu.R b/R/plotBambu.R index f5b2ac39..282b24a1 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -15,10 +15,16 @@ #' @importFrom ggbio autoplot #' @importFrom gridExtra grid.arrange #' @noRd + plot <- function(se, ..., group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ UseMethod("plot") } #' @export +#' @examples +#' se <- readRDS(system.file("extdata", "seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +#' colnames(se) <- colData(se)$name <- c("sample1","sample2") +#' assays(se)$CPM[,2] <- pmax(0, rnorm(length(assays(se)$CPM[,2]),assays(se)$CPM[,2],10)) +#' plot.bambu(se, type = "heatmap") plot.bambu <- function(se,group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ if(type == "annotation"){ diff --git a/R/readWrite.R b/R/readWrite.R index 6fc7314b..92b705f1 100644 --- a/R/readWrite.R +++ b/R/readWrite.R @@ -5,6 +5,10 @@ #' @return The function will generate three files, a .gtf file for the annotations, #' two .txt files for transcript and gene counts respectively. #' @export +#' @examples +#' se <- readRDS(system.file("extdata", "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +#' path <- tempdir() +#' writeBambuOutput(se,path) writeBambuOutput <- function(se,path){ if (missing(se) | missing(path)){ stop('Both summarizedExperiment object from bambu and the path for the output files are required.') @@ -35,6 +39,9 @@ writeBambuOutput <- function(se,path){ #' @param geneIDs an optional dataframe of geneIDs (column 2) with the corresponding transcriptIDs (column 1) #' @return gtf a GTF dataframe #' @export +#' outputGtfFile <- tempfile() +#' gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) +#' writeToGTF(gr, outputGtfFile) writeToGTF <- function (annotation,file,geneIDs=NULL) { if (missing(annotation) | missing(file)){ stop('Both GRangesList and the name of the output file are required.') @@ -89,6 +96,9 @@ writeToGTF <- function (annotation,file,geneIDs=NULL) { #' \item GENEID indicating whether filter to remove read classes which are a subset of known transcripts(), defaults to TRUE #' } #' @export +#' @examples +#' gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") +#' readFromGTF(gtf.file) readFromGTF <- function(file){ if (missing(file)){ stop('A GTF file is required.') diff --git a/R/transcriptToGeneExpression.R b/R/transcriptToGeneExpression.R index 388d9358..5551aa90 100644 --- a/R/transcriptToGeneExpression.R +++ b/R/transcriptToGeneExpression.R @@ -3,6 +3,9 @@ #' @param se a summarizedExperiment object from \code{\link{bambu}} #' @return A SummarizedExperiment object #' @export +#' @examples +#' se <- readRDS(system.file("extdata", "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +#' transcriptToGeneExpression(se) transcriptToGeneExpression<- function(se){ counts <- as.data.table(assays(se)$counts,keep.rownames = TRUE) runnames <- colnames(counts)[-1] From 5abe291109e3730806c5eaffe1d7a504bb19e1a6 Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:05:41 +0000 Subject: [PATCH 36/98] Full spelling of fun.agg to fix this warning in checking run_parallel: warning in dcast(tmp[order(nobs)], tx_sid ~ read_class_sid, fun.agg = length, value.var = "nobs"): partial argument match of 'fun.agg' to 'fun.aggregate' Former-commit-id: abfe29bd768debf840c174109fe3a41612e9020f --- R/abundance_quantification.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/R/abundance_quantification.R b/R/abundance_quantification.R index 3ffb9813..f7ac0825 100755 --- a/R/abundance_quantification.R +++ b/R/abundance_quantification.R @@ -54,7 +54,7 @@ run_parallel <- function(g,conv,bias,maxiter, readClassDt){ gene_sid = g, ntotal = sum(tmp$nobs))) }else{ - tmp_wide <- dcast(tmp[order(nobs)], tx_sid~read_class_sid, fun.agg = length, + tmp_wide <- dcast(tmp[order(nobs)], tx_sid~read_class_sid, fun.aggregate = length, value.var = 'nobs') a_mat <- tmp_wide[,-1,with=FALSE] setDF(a_mat) From f5b569d0fd10142c5603d5a748c419e07879bd1c Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:13:32 +0000 Subject: [PATCH 37/98] Updated documentation Former-commit-id: 403c5fec48713d4104145a1be984af533fb76216 --- R/annotationFunctions.R | 1 - R/plotBambu.R | 1 - man/bambu.Rd | 13 ++++++------ man/plot.bambu.Rd | 34 ------------------------------- man/prepareAnnotations.Rd | 2 -- man/prepareAnnotationsFromGTF.Rd | 4 ++++ man/readFromGTF.Rd | 4 ++++ man/transcriptToGeneExpression.Rd | 4 ++++ man/writeBambuOutput.Rd | 5 +++++ 9 files changed, 24 insertions(+), 44 deletions(-) delete mode 100644 man/plot.bambu.Rd diff --git a/R/annotationFunctions.R b/R/annotationFunctions.R index 7a48f2c7..8c6fc418 100755 --- a/R/annotationFunctions.R +++ b/R/annotationFunctions.R @@ -43,7 +43,6 @@ prepareAnnotations <- function(txdb) { #' \item GENEID indicating whether filter to remove read classes which are a subset of known transcripts(), defaults to TRUE #' \item eqClass specifying minimun read count to consider a read class valid in a sample, defaults to 2 #' } -#' #' @export #' @examples #' gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") diff --git a/R/plotBambu.R b/R/plotBambu.R index 282b24a1..216f841b 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -15,7 +15,6 @@ #' @importFrom ggbio autoplot #' @importFrom gridExtra grid.arrange #' @noRd - plot <- function(se, ..., group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ UseMethod("plot") } diff --git a/man/bambu.Rd b/man/bambu.Rd index 9de214a9..728416b1 100644 --- a/man/bambu.Rd +++ b/man/bambu.Rd @@ -70,12 +70,13 @@ When multiple samples are provided, extended annotations will be combined across Main function } \examples{ -\dontrun{ + ## ===================== -## More stringent new gene/isoform discovery: new isoforms are identified with at least 5 read count in 1 sample +## Minimum read support 5 ## Increase EM convergence threshold to 10^(-6) -seOutput <- bambu(reads, annotationGrangesList, -genomeSequence, isoreParameters = list(min.readCount=5), -emParameters = list(conv = 10^(-6)) -} + test.bam <- system.file("extdata", "SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.bam", package = "bambu") + gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) + fa.file <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", package = "bambu") + se = bambu(reads = test.bam, annotations = gr, genomeSequence = fa.file, extendAnnotations = FALSE) + } diff --git a/man/plot.bambu.Rd b/man/plot.bambu.Rd deleted file mode 100644 index a26a5012..00000000 --- a/man/plot.bambu.Rd +++ /dev/null @@ -1,34 +0,0 @@ -% Generated by roxygen2: do not edit by hand -% Please edit documentation in R/plotBambu.R -\name{plot.bambu} -\alias{plot.bambu} -\title{plot.bambu} -\usage{ -\method{plot}{bambu}( - se, - group.variable = NULL, - type = c("annotation", "pca", "heatmap"), - gene_id = NULL, - transcript_id = NULL -) -} -\arguments{ -\item{se}{An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGeneExpression}}.} - -\item{group.variable}{Variable for grouping in plot, has be to provided if choosing to plot PCA.} - -\item{type}{plot type variable, a values of annotation for a single gene with heatmap for isoform expressions, pca, or heatmap, see details.} - -\item{gene_id}{specifying the gene_id for plotting gene annotation, either gene_id or transcript_id has to be provided when type = "annotation".} - -\item{transcript_id}{specifying the transcript_id for plotting transcript annotation, either gene_id or transcript_id has to be provided when type = "annotation"} -} -\value{ -A heatmap plot for all samples -} -\description{ -plotSEOuptut -} -\details{ -\code{\link{type}} indicates the type of plots to be plotted. There are two types of plots can be chosen, PCA or heatmap. -} diff --git a/man/prepareAnnotations.Rd b/man/prepareAnnotations.Rd index 73b57568..dc4100fc 100644 --- a/man/prepareAnnotations.Rd +++ b/man/prepareAnnotations.Rd @@ -16,9 +16,7 @@ A \code{\link{GRangesList}} object Function to prepare tables and genomic ranges for transript reconstruction using a txdb object } \examples{ -\dontrun{ library(TxDb.Hsapiens.UCSC.hg38.knownGene) txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene prepareAnnotations(txdb) - } } diff --git a/man/prepareAnnotationsFromGTF.Rd b/man/prepareAnnotationsFromGTF.Rd index a0f2ee65..94fc58ed 100644 --- a/man/prepareAnnotationsFromGTF.Rd +++ b/man/prepareAnnotationsFromGTF.Rd @@ -18,3 +18,7 @@ Prepare annotation granges object from GTF file \details{ } +\examples{ +gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") +gr <- prepareAnnotationsFromGTF(gtf.file) +} diff --git a/man/readFromGTF.Rd b/man/readFromGTF.Rd index ea383f49..c1a08c62 100644 --- a/man/readFromGTF.Rd +++ b/man/readFromGTF.Rd @@ -19,3 +19,7 @@ grlist a \code{\link{GRangesList}} object, with two columns \description{ Outputs GRangesList object from reading a GTF file } +\examples{ +gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") +readFromGTF(gtf.file) +} diff --git a/man/transcriptToGeneExpression.Rd b/man/transcriptToGeneExpression.Rd index efcc1a4a..d6f9636e 100644 --- a/man/transcriptToGeneExpression.Rd +++ b/man/transcriptToGeneExpression.Rd @@ -15,3 +15,7 @@ A SummarizedExperiment object \description{ Reduce transcript expression to gene expression } +\examples{ +se <- readRDS(system.file("extdata", "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +transcriptToGeneExpression(se) +} diff --git a/man/writeBambuOutput.Rd b/man/writeBambuOutput.Rd index 8c6da71a..49533967 100644 --- a/man/writeBambuOutput.Rd +++ b/man/writeBambuOutput.Rd @@ -18,3 +18,8 @@ two .txt files for transcript and gene counts respectively. \description{ Outputs a GTF file, transcript-count file, and gene-count file from bambu } +\examples{ +se <- readRDS(system.file("extdata", "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +path <- tempdir() +writeBambuOutput(se,path) +} From 98501a4f966504d67496ab9b43c130964ed070b8 Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:16:10 +0000 Subject: [PATCH 38/98] add examples Former-commit-id: 30ffc67e214709275858276fe008b1de32c1cff5 --- R/readWrite.R | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/R/readWrite.R b/R/readWrite.R index 92b705f1..43120a83 100644 --- a/R/readWrite.R +++ b/R/readWrite.R @@ -39,8 +39,9 @@ writeBambuOutput <- function(se,path){ #' @param geneIDs an optional dataframe of geneIDs (column 2) with the corresponding transcriptIDs (column 1) #' @return gtf a GTF dataframe #' @export +#' @examples #' outputGtfFile <- tempfile() -#' gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) +#' gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) #' writeToGTF(gr, outputGtfFile) writeToGTF <- function (annotation,file,geneIDs=NULL) { if (missing(annotation) | missing(file)){ From 7ca352be1174432de3f7274895346971a39ebe11 Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:20:16 +0000 Subject: [PATCH 39/98] Updated Rd files Former-commit-id: f0717645bf01e1d86ae3710c5f76ea63f197b4b3 --- R/plotBambu.R | 9 +++++---- man/plot.bambu.Rd | 40 ++++++++++++++++++++++++++++++++++++++++ man/writeToGTF.Rd | 5 +++++ 3 files changed, 50 insertions(+), 4 deletions(-) create mode 100644 man/plot.bambu.Rd diff --git a/R/plotBambu.R b/R/plotBambu.R index 216f841b..ee65ff95 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -1,3 +1,8 @@ + +#' @noRd +plot <- function(se, ..., group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ + UseMethod("plot") + } #' plotSEOuptut #' @title plot.bambu #' @param se An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGeneExpression}}. @@ -14,10 +19,6 @@ #' @importFrom RColorBrewer brewer.pal #' @importFrom ggbio autoplot #' @importFrom gridExtra grid.arrange -#' @noRd -plot <- function(se, ..., group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ - UseMethod("plot") - } #' @export #' @examples #' se <- readRDS(system.file("extdata", "seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) diff --git a/man/plot.bambu.Rd b/man/plot.bambu.Rd new file mode 100644 index 00000000..fc34b4b7 --- /dev/null +++ b/man/plot.bambu.Rd @@ -0,0 +1,40 @@ +% Generated by roxygen2: do not edit by hand +% Please edit documentation in R/plotBambu.R +\name{plot.bambu} +\alias{plot.bambu} +\title{plot.bambu} +\usage{ +\method{plot}{bambu}( + se, + group.variable = NULL, + type = c("annotation", "pca", "heatmap"), + gene_id = NULL, + transcript_id = NULL +) +} +\arguments{ +\item{se}{An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGeneExpression}}.} + +\item{group.variable}{Variable for grouping in plot, has be to provided if choosing to plot PCA.} + +\item{type}{plot type variable, a values of annotation for a single gene with heatmap for isoform expressions, pca, or heatmap, see details.} + +\item{gene_id}{specifying the gene_id for plotting gene annotation, either gene_id or transcript_id has to be provided when type = "annotation".} + +\item{transcript_id}{specifying the transcript_id for plotting transcript annotation, either gene_id or transcript_id has to be provided when type = "annotation"} +} +\value{ +A heatmap plot for all samples +} +\description{ +plotSEOuptut +} +\details{ +\code{\link{type}} indicates the type of plots to be plotted. There are two types of plots can be chosen, PCA or heatmap. +} +\examples{ +se <- readRDS(system.file("extdata", "seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +colnames(se) <- colData(se)$name <- c("sample1","sample2") +assays(se)$CPM[,2] <- pmax(0, rnorm(length(assays(se)$CPM[,2]),assays(se)$CPM[,2],10)) +plot.bambu(se, type = "heatmap") +} diff --git a/man/writeToGTF.Rd b/man/writeToGTF.Rd index d773f051..a2b959bc 100644 --- a/man/writeToGTF.Rd +++ b/man/writeToGTF.Rd @@ -19,3 +19,8 @@ gtf a GTF dataframe \description{ Write annotation GRangesList into a GTF file } +\examples{ +outputGtfFile <- tempfile() +gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) +writeToGTF(gr, outputGtfFile) +} From f93917f53b1eebe39c5909fc52f958958607c4eb Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:28:51 +0000 Subject: [PATCH 40/98] There is a typo in the documentation for prepareAnnotationFromGTF Former-commit-id: e2dc250cebd028fa704f75051aa72aea5314985d --- R/annotationFunctions.R | 8 +++++--- 1 file changed, 5 insertions(+), 3 deletions(-) diff --git a/R/annotationFunctions.R b/R/annotationFunctions.R index 8c6fc418..34af852d 100755 --- a/R/annotationFunctions.R +++ b/R/annotationFunctions.R @@ -36,8 +36,8 @@ prepareAnnotations <- function(txdb) { #' @title Prepare annotation granges object from GTF file into a GRangesList object #' @param file a GTF file #' @return A \code{\link{GRangesList}} object -#' @details Unlike \code\link{readFromGTF}}, this function finds out the equivalence classes between the transcripts, -#' with \code{\link{mcols}} data having three columns: +#' @details Unlike \code{\link{readFromGTF}}, this function finds out the equivalence classes between the transcripts, +#' with \code{\link{mcols}} data having three columns: #' \itemize{ #' \item TXNAME specifying prefix for new gene Ids (genePrefix.number), defaults to empty #' \item GENEID indicating whether filter to remove read classes which are a subset of known transcripts(), defaults to TRUE @@ -45,7 +45,9 @@ prepareAnnotations <- function(txdb) { #' } #' @export #' @examples -#' gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") +#' gtf.file <- system.file("extdata", +#' "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", +#' package = "bambu") #' gr <- prepareAnnotationsFromGTF(gtf.file) prepareAnnotationsFromGTF <- function(file){ if (missing(file)){ From 6b39e7fdbb2621b66870484af532bf2fe334baa4 Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:29:22 +0000 Subject: [PATCH 41/98] Indented the examples to make sure it can be fully seen in manual file Former-commit-id: 07be3b94fde9194f59fcdf57343ce8b9e4f65c4e --- R/bambu.R | 15 +++++++++++---- R/plotBambu.R | 7 +++++-- R/readWrite.R | 12 +++++++++--- R/transcriptToGeneExpression.R | 4 +++- 4 files changed, 28 insertions(+), 10 deletions(-) diff --git a/R/bambu.R b/R/bambu.R index 0b537b3a..3e5a53b9 100755 --- a/R/bambu.R +++ b/R/bambu.R @@ -36,10 +36,17 @@ #' ## ===================== #' ## Minimum read support 5 #' ## Increase EM convergence threshold to 10^(-6) -#' test.bam <- system.file("extdata", "SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.bam", package = "bambu") -#' gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) -#' fa.file <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", package = "bambu") -#' se = bambu(reads = test.bam, annotations = gr, genomeSequence = fa.file, extendAnnotations = FALSE) +#' test.bam <- system.file("extdata", +#' "SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.bam", +#' package = "bambu") +#' gr <- readRDS(system.file("extdata", +#' "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", +#' package = "bambu")) +#' fa.file <- system.file("extdata", +#' "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", +#' package = "bambu") +#' se = bambu(reads = test.bam, annotations = gr, +#' genomeSequence = fa.file, extendAnnotations = FALSE) #' #' @export bambu <- function(reads = NULL, readClass.file = NULL, readClass.outputDir = NULL, diff --git a/R/plotBambu.R b/R/plotBambu.R index ee65ff95..64d732de 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -21,9 +21,12 @@ plot <- function(se, ..., group.variable = NULL, type = c("annotation","pca","he #' @importFrom gridExtra grid.arrange #' @export #' @examples -#' se <- readRDS(system.file("extdata", "seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +#' se <- readRDS(system.file("extdata", +#' "seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", +#' package = "bambu")) #' colnames(se) <- colData(se)$name <- c("sample1","sample2") -#' assays(se)$CPM[,2] <- pmax(0, rnorm(length(assays(se)$CPM[,2]),assays(se)$CPM[,2],10)) +#' assays(se)$CPM[,2] <- pmax(0, rnorm(length(assays(se)$CPM[,2]), +#' assays(se)$CPM[,2],10)) #' plot.bambu(se, type = "heatmap") plot.bambu <- function(se,group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ diff --git a/R/readWrite.R b/R/readWrite.R index 43120a83..58f32be0 100644 --- a/R/readWrite.R +++ b/R/readWrite.R @@ -6,7 +6,9 @@ #' two .txt files for transcript and gene counts respectively. #' @export #' @examples -#' se <- readRDS(system.file("extdata", "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +#' se <- readRDS(system.file("extdata", +#' "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", +#' package = "bambu")) #' path <- tempdir() #' writeBambuOutput(se,path) writeBambuOutput <- function(se,path){ @@ -41,7 +43,9 @@ writeBambuOutput <- function(se,path){ #' @export #' @examples #' outputGtfFile <- tempfile() -#' gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) +#' gr <- readRDS(system.file("extdata", +#' "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", +#' package = "bambu")) #' writeToGTF(gr, outputGtfFile) writeToGTF <- function (annotation,file,geneIDs=NULL) { if (missing(annotation) | missing(file)){ @@ -98,7 +102,9 @@ writeToGTF <- function (annotation,file,geneIDs=NULL) { #' } #' @export #' @examples -#' gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") +#' gtf.file <- system.file("extdata", +#' "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", +#' package = "bambu") #' readFromGTF(gtf.file) readFromGTF <- function(file){ if (missing(file)){ diff --git a/R/transcriptToGeneExpression.R b/R/transcriptToGeneExpression.R index 5551aa90..475e6b8e 100644 --- a/R/transcriptToGeneExpression.R +++ b/R/transcriptToGeneExpression.R @@ -4,7 +4,9 @@ #' @return A SummarizedExperiment object #' @export #' @examples -#' se <- readRDS(system.file("extdata", "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +#' se <- readRDS(system.file("extdata", +#' "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", +#' package = "bambu")) #' transcriptToGeneExpression(se) transcriptToGeneExpression<- function(se){ counts <- as.data.table(assays(se)$counts,keep.rownames = TRUE) From 95efd3a1eed45df251ab3f124b2e07dc303c65fd Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:29:40 +0000 Subject: [PATCH 42/98] Updated Rd files Former-commit-id: 41eeedb34946539257e96433b56ead2734866846 --- man/bambu.Rd | 15 +++++++++++---- man/plot.bambu.Rd | 7 +++++-- man/prepareAnnotationsFromGTF.Rd | 12 ++++++++++-- man/readFromGTF.Rd | 4 +++- man/transcriptToGeneExpression.Rd | 4 +++- man/writeBambuOutput.Rd | 4 +++- man/writeToGTF.Rd | 4 +++- 7 files changed, 38 insertions(+), 12 deletions(-) diff --git a/man/bambu.Rd b/man/bambu.Rd index 728416b1..907a1cae 100644 --- a/man/bambu.Rd +++ b/man/bambu.Rd @@ -74,9 +74,16 @@ Main function ## ===================== ## Minimum read support 5 ## Increase EM convergence threshold to 10^(-6) - test.bam <- system.file("extdata", "SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.bam", package = "bambu") - gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) - fa.file <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", package = "bambu") - se = bambu(reads = test.bam, annotations = gr, genomeSequence = fa.file, extendAnnotations = FALSE) + test.bam <- system.file("extdata", + "SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.bam", + package = "bambu") + gr <- readRDS(system.file("extdata", + "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", + package = "bambu")) + fa.file <- system.file("extdata", + "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", + package = "bambu") + se = bambu(reads = test.bam, annotations = gr, + genomeSequence = fa.file, extendAnnotations = FALSE) } diff --git a/man/plot.bambu.Rd b/man/plot.bambu.Rd index fc34b4b7..26f704b6 100644 --- a/man/plot.bambu.Rd +++ b/man/plot.bambu.Rd @@ -33,8 +33,11 @@ plotSEOuptut \code{\link{type}} indicates the type of plots to be plotted. There are two types of plots can be chosen, PCA or heatmap. } \examples{ -se <- readRDS(system.file("extdata", "seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +se <- readRDS(system.file("extdata", +"seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", + package = "bambu")) colnames(se) <- colData(se)$name <- c("sample1","sample2") -assays(se)$CPM[,2] <- pmax(0, rnorm(length(assays(se)$CPM[,2]),assays(se)$CPM[,2],10)) +assays(se)$CPM[,2] <- pmax(0, rnorm(length(assays(se)$CPM[,2]), +assays(se)$CPM[,2],10)) plot.bambu(se, type = "heatmap") } diff --git a/man/prepareAnnotationsFromGTF.Rd b/man/prepareAnnotationsFromGTF.Rd index 94fc58ed..a66c42af 100644 --- a/man/prepareAnnotationsFromGTF.Rd +++ b/man/prepareAnnotationsFromGTF.Rd @@ -16,9 +16,17 @@ A \code{\link{GRangesList}} object Prepare annotation granges object from GTF file } \details{ - +Unlike \code{\link{readFromGTF}}, this function finds out the equivalence classes between the transcripts, +with \code{\link{mcols}} data having three columns: +\itemize{ + \item TXNAME specifying prefix for new gene Ids (genePrefix.number), defaults to empty + \item GENEID indicating whether filter to remove read classes which are a subset of known transcripts(), defaults to TRUE + \item eqClass specifying minimun read count to consider a read class valid in a sample, defaults to 2 + } } \examples{ -gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") +gtf.file <- system.file("extdata", +"Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", +package = "bambu") gr <- prepareAnnotationsFromGTF(gtf.file) } diff --git a/man/readFromGTF.Rd b/man/readFromGTF.Rd index c1a08c62..7516ff1b 100644 --- a/man/readFromGTF.Rd +++ b/man/readFromGTF.Rd @@ -20,6 +20,8 @@ grlist a \code{\link{GRangesList}} object, with two columns Outputs GRangesList object from reading a GTF file } \examples{ -gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") +gtf.file <- system.file("extdata", +"Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", +package = "bambu") readFromGTF(gtf.file) } diff --git a/man/transcriptToGeneExpression.Rd b/man/transcriptToGeneExpression.Rd index d6f9636e..7b62cdac 100644 --- a/man/transcriptToGeneExpression.Rd +++ b/man/transcriptToGeneExpression.Rd @@ -16,6 +16,8 @@ A SummarizedExperiment object Reduce transcript expression to gene expression } \examples{ -se <- readRDS(system.file("extdata", "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +se <- readRDS(system.file("extdata", +"seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", +package = "bambu")) transcriptToGeneExpression(se) } diff --git a/man/writeBambuOutput.Rd b/man/writeBambuOutput.Rd index 49533967..48dffc16 100644 --- a/man/writeBambuOutput.Rd +++ b/man/writeBambuOutput.Rd @@ -19,7 +19,9 @@ two .txt files for transcript and gene counts respectively. Outputs a GTF file, transcript-count file, and gene-count file from bambu } \examples{ -se <- readRDS(system.file("extdata", "seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) +se <- readRDS(system.file("extdata", +"seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", +package = "bambu")) path <- tempdir() writeBambuOutput(se,path) } diff --git a/man/writeToGTF.Rd b/man/writeToGTF.Rd index a2b959bc..f613d853 100644 --- a/man/writeToGTF.Rd +++ b/man/writeToGTF.Rd @@ -21,6 +21,8 @@ Write annotation GRangesList into a GTF file } \examples{ outputGtfFile <- tempfile() -gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) +gr <- readRDS(system.file("extdata", +"annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", +package = "bambu")) writeToGTF(gr, outputGtfFile) } From 04f4f832868892701d41f35a93300c7263a7d619 Mon Sep 17 00:00:00 2001 From: cying111 Date: Thu, 2 Jul 2020 08:29:49 +0000 Subject: [PATCH 43/98] Updated the version Former-commit-id: 93d3512df184b225fcb901ed9d97a6d28f1533b2 --- DESCRIPTION | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/DESCRIPTION b/DESCRIPTION index 28259e6c..948e8739 100755 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -1,7 +1,7 @@ Package: bambu Type: Package Title: Reference-guided isoform reconstruction and quantification for long read RNA-Seq data -Version: 0.1.0 +Version: 0.2.0 Authors@R: c(person("Ying Chen", "Developer", role = "cre",email = "chen_ying@gis.a-star.edu.sg"), person("Jonathan Goeke", "Developer", role = "aut", email = "gokej@gis.a-star.edu.sg")) From 223b8c5e47cca8bdecb8162dabf50574a160eb63 Mon Sep 17 00:00:00 2001 From: cying111 Date: Fri, 3 Jul 2020 03:52:52 +0000 Subject: [PATCH 44/98] delete old extdata files Former-commit-id: 500ff4eb4c5809c2bdf67b8d5b7cc930fe4e61d8 --- ...ationGranges_txdbGrch38_91_chr9_1_1000000.rds | Bin 3692 -> 0 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 1504 -> 0 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 2008 -> 0 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 4440 -> 0 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 4304 -> 0 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 4412 -> 0 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 4272 -> 0 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 3184 -> 0 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 3180 -> 0 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 3184 -> 0 bytes 10 files changed, 0 insertions(+), 0 deletions(-) delete mode 100644 inst/extdata/extendedAnnotationGranges_txdbGrch38_91_chr9_1_1000000.rds delete mode 100644 inst/extdata/seIsoReCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seIsoReRef_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seOutputCombinedExtended_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seOutputExtended_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seReadClassStranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seReadClassUnstranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seReadClass_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds diff --git a/inst/extdata/extendedAnnotationGranges_txdbGrch38_91_chr9_1_1000000.rds b/inst/extdata/extendedAnnotationGranges_txdbGrch38_91_chr9_1_1000000.rds deleted file mode 100644 index d68bc6fb1c0088f157d95b5225df042c590f4794..0000000000000000000000000000000000000000 GIT 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45/98] Add new extdata files Former-commit-id: 38603107fc07e1631cead9480177251e6c989396 --- ...ationGranges_txdbGrch38_91_chr9_1_1000000.rds | Bin 0 -> 3692 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 1504 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 2008 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 4440 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 4304 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 4412 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 4272 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 3184 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 3180 bytes ..._directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 3184 bytes 10 files changed, 0 insertions(+), 0 deletions(-) create mode 100644 inst/extdata/extendedAnnotationGranges_txdbGrch38_91_chr9_1_1000000.rds create mode 100644 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zESgaXc#xfWLR+&b^wcWxH3pRo?DE@6E^`l+sME;V;}=V#iP3Xlu1;S$0EHy6dhxQvNp zEr_Ygyvc64!$!BYCx&e<>9KoEptGsNuR=*X9|m~kTJwE97d6x{5D*E$_{kQBsINw> zd@#5x2~RXZlas}vAR99_r9>Q!GI9YH~vGZ)Y7>?R@gPhQ? zAlDN(k<2r`UrKYV5RRsj9_!U=FR^4q2EUKq?Wr{D_PVm4vk&v9%m?y*45`x&uEb{V z(CuOm2iBkx9{dKguCp!->+aJ9#7cRxEczxK%!v4QI0QdcQJqW9p zq5!vIgErP2*N)Iv$u9y#00aR8x5Co==y#9aaa^OC-A}f^$F+ z%#~T~xR)T=MZU(XhBNAX_9_$Zlo7s5FP}?-PUY9=QsX^SE3Pj-4O@l@DmzTk5dg5n z2oPT}0LEZIf&vs|Yr0_M@ z|K!SY6;gt{JEP)JUKm!W!iY6v7{>f0RyvU^{^OxcXBuZS(TZ{u%nX3?h z?bS-FkGW@+2LoV#*4!vWxKs)i`*4|N2pAGY5t?X0@e%-yfCJC4DVKeb=a8ZToN}|^ zT5Up}DZ@LqNC~cM-|pFmCWyjUAWO+i`gQ4QO`3TLuu3Q&GJZ+rCvhMa<*EmQv5l6@ z`H9vnO$E~Yd7#j>Hhs%GL0BD_h>tZY$L!~jn~k{YIo)%!YT~a~Jh_p+^X}%H@L0Fu zwuNiuYVVVJL8%Hq)$ literal 0 HcmV?d00001 From 8167cc2dcad23e5852917a5131846ca47fc71c51 Mon Sep 17 00:00:00 2001 From: cying111 Date: Fri, 3 Jul 2020 08:07:56 +0000 Subject: [PATCH 50/98] Uncomment cache Former-commit-id: 9ad93542b910e77d727856948baf908a5c0368e8 --- .gitignore | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/.gitignore b/.gitignore index 0f278172..2126ec2e 100644 --- a/.gitignore +++ b/.gitignore @@ -29,8 +29,8 @@ vignettes/*.pdf .httr-oauth # knitr and R markdown default cache directories -#*_cache/ -#/cache/ +*_cache/ +/cache/ # Temporary files created by R markdown *.utf8.md From 8e84cfef148a818641bec3e29c7e199f0236d8f6 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Sun, 5 Jul 2020 18:27:30 +0800 Subject: [PATCH 51/98] replace exportPattern("^[[:alpha:]]+") MIME-Version: 1.0 Content-Type: text/plain; charset=UTF-8 Content-Transfer-Encoding: 8bit replace exportPattern("^[[:alpha:]]+") with specific exports exportPattern("^[[:alpha:]]+") exports all objects with alphabets in their names * checking for missing documentation entries ... WARNING Undocumented code objects: ‘abundance_quantification’ ‘aggReadClass’ ‘assignNewGeneIds’ ‘bambu.constructReadClass’ ‘bambu.extendAnnotations’ ‘bambu.quantDT’ ‘bambu.quantify’ ‘calculateDistToAnnotation’ ‘constructSplicedReadClassTables’ ‘constructUnsplicedReadClasses’ ‘createJunctionTable’ ‘cutStartEndFromGrangesList’ ‘data1’ ‘data2’ ‘data3’ ‘data4’ ‘data5’ ‘dropGrangesListElementsByWidth’ ‘emWithL1’ ‘em_theta’ ‘estOutput_wBC’ ‘estOutput_woBC’ ‘evalAnnotationOverlap’ ‘evalutePerformance’ ‘extendGrangesListElements’ ‘findHighConfidenceJunctions’ ‘findSpliceOverlapsByDist’ ‘findSpliceOverlapsQuick’ ‘fitBinomialModel’ ‘getEmptyClassFromSE’ ‘getMinimumEqClassByTx’ ‘helpFun’ ‘isore.combineTranscriptCandidates’ ‘isore.constructReadClasses’ ‘isore.estimateDistanceToAnnotations’ ‘isore.extendAnnotations’ ‘junctionStrandCorrection’ ‘myCompatibleTranscription’ ‘myGaps’ ‘myOneMatch’ ‘predictSpliceJunctions’ ‘prepareDataFromBam’ ‘rangesDist’ ‘run_parallel’ ‘seCombinedExtendedGeneExpected’ ‘seCombinedGeneExpected’ ‘seExtendedGeneExpected’ ‘seGeneExpected’ ‘seUCSCExpected’ ‘seWithDistExpected’ ‘selectEndExonsFromGrangesList’ ‘selectStartExonsFromGrangesList’ ‘spliceStrand’ ‘standardJunctionModels_temp’ ‘txRangesToGeneRanges’ ‘unlistIntrons’ All user-level objects in a package should have documentation entries. See chapter ‘Writing R documentation files’ in the ‘Writing R Extensions’ manual. Former-commit-id: c4cc56ee28cd70ed11faa6c57455d0882a47115c --- NAMESPACE | 10 +++++++++- 1 file changed, 9 insertions(+), 1 deletion(-) diff --git a/NAMESPACE b/NAMESPACE index 09a8c600..21544d74 100755 --- a/NAMESPACE +++ b/NAMESPACE @@ -1,5 +1,4 @@ useDynLib(bambu, .registration=TRUE) -exportPattern("^[[:alpha:]]+") importFrom(Rcpp, evalCpp) importFrom(Rcpp, show) importFrom(Rsamtools, index) @@ -18,3 +17,12 @@ import(ggplot2) import(IRanges, except=c(slice, collapse, setdiff, intersect)) import(S4Vectors, except=c(rename, setequal, setdiff, intersect)) import(SummarizedExperiment) +export(bambu) +export(plot) +export(plot.bambu) +export(prepareAnnotations) +export(prepareAnnotationsFromGTF) +export(readFromGTF) +export(transcriptToGeneExpression) +export(writeBambuOutput) +export(writeToGTF) From d012fb8871059f7b5cb3af3a1dcda5c90fc29f7f Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Sun, 5 Jul 2020 18:45:29 +0800 Subject: [PATCH 52/98] plot function with different arguments from rbase Need to export the plot function with plot.bambu arguments to eliminate the warning below: * checking S3 generic/method consistency ... WARNING plot: function(x, ...) plot.bambu: function(se, group.variable, type, gene_id, transcript_id) Former-commit-id: fb6dda099aafdd941bea0175e423c06e4109842b --- man/plot.Rd | 31 +++++++++++++++++++++++++++++++ 1 file changed, 31 insertions(+) create mode 100644 man/plot.Rd diff --git a/man/plot.Rd b/man/plot.Rd new file mode 100644 index 00000000..bf33b901 --- /dev/null +++ b/man/plot.Rd @@ -0,0 +1,31 @@ +% Generated by roxygen2: do not edit by hand +% Please edit documentation in R/plotBambu.R +\name{plot} +\alias{plot} +\title{plot} +\usage{ +plot( + se, + ..., + group.variable = NULL, + type = c("annotation", "pca", "heatmap"), + gene_id = NULL, + transcript_id = NULL +) +} +\arguments{ +\item{se}{An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGeneExpression}}.} + +\item{...}{Additional arguments} + +\item{group.variable}{Variable for grouping in plot, has be to provided if choosing to plot PCA.} + +\item{type}{plot type variable, a values of annotation for a single gene with heatmap for isoform expressions, pca, or heatmap, see details.} + +\item{gene_id}{specifying the gene_id for plotting gene annotation, either gene_id or transcript_id has to be provided when type = "annotation".} + +\item{transcript_id}{specifying the transcript_id for plotting transcript annotation, either gene_id or transcript_id has to be provided when type = "annotation"} +} +\description{ +redefine plot in bambu for plot.bambu +} From 51a90655365e0a56b1e39862084d34eebfd2746d Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Sun, 5 Jul 2020 18:48:00 +0800 Subject: [PATCH 53/98] redefine arguments of the plot function for plot.bambu * checking S3 generic/method consistency ... WARNING plot: function(x, ...) plot.bambu: function(se, group.variable, type, gene_id, transcript_id) Former-commit-id: 0afe22f2c0b432d72c9458c5d4ae775e25b79e43 --- R/plotBambu.R | 14 +++++++++++--- 1 file changed, 11 insertions(+), 3 deletions(-) diff --git a/R/plotBambu.R b/R/plotBambu.R index 64d732de..d591c988 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -1,8 +1,16 @@ - -#' @noRd -plot <- function(se, ..., group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ +#' redefine plot for plot.bambu +#' @title plot +#' @param se An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGeneExpression}}. +#' @param ... Additional arguments +#' @param group.variable Variable for grouping in plot, has be to provided if choosing to plot PCA. +#' @param type plot type variable, a values of annotation for a single gene with heatmap for isoform expressions, pca, or heatmap, see details. +#' @param gene_id specifying the gene_id for plotting gene annotation, either gene_id or transcript_id has to be provided when type = "annotation". +#' @param transcript_id specifying the transcript_id for plotting transcript annotation, either gene_id or transcript_id has to be provided when type = "annotation" +#' @export +plot <- function(se, ...,group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ UseMethod("plot") } + #' plotSEOuptut #' @title plot.bambu #' @param se An summarized experiment object obtained from \code{\link{bambu}} or \code{\link{transcriptToGeneExpression}}. From 5c8a0213008cad9037e649e29d17e81c42df4524 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Sun, 5 Jul 2020 19:59:02 +0800 Subject: [PATCH 54/98] non-empty \value Former-commit-id: ed24e9a6fc6e07a86aeae691c649fbd8d7a8f315 --- R/plotBambu.R | 1 + 1 file changed, 1 insertion(+) diff --git a/R/plotBambu.R b/R/plotBambu.R index d591c988..a5722a5c 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -6,6 +6,7 @@ #' @param type plot type variable, a values of annotation for a single gene with heatmap for isoform expressions, pca, or heatmap, see details. #' @param gene_id specifying the gene_id for plotting gene annotation, either gene_id or transcript_id has to be provided when type = "annotation". #' @param transcript_id specifying the transcript_id for plotting transcript annotation, either gene_id or transcript_id has to be provided when type = "annotation" +#' @return A heatmap plot for all samples #' @export plot <- function(se, ...,group.variable = NULL, type = c("annotation","pca","heatmap"), gene_id = NULL, transcript_id = NULL){ UseMethod("plot") From db771b9f8d533c1aae6419ff9f6e7b2009483ddc Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Sun, 5 Jul 2020 19:59:49 +0800 Subject: [PATCH 55/98] non-empty \value Former-commit-id: 8dc583f3f220eef69fe9a2fddc19a54fac4e1f22 --- man/plot.Rd | 5 ++++- 1 file changed, 4 insertions(+), 1 deletion(-) diff --git a/man/plot.Rd b/man/plot.Rd index bf33b901..ee7fce53 100644 --- a/man/plot.Rd +++ b/man/plot.Rd @@ -26,6 +26,9 @@ plot( \item{transcript_id}{specifying the transcript_id for plotting transcript annotation, either gene_id or transcript_id has to be provided when type = "annotation"} } +\value{ +A heatmap plot for all samples +} \description{ -redefine plot in bambu for plot.bambu +redefine plot for plot.bambu } From 97ec6f33328ad7df6fd3b9f1c20a71c4b715fb34 Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 6 Jul 2020 08:50:35 +0000 Subject: [PATCH 56/98] Fix test bugs for isore Former-commit-id: 01ce6885212b6f2ccaa4cf26278392faaca0cabd --- tests/testthat/test_isore.R | 10 ++++++++-- 1 file changed, 8 insertions(+), 2 deletions(-) diff --git a/tests/testthat/test_isore.R b/tests/testthat/test_isore.R index 555f1419..1b68e5de 100644 --- a/tests/testthat/test_isore.R +++ b/tests/testthat/test_isore.R @@ -19,6 +19,9 @@ test_that("isore.constructReadClasses completes successfully", { stranded = FALSE, ncore = 1, verbose = FALSE) + ## in case of testing on Mac + names(seReadClassUnstranded@rowRanges@elementMetadata@listData$intronStarts) <- NULL + names(seReadClassUnstranded@rowRanges@elementMetadata@listData$intronEnds) <- NULL expect_equal(seReadClassUnstranded, seReadClassUnstrandedExpected) @@ -29,6 +32,8 @@ test_that("isore.constructReadClasses completes successfully", { stranded=TRUE, ncore = 1, verbose = FALSE) + names(seReadClassStranded@rowRanges@elementMetadata@listData$intronStarts) <- NULL + names(seReadClassStranded@rowRanges@elementMetadata@listData$intronEnds) <- NULL expect_equal(seReadClassStranded, seReadClassStrandedExpected) seReadClassFromBsgenome <- isore.constructReadClasses(readGrgList = readGrgList, @@ -38,7 +43,8 @@ test_that("isore.constructReadClasses completes successfully", { stranded = TRUE, ncore = 1, verbose = FALSE) - + names(seReadClassFromBsgenome@rowRanges@elementMetadata@listData$intronStarts) <- NULL + names(seReadClassFromBsgenome@rowRanges@elementMetadata@listData$intronEnds) <- NULL expect_equal(seReadClassFromBsgenome,seReadClassFromBsgenomeExpected) }) @@ -97,6 +103,6 @@ test_that("isore.estimateDistanceToAnnotations completes successfully", { seWithDist <- isore.estimateDistanceToAnnotations(seReadClass = seReadClass1, annotationGrangesList = extendedAnnotations, min.exonDistance = 35) - + names(seWithDist@metadata$distTable$readCount) <- NULL expect_equal(seWithDist, seWithDistExpected) }) From dc45806df3f1c2dffd54e47eeb34cef4990cb2d6 Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 6 Jul 2020 10:18:49 +0000 Subject: [PATCH 57/98] Modify data-raw file Former-commit-id: b28f08eb5f378839e80c9772d1b1de681fb650fa --- data-raw/DATASET.R | 1 + 1 file changed, 1 insertion(+) diff --git a/data-raw/DATASET.R b/data-raw/DATASET.R index 180f785c..0ec1c8c5 100755 --- a/data-raw/DATASET.R +++ b/data-raw/DATASET.R @@ -43,6 +43,7 @@ estOutput_wBC <- lapply(1:5, function(s){ seReadClass1 <- readRDS(system.file("extdata", "seReadClassUnstranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds", package = "bambu")) extendedAnnotations <- readRDS(system.file("extdata", "extendedAnnotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) +gr <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) seWithDistExpected <- isore.estimateDistanceToAnnotations(seReadClass=seReadClass1, From 688f210cc5bca1d4c46cb2e6a81caa51ec087576 Mon Sep 17 00:00:00 2001 From: cying111 Date: Tue, 7 Jul 2020 06:19:28 +0000 Subject: [PATCH 58/98] Delete old rds and rda files Former-commit-id: 93dc3ca951ca4a94bb7c1d5f160fd584071369fe --- R/sysdata.rda | Bin 38207 -> 0 bytes ...tionGranges_txdbGrch38_91_chr9_1_1000000.rds | Bin 3692 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 1504 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 2008 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 4440 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 4304 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 4412 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 4272 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 3240 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 3184 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 3180 -> 0 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 3184 -> 0 bytes 12 files changed, 0 insertions(+), 0 deletions(-) delete mode 100755 R/sysdata.rda delete mode 100644 inst/extdata/extendedAnnotationGranges_txdbGrch38_91_chr9_1_1000000.rds delete mode 100644 inst/extdata/seIsoReCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seIsoReRef_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seOutputCombinedExtended_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seOutputExtended_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seReadClassBsgenomeStranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 100644 inst/extdata/seReadClassStranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds delete mode 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z@|8u!Lz!&3hkc6KyzzasM};zsQb^4I`{`8L7P_J#j)shxD(wm@uOs@B=|q z1+pXX#BP?Dje(@OIHF6vD-?%EC-wTpj~W7g(c%00k!v_aqGDr$a}F$EwqOWclxn{W~+*nTsAxPw*&FJsg3#jZk^eOGMiM=O-Yla8_0002%x-qB#0m&HK W%K-qIF_zFiFb#_W000000a;oqAtXrv literal 0 HcmV?d00001 From 8ffbd36ccdb47fb0694b3b6f5dc2a715659af79c Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Thu, 9 Jul 2020 11:21:20 +0800 Subject: [PATCH 60/98] update RData names from NanoporeRNASeq Former-commit-id: b0ebaa8424b41d42501248d80559bd0fa25d23d7 --- vignettes/bambu.Rmd | 14 ++++++-------- 1 file changed, 6 insertions(+), 8 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 49210f36..4376c7e7 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -42,27 +42,25 @@ sample_info ``` -Name of bamfiles can be loaded as follows +Bamfiles can be loaded as follows ```{r} -data("bamFileNames") -bamFileNames +data("bamFiles") ``` -We then loaded bam.files. +We then loaded annotation. ```{r} -bam.file <- system.file("extdata",bamFileNames, package = "NanoporeRNASeq") -data("annotationGrangesList_chr22_1_25409234") +data("annotation") ``` We applied *bambu* to perform EM on extended annotations ```{r} -seExtended <- bambu(reads = bam.file, annotations = annotationGrangesList_chr22_1_25409234, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE, verbose = FALSE, ncore = 6) +seExtended <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE, verbose = FALSE, ncore = 6) seExtended ``` *bambu* allows quantification without isoform discovery ```{r} -se <- bambu(reads = bam.file, annotations = annotationGrangesList_chr22_1_25409234, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = FALSE, verbose = FALSE, ncore = 6) +se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = FALSE, verbose = FALSE, ncore = 6) se ``` From 15267ce3b2f2dca6025262e4cd689c2e08dbcb76 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Thu, 9 Jul 2020 11:25:15 +0800 Subject: [PATCH 61/98] Update bambu.Rmd Former-commit-id: bf078f3e58d7df2f6ef6e360b7b6109877ab4cf3 --- vignettes/bambu.Rmd | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 4376c7e7..7ccf5461 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -47,7 +47,7 @@ Bamfiles can be loaded as follows data("bamFiles") ``` -We then loaded annotation. +We then load annotation. ```{r} data("annotation") ``` From 0c072ccb342c72157c59d060971a32d783ebee58 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 15 Jul 2020 13:32:58 +0800 Subject: [PATCH 62/98] Update bambu.Rmd Former-commit-id: 8ffee27f27e08fbad7bb633ae98a569f8a3c2cf0 --- vignettes/bambu.Rmd | 38 +++++++++++++++++++++++++++----------- 1 file changed, 27 insertions(+), 11 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 7ccf5461..0aa4f958 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -1,5 +1,6 @@ --- -title: "bambu" +title: "Analysing Long Read RNA-Seq data with bambu" +author: Chen Ying, Yuk Kei Wan, Jonathan Göke output: rmarkdown::html_vignette vignette: > %\VignetteIndexEntry{bambu} @@ -17,10 +18,25 @@ knitr::opts_chunk$set( ## Introduction -*[Bambu](https://github.com/GoekeLab/bambu)* can be used for transcript discovery and quantification from long read RNA-Seq data. Here, we present an example usage of bambu on Nanopore long read RNA-Sequencing from 2 human cancer cell lines - - -## Data description +*[Bambu](https://github.com/GoekeLab/bambu)* can be used for transcript discovery and quantification from long read RNA-Seq data. Here, we present an example workflow for analysing Nanopore long read RNA-Sequencing data from 2 human cancer cell lines + + +### Content +* [Quick Start: Transcript discovery and quantification with bambu](#quick-start) +* [Input data](#input-data) + + [Aligned reads (bam files)](#bam-files) + + Genome sequence (fasta file/ BSGenome object) + + [Genome annotations (bambu annotations object/ gtf file / TxDb object)](#annotations) +* [Transcript discovery and quantification](#transcript-discovery-quantification) + + [Run bambu](#run-bambu) + + [Visualise results](#visualise-results) + + [Obtain gene expression estimates from transcript expression]() + + [Save data (gtf/text)] +* Identify differentially expressed genes +* Identify differential transcript usage +* Running bambu with large sample numbers + +## Data description {#quick-start} To demonstrate the usage of *Bambu*, we used the data in NanoporeRNASeq, which contains single chromosome RNA-Seq data from two common cell lines K562 and MCF7. Each of these cell line has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. @@ -41,19 +57,19 @@ data("sample_info") sample_info ``` - -Bamfiles can be loaded as follows +## Input data {#input-data} +Bamfiles can be loaded as follows {#bam-files} ```{r} data("bamFiles") ``` -We then load annotation. +We then load annotation. {#annotation} ```{r} data("annotation") ``` - -We applied *bambu* to perform EM on extended annotations +## Transcript discovery and quantification {#transcript-discovery-quantification} +We applied *bambu* to perform EM on extended annotations {#run-bambu} ```{r} seExtended <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE, verbose = FALSE, ncore = 6) seExtended @@ -64,7 +80,7 @@ se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgeno se ``` -## Check expression estimates +## Check expression estimates We can check the estimated transcript expression using heatmap: ```{r, fig.width = 8, fig.height = 6} colData(seExtended)$groupVar <- unlist(lapply(colnames(seExtended),function(x) unlist(strsplit(x,"_"))[2])) From 40ed4783e46e9be31cb2863201413880df2bed90 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 15 Jul 2020 14:34:37 +0800 Subject: [PATCH 63/98] seExtended -> se and se -> seUnextended Former-commit-id: 0211c10a9f3b4cefcd1c1faa02592c4736c6666d --- vignettes/bambu.Rmd | 34 +++++++++++++++++----------------- 1 file changed, 17 insertions(+), 17 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 0aa4f958..d7dee5c3 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -31,7 +31,7 @@ knitr::opts_chunk$set( + [Run bambu](#run-bambu) + [Visualise results](#visualise-results) + [Obtain gene expression estimates from transcript expression]() - + [Save data (gtf/text)] + + [Save data (gtf/text)]() * Identify differentially expressed genes * Identify differential transcript usage * Running bambu with large sample numbers @@ -71,28 +71,28 @@ data("annotation") ## Transcript discovery and quantification {#transcript-discovery-quantification} We applied *bambu* to perform EM on extended annotations {#run-bambu} ```{r} -seExtended <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE, verbose = FALSE, ncore = 6) -seExtended +se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE) +se ``` *bambu* allows quantification without isoform discovery ```{r} -se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = FALSE, verbose = FALSE, ncore = 6) -se +seUnextended <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38") +seUnextended ``` ## Check expression estimates We can check the estimated transcript expression using heatmap: ```{r, fig.width = 8, fig.height = 6} -colData(seExtended)$groupVar <- unlist(lapply(colnames(seExtended),function(x) unlist(strsplit(x,"_"))[2])) -colnames(seExtended) <- gsub("_genome_chr22_1_25409234","",colnames(seExtended)) -colData(seExtended)$name <- gsub("_genome_chr22_1_25409234","",colData(seExtended)$name) -plot.bambu(seExtended, group.variable = "groupVar", type = "heatmap") +colData(se)$groupVar <- unlist(lapply(colnames(se),function(x) unlist(strsplit(x,"_"))[2])) +colnames(se) <- gsub("_genome_chr22_1_25409234","",colnames(se)) +colData(se)$name <- gsub("_genome_chr22_1_25409234","",colData(se)$name) +plot.bambu(se, group.variable = "groupVar", type = "heatmap") ``` or with PCA plot ```{r, fig.width = 8, fig.height = 6} -colData(seExtended)$groupVar <- unlist(lapply(colnames(seExtended),function(x) unlist(strsplit(x,"_"))[2])) -plot.bambu(seExtended, group.variable = "groupVar", type = "pca") +colData(se)$groupVar <- unlist(lapply(colnames(se),function(x) unlist(strsplit(x,"_"))[2])) +plot.bambu(se, group.variable = "groupVar", type = "pca") ``` @@ -100,14 +100,14 @@ plot.bambu(seExtended, group.variable = "groupVar", type = "pca") Single gene examples can also be checked using plot functions from *bambu* ```{r, fig.width = 8, fig.height = 10} -plot.bambu(seExtended, type = "annotation", gene_id = unique(rowData(seExtended)$GENEID)[10]) +plot.bambu(se, type = "annotation", gene_id = unique(rowData(seExtended)$GENEID)[10]) ``` ### Transcript to Gene expression Gene expression can be obtained from transcript expression using this function: ```{r} -seGene <- transcriptToGeneExpression(seExtended) +seGene <- transcriptToGeneExpression(se) seGene ``` @@ -128,7 +128,7 @@ We used *DESeq2* to find the differentially expressed genes: ```{r} library(DESeq2) dds <- DESeqDataSetFromMatrix(apply(assays(seGene)$counts,c(1,2),round),#tmp_wide[,-1], - colData = colData(seExtended), + colData = colData(se), design = ~ groupVar) system.time(dds.deseq <- DESeq(dds)) @@ -158,14 +158,14 @@ plotMA(resLFC, ylim = c(-3,3)) We used *DEXSeq* to detect alternative used isoforms. ```{r} library(DRIMSeq) -count.data <- as.data.frame(rowData(seExtended)) +count.data <- as.data.frame(rowData(se)) count.data$gene_id <- count.data$GENEID count.data$feature_id <- count.data$TXNAME count.data$GENEID <- count.data$TXNAME <- NULL -count.data <- cbind(count.data, assays(seExtended)$counts) +count.data <- cbind(count.data, assays(se)$counts) -sample.info <- as.data.frame(colData(seExtended)) +sample.info <- as.data.frame(colData(se)) sample.info$sample_id <- sample.info$name sample.info$name <- NULL d <- dmDSdata(counts=count.data, samples=sample.info) From 0027224745b0c5c614303484bbc9561a6143b8f3 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 15 Jul 2020 18:00:53 +0800 Subject: [PATCH 64/98] colData(se) using sample_info$protocol Former-commit-id: 9019ab7dd012bafdb1792736f75d8dbc696013f4 --- vignettes/bambu.Rmd | 14 +++++++------- 1 file changed, 7 insertions(+), 7 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index d7dee5c3..cdebcfbc 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -83,15 +83,15 @@ seUnextended ## Check expression estimates We can check the estimated transcript expression using heatmap: ```{r, fig.width = 8, fig.height = 6} -colData(se)$groupVar <- unlist(lapply(colnames(se),function(x) unlist(strsplit(x,"_"))[2])) -colnames(se) <- gsub("_genome_chr22_1_25409234","",colnames(se)) -colData(se)$name <- gsub("_genome_chr22_1_25409234","",colData(se)$name) +colData(se)$groupVar <- sample_info$protocol +colnames(se) <- sample_info$sample_id +colData(se)$name <- sample_info$sample_id plot.bambu(se, group.variable = "groupVar", type = "heatmap") ``` or with PCA plot ```{r, fig.width = 8, fig.height = 6} -colData(se)$groupVar <- unlist(lapply(colnames(se),function(x) unlist(strsplit(x,"_"))[2])) +colData(se)$groupVar <- sample_info$protocol plot.bambu(se, group.variable = "groupVar", type = "pca") ``` @@ -100,7 +100,7 @@ plot.bambu(se, group.variable = "groupVar", type = "pca") Single gene examples can also be checked using plot functions from *bambu* ```{r, fig.width = 8, fig.height = 10} -plot.bambu(se, type = "annotation", gene_id = unique(rowData(seExtended)$GENEID)[10]) +plot.bambu(se, type = "annotation", gene_id = unique(rowData(se)$GENEID)[10]) ``` @@ -113,13 +113,13 @@ seGene Gene expression heatmap ```{r, fig.width = 8, fig.height = 6} -colData(seGene)$groupVar <- unlist(lapply(colnames(seGene),function(x) unlist(strsplit(x,"_"))[2])) +colData(seGene)$groupVar <- sample_info$protocol plot.bambu(seGene, group.variable = "groupVar", type = "heatmap") ``` Gene expression PCA plot ```{r, fig.width = 8, fig.height = 6} -colData(seGene)$groupVar <- unlist(lapply(colnames(seGene),function(x) unlist(strsplit(x,"_"))[2])) +colData(seGene)$groupVar <- sample_info$protocol plot.bambu(seGene, group.variable = "groupVar", type = "pca") ``` From 99df5bc486593793db09dd2b18a0e2798dc6c59c Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 15 Jul 2020 19:26:56 +0800 Subject: [PATCH 65/98] protocols to cellLine Former-commit-id: 216e6c08fa22964ee56bb88a76469df91e7738bc --- vignettes/bambu.Rmd | 52 +++++++++++++++++++++++++++------------------ 1 file changed, 31 insertions(+), 21 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index cdebcfbc..28cf68e3 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -25,16 +25,16 @@ knitr::opts_chunk$set( * [Quick Start: Transcript discovery and quantification with bambu](#quick-start) * [Input data](#input-data) + [Aligned reads (bam files)](#bam-files) - + Genome sequence (fasta file/ BSGenome object) + + [Genome sequence (fasta file/ BSGenome object)](#genome-sequences) + [Genome annotations (bambu annotations object/ gtf file / TxDb object)](#annotations) * [Transcript discovery and quantification](#transcript-discovery-quantification) + [Run bambu](#run-bambu) + [Visualise results](#visualise-results) - + [Obtain gene expression estimates from transcript expression]() - + [Save data (gtf/text)]() -* Identify differentially expressed genes -* Identify differential transcript usage -* Running bambu with large sample numbers + + [Obtain gene expression estimates from transcript expression](#gene-expression) + + [Save data (gtf/text)](#save-data) +* [Identify differentially expressed genes](#DESeq2) +* [Identify differential transcript usage](#DEXSeq) +* [Running bambu with large sample numbers](#large-sample-num) ## Data description {#quick-start} To demonstrate the usage of *Bambu*, we used the data in NanoporeRNASeq, which contains single chromosome RNA-Seq data from two common cell lines K562 and MCF7. Each of these cell line has three @@ -58,18 +58,24 @@ sample_info ``` ## Input data {#input-data} -Bamfiles can be loaded as follows {#bam-files} +### Bamfiles can be loaded as follows {#bam-files} ```{r} data("bamFiles") ``` -We then load annotation. {#annotation} +### Loading genome sequences {#genome-sequences} +```{r} +# BSgenome +library(library(BSgenome.Hsapiens.NCBI.GRCh38)) +``` + +### We then load annotation. {#annotation} ```{r} data("annotation") ``` ## Transcript discovery and quantification {#transcript-discovery-quantification} -We applied *bambu* to perform EM on extended annotations {#run-bambu} +### We applied *bambu* to perform EM on extended annotations {#run-bambu} ```{r} se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE) se @@ -80,10 +86,10 @@ seUnextended <- bambu(reads = bamFiles, annotations = annotation, genomeSequence seUnextended ``` -## Check expression estimates +### Check expression estimates {#visualise-results} We can check the estimated transcript expression using heatmap: ```{r, fig.width = 8, fig.height = 6} -colData(se)$groupVar <- sample_info$protocol +colData(se)$groupVar <- sample_info$cellLine colnames(se) <- sample_info$sample_id colData(se)$name <- sample_info$sample_id plot.bambu(se, group.variable = "groupVar", type = "heatmap") @@ -91,12 +97,12 @@ plot.bambu(se, group.variable = "groupVar", type = "heatmap") or with PCA plot ```{r, fig.width = 8, fig.height = 6} -colData(se)$groupVar <- sample_info$protocol +colData(se)$groupVar <- sample_info$cellLine plot.bambu(se, group.variable = "groupVar", type = "pca") ``` -### Check for gene examples +### Check for gene examples Single gene examples can also be checked using plot functions from *bambu* ```{r, fig.width = 8, fig.height = 10} @@ -104,7 +110,7 @@ plot.bambu(se, type = "annotation", gene_id = unique(rowData(se)$GENEID)[10]) ``` -### Transcript to Gene expression +### Transcript to Gene expression {#gene-expression} Gene expression can be obtained from transcript expression using this function: ```{r} seGene <- transcriptToGeneExpression(se) @@ -113,17 +119,22 @@ seGene Gene expression heatmap ```{r, fig.width = 8, fig.height = 6} -colData(seGene)$groupVar <- sample_info$protocol +colData(seGene)$groupVar <- sample_info$cellLine plot.bambu(seGene, group.variable = "groupVar", type = "heatmap") ``` Gene expression PCA plot ```{r, fig.width = 8, fig.height = 6} -colData(seGene)$groupVar <- sample_info$protocol +colData(seGene)$groupVar <- sample_info$cellLine plot.bambu(seGene, group.variable = "groupVar", type = "pca") ``` -## Differentially expressed genes +### Save data (gtf/text){#save-data} +```{r} +writeBambuOutput(se, path = "./bambu/") +``` + +## Differentially expressed genes {#DESeq2} We used *DESeq2* to find the differentially expressed genes: ```{r} library(DESeq2) @@ -148,13 +159,12 @@ plotMA(deGeneRes, ylim = c(-3,3)) Plotting shrinked lFC results ```{r, fig.width = 8, fig.height = 6} +library(apeglm) resLFC <- lfcShrink(dds.deseq, coef="groupVar_MCF7_vs_K562", type="apeglm") plotMA(resLFC, ylim = c(-3,3)) ``` - - -## Differential expression for isoform detection +## Differential expression for isoform detection {#DEXSeq} We used *DEXSeq* to detect alternative used isoforms. ```{r} library(DRIMSeq) @@ -267,10 +277,10 @@ ggplot(dxrDT, aes(y = log2fold_MCF7_K562, x = exonBaseMean, color = as.factor(pa ``` - ```{r} dxrDT[padj<0.5,.(geneID, txID, log2fold_MCF7_K562,K562,MCF7)] ``` +### Running bambu with large sample numbers {#large-sample-num} ```{r} From 5ea8de063ad1d45ece8492c0dbdd6a94ff35e0fc Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 15 Jul 2020 19:42:16 +0800 Subject: [PATCH 66/98] install_github when package go public Former-commit-id: eeaaa86ca8f27888ee2fbd9b073e44a63f404edb --- vignettes/bambu.Rmd | 5 +---- 1 file changed, 1 insertion(+), 4 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 28cf68e3..b07e49d6 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -41,10 +41,7 @@ To demonstrate the usage of *Bambu*, we used the data in NanoporeRNASeq, which c replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. ```{r, include = FALSE} -# To be removed later -devtools::load_all("/mnt/ont/s3.ontdata.store.genome.sg/projects/bambuAnddatapackage/PrepareForBambu/bambu/") -#To be removed later -devtools::load_all("/mnt/ont/s3.ontdata.store.genome.sg/projects/bambuAnddatapackage/PrepareDataPackage/NanoporeRNASeq/") +devtools::install_github("GoekeLab/NanoporeRNASeq") ``` ```{r setup} From 2a0618b04da6c22640523373309cdc42509e9fcd Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 15 Jul 2020 22:09:50 +0800 Subject: [PATCH 67/98] Update bambu.Rmd Former-commit-id: de7aaae4e1bf1dc3a0dd9d4be83444f260bb1c9d --- vignettes/bambu.Rmd | 8 +++++--- 1 file changed, 5 insertions(+), 3 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index b07e49d6..b64f03a8 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -37,8 +37,7 @@ knitr::opts_chunk$set( * [Running bambu with large sample numbers](#large-sample-num) ## Data description {#quick-start} -To demonstrate the usage of *Bambu*, we used the data in NanoporeRNASeq, which contains single chromosome RNA-Seq data from two common cell lines K562 and MCF7. Each of these cell line has three -replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. +To demonstrate the usage of *Bambu*, we used long-read RNA-Seq data generated using Oxford Nanopore Sequencing from the NanoporeRNASeq package, which consists of 6 samples from two human cell lines (K562 and MCF7). Each of these cell lines has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. Reads are aligned to chromosome 22 (Grch38) and stored as bam files. ```{r, include = FALSE} devtools::install_github("GoekeLab/NanoporeRNASeq") @@ -49,12 +48,13 @@ library(NanoporeRNASeq) library(bambu) ``` +## Input data {#input-data} +load sample information ```{r} data("sample_info") sample_info ``` -## Input data {#input-data} ### Bamfiles can be loaded as follows {#bam-files} ```{r} data("bamFiles") @@ -62,6 +62,8 @@ data("bamFiles") ### Loading genome sequences {#genome-sequences} ```{r} +# get path to fasta file +fasta_path <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr22_1_25409234.fa", package="NanoporeRNASeq") # BSgenome library(library(BSgenome.Hsapiens.NCBI.GRCh38)) ``` From 580c33fe714da5305f42634008c4dacc0363d800 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 15 Jul 2020 22:25:21 +0800 Subject: [PATCH 68/98] Update bambu.Rmd Former-commit-id: 3ab18dae1c34cb863e185c91f1eb9dc5fc4cc544 --- vignettes/bambu.Rmd | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index b64f03a8..9cacad14 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -55,12 +55,12 @@ data("sample_info") sample_info ``` -### Bamfiles can be loaded as follows {#bam-files} +### Load Bam files {#bam-files} ```{r} data("bamFiles") ``` -### Loading genome sequences {#genome-sequences} +### Load genome sequences {#genome-sequences} ```{r} # get path to fasta file fasta_path <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr22_1_25409234.fa", package="NanoporeRNASeq") @@ -68,13 +68,13 @@ fasta_path <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembl library(library(BSgenome.Hsapiens.NCBI.GRCh38)) ``` -### We then load annotation. {#annotation} +### Load Annotation {#annotation} ```{r} data("annotation") ``` ## Transcript discovery and quantification {#transcript-discovery-quantification} -### We applied *bambu* to perform EM on extended annotations {#run-bambu} +### Apply *bambu* to perform EM on extended annotations {#run-bambu} ```{r} se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE) se From e92d51d054b36d1566aac6f72d94b25b11f104e0 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Wed, 15 Jul 2020 22:27:56 +0800 Subject: [PATCH 69/98] Update bambu.Rmd Former-commit-id: 9c49a98c54eaf656e29d097d174258676505a10c --- vignettes/bambu.Rmd | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 9cacad14..876c0f10 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -34,7 +34,7 @@ knitr::opts_chunk$set( + [Save data (gtf/text)](#save-data) * [Identify differentially expressed genes](#DESeq2) * [Identify differential transcript usage](#DEXSeq) -* [Running bambu with large sample numbers](#large-sample-num) +* [Running bambu with large number of samples](#large-sample-num) ## Data description {#quick-start} To demonstrate the usage of *Bambu*, we used long-read RNA-Seq data generated using Oxford Nanopore Sequencing from the NanoporeRNASeq package, which consists of 6 samples from two human cell lines (K562 and MCF7). Each of these cell lines has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. Reads are aligned to chromosome 22 (Grch38) and stored as bam files. @@ -279,7 +279,7 @@ ggplot(dxrDT, aes(y = log2fold_MCF7_K562, x = exonBaseMean, color = as.factor(pa ```{r} dxrDT[padj<0.5,.(geneID, txID, log2fold_MCF7_K562,K562,MCF7)] ``` -### Running bambu with large sample numbers {#large-sample-num} +### Running bambu with large number of samples {#large-sample-num} ```{r} From 63cb4c3d2dfbb32145cbdedadc555be403d8a4bc Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Thu, 16 Jul 2020 17:14:52 +0800 Subject: [PATCH 70/98] Update bambu.Rmd Former-commit-id: 36953ef6277dcadee436f438f40852c0e9a4c414 --- vignettes/bambu.Rmd | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 876c0f10..d90c6451 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -65,10 +65,10 @@ data("bamFiles") # get path to fasta file fasta_path <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr22_1_25409234.fa", package="NanoporeRNASeq") # BSgenome -library(library(BSgenome.Hsapiens.NCBI.GRCh38)) +library(BSgenome.Hsapiens.NCBI.GRCh38) ``` -### Load Annotation {#annotation} +### Load Annotation {#annotations} ```{r} data("annotation") ``` From 376b6a2058c11fe02b5d2528bba244b5187aa2f1 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Fri, 17 Jul 2020 10:10:48 +0800 Subject: [PATCH 71/98] update introduction and content table Former-commit-id: c673fdbd0b9dd5745b5c6cec27ab8bb134ac5fd7 --- vignettes/bambu.Rmd | 34 +++++++++++++++++++--------------- 1 file changed, 19 insertions(+), 15 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index d90c6451..75493231 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -1,6 +1,6 @@ --- title: "Analysing Long Read RNA-Seq data with bambu" -author: Chen Ying, Yuk Kei Wan, Jonathan Göke +author: Ying Chen, Yuk Kei Wan, Jonathan Göke output: rmarkdown::html_vignette vignette: > %\VignetteIndexEntry{bambu} @@ -17,24 +17,28 @@ knitr::opts_chunk$set( ``` -## Introduction -*[Bambu](https://github.com/GoekeLab/bambu)* can be used for transcript discovery and quantification from long read RNA-Seq data. Here, we present an example workflow for analysing Nanopore long read RNA-Sequencing data from 2 human cancer cell lines +# Introduction +*[Bambu](https://github.com/GoekeLab/bambu)* is a method for transcript discovery and quantification from long read RNA-Seq data. Bambu uses aligned reads and genome reference annotations as input, and will return abundance estimates for all known transcripts and for newly discovered transcripts. Bambu uses the information from the reference annotations to correct misalignment at splice junctions, then reduces the aligned reads to read equivalent classes, and uses this information to identify novel transcripts across all samples of interest. Reads are then assigned to transcripts, and expression estimates are obtained using an expectation maximisation algorithm. Here, we present an example workflow for analysing Nanopore long read RNA-Sequencing data from two human cancer cell lines from the Singapore Nanopore Expression Project (SG-NEx). -### Content +# Content * [Quick Start: Transcript discovery and quantification with bambu](#quick-start) -* [Input data](#input-data) - + [Aligned reads (bam files)](#bam-files) - + [Genome sequence (fasta file/ BSGenome object)](#genome-sequences) - + [Genome annotations (bambu annotations object/ gtf file / TxDb object)](#annotations) -* [Transcript discovery and quantification](#transcript-discovery-quantification) - + [Run bambu](#run-bambu) - + [Visualise results](#visualise-results) - + [Obtain gene expression estimates from transcript expression](#gene-expression) - + [Save data (gtf/text)](#save-data) -* [Identify differentially expressed genes](#DESeq2) -* [Identify differential transcript usage](#DEXSeq) +* [A complete workflow to identify and quantify transcript expression from Nanopore RNA-Seq data](#complete-workflow) + + [Input data](#input-data) + - [Aligned reads (bam files)](#bam-files) + - [Genome sequence (fasta file/ BSGenome object)](#genome-sequences) + - [Genome annotations (bambu annotations object/ gtf file / TxDb object)](#annotations) + + [Transcript discovery and quantification](#transcript-discovery-quantification) + - [Run bambu](#run-bambu) + - [Visualise results](#visualise-results) + - [Obtain gene expression estimates from transcript expression](#gene-expression) + - [Save data (gtf/text)](#save-data) + + [Identify differentially expressed genes](#DESeq2) + + [Identify differential transcript usage](#DEXSeq) * [Running bambu with large number of samples](#large-sample-num) +* [Getting Help](#get-help) +* [Session Information](#session-info) +* [Citing bambu](#cite-bambu) ## Data description {#quick-start} To demonstrate the usage of *Bambu*, we used long-read RNA-Seq data generated using Oxford Nanopore Sequencing from the NanoporeRNASeq package, which consists of 6 samples from two human cell lines (K562 and MCF7). Each of these cell lines has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. Reads are aligned to chromosome 22 (Grch38) and stored as bam files. From 3e9fec9122a284899fa168f76f7cf15e6155adf4 Mon Sep 17 00:00:00 2001 From: cying111 Date: Fri, 17 Jul 2020 02:18:10 +0000 Subject: [PATCH 72/98] Update badges Former-commit-id: 31e05aaf1bcc414a3cac73244ce5de965b926234 --- README.md | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/README.md b/README.md index 4ca79925..9a0f7af3 100755 --- a/README.md +++ b/README.md @@ -5,10 +5,11 @@ # bambu: reference-guided transcript discovery and quantification for long read RNA-Seq data -[![GitHub release (latest by date)](https://img.shields.io/github/v/release/GoekeLab/bambu?style=plastic)](https://zenodo.org/record/3900025#.XwPOMigzbIU) +[![GitHub release (latest by date)](https://img.shields.io/github/v/release/GoekeLab/bambu?style=plastic)](https://github.com/GoekeLab/bambu/releases/tag/v0.2.0) [![Maintained?](https://img.shields.io/badge/Maintained%3F-Yes-brightgreen)](https://github.com/GoekeLab/bambu/graphs/contributors) [![Install](https://img.shields.io/badge/Install-Github-brightgreen)](#installation) [![License: GPL v3](https://img.shields.io/badge/License-GPLv3-blue.svg)](https://www.gnu.org/licenses/gpl-3.0) +[![DOI](https://zenodo.org/badge/DOI/10.5281/zenodo.3900025.svg)](https://doi.org/10.5281/zenodo.3900025) ***bambu*** is a R package for multi-sample transcript discovery and quantification using long read RNA-Seq data. You can use ***bambu*** after read alignment to obtain expression estimates for known and novel transcripts and genes. The output from ***bambu*** can directly be used for visualisation and downstream analysis such as differential gene expression or transcript usage. From be196acbffe2916214df606b59b15da1adf58aef Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Fri, 17 Jul 2020 10:51:14 +0800 Subject: [PATCH 73/98] Update bambu.Rmd Former-commit-id: ebddb9fa32c7c8d2ad3e76d5f3b8f1722d1db17b --- vignettes/bambu.Rmd | 103 +++++++++++++++++++++++++++++++++++--------- 1 file changed, 82 insertions(+), 21 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 75493231..6d2786a5 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -37,34 +37,62 @@ knitr::opts_chunk$set( + [Identify differential transcript usage](#DEXSeq) * [Running bambu with large number of samples](#large-sample-num) * [Getting Help](#get-help) -* [Session Information](#session-info) * [Citing bambu](#cite-bambu) +* [Session Information](#session-info) -## Data description {#quick-start} -To demonstrate the usage of *Bambu*, we used long-read RNA-Seq data generated using Oxford Nanopore Sequencing from the NanoporeRNASeq package, which consists of 6 samples from two human cell lines (K562 and MCF7). Each of these cell lines has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. Reads are aligned to chromosome 22 (Grch38) and stored as bam files. -```{r, include = FALSE} -devtools::install_github("GoekeLab/NanoporeRNASeq") +# Quick start: Transcript discovery and quantification with bambu {#quick-start} + +## Installation +You can install bambu from github: +```{r, eval = FALSE} +if (!requireNamespace("devtools", quietly = TRUE)) + install.packages("devtools") +devtools::install_github("GoekeLab/bambu") ``` -```{r setup} -library(NanoporeRNASeq) +## General Usage +The default mode to run *bambu is using a set of aligned reads (bam files), reference genome annotations (gtf file, TxDb object, or bambuAnnotation object), and reference genome sequence (fasta file or BSgenome). bambu will return a summarizedExperiment object with the genomic coordinates for annotated and new transcripts and transcript expression estimates: +```{r, eval = FALSE} library(bambu) + +test.bam <- system.file("extdata", "SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.bam", package = "bambu") + +se <- bambu(reads = test.bam, annotations = "TxDb.Hsapiens.UCSC.hg38.knownGene", genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38") ``` +We highly recommend to use the same annotations that were used for genome alignment. If you have a gtf file and fasta file you can run bambu with the following options: +```{r, eval = FALSE} +test.bam <- system.file("extdata", "SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.bam", package = "bambu") + +fa.file <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa", package = "bambu") + +gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") + +bambuAnnotations <- prepareAnnotationsFromGTF(gtf.file) + +se <- bambu(reads = test.bam, annotations = bambuAnnotations, genomeSequence = fa.file) +``` +# A complete workflow to identify and quantify transcript expression from Nanopore RNA-Seq data {#complete-workflow} +To demonstrate the usage of Bambu, we used long-read RNA-Seq data generated using Oxford Nanopore Sequencing from the NanoporeRNASeq package, which consists of 6 samples from two human cell lines (K562 and MCF7). Each of these cell lines has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. Reads are aligned to chromosome 22 (Grch38) and stored as bam files. + ## Input data {#input-data} -load sample information +### Aligned reads (bam files) {#bam-files} +```{r} +devtools::install_github("GoekeLab/NanoporeRNASeq") +library(NanoporeRNASeq) +devtools::load_all() +library(bambu) +``` ```{r} data("sample_info") sample_info ``` - -### Load Bam files {#bam-files} ```{r} data("bamFiles") ``` -### Load genome sequences {#genome-sequences} +### Genome sequence (fasta file/ BSGenome object) {#genome-sequences} ```{r} # get path to fasta file fasta_path <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr22_1_25409234.fa", package="NanoporeRNASeq") @@ -72,13 +100,14 @@ fasta_path <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembl library(BSgenome.Hsapiens.NCBI.GRCh38) ``` -### Load Annotation {#annotations} +### Genome annotations (bambu annotations object/ gtf file / TxDb object) {#annotations} ```{r} data("annotation") ``` ## Transcript discovery and quantification {#transcript-discovery-quantification} -### Apply *bambu* to perform EM on extended annotations {#run-bambu} +### Running bambu {#run-bambu} +#### Apply *bambu* to perform EM on extended annotations ```{r} se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE) se @@ -89,7 +118,7 @@ seUnextended <- bambu(reads = bamFiles, annotations = annotation, genomeSequence seUnextended ``` -### Check expression estimates {#visualise-results} +### Visualise results {#visualise-results} We can check the estimated transcript expression using heatmap: ```{r, fig.width = 8, fig.height = 6} colData(se)$groupVar <- sample_info$cellLine @@ -104,16 +133,14 @@ colData(se)$groupVar <- sample_info$cellLine plot.bambu(se, group.variable = "groupVar", type = "pca") ``` - -### Check for gene examples - +Check for gene examples Single gene examples can also be checked using plot functions from *bambu* ```{r, fig.width = 8, fig.height = 10} plot.bambu(se, type = "annotation", gene_id = unique(rowData(se)$GENEID)[10]) ``` -### Transcript to Gene expression {#gene-expression} +### Obtain gene expression estimates from transcript expression {#gene-expression} Gene expression can be obtained from transcript expression using this function: ```{r} seGene <- transcriptToGeneExpression(se) @@ -137,7 +164,7 @@ plot.bambu(seGene, group.variable = "groupVar", type = "pca") writeBambuOutput(se, path = "./bambu/") ``` -## Differentially expressed genes {#DESeq2} +## Identifying differentially expressed genes {#DESeq2} We used *DESeq2* to find the differentially expressed genes: ```{r} library(DESeq2) @@ -167,7 +194,7 @@ resLFC <- lfcShrink(dds.deseq, coef="groupVar_MCF7_vs_K562", type="apeglm") plotMA(resLFC, ylim = c(-3,3)) ``` -## Differential expression for isoform detection {#DEXSeq} +## Identifying differential expression for isoform detection {#DEXSeq} We used *DEXSeq* to detect alternative used isoforms. ```{r} library(DRIMSeq) @@ -283,9 +310,43 @@ ggplot(dxrDT, aes(y = log2fold_MCF7_K562, x = exonBaseMean, color = as.factor(pa ```{r} dxrDT[padj<0.5,.(geneID, txID, log2fold_MCF7_K562,K562,MCF7)] ``` -### Running bambu with large number of samples {#large-sample-num} +# Running bambu with large number of samples {#large-sample-num} +For larger sample numbers we recommend to write the processed data to a file: +```{r, eval = FALSE} +bambu(reads = test.bam, readClass.outputDir = "./bambu/", annotations = bambuAnnotations, genomeSequence = fa.file) +``` +# Getting Help {#get-help} +## Advanced Options +More stringent filtering thresholds imposed on potential novel transcripts +* Keep novel transcripts with min 5 read count in at least 1 sample: +```{r, eval = FALSE} +bambu(reads, annotations, genomeSequence, isoreParameters = list(min.readCount = 5)) +``` +* Keep novel transcripts with min 5 samples having at least 2 counts: +```{r, eval = FALSE} +bambu(reads, annotations, genomeSequence, isoreParameters = list(min.sampleNumber = 5)) +``` +* Filter out transcripts with relative abundance within gene lower than 10%: +```{r, eval = FALSE} +bambu(reads, annotations, genomeSequence, isoreParameters = list(min.readFractionByGene = 0.1)) +``` +Quantification without bias correction +The default estimation automatically does bias correction for expression estimates. However, you can choose to perform the quantification without bias correction. +```{r, eval = FALSE} +bambu(reads, annotations, genomeSequence, emParameters = list(bias = FALSE)) +``` +Parallel computation +bambu allows parallel computation. +```{r, eval = FALSE} +bambu(reads, annotations, genomeSequence, ncore = 8) +``` +See *[manual](https://github.com/GoekeLab/bambu/blob/master/docs/bambu_0.1.0.pdf)* for details to customize other conditions. + +# Citing bambu {#cite-bambu} +A manuscript describing bambu is currently in preparation. If you use bambu for your research, please cite using the following doi: 10.5281/zenodo.3900025. +# Session Information {#session-info} ```{r} sessionInfo() ``` From 79ee4932203f78e78f8f0ac7e6462d3375fc090a Mon Sep 17 00:00:00 2001 From: cying111 Date: Fri, 17 Jul 2020 02:56:45 +0000 Subject: [PATCH 74/98] Update release link Former-commit-id: 28f3980bd8f87e196b2f0520b59ad4ad835ac9c4 --- README.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/README.md b/README.md index 9a0f7af3..2cb67b32 100755 --- a/README.md +++ b/README.md @@ -5,7 +5,7 @@ # bambu: reference-guided transcript discovery and quantification for long read RNA-Seq data -[![GitHub release (latest by date)](https://img.shields.io/github/v/release/GoekeLab/bambu?style=plastic)](https://github.com/GoekeLab/bambu/releases/tag/v0.2.0) +[![GitHub release (latest by date)](https://img.shields.io/github/v/release/GoekeLab/bambu?style=plastic)](https://github.com/GoekeLab/bambu/releases/) [![Maintained?](https://img.shields.io/badge/Maintained%3F-Yes-brightgreen)](https://github.com/GoekeLab/bambu/graphs/contributors) [![Install](https://img.shields.io/badge/Install-Github-brightgreen)](#installation) [![License: GPL v3](https://img.shields.io/badge/License-GPLv3-blue.svg)](https://www.gnu.org/licenses/gpl-3.0) From df22a158719cea84571e1f72f08e7bf4d7638f10 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Fri, 17 Jul 2020 11:14:23 +0800 Subject: [PATCH 75/98] Update bambu.Rmd Former-commit-id: f30520163c4b5587233a5faee4014a45907d956a --- vignettes/bambu.Rmd | 22 +++++++++++----------- 1 file changed, 11 insertions(+), 11 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 6d2786a5..5211e3bb 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -24,17 +24,17 @@ knitr::opts_chunk$set( # Content * [Quick Start: Transcript discovery and quantification with bambu](#quick-start) * [A complete workflow to identify and quantify transcript expression from Nanopore RNA-Seq data](#complete-workflow) - + [Input data](#input-data) - - [Aligned reads (bam files)](#bam-files) - - [Genome sequence (fasta file/ BSGenome object)](#genome-sequences) - - [Genome annotations (bambu annotations object/ gtf file / TxDb object)](#annotations) - + [Transcript discovery and quantification](#transcript-discovery-quantification) - - [Run bambu](#run-bambu) - - [Visualise results](#visualise-results) - - [Obtain gene expression estimates from transcript expression](#gene-expression) - - [Save data (gtf/text)](#save-data) - + [Identify differentially expressed genes](#DESeq2) - + [Identify differential transcript usage](#DEXSeq) + + [Input data](#input-data) + - [Aligned reads (bam files)](#bam-files) + - [Genome sequence (fasta file/ BSGenome object)](#genome-sequences) + - [Genome annotations (bambu annotations object/ gtf file / TxDb object)](#annotations) + + [Transcript discovery and quantification](#transcript-discovery-quantification) + - [Run bambu](#run-bambu) + - [Visualise results](#visualise-results) + - [Obtain gene expression estimates from transcript expression](#gene-expression) + - [Save data (gtf/text)](#save-data) + + [Identify differentially expressed genes](#DESeq2) + + [Identify differential transcript usage](#DEXSeq) * [Running bambu with large number of samples](#large-sample-num) * [Getting Help](#get-help) * [Citing bambu](#cite-bambu) From 655f873800dcbef2988a7a840663230457adf945 Mon Sep 17 00:00:00 2001 From: Yuk Kei Wan <41866052+yuukiiwa@users.noreply.github.com> Date: Fri, 17 Jul 2020 11:29:23 +0800 Subject: [PATCH 76/98] *bambu to *bambu* Former-commit-id: 99b06d761bd77bdb5ed2996d13cf7bb69db03305 --- vignettes/bambu.Rmd | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 5211e3bb..ae219d62 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -52,7 +52,7 @@ devtools::install_github("GoekeLab/bambu") ``` ## General Usage -The default mode to run *bambu is using a set of aligned reads (bam files), reference genome annotations (gtf file, TxDb object, or bambuAnnotation object), and reference genome sequence (fasta file or BSgenome). bambu will return a summarizedExperiment object with the genomic coordinates for annotated and new transcripts and transcript expression estimates: +The default mode to run *bambu* is using a set of aligned reads (bam files), reference genome annotations (gtf file, TxDb object, or bambuAnnotation object), and reference genome sequence (fasta file or BSgenome). bambu will return a summarizedExperiment object with the genomic coordinates for annotated and new transcripts and transcript expression estimates: ```{r, eval = FALSE} library(bambu) From bc83bbd3be7ef20ac813327d7407d10f9c789834 Mon Sep 17 00:00:00 2001 From: cying111 Date: Fri, 17 Jul 2020 03:37:21 +0000 Subject: [PATCH 77/98] update rdata files 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z4>o+#r+=6>dB(P@<_`lv1=zm%UiA8@=PUj<&}ep+|HV>NS2Qt&rkAueHgN}NF#wp@ zS#_Z4l`K4e%use#XnHk(79)Te!1$A>XlLjAlgRW_h2np%Ftz)M{l{wmPfP@$#ly(R zz`@AOD#j)zEXK;l#=$Bm{KL@CBFx6Y$il?N$ixTu|1a{>&VPfn0c^}1|81fEADF0k zNs?|d3aDZ;KFP>Bzw!!E=2AourG%V|q#Fgpx{t+;O@2n;H(4yrS90IL`0!l~vM7)D z%nO(6GS6eScQ1OPJv}r9(ePVU1}+ui5lpn-0alBy%$EN6_K|B81!U?Fx(YZ3i!e*e zh_X&;r{J~mn*2?K#hJaPmW@BufbFiHyZ2tzX}-eGs`G-xp58^p_g#thi&P(c@X|8Atyk#=vG35jLu}pigV2D` z7-q&ueXd-X;U_Lgl8GuW(#(Bb5sZdrNz$px!MDbWCI&gc4ZJyQ(ns3POsic#S$C6(5nEFQP z&LS0n{Al7~{6dtXs`#0mlH1r!3bLM`hjOyImDeK0?S7^>Z&6g0$0Ci1!91~0;);|t zDF`xfxW9 Date: Fri, 17 Jul 2020 09:00:37 +0000 Subject: [PATCH 80/98] Use the top half varied genes/transcripts for correlation plot Former-commit-id: f86b3acea3d513fc1868aefbeec6f0055d24ee88 --- R/plotBambu.R | 7 ++----- 1 file changed, 2 insertions(+), 5 deletions(-) diff --git a/R/plotBambu.R b/R/plotBambu.R index a5722a5c..f495a665 100644 --- a/R/plotBambu.R +++ b/R/plotBambu.R @@ -96,13 +96,10 @@ plot.bambu <- function(se,group.variable = NULL, type = c("annotation","pca","he #= count.data <- assays(se)$CPM - if(length(apply(count.data,1,sum)>10)>100){ - count.data <- count.data[apply(count.data,1,sum)>10,] - } - + count.data <- count.data[apply(count.data,1,sd) > quantile(apply(count.data,1,sd), 0.50),] count.data <- log2(count.data+1) - + if(type == "pca"){ From 5b42c5fb2a40cb18aabd439e5d597823977b5479 Mon Sep 17 00:00:00 2001 From: cying111 Date: Fri, 17 Jul 2020 09:11:21 +0000 Subject: [PATCH 81/98] updated vignettes Former-commit-id: fa1e93190c18aa5daaa43ddd01dfa6c837b676e4 --- vignettes/bambu.Rmd | 198 ++++++++++++++------------------------------ 1 file changed, 60 insertions(+), 138 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index ae219d62..9a045dfb 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -10,7 +10,7 @@ vignette: > ```{r, include = FALSE} knitr::opts_chunk$set( - collapse = TRUE, + collapse = TRUE,tidy = TRUE, warning=FALSE, message=FALSE, comment = "##>" ) @@ -74,25 +74,28 @@ bambuAnnotations <- prepareAnnotationsFromGTF(gtf.file) se <- bambu(reads = test.bam, annotations = bambuAnnotations, genomeSequence = fa.file) ``` # A complete workflow to identify and quantify transcript expression from Nanopore RNA-Seq data {#complete-workflow} -To demonstrate the usage of Bambu, we used long-read RNA-Seq data generated using Oxford Nanopore Sequencing from the NanoporeRNASeq package, which consists of 6 samples from two human cell lines (K562 and MCF7). Each of these cell lines has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. Reads are aligned to chromosome 22 (Grch38) and stored as bam files. +To demonstrate the usage of Bambu, we used long-read RNA-Seq data generated using Oxford Nanopore Sequencing from the NanoporeRNASeq package, which consists of 6 samples from two human cell lines (K562 and MCF7). Each of these cell lines has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. Reads are aligned to chromosome 22 (Grch38) and stored as bam files. We applied *bambu* to these bam files, visualized the results, and performed differential analysis to identify differentially expressed genes/transcripts. ## Input data {#input-data} -### Aligned reads (bam files) {#bam-files} +### Aligned reads (bam files) {#bam-files} +*bambu* takes genomic alignments saved in bam files. We used bamb-files from *NanoporeRNASeq* package, which contains reads aligned to the first half of the human chromosome 22 using *minimap2*. ```{r} -devtools::install_github("GoekeLab/NanoporeRNASeq") +devtools::load_all("/mnt/ont/s3.ontdata.store.genome.sg/projects/NanoporeRNASeq") +#devtools::install_github("GoekeLab/NanoporeRNASeq") library(NanoporeRNASeq) devtools::load_all() library(bambu) -``` -```{r} + data("sample_info") sample_info -``` -```{r} + data("bamFiles") +bamFiles <- Rsamtools::BamFileList(system.file("extdata",bamFiles,package = "NanoporeRNASeq")) +bamFiles ``` -### Genome sequence (fasta file/ BSGenome object) {#genome-sequences} +### Genome sequence (fasta file/ BSGenome object) {#genome-sequences} +*bambu* additionally requires genome sequence, which is used to correct splicing junctions in read alignments. Ideally, we recommend the same genome seqeunce file that is used for alignment to be used for bambu. As an option, users can also choose to use BS genome object. ```{r} # get path to fasta file fasta_path <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr22_1_25409234.fa", package="NanoporeRNASeq") @@ -100,26 +103,34 @@ fasta_path <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembl library(BSgenome.Hsapiens.NCBI.GRCh38) ``` -### Genome annotations (bambu annotations object/ gtf file / TxDb object) {#annotations} +### Genome annotations (bambu annotations object/ gtf file / TxDb object) {#annotations} +*bambu* also requires annotation object, which is used in *bambu* to correct for start or end sites of matched read classes as well as determing the annotation type of identified transcripts. Here we used the annotation object from *NanoporeRNASeq* package that is prepared by \code{prepareAnnotation} function in *bambu*. ```{r} data("annotation") +annotation ``` -## Transcript discovery and quantification {#transcript-discovery-quantification} -### Running bambu {#run-bambu} -#### Apply *bambu* to perform EM on extended annotations -```{r} +## Transcript discovery and quantification {#transcript-discovery-quantification} +### Running bambu {#run-bambu} +Applying *bambu* to perform isoform discovery to extend provided annotation, and then perform EM quantification on extended annotation. +```{r, results = "hide"} se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE) -se ``` -*bambu* allows quantification without isoform discovery ```{r} +se +``` + +Optionally, users can choose to apply *bambu* to do quantification without isoform discovery +```{r, results = "hide"} seUnextended <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38") +``` +```{r} seUnextended ``` -### Visualise results {#visualise-results} -We can check the estimated transcript expression using heatmap: + +### Visualise results {#visualise-results} +*bambu* provides quick visualisation of transcript estimation results with \code{plot.bambu} function. With multiple samples, we can visualise the correlation clustering between cell lines with heatmap: ```{r, fig.width = 8, fig.height = 6} colData(se)$groupVar <- sample_info$cellLine colnames(se) <- sample_info$sample_id @@ -127,67 +138,65 @@ colData(se)$name <- sample_info$sample_id plot.bambu(se, group.variable = "groupVar", type = "heatmap") ``` -or with PCA plot +Additionally, we can also visualise the correlation with a 2-dimmension PCA plot. ```{r, fig.width = 8, fig.height = 6} colData(se)$groupVar <- sample_info$cellLine plot.bambu(se, group.variable = "groupVar", type = "pca") ``` -Check for gene examples -Single gene examples can also be checked using plot functions from *bambu* +In addition to visualising correlation between samples, we also provide quick look into the extended annotation by looking at gene examples. Here we looked at gene ENSG00000099968 and visualised the genomic annotations for annotated and novel isoforms for this gene and compared the expression levels for these isoforms across samples. ```{r, fig.width = 8, fig.height = 10} -plot.bambu(se, type = "annotation", gene_id = unique(rowData(se)$GENEID)[10]) +plot.bambu(se, type = "annotation", gene_id = "ENSG00000099968") ``` ### Obtain gene expression estimates from transcript expression {#gene-expression} -Gene expression can be obtained from transcript expression using this function: +Gene expression can be obtained from transcript expression obtained from *bambu* using this function. Looking at the output, we can see there are novel genes identified as well ```{r} seGene <- transcriptToGeneExpression(se) seGene ``` -Gene expression heatmap +We compared the gene expression across the 6 samples. Generally, samples from the cell line showed higher correlation than across the cell lines for gene expression. ```{r, fig.width = 8, fig.height = 6} colData(seGene)$groupVar <- sample_info$cellLine plot.bambu(seGene, group.variable = "groupVar", type = "heatmap") ``` -Gene expression PCA plot +Similarly, when we visualise using PCA plot, we see that samples for the same cell line are more clustered. ```{r, fig.width = 8, fig.height = 6} colData(seGene)$groupVar <- sample_info$cellLine plot.bambu(seGene, group.variable = "groupVar", type = "pca") ``` ### Save data (gtf/text){#save-data} +*bambu* allows write annotation output to gtf file and gene and transcript estimates to txt files. ```{r} writeBambuOutput(se, path = "./bambu/") ``` ## Identifying differentially expressed genes {#DESeq2} -We used *DESeq2* to find the differentially expressed genes: +We used *DESeq2* to find the differentially expressed genes. Similar to using results from *Salmon*, estimates from *bambu* will first be rounded. ```{r} library(DESeq2) -dds <- DESeqDataSetFromMatrix(apply(assays(seGene)$counts,c(1,2),round),#tmp_wide[,-1], +dds <- DESeqDataSetFromMatrix(apply(assays(seGene)$counts,c(1,2),round), colData = colData(se), design = ~ groupVar) -system.time(dds.deseq <- DESeq(dds)) - +dds.deseq <- DESeq(dds) deGeneRes <- DESeq2::results(dds.deseq, independentFiltering=FALSE) -``` - -```{r} head(deGeneRes[order(deGeneRes$padj),]) ``` - +A quick summary of differentially expressed genes ```{r} summary(deGeneRes) ``` + +We can also visualise the MA-plot for differentially used isoforms ```{r, fig.width = 8, fig.height = 6} plotMA(deGeneRes, ylim = c(-3,3)) ``` -Plotting shrinked lFC results +However, as recommended by [*DESeq2* tutorial](http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#alternative-shrinkage-estimators), visualizing the MA-plots using the original log-fold change results will be affected by the noise associated with log2 fold changes from low count genes without requiring arbitrary filtering thresholds. So we applied same shrinkage to effect sizes to improve the visualization. ```{r, fig.width = 8, fig.height = 6} library(apeglm) resLFC <- lfcShrink(dds.deseq, coef="groupVar_MCF7_vs_K562", type="apeglm") @@ -197,119 +206,31 @@ plotMA(resLFC, ylim = c(-3,3)) ## Identifying differential expression for isoform detection {#DEXSeq} We used *DEXSeq* to detect alternative used isoforms. ```{r} -library(DRIMSeq) -count.data <- as.data.frame(rowData(se)) -count.data$gene_id <- count.data$GENEID -count.data$feature_id <- count.data$TXNAME -count.data$GENEID <- count.data$TXNAME <- NULL - -count.data <- cbind(count.data, assays(se)$counts) - -sample.info <- as.data.frame(colData(se)) -sample.info$sample_id <- sample.info$name -sample.info$name <- NULL -d <- dmDSdata(counts=count.data, samples=sample.info) - -n_samp_gene <- 1 -n_samp_feature <- 1 -min_count_gene <- 1 -min_count_feature <- 1 -dFilter <- dmFilter(d, - min_samps_feature_expr = n_samp_feature, - min_samps_feature_prop = n_samp_feature, - min_samps_gene_expr = n_samp_gene, - min_feature_expr = min_count_feature, - min_gene_expr = min_count_gene, - min_feature_prop=0.1) -table(table(counts(dFilter)$gene_id)) ## number of isoforms - - - +count.data <- cbind(data.frame(gene_id = as.data.frame(rowData(se))$GENEID, + feature_id = as.data.frame(rowData(se))$TXNAME), + assays(se)$counts) ``` ```{r} library(DEXSeq) -formulaFullModel <- as.formula("~sample + exon + groupVar:exon") - - -dxd <- DEXSeqDataSet(countData=round(as.matrix(counts(dFilter)[,-c(1:2)])), - sampleData=DRIMSeq::samples(dFilter), - design=formulaFullModel, - featureID = counts(dFilter)$feature_id, - groupID=counts(dFilter)$gene_id) - - -system.time({ - dxd <- estimateSizeFactors(dxd) - print('Size factor estimated') - dxd <- estimateDispersions(dxd, formula = formulaFullModel) - print('Dispersion estimated') - #dxd <- estimateExonFoldChanges( dxd ) - dxd <- testForDEU(dxd, fullModel = formulaFullModel) - print('DEU tested') - dxd <- estimateExonFoldChanges(dxd, fitExpToVar="groupVar") - print('Exon fold changes estimated') -}) -``` +formula <- as.formula("~sample + exon + groupVar:exon") +dxd <- DEXSeqDataSet(countData=round(as.matrix(count.data[,-c(1:2)])), + sampleData=as.data.frame(colData(se)), + design=formula, + featureID = count.data$feature_id, + groupID=count.data$gene_id) +dxd <- estimateExonFoldChanges(testForDEU(estimateDispersions(estimateSizeFactors(dxd), formula = formula), fullModel = formula), fitExpToVar="groupVar") -```{r} dxr <- DEXSeqResults(dxd, independentFiltering=FALSE) head(dxr) ``` - - -```{r} -library(stageR) -strp <- function(x) substr(x,1,15) -qval <- perGeneQValue(dxr) -dxr.g <- data.frame(gene=names(qval),qval) - -columns <- c("featureID","groupID","pvalue") -dxr_pval <- as.data.frame(dxr[,columns]) -head(dxr_pval) - -pConfirmation <- matrix(dxr_pval$pvalue,ncol=1) -dimnames(pConfirmation) <- list(strp(dxr_pval$featureID),"transcript") -pScreen <- qval -names(pScreen) <- strp(names(pScreen)) -tx2gene <- as.data.frame(dxr_pval[,c("featureID", "groupID")]) -for (i in 1:2) tx2gene[,i] <- strp(tx2gene[,i]) - - -stageRObj <- stageRTx(pScreen=pScreen, pConfirmation=pConfirmation, - pScreenAdjusted=TRUE, tx2gene=tx2gene) -stageRObj <- stageWiseAdjustment(stageRObj, method="dtu", alpha=0.5) -suppressWarnings({ - dex.padj <- getAdjustedPValues(stageRObj, order=FALSE, - onlySignificantGenes=TRUE) -}) -``` - -```{r} -dxrDT <- data.table(as.data.frame(dxr)) -setnames(dxrDT, old = c('groupID','featureID'), new = c('geneID','txID')) -dex.padj <- data.table(dex.padj) - -dxrDT <- dex.padj[dxrDT, on = c('geneID','txID')] -head(dxrDT) +We can visualize the MA-plot +```{r,fig.width = 8, fig.height = 6} +plotMA(dxr, cex=0.8 ) ``` -```{r, fig.width = 8, fig.height = 6} -dxrDT[,sigLFC2:=(padj < 0.5&(abs(log2fold_MCF7_K562)>=2))] -ggplot(dxrDT, aes(y = log2fold_MCF7_K562, x = exonBaseMean, color = as.factor(padj<0.5)))+ - geom_point(size = 0.5)+ - scale_x_log10()+ - scale_color_manual(values = c('grey','indianred'), name = "Significant")+ - xlab("Mean of normalized counts")+ - ylab("Log2 Fold change")+ - theme_minimal() - -``` -```{r} -dxrDT[padj<0.5,.(geneID, txID, log2fold_MCF7_K562,K562,MCF7)] -``` # Running bambu with large number of samples {#large-sample-num} For larger sample numbers we recommend to write the processed data to a file: ```{r, eval = FALSE} @@ -317,7 +238,8 @@ bambu(reads = test.bam, readClass.outputDir = "./bambu/", annotations = bambuAnn ``` # Getting Help {#get-help} ## Advanced Options -More stringent filtering thresholds imposed on potential novel transcripts +More stringent filtering thresholds imposed on potential novel transcripts + * Keep novel transcripts with min 5 read count in at least 1 sample: ```{r, eval = FALSE} bambu(reads, annotations, genomeSequence, isoreParameters = list(min.readCount = 5)) @@ -330,12 +252,12 @@ bambu(reads, annotations, genomeSequence, isoreParameters = list(min.sampleNumbe ```{r, eval = FALSE} bambu(reads, annotations, genomeSequence, isoreParameters = list(min.readFractionByGene = 0.1)) ``` -Quantification without bias correction +Quantification without bias correction The default estimation automatically does bias correction for expression estimates. However, you can choose to perform the quantification without bias correction. ```{r, eval = FALSE} bambu(reads, annotations, genomeSequence, emParameters = list(bias = FALSE)) ``` -Parallel computation +Parallel computation bambu allows parallel computation. ```{r, eval = FALSE} bambu(reads, annotations, genomeSequence, ncore = 8) From 01a8080c82ea7763b388785417765833610a324a Mon Sep 17 00:00:00 2001 From: cying111 Date: Fri, 17 Jul 2020 10:54:54 +0000 Subject: [PATCH 82/98] Added a fix for windows issue with fasta file Former-commit-id: c992be40677c1a295ecd4dd404ade317693ebb28 --- R/junctionCorrection.R | 9 +++++++-- 1 file changed, 7 insertions(+), 2 deletions(-) diff --git a/R/junctionCorrection.R b/R/junctionCorrection.R index 13f8648f..ebc8cbc3 100755 --- a/R/junctionCorrection.R +++ b/R/junctionCorrection.R @@ -13,12 +13,17 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, if(!is(genomeSequence,'FaFile')){ if(grepl('.fa',genomeSequence)){ - genomeSequence <- Rsamtools::FaFile(genomeSequence) + if(.Platform$OS.type == "windows"){ + genomeSequence <- Biostring::readDNAStringSet(genomeSequence) + }else{ + genomeSequence <- Rsamtools::FaFile(genomeSequence) + } if(seqlevelsStyle(genomeSequence)[1] != seqlevelsStyle(unlisted_junction_granges)[1]){ seqlevelsStyle(unlisted_junction_granges) <- seqlevelsStyle(genomeSequence)[1] } + }else { if (!suppressWarnings(require(BSgenome, quietly=TRUE))) stop("Please install the BSgenome package") @@ -39,7 +44,7 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, value = seqlevels(unlisted_junction_granges)[seqlevels(unlisted_junction_granges) %in% seqlevels(genomeSequence)], pruning.mode = 'coarse') } - + ##Todo: don't create junction names, instead work with indices/intergers (names are memory intensive) unstranded_unlisted_junctions <- unstrand(unlisted_junction_granges) From 104ed30ef88c5dac665feff42657416dc7672ce0 Mon Sep 17 00:00:00 2001 From: jonathangoeke <10560051+jonathangoeke@users.noreply.github.com> Date: Wed, 22 Jul 2020 11:48:18 +0800 Subject: [PATCH 83/98] bambu can now run diretly with a BSgenome object Former-commit-id: ad9993ac9c6a69f62e112035b8e0201be809f913 --- R/junctionCorrection.R | 10 +++++----- 1 file changed, 5 insertions(+), 5 deletions(-) diff --git a/R/junctionCorrection.R b/R/junctionCorrection.R index ebc8cbc3..2b678219 100755 --- a/R/junctionCorrection.R +++ b/R/junctionCorrection.R @@ -11,7 +11,7 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, original_seqlevelstyle <- seqlevelsStyle(unlisted_junction_granges)[1] - if(!is(genomeSequence,'FaFile')){ + if(class(genomeSequence)=='character'){ if(grepl('.fa',genomeSequence)){ if(.Platform$OS.type == "windows"){ @@ -22,16 +22,16 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, if(seqlevelsStyle(genomeSequence)[1] != seqlevelsStyle(unlisted_junction_granges)[1]){ seqlevelsStyle(unlisted_junction_granges) <- seqlevelsStyle(genomeSequence)[1] } - - - }else { + } else { if (!suppressWarnings(require(BSgenome, quietly=TRUE))) stop("Please install the BSgenome package") genomeSequence <- BSgenome::getBSgenome(genomeSequence) seqlevelsStyle(genomeSequence) <- seqlevelsStyle(unlisted_junction_granges)[1] } - } + } else if(class(genomeSequence)=='BSgenome'){ + seqlevelsStyle(genomeSequence) <- seqlevelsStyle(unlisted_junction_granges)[1] + } if(is(genomeSequence,'FaFile')){ if(seqlevelsStyle(genomeSequence)[1] != seqlevelsStyle(unlisted_junction_granges)[1]){ From d3c2795cba14b73f4403df11d61f85b8ad64194c Mon Sep 17 00:00:00 2001 From: jonathangoeke <10560051+jonathangoeke@users.noreply.github.com> Date: Wed, 22 Jul 2020 14:36:12 +0800 Subject: [PATCH 84/98] enable the use of a prefix when saving bambu objects to text Former-commit-id: d34d8a9efd8167dbba5b81499f9cecff966d42b3 --- R/readWrite.R | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/R/readWrite.R b/R/readWrite.R index 58f32be0..7e0d3295 100644 --- a/R/readWrite.R +++ b/R/readWrite.R @@ -11,7 +11,7 @@ #' package = "bambu")) #' path <- tempdir() #' writeBambuOutput(se,path) -writeBambuOutput <- function(se,path){ +writeBambuOutput <- function(se, path, prefix=''){ if (missing(se) | missing(path)){ stop('Both summarizedExperiment object from bambu and the path for the output files are required.') }else{ @@ -20,17 +20,17 @@ writeBambuOutput <- function(se,path){ dir.create(outdir, recursive = TRUE) } transcript_grList <- rowRanges(se) - transcript_gtffn <- paste(path,"transcript_exon.gtf",sep="") + transcript_gtffn <- paste(outdir, prefix, "extended_annotations.gtf",sep="") gtf <- writeToGTF(annotation=transcript_grList,file=transcript_gtffn) transcript_counts <- as.data.frame(assays(se)$counts) geneIDs <- data.frame(mcols(transcript_grList, use.names=FALSE)$TXNAME,mcols(transcript_grList, use.names=FALSE)$GENEID) colnames(geneIDs) <- c("TXNAME","GENEID") transcript_counts <- cbind(geneIDs,transcript_counts) - transcript_countsfn <- paste(path,"counts_transcript.txt",sep="") + transcript_countsfn <- paste(outdir,prefix, "counts_transcript.txt",sep="") write.table(transcript_counts, file= transcript_countsfn, sep="\t",quote=FALSE,row.names= FALSE) gene_se <- transcriptToGeneExpression(se) gene_counts <- as.data.frame(assays(gene_se)$counts) - gene_countsfn <- paste(path,"counts_gene.txt",sep="") + gene_countsfn <- paste(outdir, prefix, "counts_gene.txt",sep="") write.table(gene_counts, file= gene_countsfn, sep="\t", quote=FALSE) } } From 41e95e4b756b9f1b0575d4544e3a1380750280b1 Mon Sep 17 00:00:00 2001 From: jonathangoeke <10560051+jonathangoeke@users.noreply.github.com> Date: Wed, 22 Jul 2020 14:56:02 +0800 Subject: [PATCH 85/98] set options for scipen back to 0 after setting it to 999 (to avoid a problem with integer coordinates) Former-commit-id: 5a853a03a8771e77dab3053dc349ecb8580c21c7 --- R/constructReadClassTables.R | 1 + 1 file changed, 1 insertion(+) diff --git a/R/constructReadClassTables.R b/R/constructReadClassTables.R index 5bca2eff..4e0d5154 100755 --- a/R/constructReadClassTables.R +++ b/R/constructReadClassTables.R @@ -112,6 +112,7 @@ constructSplicedReadClassTables <- function(uniqueJunctions, unlisted_junctions, mcols(exonsByReadClass) <- readTable # seqlevels(exonsByReadClass) <- seqLevelList + options(scipen = 0) return(exonsByReadClass) } From f5e19a838c4b2cbdbe40120827de1b75889e38ae Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 02:02:33 +0000 Subject: [PATCH 86/98] update prepareAnnotations function such that it takes both gtf.file or txdb Former-commit-id: ae3c56dbab49c290f1ba98c317730afa2b674c6e --- R/annotationFunctions.R | 65 ++++++++++++++++++++++------------------- 1 file changed, 35 insertions(+), 30 deletions(-) diff --git a/R/annotationFunctions.R b/R/annotationFunctions.R index 34af852d..e410a5ed 100755 --- a/R/annotationFunctions.R +++ b/R/annotationFunctions.R @@ -1,34 +1,44 @@ #' Function to prepare tables and genomic ranges for transript reconstruction using a txdb object -#' @title prepare annotations from txdb object -#' @param txdb a \code{\link{TxDb}} object +#' @title prepare annotations from txdb object or gtf file +#' @param x A \code{\link{TxDb}} object or a gtf file #' @return A \code{\link{GRangesList}} object #' @export #' @examples #' library(TxDb.Hsapiens.UCSC.hg38.knownGene) #' txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene -#' prepareAnnotations(txdb) -prepareAnnotations <- function(txdb) { - exonsByTx = exonsBy(txdb,by='tx', use.names=TRUE) - if(any(duplicated(names(exonsByTx)))) { - warning('transcript names are not unique, only one transcript per ID will be kept') - exonsByTx <- exonsByTx[!duplicated(exonsByTx)] +#' prepareAnnotations(x = txdb) +#' gtf.file <- system.file("extdata", +#' "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", +#' package = "bambu") +#' gr <- prepareAnnotationsFrom(x = gtf.file) +prepareAnnotations <- function(x) { + if(grepl(".gtf",x)){ + return(prepareAnnotationsFromGTF(x)) } - - unlistedExons <- unlist(exonsByTx, use.names = FALSE) - partitioning <- PartitioningByEnd(cumsum(elementNROWS(exonsByTx)), names=NULL) - txIdForReorder <- togroup(PartitioningByWidth(exonsByTx)) - unlistedExons <- unlistedExons[order(txIdForReorder, unlistedExons$exon_rank)] #'exonsByTx' is always sorted by exon rank, not by strand, make sure that this is the case here - unlistedExons$exon_endRank <- unlist(sapply(elementNROWS(exonsByTx),seq,to=1), use.names=FALSE) - unlistedExons <- unlistedExons[order(txIdForReorder, start(unlistedExons))] - mcols(unlistedExons) <- mcols(unlistedExons)[,c('exon_rank','exon_endRank')] - exonsByTx <- relist(unlistedExons, partitioning) - - mcols(exonsByTx) <- suppressMessages(AnnotationDbi::select(txdb, names(exonsByTx), - columns=c("TXNAME", "GENEID"), - keytype="TXNAME")) - minEqClasses <- getMinimumEqClassByTx(exonsByTx) - mcols(exonsByTx)$eqClass <- minEqClasses$eqClass[match(names(exonsByTx),minEqClasses$queryTxId)] - return(exonsByTx) + if(class(x) == "TxDb"){ + exonsByTx = exonsBy(txdb,by='tx', use.names=TRUE) + if(any(duplicated(names(exonsByTx)))) { + warning('transcript names are not unique, only one transcript per ID will be kept') + exonsByTx <- exonsByTx[!duplicated(exonsByTx)] + } + + unlistedExons <- unlist(exonsByTx, use.names = FALSE) + partitioning <- PartitioningByEnd(cumsum(elementNROWS(exonsByTx)), names=NULL) + txIdForReorder <- togroup(PartitioningByWidth(exonsByTx)) + unlistedExons <- unlistedExons[order(txIdForReorder, unlistedExons$exon_rank)] #'exonsByTx' is always sorted by exon rank, not by strand, make sure that this is the case here + unlistedExons$exon_endRank <- unlist(sapply(elementNROWS(exonsByTx),seq,to=1), use.names=FALSE) + unlistedExons <- unlistedExons[order(txIdForReorder, start(unlistedExons))] + mcols(unlistedExons) <- mcols(unlistedExons)[,c('exon_rank','exon_endRank')] + exonsByTx <- relist(unlistedExons, partitioning) + + mcols(exonsByTx) <- suppressMessages(AnnotationDbi::select(txdb, names(exonsByTx), + columns=c("TXNAME", "GENEID"), + keytype="TXNAME")) + minEqClasses <- getMinimumEqClassByTx(exonsByTx) + mcols(exonsByTx)$eqClass <- minEqClasses$eqClass[match(names(exonsByTx),minEqClasses$queryTxId)] + return(exonsByTx) + } + } @@ -43,12 +53,7 @@ prepareAnnotations <- function(txdb) { #' \item GENEID indicating whether filter to remove read classes which are a subset of known transcripts(), defaults to TRUE #' \item eqClass specifying minimun read count to consider a read class valid in a sample, defaults to 2 #' } -#' @export -#' @examples -#' gtf.file <- system.file("extdata", -#' "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", -#' package = "bambu") -#' gr <- prepareAnnotationsFromGTF(gtf.file) +#' @noRd prepareAnnotationsFromGTF <- function(file){ if (missing(file)){ stop('A GTF file is required.') From d34b95591f7357593aaa1762a64ec4921ec5a272 Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 02:03:20 +0000 Subject: [PATCH 87/98] remove lines require BSgenome installation Former-commit-id: fc0931f9879929197df19cb8ffbc63333740e42d --- R/junctionCorrection.R | 3 --- 1 file changed, 3 deletions(-) diff --git a/R/junctionCorrection.R b/R/junctionCorrection.R index 2b678219..41c017b6 100755 --- a/R/junctionCorrection.R +++ b/R/junctionCorrection.R @@ -23,9 +23,6 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, seqlevelsStyle(unlisted_junction_granges) <- seqlevelsStyle(genomeSequence)[1] } } else { - if (!suppressWarnings(require(BSgenome, quietly=TRUE))) - stop("Please install the BSgenome package") - genomeSequence <- BSgenome::getBSgenome(genomeSequence) seqlevelsStyle(genomeSequence) <- seqlevelsStyle(unlisted_junction_granges)[1] } From b2c99e9fb8faa68afc04191635eb4f2ac329c220 Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 02:03:45 +0000 Subject: [PATCH 88/98] add back rds and rda files Former-commit-id: 8f444a1fd2ad9026f33e25f5aeb72c953eeaf363 --- R/sysdata.rda | Bin 0 -> 38207 bytes ...tionGranges_txdbGrch38_91_chr9_1_1000000.rds | Bin 0 -> 3692 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 1504 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 2008 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 4440 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 4304 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 4412 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 4272 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 3240 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 3184 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 3180 bytes ...directRNA_replicate5_run1_chr9_1_1000000.rds | Bin 0 -> 3184 bytes 12 files changed, 0 insertions(+), 0 deletions(-) create mode 100755 R/sysdata.rda create mode 100644 inst/extdata/extendedAnnotationGranges_txdbGrch38_91_chr9_1_1000000.rds create mode 100644 inst/extdata/seIsoReCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds create mode 100644 inst/extdata/seIsoReRef_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds create mode 100644 inst/extdata/seOutputCombinedExtended_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds create mode 100644 inst/extdata/seOutputCombined_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds create mode 100644 inst/extdata/seOutputExtended_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds create mode 100644 inst/extdata/seOutput_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds create mode 100644 inst/extdata/seReadClassBsgenomeStranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds create mode 100644 inst/extdata/seReadClassStranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds create mode 100644 inst/extdata/seReadClassUnstranded_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds create mode 100644 inst/extdata/seReadClass_SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.rds diff --git a/R/sysdata.rda b/R/sysdata.rda new file mode 100755 index 0000000000000000000000000000000000000000..d63cc3790a223ffebd6a509fe7174301c13d1a71 GIT binary patch literal 38207 zcmbrEMOPdSjKGIsfWZfM7~I``(BiJei&Lx=mom7!yB4QNaVr$36nFRHP}~ap-%qfI zO%6#8c`tdnBzZsS_jbbEQicrLhLutr!+=HSxBpYN0s^);zcy>(0{}?sogBs9%p7HOLeS#;0tf2)fG1bhtm z)3Z*bcD>GDjpsSPU@)F{=X>E%)6TB%|8T$9Epy?eVX}6}?m%XZ;)&>4BJd=*aM*KR zaxT#{Qjx4nld82`Ra$g+w%Rz`^01LFo_870)xt@Zp6BMd+PF^M>g-WNxazq+oVOeP zEP_(k({s^sW$ah|akW77kAy+Dc=Pg!{EANi>xvSCD!$X#TBRzSmaqIl+D z1hNZK_6;rT3-c5e#CrSfwc1X(l=Ht<^?vD%eC=-k4Q|0ywyLQuU7vOA%&_bvop&n! 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z@|8u!Lz!&3hkc6KyzzasM};zsQb^4I`{`8L7P_J#j)shxD(wm@uOs@B=|q z1+pXX#BP?Dje(@OIHF6vD-?%EC-wTpj~W7g(c%00k!v_aqGDr$a}F$EwqOWclxn{W~+*nTsAxPw*&FJsg3#jZk^eOGMiM=O-Yla8_0002%x-qB#0m&HK W%K-qIF_zFiFb#_W000000a;oqAtXrv literal 0 HcmV?d00001 From 1368f3182d739fbda03132ab091de6128e70e4ad Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 02:04:54 +0000 Subject: [PATCH 89/98] update unit test for prepareAnnotations to test for gtf file Former-commit-id: 233c880eedd369c97e5754c45d48955c42037a3f --- tests/testthat/test_prepareAnnotations.R | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/tests/testthat/test_prepareAnnotations.R b/tests/testthat/test_prepareAnnotations.R index b184aab7..91568ad1 100644 --- a/tests/testthat/test_prepareAnnotations.R +++ b/tests/testthat/test_prepareAnnotations.R @@ -5,7 +5,7 @@ test_that("prepareAnnotations of txdb object is a GRangesList",{ expectedGR <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) - gr <- prepareAnnotations(txdb) + gr <- prepareAnnotations(x = txdb) expect_equal(gr, expectedGR) expect_s4_class(gr, class = 'CompressedGRangesList') @@ -24,12 +24,12 @@ test_that("prepareAnnotations of genome library is a GRangesList",{ -test_that("prepareAnnotationsFromGTF is GRangesList",{ +test_that("prepareAnnotations from gtf file is GRangesList",{ gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") expectedGR <- readRDS(system.file("extdata", "annotationGranges_txdbGrch38_91_chr9_1_1000000.rds", package = "bambu")) - gr <- prepareAnnotationsFromGTF(gtf.file) + gr <- prepareAnnotations(x = gtf.file) expect_equal(gr[order(names(gr))], expectedGR[order(names(expectedGR))]) expect_s4_class(gr, class = 'CompressedGRangesList') From 19bc8cae82f09e62ed98f5d144b5a99621dbe4cc Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 02:05:25 +0000 Subject: [PATCH 90/98] update vignettes Former-commit-id: 131f68829dd8ac2352d57cc995b8a71f014aff97 --- vignettes/bambu.Rmd | 176 +++++++++++++++++++++++++------------------- 1 file changed, 101 insertions(+), 75 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index 9a045dfb..a8f6666f 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -18,7 +18,7 @@ knitr::opts_chunk$set( # Introduction -*[Bambu](https://github.com/GoekeLab/bambu)* is a method for transcript discovery and quantification from long read RNA-Seq data. Bambu uses aligned reads and genome reference annotations as input, and will return abundance estimates for all known transcripts and for newly discovered transcripts. Bambu uses the information from the reference annotations to correct misalignment at splice junctions, then reduces the aligned reads to read equivalent classes, and uses this information to identify novel transcripts across all samples of interest. Reads are then assigned to transcripts, and expression estimates are obtained using an expectation maximisation algorithm. Here, we present an example workflow for analysing Nanopore long read RNA-Sequencing data from two human cancer cell lines from the Singapore Nanopore Expression Project (SG-NEx). +*[Bambu](https://github.com/GoekeLab/bambu)* is a method for transcript discovery and quantification from long read RNA-Seq data. Bambu uses aligned reads and genome reference annotations as input, and will return abundance estimates for all known transcripts and for newly discovered transcripts. Bambu uses the information from the reference annotations to correct misalignment at splice junctions, then reduces the aligned reads to read equivalent classes, and uses this information to identify novel transcripts across all samples of interest. Reads are then assigned to transcripts, and expression estimates are obtained using an expectation maximisation algorithm. Here, we present an example workflow for analysing Nanopore long read RNA-Sequencing data from two human cancer cell lines from the Singapore Nanopore Expression Project (SG-NEx). # Content @@ -36,9 +36,10 @@ knitr::opts_chunk$set( + [Identify differentially expressed genes](#DESeq2) + [Identify differential transcript usage](#DEXSeq) * [Running bambu with large number of samples](#large-sample-num) -* [Getting Help](#get-help) -* [Citing bambu](#cite-bambu) -* [Session Information](#session-info) +* [Bambu parameters](#bambu-parameters) +* [Getting help](#get-help) +* [Citing bambu](#citing-bambu) +* [Session Information](#session-information) # Quick start: Transcript discovery and quantification with bambu {#quick-start} @@ -52,7 +53,7 @@ devtools::install_github("GoekeLab/bambu") ``` ## General Usage -The default mode to run *bambu* is using a set of aligned reads (bam files), reference genome annotations (gtf file, TxDb object, or bambuAnnotation object), and reference genome sequence (fasta file or BSgenome). bambu will return a summarizedExperiment object with the genomic coordinates for annotated and new transcripts and transcript expression estimates: +The default mode to run *bambu* is using a set of aligned reads (bam files), reference genome annotations (gtf file, TxDb object, or bambuAnnotation object), and reference genome sequence (fasta file or BSgenome). *bambu* will return a summarizedExperiment object with the genomic coordinates for annotated and new transcripts and transcript expression estimates: ```{r, eval = FALSE} library(bambu) @@ -69,42 +70,63 @@ fa.file <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_c gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") -bambuAnnotations <- prepareAnnotationsFromGTF(gtf.file) +bambuAnnotations <- prepareAnnotations(gtf.file) se <- bambu(reads = test.bam, annotations = bambuAnnotations, genomeSequence = fa.file) ``` +*bambu* returns a SummarizedExperiment object which can be accessed as follows: +* assays(se) returns the transcript abundance estimates as counts or CPM +* rowRanges(se) returns a GRangesList with all annotated and newly discovered transcripts +* rowData(se) returns additional information about each transcript such as the gene name and the class of newly discovered transcript + # A complete workflow to identify and quantify transcript expression from Nanopore RNA-Seq data {#complete-workflow} -To demonstrate the usage of Bambu, we used long-read RNA-Seq data generated using Oxford Nanopore Sequencing from the NanoporeRNASeq package, which consists of 6 samples from two human cell lines (K562 and MCF7). Each of these cell lines has three replicates, with 1 direct RNA sequencing data and 2 cDNA sequencing data. Reads are aligned to chromosome 22 (Grch38) and stored as bam files. We applied *bambu* to these bam files, visualized the results, and performed differential analysis to identify differentially expressed genes/transcripts. +To demonstrate the usage of Bambu, we used long-read RNA-Seq data generated using Oxford Nanopore Sequencing from the NanoporeRNASeq package, which consists of 6 samples from two human cell lines (K562 and MCF7) that were generated by the [SG-NEx project](https://github.com/GoekeLab/sg-nex-data). Each of these cell lines has three replicates, with 1 direct RNA sequencing run and 2 cDNA sequencing runs. Reads are aligned to chromosome 22 (Grch38) and stored as bam files. In this workflow, we will demonstrate how to apply *bambu* to these bam files to identify novel transcripts and estimates transcript expression, visualize the results, and identify differentially expressed genes and transcripts. ## Input data {#input-data} ### Aligned reads (bam files) {#bam-files} -*bambu* takes genomic alignments saved in bam files. We used bamb-files from *NanoporeRNASeq* package, which contains reads aligned to the first half of the human chromosome 22 using *minimap2*. +*bambu* takes genomic alignments saved in bam files. Here we use bam-files from the *NanoporeRNASeq* package, which contains reads aligned to the first half of the human chromosome 22 using *minimap2*. ```{r} -devtools::load_all("/mnt/ont/s3.ontdata.store.genome.sg/projects/NanoporeRNASeq") -#devtools::install_github("GoekeLab/NanoporeRNASeq") -library(NanoporeRNASeq) -devtools::load_all() library(bambu) +devtools::install_github("GoekeLab/NanoporeRNASeq") +library(NanoporeRNASeq) data("sample_info") sample_info -data("bamFiles") -bamFiles <- Rsamtools::BamFileList(system.file("extdata",bamFiles,package = "NanoporeRNASeq")) +bamFiles <- Rsamtools::BamFileList(system.file("extdata", sample_info$fileNames, package = "NanoporeRNASeq")) +names(bamFiles) <- sample_info$sample_id + bamFiles ``` ### Genome sequence (fasta file/ BSGenome object) {#genome-sequences} -*bambu* additionally requires genome sequence, which is used to correct splicing junctions in read alignments. Ideally, we recommend the same genome seqeunce file that is used for alignment to be used for bambu. As an option, users can also choose to use BS genome object. +*bambu* additionally requires a genome sequence, which is used to correct splicing junctions in read alignments. Ideally, we recommend to use the same genome seqeunce file that was used for alignment to be used for bambu. ```{r} # get path to fasta file -fasta_path <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr22_1_25409234.fa", package="NanoporeRNASeq") -# BSgenome +genomeSequence <- system.file("extdata", "Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr22_1_25409234.fa", package="NanoporeRNASeq") + +``` +As an option, users can also choose to use a BSgenome object: +```{r} library(BSgenome.Hsapiens.NCBI.GRCh38) +genomeSequence <- BSgenome.Hsapiens.NCBI.GRCh38 ``` ### Genome annotations (bambu annotations object/ gtf file / TxDb object) {#annotations} -*bambu* also requires annotation object, which is used in *bambu* to correct for start or end sites of matched read classes as well as determing the annotation type of identified transcripts. Here we used the annotation object from *NanoporeRNASeq* package that is prepared by \code{prepareAnnotation} function in *bambu*. +*bambu* also requires a reference transcript annotations object, which is used to correct read alignments, to identify for transcripts and genes (and the type for novel transcripts), and for quantification. The annotation object can be created from a gtf file: +```{r} +gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") + +annotation <- prepareAnnotations(gtf.file) +``` +The annotation object can also be created from a TxDb object: +```{r} +txdb <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", package = "bambu") + +annotation <- prepareAnnotations(txdb) +``` + +The annotation object can be stored and used again for re-running bambu. Here we will used the annotation object from the *NanoporeRNASeq* package that wasis prepared from a gtf file using the \code{prepareAnnotations} function in by function in *bambu*. ```{r} data("annotation") annotation @@ -112,17 +134,24 @@ annotation ## Transcript discovery and quantification {#transcript-discovery-quantification} ### Running bambu {#run-bambu} -Applying *bambu* to perform isoform discovery to extend provided annotation, and then perform EM quantification on extended annotation. +Next we apply *bambu* on the input data (bam files, annotations, genomeSequence). Bambu willto perform isoform discovery to extend the provided annotation, and then quantify the transcript expression from these extended annotation using an Expectation-Maximisation algorithm. Here we will use 1 core, which can be changed to process multiple files in parallel. + ```{r, results = "hide"} -se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38", extendAnnotations = TRUE) +se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = genomeSequence, ncore = 1) ``` ```{r} se ``` +For the downstream analysis we will add the condition of interest to the \code{colData} object that describes the samples. Here we are interested in a comparison of the 2 cell lines: +```{r} +colData(se)$condition <- as.factor(sample_info$cellLine) +``` + -Optionally, users can choose to apply *bambu* to do quantification without isoform discovery + +Optionally, users can choose to apply *bambu* to do quantification only (without isoform discovery) ```{r, results = "hide"} -seUnextended <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = "BSgenome.Hsapiens.NCBI.GRCh38") +seUnextended <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = genomeSequence, extendAnnotations = FALSE) ``` ```{r} seUnextended @@ -130,58 +159,57 @@ seUnextended ### Visualise results {#visualise-results} -*bambu* provides quick visualisation of transcript estimation results with \code{plot.bambu} function. With multiple samples, we can visualise the correlation clustering between cell lines with heatmap: +*bambu* provides functions to visualise and explore the results. When multiple samples are used, we can visualise the correlation and clustering of all samples with a heatmap: + ```{r, fig.width = 8, fig.height = 6} -colData(se)$groupVar <- sample_info$cellLine -colnames(se) <- sample_info$sample_id -colData(se)$name <- sample_info$sample_id -plot.bambu(se, group.variable = "groupVar", type = "heatmap") +plot.bambu(se, group.variable = "condition", type = "heatmap") ``` -Additionally, we can also visualise the correlation with a 2-dimmension PCA plot. +Additionally, we can also visualise the correlation with a 2-dimmensional PCA plot. ```{r, fig.width = 8, fig.height = 6} -colData(se)$groupVar <- sample_info$cellLine -plot.bambu(se, group.variable = "groupVar", type = "pca") +plot.bambu(se, group.variable = "condition", type = "pca") ``` -In addition to visualising correlation between samples, we also provide quick look into the extended annotation by looking at gene examples. Here we looked at gene ENSG00000099968 and visualised the genomic annotations for annotated and novel isoforms for this gene and compared the expression levels for these isoforms across samples. +In addition to visualising the correlation between samples, *bambu* also provide a function to visualise the extended annotation and expression estimation for individual genes. Here we look at gene ENSG00000099968 and visualise the transcript coordinates for annotated and novel isoforms and expression levels for these isoforms across all samples. ```{r, fig.width = 8, fig.height = 10} plot.bambu(se, type = "annotation", gene_id = "ENSG00000099968") - ``` ### Obtain gene expression estimates from transcript expression {#gene-expression} -Gene expression can be obtained from transcript expression obtained from *bambu* using this function. Looking at the output, we can see there are novel genes identified as well +Gene expression can be calculated from the transcript expression estimates returned by *bambu* using the \code{transcriptToGeneExpression} function. Looking at the output, we can see there are novel genes identified as well ```{r} seGene <- transcriptToGeneExpression(se) seGene ``` -We compared the gene expression across the 6 samples. Generally, samples from the cell line showed higher correlation than across the cell lines for gene expression. +We can again use the \code{plot.bambu} function to visualise the gene expression data across the 6 samples with a heatmap or PCA plot. As expected, samples from the same cell line showed higher correlation than across the cell lines. ```{r, fig.width = 8, fig.height = 6} colData(seGene)$groupVar <- sample_info$cellLine -plot.bambu(seGene, group.variable = "groupVar", type = "heatmap") +plot.bambu(seGene, group.variable = "condition", type = "heatmap") ``` -Similarly, when we visualise using PCA plot, we see that samples for the same cell line are more clustered. -```{r, fig.width = 8, fig.height = 6} -colData(seGene)$groupVar <- sample_info$cellLine -plot.bambu(seGene, group.variable = "groupVar", type = "pca") -``` ### Save data (gtf/text){#save-data} -*bambu* allows write annotation output to gtf file and gene and transcript estimates to txt files. -```{r} -writeBambuOutput(se, path = "./bambu/") +*bambu* includes a function to write the extended annotations, the transcript and the gene expression estimates that include any newly discovered genes and transcripts to text files. +```{r， eval = FALSE} +writeBambuOutput(se, path = "./bambu/", prefix="NanoporeRNASeq_") +``` + +*bambu* also includes a function that only exports the extended annotations to gtf file: +```{r, eval = FALSE} +writeToGTF(rowRanges(se), file="./bambu/NanoporeRNASeq_extendedAnnotations.gtf") ``` + + + ## Identifying differentially expressed genes {#DESeq2} -We used *DESeq2* to find the differentially expressed genes. Similar to using results from *Salmon*, estimates from *bambu* will first be rounded. +One of the most common tasks when analysing RNA-Seq data is the analysis of differential gene expression across a condition of intertest. Here we use *DESeq2* to find the differentially expressed genes between MCF7 and K562 cell lines. Similar to using results from *Salmon*, estimates from *bambu* will first be rounded. ```{r} library(DESeq2) -dds <- DESeqDataSetFromMatrix(apply(assays(seGene)$counts,c(1,2),round), +dds <- DESeqDataSetFromMatrix(round(assays(seGene)$counts), colData = colData(se), - design = ~ groupVar) + design = ~ condition) dds.deseq <- DESeq(dds) deGeneRes <- DESeq2::results(dds.deseq, independentFiltering=FALSE) head(deGeneRes[order(deGeneRes$padj),]) @@ -191,39 +219,28 @@ A quick summary of differentially expressed genes summary(deGeneRes) ``` -We can also visualise the MA-plot for differentially used isoforms -```{r, fig.width = 8, fig.height = 6} -plotMA(deGeneRes, ylim = c(-3,3)) -``` - -However, as recommended by [*DESeq2* tutorial](http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#alternative-shrinkage-estimators), visualizing the MA-plots using the original log-fold change results will be affected by the noise associated with log2 fold changes from low count genes without requiring arbitrary filtering thresholds. So we applied same shrinkage to effect sizes to improve the visualization. +We can also visualise the MA-plot for differentially used isoforms using \code{plotMA(deGeneRes)}. However, visualizing the MA-plots using the original log-fold change results will be affected by the noise associated with log2 fold changes from low count genes without requiring arbitrary filtering thresholds. As recommended in the [*DESeq2* tutorial](http://bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#alternative-shrinkage-estimators). we applied the same shrinkage to effect sizes to improve the visualization. ```{r, fig.width = 8, fig.height = 6} library(apeglm) -resLFC <- lfcShrink(dds.deseq, coef="groupVar_MCF7_vs_K562", type="apeglm") +resLFC <- lfcShrink(dds.deseq, coef="condition_MCF7_vs_K562", type="apeglm") plotMA(resLFC, ylim = c(-3,3)) ``` -## Identifying differential expression for isoform detection {#DEXSeq} +## Identifying differential transcript usage {#DEXSeq} We used *DEXSeq* to detect alternative used isoforms. -```{r} -count.data <- cbind(data.frame(gene_id = as.data.frame(rowData(se))$GENEID, - feature_id = as.data.frame(rowData(se))$TXNAME), - assays(se)$counts) -``` - ```{r} library(DEXSeq) -formula <- as.formula("~sample + exon + groupVar:exon") -dxd <- DEXSeqDataSet(countData=round(as.matrix(count.data[,-c(1:2)])), - sampleData=as.data.frame(colData(se)), - design=formula, - featureID = count.data$feature_id, - groupID=count.data$gene_id) -dxd <- estimateExonFoldChanges(testForDEU(estimateDispersions(estimateSizeFactors(dxd), formula = formula), fullModel = formula), fitExpToVar="groupVar") - -dxr <- DEXSeqResults(dxd, independentFiltering=FALSE) +dxd <- DEXSeqDataSet(countData = round(assays(se)$counts), +sampleData = as.data.frame(colData(se)), +design = ~sample + exon + condition:exon, +featureID = rowData(se)$TXNAME, +groupID = rowData(se)$GENEID) + +dxr <- DEXSeq(dxd) + head(dxr) ``` + We can visualize the MA-plot ```{r,fig.width = 8, fig.height = 6} plotMA(dxr, cex=0.8 ) @@ -232,13 +249,14 @@ plotMA(dxr, cex=0.8 ) # Running bambu with large number of samples {#large-sample-num} -For larger sample numbers we recommend to write the processed data to a file: +For larger sample numbers we recommend to write the processed data to a file. This can be done by providing the readClass.outputDir: ```{r, eval = FALSE} -bambu(reads = test.bam, readClass.outputDir = "./bambu/", annotations = bambuAnnotations, genomeSequence = fa.file) +se <- bambu(reads = bamFiles, readClass.outputDir = "./bambu/", annotations = annotaiton, + genomeSequence = genomeSequence) ``` -# Getting Help {#get-help} +# Bambu parameters {#bambu-parameters} ## Advanced Options -More stringent filtering thresholds imposed on potential novel transcripts +### More stringent filtering thresholds imposed on potential novel transcripts * Keep novel transcripts with min 5 read count in at least 1 sample: ```{r, eval = FALSE} @@ -252,18 +270,26 @@ bambu(reads, annotations, genomeSequence, isoreParameters = list(min.sampleNumbe ```{r, eval = FALSE} bambu(reads, annotations, genomeSequence, isoreParameters = list(min.readFractionByGene = 0.1)) ``` -Quantification without bias correction +### Quantification without bias correction + The default estimation automatically does bias correction for expression estimates. However, you can choose to perform the quantification without bias correction. ```{r, eval = FALSE} bambu(reads, annotations, genomeSequence, emParameters = list(bias = FALSE)) ``` -Parallel computation +### Parallel computation + bambu allows parallel computation. ```{r, eval = FALSE} bambu(reads, annotations, genomeSequence, ncore = 8) ``` See *[manual](https://github.com/GoekeLab/bambu/blob/master/docs/bambu_0.1.0.pdf)* for details to customize other conditions. +# Getting help {#get-help} + +Questions and issues can be raised at the Bioconductor support site (once bambu is available through bioconductor): https://support.bioconductor.org. Please tag your your posts with bambu. + +Alternatively, issues can be raised at the bambu Github repository: https://github.com/GoekeLab/bambu. + # Citing bambu {#cite-bambu} A manuscript describing bambu is currently in preparation. If you use bambu for your research, please cite using the following doi: 10.5281/zenodo.3900025. 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zw7SZFv`?M;q^D~BQ`HE`!(pj~F)B)qjPllAXitEmiV~yzJ%9rU00w|4K`Rt0h!Rw! zusy68<&7xxR*DDyUk?Uw&;)@LR6$^+JIZ(T?z?mOdAPdOd0ye#LM%!RQVLxU&@+V@bW3Zb${G=_DjZ-_6YC}jm_2NNAT9^4wNJU;x14QSxmqxE^M zPQ@D2@6rle%brz*>9FSSX?JcZt1`U7^7zb#_B`FD4;yC7#(F7TO`}P(zj=*Fx}r)e zW_&CcF~Z7p)`#qoQN@^9+iU~BWsRTVyiTa^UkL=BceQ@B^Jgu(#?u4z=Ubv)=A^}3 zY}L33$zXR$bgc}2O$GHwa;?`6V#Qq=2V7b|a)$LBG}@oBPS|SEq?5m=X7(N!^ugY& zUuemK&KK*PVpT}8Vb@lUbZBH~WZVkVj4$W*9tdRKn16Y~K-v_sb6N-%F5ay+2 zaGQ0x0n-6fL6ddkb>leu0cahkwe$hJ9QJQ3fEBH>fdf z@qRBIPOnGD|3%2zzBsAuQAvB-Y|>G27ho}*y^%>wwL-vmP}ZR%Y)8o~~23q1kiBK0q2y>SQcT_xyHj`~dVEI0!8TiBm~s<$*ae=nXN6 t4H>qEGNL!$T91y7qt|Y2t?8I4*H9z!YGbKyGO2+eAVwh2z~T<$e*uvwwZQ-Y From a0a2a2d89fedefe48fad5513df993101677f7ece Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 02:11:00 +0000 Subject: [PATCH 92/98] update documentation for prepareAnnotations function Former-commit-id: ea36463698b76acc07597d1609c8c6ab07ac283f --- man/prepareAnnotations.Rd | 12 ++++++++---- 1 file changed, 8 insertions(+), 4 deletions(-) diff --git a/man/prepareAnnotations.Rd b/man/prepareAnnotations.Rd index dc4100fc..3fa9f205 100644 --- a/man/prepareAnnotations.Rd +++ b/man/prepareAnnotations.Rd @@ -2,12 +2,12 @@ % Please edit documentation in R/annotationFunctions.R \name{prepareAnnotations} \alias{prepareAnnotations} -\title{prepare annotations from txdb object} +\title{prepare annotations from txdb object or gtf file} \usage{ -prepareAnnotations(txdb) +prepareAnnotations(x) } \arguments{ -\item{txdb}{a \code{\link{TxDb}} object} +\item{x}{A \code{\link{TxDb}} object or a gtf file} } \value{ A \code{\link{GRangesList}} object @@ -18,5 +18,9 @@ Function to prepare tables and genomic ranges for transript reconstruction using \examples{ library(TxDb.Hsapiens.UCSC.hg38.knownGene) txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene - prepareAnnotations(txdb) + prepareAnnotations(x = txdb) + gtf.file <- system.file("extdata", + "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", + package = "bambu") + gr <- prepareAnnotationsFrom(x = gtf.file) } From a65717e48a037d7147813b5664b4dc40f00986e6 Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 02:11:18 +0000 Subject: [PATCH 93/98] remove documentation for prepareAnnotationsFromGTF Former-commit-id: 4d7ab0801c588bc03dc0ad5f60da95801c1e352e --- man/prepareAnnotationsFromGTF.Rd | 32 -------------------------------- 1 file changed, 32 deletions(-) delete mode 100644 man/prepareAnnotationsFromGTF.Rd diff --git a/man/prepareAnnotationsFromGTF.Rd b/man/prepareAnnotationsFromGTF.Rd deleted file mode 100644 index a66c42af..00000000 --- a/man/prepareAnnotationsFromGTF.Rd +++ /dev/null @@ -1,32 +0,0 @@ -% Generated by roxygen2: do not edit by hand -% Please edit documentation in R/annotationFunctions.R -\name{prepareAnnotationsFromGTF} -\alias{prepareAnnotationsFromGTF} -\title{Prepare annotation granges object from GTF file into a GRangesList object} -\usage{ -prepareAnnotationsFromGTF(file) -} -\arguments{ -\item{file}{a GTF file} -} -\value{ -A \code{\link{GRangesList}} object -} -\description{ -Prepare annotation granges object from GTF file -} -\details{ -Unlike \code{\link{readFromGTF}}, this function finds out the equivalence classes between the transcripts, -with \code{\link{mcols}} data having three columns: -\itemize{ - \item TXNAME specifying prefix for new gene Ids (genePrefix.number), defaults to empty - \item GENEID indicating whether filter to remove read classes which are a subset of known transcripts(), defaults to TRUE - \item eqClass specifying minimun read count to consider a read class valid in a sample, defaults to 2 - } -} -\examples{ -gtf.file <- system.file("extdata", -"Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf", -package = "bambu") -gr <- prepareAnnotationsFromGTF(gtf.file) -} From d466fede9254a21e3725946edbf81409307e13ff Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 02:11:40 +0000 Subject: [PATCH 94/98] update usage example for writeBambuOutput in documentation Former-commit-id: 323311561235f83ab1a4f6f37c5ca54d236a5552 --- man/writeBambuOutput.Rd | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/man/writeBambuOutput.Rd b/man/writeBambuOutput.Rd index 48dffc16..1639a595 100644 --- a/man/writeBambuOutput.Rd +++ b/man/writeBambuOutput.Rd @@ -4,7 +4,7 @@ \alias{writeBambuOutput} \title{Write bambu results to GTF and transcript/gene-count files} \usage{ -writeBambuOutput(se, path) +writeBambuOutput(se, path, prefix = "") } \arguments{ \item{se}{a \code{\link{SummarizedExperiment}} object from \code{\link{bambu}}} From 6f400d0b78c25ddd494a52f9542624dbde22a683 Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 09:10:10 +0000 Subject: [PATCH 95/98] when readDNAStringSet is used, correct the names to match to the annotations Former-commit-id: dd86eced8417470b0ea6061cf0f83f5433122126 --- R/junctionCorrection.R | 2 ++ 1 file changed, 2 insertions(+) diff --git a/R/junctionCorrection.R b/R/junctionCorrection.R index 41c017b6..99fa697e 100755 --- a/R/junctionCorrection.R +++ b/R/junctionCorrection.R @@ -16,6 +16,8 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, if(.Platform$OS.type == "windows"){ genomeSequence <- Biostring::readDNAStringSet(genomeSequence) + newlevels <- unlist(lapply(strsplit(names(genomeSequence)," "),"[[",1)) + names(genomeSequence) <- newlevels }else{ genomeSequence <- Rsamtools::FaFile(genomeSequence) } From b22dae30536babc7fe4a83825dcebb9d1a6790e7 Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 09:10:38 +0000 Subject: [PATCH 96/98] typo for biostrings Former-commit-id: 2fdd967985c69158bcd1f6553aab85a8afcd6e12 --- R/junctionCorrection.R | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/R/junctionCorrection.R b/R/junctionCorrection.R index 99fa697e..7ddf7046 100755 --- a/R/junctionCorrection.R +++ b/R/junctionCorrection.R @@ -15,7 +15,7 @@ createJunctionTable <- function(unlisted_junction_granges, genomeSequence=NULL, if(grepl('.fa',genomeSequence)){ if(.Platform$OS.type == "windows"){ - genomeSequence <- Biostring::readDNAStringSet(genomeSequence) + genomeSequence <- Biostrings::readDNAStringSet(genomeSequence) newlevels <- unlist(lapply(strsplit(names(genomeSequence)," "),"[[",1)) names(genomeSequence) <- newlevels }else{ From 439fdae5a257e9e01f49c36b2f302e24e523e24b Mon Sep 17 00:00:00 2001 From: cying111 Date: Mon, 27 Jul 2020 14:30:48 +0000 Subject: [PATCH 97/98] added double tab indent before subset points to show indent of sublevels correctly and corrected some typos Former-commit-id: edbaec452b73fd444dd21772c4999bb10ff282e7 --- vignettes/bambu.Rmd | 48 ++++++++++++++++++++++++--------------------- 1 file changed, 26 insertions(+), 22 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index a8f6666f..b3122b18 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -21,25 +21,26 @@ knitr::opts_chunk$set( *[Bambu](https://github.com/GoekeLab/bambu)* is a method for transcript discovery and quantification from long read RNA-Seq data. Bambu uses aligned reads and genome reference annotations as input, and will return abundance estimates for all known transcripts and for newly discovered transcripts. Bambu uses the information from the reference annotations to correct misalignment at splice junctions, then reduces the aligned reads to read equivalent classes, and uses this information to identify novel transcripts across all samples of interest. Reads are then assigned to transcripts, and expression estimates are obtained using an expectation maximisation algorithm. Here, we present an example workflow for analysing Nanopore long read RNA-Sequencing data from two human cancer cell lines from the Singapore Nanopore Expression Project (SG-NEx). -# Content -* [Quick Start: Transcript discovery and quantification with bambu](#quick-start) -* [A complete workflow to identify and quantify transcript expression from Nanopore RNA-Seq data](#complete-workflow) - + [Input data](#input-data) - - [Aligned reads (bam files)](#bam-files) - - [Genome sequence (fasta file/ BSGenome object)](#genome-sequences) - - [Genome annotations (bambu annotations object/ gtf file / TxDb object)](#annotations) - + [Transcript discovery and quantification](#transcript-discovery-quantification) - - [Run bambu](#run-bambu) - - [Visualise results](#visualise-results) - - [Obtain gene expression estimates from transcript expression](#gene-expression) - - [Save data (gtf/text)](#save-data) - + [Identify differentially expressed genes](#DESeq2) - + [Identify differential transcript usage](#DEXSeq) +# Content + +* [Quick Start: Transcript discovery and quantification with bambu](#quick-start) +* [A complete workflow to identify and quantify transcript expression from Nanopore RNA-Seq data](#complete-workflow) + + [Input data](#input-data) + - [Aligned reads (bam files)](#bam-files) + - [Genome sequence (fasta file/ BSGenome object)](#genome-sequences) + - [Genome annotations (bambu annotations object/ gtf file / TxDb object)](#annotations) + + [Transcript discovery and quantification](#transcript-discovery-quantification) + - [Run bambu](#run-bambu) + - [Visualise results](#visualise-results) + - [Obtain gene expression estimates from transcript expression](#gene-expression) + - [Save data (gtf/text)](#save-data) + + [Identify differentially expressed genes](#DESeq2) + + [Identify differential transcript usage](#DEXSeq) * [Running bambu with large number of samples](#large-sample-num) * [Bambu parameters](#bambu-parameters) * [Getting help](#get-help) -* [Citing bambu](#citing-bambu) -* [Session Information](#session-information) +* [Citing bambu](#cite-bambu) +* [Session Information](#session-info) # Quick start: Transcript discovery and quantification with bambu {#quick-start} @@ -74,9 +75,10 @@ bambuAnnotations <- prepareAnnotations(gtf.file) se <- bambu(reads = test.bam, annotations = bambuAnnotations, genomeSequence = fa.file) ``` -*bambu* returns a SummarizedExperiment object which can be accessed as follows: +*bambu* returns a SummarizedExperiment object which can be accessed as follows: + * assays(se) returns the transcript abundance estimates as counts or CPM -* rowRanges(se) returns a GRangesList with all annotated and newly discovered transcripts +* rowRanges(se) returns a GRangesList with all annotated and newly discovered transcripts * rowData(se) returns additional information about each transcript such as the gene name and the class of newly discovered transcript # A complete workflow to identify and quantify transcript expression from Nanopore RNA-Seq data {#complete-workflow} @@ -134,7 +136,7 @@ annotation ## Transcript discovery and quantification {#transcript-discovery-quantification} ### Running bambu {#run-bambu} -Next we apply *bambu* on the input data (bam files, annotations, genomeSequence). Bambu willto perform isoform discovery to extend the provided annotation, and then quantify the transcript expression from these extended annotation using an Expectation-Maximisation algorithm. Here we will use 1 core, which can be changed to process multiple files in parallel. +Next we apply *bambu* on the input data (bam files, annotations, genomeSequence). Bambu will perform isoform discovery to extend the provided annotation, and then quantify the transcript expression from these extended annotation using an Expectation-Maximisation algorithm. Here we will use 1 core, which can be changed to process multiple files in parallel. ```{r, results = "hide"} se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = genomeSequence, ncore = 1) @@ -142,7 +144,7 @@ se <- bambu(reads = bamFiles, annotations = annotation, genomeSequence = genomeS ```{r} se ``` -For the downstream analysis we will add the condition of interest to the \code{colData} object that describes the samples. Here we are interested in a comparison of the 2 cell lines: +For the downstream analysis, we will add the condition of interest to the \code{colData} object that describes the samples. Here we are interested in a comparison of the 2 cell lines: ```{r} colData(se)$condition <- as.factor(sample_info$cellLine) ``` @@ -190,8 +192,8 @@ plot.bambu(seGene, group.variable = "condition", type = "heatmap") ### Save data (gtf/text){#save-data} -*bambu* includes a function to write the extended annotations, the transcript and the gene expression estimates that include any newly discovered genes and transcripts to text files. -```{r， eval = FALSE} +*bambu* includes a function to write the extended annotations, the transcript and the gene expression estimates that include any newly discovered genes and transcripts to text files. +```{r, eval = FALSE} writeBambuOutput(se, path = "./bambu/", prefix="NanoporeRNASeq_") ``` @@ -284,6 +286,8 @@ bambu(reads, annotations, genomeSequence, ncore = 8) ``` See *[manual](https://github.com/GoekeLab/bambu/blob/master/docs/bambu_0.1.0.pdf)* for details to customize other conditions. +For transcript discovery we recommend to adjust the parameters according to the number of replicates and the sequencing throughput. The most relevant parameters are explained here. You can use any combination of these parameters. + # Getting help {#get-help} Questions and issues can be raised at the Bioconductor support site (once bambu is available through bioconductor): https://support.bioconductor.org. Please tag your your posts with bambu. From ed21e444ada88f960713be39329cc254ef327098 Mon Sep 17 00:00:00 2001 From: Chen Ying Date: Tue, 28 Jul 2020 10:47:08 +0800 Subject: [PATCH 98/98] moved the paragraph on parameters to below advanced options Former-commit-id: e24b800b3f0dd80b6c0b9e7d3a69e72289342176 --- vignettes/bambu.Rmd | 3 +-- 1 file changed, 1 insertion(+), 2 deletions(-) diff --git a/vignettes/bambu.Rmd b/vignettes/bambu.Rmd index b3122b18..bae6b826 100644 --- a/vignettes/bambu.Rmd +++ b/vignettes/bambu.Rmd @@ -258,6 +258,7 @@ se <- bambu(reads = bamFiles, readClass.outputDir = "./bambu/", annotations = an ``` # Bambu parameters {#bambu-parameters} ## Advanced Options +For transcript discovery we recommend to adjust the parameters according to the number of replicates and the sequencing throughput. The most relevant parameters are explained here. You can use any combination of these parameters. ### More stringent filtering thresholds imposed on potential novel transcripts * Keep novel transcripts with min 5 read count in at least 1 sample: @@ -286,8 +287,6 @@ bambu(reads, annotations, genomeSequence, ncore = 8) ``` See *[manual](https://github.com/GoekeLab/bambu/blob/master/docs/bambu_0.1.0.pdf)* for details to customize other conditions. -For transcript discovery we recommend to adjust the parameters according to the number of replicates and the sequencing throughput. The most relevant parameters are explained here. You can use any combination of these parameters. - # Getting help {#get-help} Questions and issues can be raised at the Bioconductor support site (once bambu is available through bioconductor): https://support.bioconductor.org. Please tag your your posts with bambu.