Photoactivatable primer based Transcriptome In Vivo Analysis (TIVA) has shown successful application in profiling the transcriptome in live single cells [1]. As one of its successors, CHEX-seq employs 3' blocked, light-activatable probes, which anneal to single-stranded DNA and get laser activated in selected cells (fixed tissue or primary culture) at specified foci. Activated probes serve as primers to in situ copy the single-stranded DNA into amplicons that can be finally sequenced. As such, CHEX-seq enables genome-wide mapping of the single-stranded open-chromatin landscape while preserving the local tissue context [2].
This is the computational pipeline for converting raw reads from next-generation sequencing to single-stranded chromatin sites (in BED format) as well as the gene-wise count matrix in various subgenic regions.
This pipeline takes in the PennSCAP-T NGS pipeline preprocessed data, evaluates each read based on the barcode/primer quality as well as the mapping quality, then tracks the start site of a primer-extension event (i.e the probe annealing site) and removes multiple start sites in a small genomic range (20bp by default) as biochemically unlikely. Next, it summarizes the primer annealing sites (as single bases) as well as the primer-extended regions (as genomic intervals). It also searches and removes technical artifacts, including ENCODE low-mappability loci, cross-species contaminants, and low mapping-quality reads.
For complete manual please refer to the Wiki Page.
- PennSCAP-T pipeline (>=2.3)
- GNU parallel (>=20190122)
- Perl (>=5.010), Log::Log4perl (>1.01)
- R (>=3.5.0), GenomicRanges (>=1.34), GenomicAlignments (>=1.18), rtracklayer (>=1.42), GenomeInfoDb (>=1.29), ensembldb (>=2.6), EnsDb.Hsapiens.v86 (>=2.99), EnsDb.Mmusculus.v79 (>=2.99)
- Picard (>=2.17)
- bedtools (>=2.26)
- samtools (>=1.12)
- Install the dependencies as listed above;
- This package is designed to work as a
libhence doesn't need to be installed to your system or localbin. You just need to download and place the source code folder (src) to the workplace where you want to run the pipeline. Make sure the folderdatais the same level assrc. If you want to use some module as standalone, you may simply source the corresponding file in bash or R environment.
- Prepare the Sequencing Configuration (
data/info/config.csv), the sample sheet (data/SampleInfo.csv) - Generate the sample grouping info in R session
source("src/1.groupinfo.R")
- Run the pipeline by
$ bash src/2.pipeline.bash
- Get QC statistics in R session
source("src/3.qc.R")
- Extract the priming sites of desired quality in R session
source("src/4.sites.R")
- Generate genomic features and store for count quantification in R session
source("src/5.features.R")
- Generate priming count matrices per genomic feature type in R session
source("src/6.cntmatrix.R")
Copyright (c) 2017-2023, Jaehee Lee, Jifen Li, Jinhui Wang, Hyun-Bum Kim and James Eberwine, Perelman School of Medicine, University of Pennsylvania
Copyright (c) 2017-2023, Youtao Lu, Kevin L. Bullaughey, Jean G. Rosario, C. Erik Nordgren, Stephen A. Fisher and Junhyong Kim, Department of Biology, University of Pennsylvania
- Ditte Lovatt, Brittani K Ruble, Jaehee Lee, Hannah Dueck, Tae Kyung Kim, Stephen Fisher, Chantal Francis, Jennifer M Spaethling, John A Wolf, M Sean Grady, Alexandra V Ulyanova, Sean B Yeldell, Julianne C Griepenburg, Peter T Buckley, Junhyong Kim, Jai-Yoon Sul, Ivan J Dmochowski and James Eberwine. "Transcriptome in Vivo Analysis (TIVA) of Spatially Defined Single Cells in Live Tissue." (Nature Methods 11, no. 2 (February 2014): 190-96)
- Youtao Lu, Jaehee Lee, Jifen Li, Srinivasa Rao Allu, Jinhui Wang, Hyun-Bum Kim, Kevin L. Bullaughey, Stephen A. Fisher, C. Erik Nordgren, Jean G. Rosario, Stewart A. Anderson, Alexandra V. Ulyanova, Steven Brem, H. Isaac Chen, John A. Wolf, M. Sean Grady, Sergei A. Vinogradov, Junhyong Kim and James Eberwine. "CHEX-seq Detects Single-Cell Genomic Single-Strand DNA with Catalytical Potential." (Nature Communications 14, 7346 (2023))