README.mkdn

Re-analysis of a combined ChIP-Seq & RNA-Seq data set

This is the code for a re-analysis of a GEO dataset that I originally analyzed for this paper using statistical methods that were not yet available at the time, such as the csaw Bioconductor package, which provides a principled way to normalize windowed counts of ChIP-Seq reads and test them for differential binding. The original paper only analyzed binding within pre-defined promoter regions. In addition, some improvements have also been made to the RNA-seq analysis using newer features of limma such as quality weights.

This workflow downloads the sequence data and sample metadata from the public GEO/SRA release, so anyone can download and run this code to reproduce the full analysis.

Workflow

Completed components

• ChIP-seq
  • Mapping with bowtie2
  • Peak calling with MACS2 and Epic
  • Fetching of blacklists from UCSC
  • Generation of greylists from ChIP-Seq input samples
  • IDR analysis of blacklist-filtered peak calls
  • Computation of cross-correlation function for ChIP-Seq samples, excluding blacklisted regions
  • Counting in windows across the genome
• RNA-seq
  • Mapping with STAR & HISAT2
  • Counting reads aligned to genes
  • Alignment-free bias-corrected transcript quantification using Salmon & Kallisto
  • Differential gene expression

Possible TODO components

• Integrating RNA-seq and ChIP-seq
  • hiAnnotator: http://bioconductor.org/packages/devel/bioc/html/hiAnnotator.html
  • ChIPseeker: http://bioconductor.org/packages/devel/bioc/html/ChIPseeker.html
  • mogsa: http://bioconductor.org/packages/release/bioc/html/mogsa.html
• Gene set tests
  • ToPASeq: http://bioconductor.org/packages/devel/bioc/html/ToPASeq.html
  • mvGST: http://bioconductor.org/packages/devel/bioc/html/mvGST.html
  • mgsa: http://bioconductor.org/packages/release/bioc/html/mgsa.html
• QC Stuff
  • ChIPQC: http://bioconductor.org/packages/release/bioc/html/ChIPQC.html
  • MultiQC: http://multiqc.info/
  • Rqc: http://www.bioconductor.org/packages/devel/bioc/html/Rqc.html
• mixOmics: http://mixomics.org/
• ica: https://cran.rstudio.com/web/packages/ica/index.html
• Motif enrichment
• pcaExplorer: https://bioconductor.org/packages/release/bioc/html/pcaExplorer.html

TODO Code cleanup

• Remove unnecessary library() calls
• Put spaces around equals signs

TODO Other

• Document how to run the pipeline
• Provide install script for R & Python packages.

Dependencies

Command-line tools

• ascp Aspera download client for downloading SRA files
• Bedtools
• Bowtie2 aligner
• Epic peak caller
• fastq-tools
• HISAT2 aligner
• IDR python script
• Kallisto RNA-seq quantifier
• MACS2 peak caller
• Picard tools for various file manipulation utilities
• Salmon RNA-seq quantifier (devel version 0.7.3)
• Shoal
• Snakemake for running the workflow
• SRA toolkit for extracting reads from SRA files
• STAR aligner
• UCSC command-line tools (e.g. liftOver)

Programming languages and packages

• R, Bioconductor, and the following R packages:
  • From CRAN: assertthat, doParallel, dplyr, future, getopt, GGally, ggforce, ggfortify, ggplot2, ks, lazyeval, lubridate, magrittr, MASS, Matrix, openxlsx, optparse, parallel, purrr, RColorBrewer, readr, reshape2, rex, scales, stringi, stringr
  • From Bioconductor: annotate, Biobase, BiocParallel, BSgenome.Hsapiens.UCSC.hg19, BSgenome.Hsapiens.UCSC.hg38, ChIPQC, csaw, edgeR, GenomicFeatures, GenomicRanges, GEOquery, limma, org.Hs.eg.db, Rsamtools, Rsubread, rtracklayer, S4Vectors, SRAdb, SummarizedExperiment, TxDb.Hsapiens.UCSC.hg19.knownGene, tximport
  • Installed manually: sleuth, wasabi
• Python 3 and the following Python packages: biopython, atomicwrites, numpy, pandas, plac, pysam, rpy2, snakemake