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https://github.com/darwinawardwinner/cd4-csaw

Reproducible reanalysis of a combined ChIP-Seq & RNA-Seq data set
https://github.com/darwinawardwinner/cd4-csaw

bioconductor bioinformatics-pipeline chip-seq r reproducible-research rna-seq

Last synced: 19 days ago
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Reproducible reanalysis of a combined ChIP-Seq & RNA-Seq data set

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



This is the code for a re-analysis of a [GEO dataset][1] that I

originally analyzed for [this paper][2] using statistical methods that

were not yet available at the time, such as the

[csaw Bioconductor package][3], 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][4]

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



![Rule Graph](rulegraphs/rulegraph-all.png "Rule graph of currently implemented workflow")



### Completed components



* ChIP-seq

  * Mapping with bowtie2

  * Peak calling with MACS2 and Epic

  * Fetching of [blacklists][5] 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](http://downloads.asperasoft.com/en/downloads/50) Aspera

  download client for downloading SRA files

* [Bedtools](http://bedtools.readthedocs.io/en/latest/)

* [Bowtie2](http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)

  aligner

* [Epic](https://github.com/endrebak/epic) peak caller

* [fastq-tools](http://homes.cs.washington.edu/~dcjones/fastq-tools/)

* [HISAT2](https://ccb.jhu.edu/software/hisat2/index.shtml) aligner

* [IDR python script](https://github.com/nboley/idr)

* [Kallisto](https://pachterlab.github.io/kallisto/about) RNA-seq

  quantifier

* [MACS2](https://github.com/taoliu/MACS) peak caller

* [Picard tools](https://broadinstitute.github.io/picard/) for various

  file manipulation utilities

* [Salmon](http://salmon.readthedocs.io/en/latest/) RNA-seq quantifier

  (devel version 0.7.3)

* [Shoal](https://github.com/COMBINE-lab/shoal)

* [Snakemake](https://bitbucket.org/snakemake/snakemake/wiki/Home) for

  running the workflow

* [SRA toolkit](https://github.com/ncbi/sra-tools) for extracting

  reads from SRA files

* [STAR](https://github.com/alexdobin/STAR) aligner

* [UCSC command-line tools](http://hgdownload.cse.ucsc.edu/downloads.html#source_downloads)

  (e.g. liftOver)



### Programming languages and packages



* [R](https://www.r-project.org/),

  [Bioconductor](http://bioconductor.org/), and the following R

  packages:

    * From [CRAN](http://cran.r-project.org/): 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](http://bioconductor.org/): 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](http://pachterlab.github.io/sleuth/about),

      [wasabi](https://github.com/COMBINE-lab/wasabi)

* [Python 3](https://www.python.org/) and the following Python

  packages: biopython, atomicwrites, numpy, pandas, plac, pysam, rpy2,

  snakemake



[1]: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73214

[2]: http://www.ncbi.nlm.nih.gov/pubmed/27170561

[3]: https://bioconductor.org/packages/release/bioc/html/csaw.html

[4]: https://bioconductor.org/packages/release/bioc/html/limma.html

[5]: http://www.broadinstitute.org/~anshul/projects/encode/rawdata/blacklists/hg19-blacklist-README.pdf