CCBR DNAnexus Workflows

Table of Contents

1. RNA-seq
2. ChIP-seq
3. EXOME-seq

Description:

These workflows provides access to some of the NGS pipelines used and developed by CCBR on Biowulf. They allow the user to select a set of fastq files, and process them on DNAnexus platform to reach an endpoint, such as a list of mutations with accompanying annotations, a set of annotated peaks, or a raw counts matrix.

1. RNA-seq

Overview of the RNA-seq pipeline:

1. FastQC: a quality control tool for high throughput sequence data, written by Simon Andrews at the Babraham Institute in Cambridge. It provides a modular set of analyses which can be used to give a quick impression of whether your data has any problems of which you should be aware before doing any further analysis.
2. Cutadapt: a tool to find and remove adapter sequences, primers, poly-A tails and other types of unwanted sequence from your high-throughput sequencing reads. Cutadapt is being used to remove adapter sequences.
3. FastQ Screen: a tool to screen a library of sequences against a set of sequence databases so one can see if the composition of the library matches with what is expected.
4. STAR: an ultrafast splice-aware RNA-seq aligner.
5. RSEM: quantifies gene and isoform expression levels from RNA-Seq data.
6. MultiQC: aggregate results from bioinformatics analyses across many samples into a single report

USAGE

RNA-seq Workflow
Center for Cancer Research
Collaborative Bioinformatics Resource

usage: dx run ccbr_RNA-seq_WF [-iARG_NAME=VALUE ...]


Inputs:
  Input Fastq File Array: -iFastqFiles=(file) [-iFastqFiles=... [...]]
        Please provide a series of paired-end FastQ files:
            -iFastqFiles=sample1.R1.fastq.gz -iFastqFiles=sample1.R2.fastq.gz
            -iFastqFiles=sample2.R1.fastq.gz -iFastqFiles=sample2.R2.fastq.gz

  Reference Files for each Genome: -iReferenceFiles=(file)
        Genomic Resource file: 'genome2resources.tsv':
            See Example (file-FbJJz300xx3qbfp80v1vVKKp)

  Version of STAR to use for alignment: '2.6.0a' or '2.7.0f' [default]: [-iStarVersion=(string, default="2.7.0f")]
        Choices: 2.6.0a, 2.7.0f

        Version of STAR to Run: '2.6.0a' or '2.7.0f'. Optional Argument.
            Default is set to latest supported verison of STAR: '2.7.0f'.

  Reference Genome: -iRefGenome=(string)
        Reference Genome: Needed for dynamically resolving the correct reference files.
        Choices: [mm9, mm10, mm10_M18,
                   hg38, hg38_v28, hg19,
                   hg38_HPV16, hg19_KSHV,
                   hs38d1, hs37d5, Mmul_8.0.1]
	Note: mm10 was built using M21 GENCODE annotations and hg38 was built using version 30 GENCODE annotations.

Running the RNA-seq Workflow

module load DNAnexus # or download dx-toolkit and add to $PATH

dx login # optional step (if you have not logged in the last 30 days)
         # prompt will ask for Username and Password
         # select this Project: 'CCBR_RNASeq_Pipeline'

# Data upload (optional, if data not on DNAnexus)
## Assumes data exists in present working directory
dx upload control_1.R1.fastq.gz --destination=/Testing/
dx upload control_1.R2.fastq.gz --destination=/Testing/
dx upload treatment_1.R1.fastq.gz --destination=/Testing/
dx upload treatment_1.R2.fastq.gz --destination=/Testing/

# Run the RNA-seq Pipeline
## Supports mm9, mm10, hg38, hg38_HPV16, hg19, hg19_KSHV, hs38d1, hs37d5, Mmul_8.0.1
echo "Y n" | dx run /Workflow/ccbr_RNA-seq_WF \
  -iFastqFiles=/Testing/control_1.R1.fastq.gz \
  -iFastqFiles=/Testing/control_1.R2.fastq.gz \
  -iFastqFiles=/Testing/treatment_1.R1.fastq.gz \
  -iFastqFiles=/Testing/treatment_1.R2.fastq.gz \
  -iRefGenome=hg19 \
  --destination=/Testing/Example/ \
  --priority=high

For more detailed documentation about RNA-seq and the pipeline, please checkout our user documentation page.

2. ChIP-seq

Description:

Under Constuction: Coming Soon!

3. EXOME-seq

Description:

Under Constuction: Coming Soon!

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