A set of NGS data analysis tools and pipelines developed and utilised at the Institute of Molecular Biology gGmbH in Mainz (https://www.imb.de/).

A flowchart for the RNA-seq pipeline is given here.

• FastQC
• STAR
• Samtools
• Bedtools
• Subread
• Picard
• UCSC utilities
• RSeQC
• DEseq2
• dupRadar (provided by another project from imbforge)

• targets.txt (sample names)
• contrasts.txt (pairwise comparisons)
• raw reads (.fastq.gz) or mapped data (.bam)

A flowchart for the ChIP-seq pipeline is available for single-read and paired-end sequencing.

• FastQC
• Bowtie
• Samtools
• Bedtools
• Picard
• UCSC utilities
• MACS2
• ChIPSeeker
• encodeChIPqc (provided by another project from imbforge)

• targets.txt (sample names)
• raw reads (.fastq.gz) or mapped data (.bam)

A flowchart for the DNA-seq pipeline is available here.

• FastQC
• BWA
• Samtools
• Bedtools
• Picard
• dupRadar (provided by another project from imbforge)
• GATK

• raw reads (.fastq.gz) or mapped data (.bam)

GATK requires chromosomes in bam files to be karyotypically ordered. Best you use an ordered genome fasta file as reference for the pipeline (assigned in essential.vars.groovy, see below).

NGS projects should be run in a consistant and reproducible way, hence NGSpipe2go asks you to copy all tools into the project folder, which will ensure that you always use the same program versions at a later time point. This can be done either from a local NGSpipe2go copy, a version from the GitHub releases (https://github.com/imbforge/NGSpipe2go/releases) or using the most recent development version from the GitHub repository

git clone https://github.com/imbforge/NGSpipe2go.git <project_dir>/NGSpipe2go

Select a pipeline to run and make symlinks in the main project dir, e.g. for RNA-seq project

ln -s NGSpipe2go/pipelines/RNAseq/* .
ln -s NGSpipe2go/modules/RNAseq/essential.vars.groovy .
ln -s NGSpipe2go/modules/RNAseq/tool.locations.groovy .

or for single-read (SR) ChIP-seq project

ln -s NGSpipe2go/pipelines/ChIPseq/* .
ln -s NGSpipe2go/modules/ChIPseq/essential.vars.groovy .
ln -s NGSpipe2go/modules/ChIPseq/tool.locations.groovy .

or for paired-end (PE) ChIP-seq project

ln -s NGSpipe2go/pipelines/ChIPseq_pe/* .
ln -s NGSpipe2go/modules/ChIPseq/essential.vars.groovy .
ln -s NGSpipe2go/modules/ChIPseq/tool.locations.groovy .

Adjust the project-specific information in the following files:

• essential.vars.groovy specifies the main project variables like project dir and reference genome
• xxx.pipeline.groovy describes the pipeline steps and the location of the respective modules
• targets.txt and contrasts.txt contain the sample names and the differential group comparisons
• tool.location.groovy and bpipe.config specify the paths and resource allocation for the tools

Additional software parameters can be customised in the xxx.vars.groovy files accompanying each bpipe module.

Copy the input FastQ files into the <project_dir>/rawdata folder.

Using GNU Screen (for persistence) load the bpipe module customised for the Slurm job manager, e.g.

screen
module load bpipe/0.9.9.3.slurm

Start running the pipeline of choice, e.g.

bpipe run rnaseq.pipeline.groovy rawdata/*.fastq.gz

or

bpipe run chipseq.pipeline.groovy rawdata/*.fastq.gz    

or

bpipe run chipseq_pe.pipeline.groovy rawdata/*.fastq.gz

The final result of the provided pipelines will be saved in the ./reports folder. The Rmd file can be edited or customised using a text editor and then converted into HTML report using knitr

R usage:
rmarkdown::render("DEreport.Rmd")
or
rmarkdown::render("ChIPreport.Rmd")