LOGAN 🔬

LOGAN-whoLe genOme-sequencinG Analysis pipeliNe. Call germline and somatic variants, CNVs, and SVs and annotate variants!

Overview

Welcome to LOGAN! Before getting started, we highly recommend reading through LOGAN's documentation.

LOGAN is a comprehensive whole genome-sequencing pipeline following the Broad's set of best practices. It relies on technologies like Singularity¹ to maintain the highest-level of reproducibility. The pipeline consists of a series of data processing and quality-control steps orchestrated by Nextflow², a flexible and scalable workflow management system, to submit jobs to a cluster or cloud provider.

Before getting started, we highly recommend reading through the usage section of each available sub command.

For more information about issues or trouble-shooting a problem, please checkout our FAQ prior to opening an issue on Github.

Original pipelining and code forked from the CCBR Exome-seek Pipeline Exome-seek and OpenOmics

Dependencies

Requires: singularity>=3.5 nextflow>=22.10.2

singularity must be installed on the target system. Snakemake orchestrates the execution of each step in the pipeline. To guarantee the highest level of reproducibility, each step relies on versioned images from DockerHub. Nextflow uses singularity to pull these images onto the local filesystem prior to job execution, and as so, nextflow and singularity are the only two dependencies.

Setup

LOGAN can be used with the Nextflow pipelining software Please clone this repository to your local filesystem using the following command on Biowulf:

# start an interactive node
sinteractive --mem=2g --cpus-per-task=2 --gres=lscratch:200
git clone https://github.com/CCBR/LOGAN
module load nextflow
##Example run 
nextflow run /data/LOGAN//main.nf

Usage

LOGAN supports

Input Files

LOGAN supports inputs of either

1. paired end fastq files

--fastq_input- A glob can be used to include all FASTQ files. Like --fastq_input "*R{1,2}.fastq.gz". Globbing requires quotes.

2. Pre aligned BAM files with BAI indices

--bam_input- A glob can be used to include all FASTQ files. Like --bam_input "*.bam". Globbing requires quotes.

3. A sheet that indicates the sample name and either FASTQs or BAM file locations

--fastq_file_input- A headerless tab delimited sheet that has the sample name, R1, and R2 file locations

Example

c130863309_TUMOR   /data/nousomedr/c130863309_TUMOR.R1_001.fastq.gz  /data/nousomedr/c130863309_TUMOR.R2_001.fastq.gz
c130889189_PBMC  /data/nousomedr/c130889189_PBMC.R1_001.fastq.gz  /data/nousomedr/c130889189_PBMC.R2_001.fastq.gz

--bam_file_input - A headerless tab delimited sheet that has the sample name, bam, and bam index (bai) file locations

Example

c130863309_TUMOR   /data/nousomedr/c130863309_TUMOR.bam  /data/nousomedr/c130863309_TUMOR.bam.bai
c130889189_PBMC  /data/nousomedr/c130889189_PBMC.bam  /data/nousomedr/c130889189_PBMC.bam.bai

Genome

--genome - A flag to indicate which genome to run for alignment/variant calling/etc. Like --genome hg38 to run the hg38 genome

--genome hg19 and --genome mm10 are also supported

hg38 has options for either

--genome hg38 - Based off the GRCh38.d1.vd1.fa which is consistent with TCGA and other GDC processing pipelines

--genome hg38_sf - Based off the Homo_sapiens_assembly38.fasta which is derived from the Broad Institute/NCI Sequencing Facility The biggest difference between the two is that GRCh38.d1.vd1.fa has fewer contigs (especially related to HLA regions), so reads should map to chr6 vs the HLA contig directly

Operating Modes

1. Paired Tumor/Normal Mode

Required for Paired Tumor/Normal Mode

--sample_sheet In Paired mode a sample sheet must be provided with the basename of the Tumor and Normal samples. This sheet must be Tab separated with a header for Tumor and Normal.

Example

Tumor  Normal
c130863309_TUMOR  c130863309_PBMC
c130889189_TUMOR  c130889189_PBMC

2. Tumor only mode

No addtional flags for sample sheet are required as all samples will be used to call variants

Calling Mode

Adding flags determines SNV (germline and/or somatic), SV, and/or CNV calling modes

--vc- Enables somatic SNV calling using mutect2, vardict, varscan, octopus, strelka (TN only), MUSE (TN only), and lofreq (TN only)

--germline- Enables germline using Deepvariant

--sv- Enables somatic SV calling using Manta, SVABA, and GRIDSS (coming soon)

--cnv- Enables somatic CNV calling using FREEC, Sequenza, and Purple (hg19/hg38 only)

Optional Arguments

--indelrealign - Enables indel realignment when running alignment steps. May be helpful for certain callers (VarScan, VarDict)

--callers- Comma separated argument for callers, the default is to use all available.
Example: --callers mutect2,octopus

--cnvcallers- - Comma separated argument for CNV callers to use. Adding flag allows only certain callers to run.
Example: --cnvcallers purple

--svcallers- - Comma separated argument for SV callers. Adding flag allows only certain callers to run.
Example: --cnvcallers manta

Running LOGAN

Example of Tumor_Normal calling mode

# preview the logan jobs that will run 
nextflow run /data/LOGAN/main.nf --mode local -profile ci_stub --genome hg38 --sample_sheet samplesheet.tsv --outdir out --fastq_input "*R{1,2}.fastq.gz" -preview --vc --sv --cnv
# run a stub/dryrun of the logan jobs 
nextflow run /data/LOGAN/main.nf --mode local -profile ci_stub --genome hg38 --sample_sheet samplesheet.tsv --outdir out --fastq_input "*R{1,2}.fastq.gz" -stub --vc --sv --cnv
# launch a logan run on slurm with the test dataset
nextflow run /data/LOGAN/main.nf --mode slurm -profile biowulf,slurm --genome hg38 --sample_sheet samplesheet.tsv --outdir out --fastq_input "*R{1,2}.fastq.gz" --vc --sv --cnv 

Example of Tumor only calling mode

# preview the logan jobs that will run 
nextflow run /data/LOGAN/main.nf --mode local -profile ci_stub --genome hg38 --outdir out --fastq_input "*R{1,2}.fastq.gz" --callers octopus,mutect2 -preview --vc --sv --cnv
# run a stub/dryrun of the logan jobs 
nextflow run /data/LOGAN/main.nf --mode local -profile ci_stub --genome hg38 --outdir out --fastq_input "*R{1,2}.fastq.gz" --callers octopus,mutect2 -stub --vc --sv --cnv
# launch a logan run on slurm with the test dataset
nextflow run /data/LOGAN/main.nf --mode slurm -profile biowulf,slurm --genome hg38 --outdir out --fastq_input "*R{1,2}.fastq.gz" --callers octopus,mutect2 --vc --sv --cnv

Contribute

This site is a living document, created for and by members like you. LOGAN is maintained by the members of CCBR and is improved by continous feedback! We encourage you to contribute new content and make improvements to existing content via pull request to our repository.

References

This repo was originally generated from the CCBR Nextflow Template.

^{1. Kurtzer GM, Sochat V, Bauer MW (2017). Singularity: Scientific containers for mobility of compute. PLoS ONE 12(5): e0177459.}