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 in Please clone this repository to your local filesystem using the following command on Biowulf:

```bash

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 LOGAN/main.nf -profile ci_stub -preview ```

Usage

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.

1. 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.

1. 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

bash 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

bash 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. hg38, hg19 and mm10 are supported.
Example: --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/GDC processing pipelines

--genome hg38_sf - Based off the Homosapiensassembly38.fasta which is derived from the Broad Institute/NCI Sequencing Facility The biggest difference between the two is that GRCh38.d1.vd1.fa only the GCA000001405.15GRCh38noaltanalysisset, Sequence Decoys (GenBank Accession GCA000786075), and Virus Sequences. Homosapiens_assembly38.fasta has HLA specific contigs which may not be compatible with certain downstream tools.

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

bash Tumor Normal c130863309_TUMOR c130863309_PBMC c130889189_TUMOR c130889189_PBMC

2. Tumor only mode

No additional 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 or --snv - Enables somatic SNV calling using mutect2, vardict, varscan, octopus, deepsomatic, strelka (TN only), MUSE (TN only), and lofreq (TN only)

--gl or --germline - Enables germline calling using Deepvariant

--sv or --structural- Enables somatic SV calling using Manta, GRIDSS, and SVABA

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

Optional Arguments

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

--cnvcallers - Comma separated argument for selecting only specified CNV callers, the default is to use all. Example: --cnvcallers purple

--svcallers - Comma separated argument for selecting only specified SV callers, the default is to use all. Example: --svcallers gridss

--ffpe - Adds additional filtering for FFPE by detecting strand orientation bias using SOBDetector.

--exome - When using exome data, this flag limits calling to intervals provided in target bed to reduce time and to account for exome sequencing specific parameters. An intervals file is required.

--indelrealign - Enables indel realignment using the GATK pipeline when running alignment steps. May be helpful for certain callers (VarScan, VarDict) that do not have local haplotype reassembly.

Running LOGAN

Example of Tumor_Normal calling mode

```bash

preview the logan jobs that will run

nextflow run LOGAN/main.nf --mode local -profile cistub --genome hg38 --samplesheet 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 LOGAN/main.nf --mode local -profile cistub --genome hg38 --samplesheet 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 LOGAN/main.nf --mode slurm -profile biowulf,slurm --genome hg38 --samplesheet samplesheet.tsv --outdir out --fastqinput "*R{1,2}.fastq.gz" --vc --sv --cnv ```

Example of Tumor only calling mode

```bash

preview the logan jobs that will run

nextflow run LOGAN/main.nf --mode local -profile cistub --genome hg38 --outdir out --fastqinput "*R{1,2}.fastq.gz" --callers octopus,mutect2 -preview --vc --sv --cnv

run a stub/dryrun of the logan jobs

nextflow run LOGAN/main.nf --mode local -profile cistub --genome hg38 --outdir out --fastqinput "*R{1,2}.fastq.gz" --callers octopus,mutect2 -stub --vc --sv --cnv

launch a logan run on slurm with the test dataset

nextflow run 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 ```

Pipeline Tools and Overview

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 continuous 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.

<sup>1. Kurtzer GM, Sochat V, Bauer MW (2017). Singularity: Scientific containers for mobility of compute. PLoS ONE 12(5): e0177459.</sup>