alignment-nf

Nextflow pipeline for BAM realignment or fastq alignment

Description

Nextflow pipeline to perform BAM realignment or fastq alignment and QC, with/without local indel realignment and base quality score recalibration.

Dependencies

Nextflow : for common installation procedures see the IARC-nf repository.

Basic fastq alignment

BAM files realignment

samtools

Adapter sequence trimming

AdapterRemoval

ALT contigs handling

the k8 javascript execution shell (e.g., available in the bwakit archive); must be in the PATH
javascript bwa-postalt.js and the additional fasta reference .alt file from bwakit must be in the same directory as the reference genome file.

QC

Qualimap.
Multiqc.

Base quality score recalibration

GATK4; wrapper 'gatk' must be in the path
GATK bundle VCF files with lists of indels and SNVs (recommended: Mills gold standard indels VCFs, dbsnp VCF), and corresponding tabix indexes (.tbi)

A conda receipe, and docker and singularity containers are available with all the tools needed to run the pipeline (see "Usage")

Input

Type	Description
--input_folder	a folder with fastq files or bam files

Parameters

Mandatory

Name	Example value	Description
--ref	hg19.fasta	genome reference with its index files (.fai, .sa, .bwt, .ann, .amb, .pac, and .dict; in the same directory)

Optional

Name	Default value	Description
--input_file	null	Input file (comma-separated) with 4 columns: SM (sample name), RG (read group ID), pair1 (first fastq of the pair), and pair2 (second fastq of the pair).
--output_folder	.	Output folder for aligned BAMs
--cpu	8	number of CPUs
--cpu_BQSR	2	number of CPUs for GATK base quality score recalibration
--mem	32	memory
--mem_BQSR	10	memory for GATK base quality score recalibration
--RG	PL:ILLUMINA	sequencing information for aligned (for bwa)
--fastq_ext	fastq.gz	extension of fastq files
--suffix1	_1	suffix for second element of read files pair
--suffix2	_2	suffix for second element of read files pair
--bed		bed file with interval list
--snp_vcf	dbsnp.vcf	path to SNP VCF from GATK bundle (default : dbsnp.vcf)
--indel_vcf	Mills_1000G_indels.vcf	path to indel VCF from GATK bundle (default : Mills_1000G_indels.vcf)
--postaltjs	bwa-postalt.js"	path to postalignment javascript bwa-postalt.js
--feature_file	null	Path to feature file for qualimap
--multiqc_config	null	config yaml file for multiqc
--adapterremoval_opt	null	Command line options for AdapterRemoval
--bwa_mem	bwa-mem2 mem	bwa-mem command; use "bwa mem" to switch to regular bwa-mem (both are in the docker and singularity containers)

Flags

Flags are special parameters without value.

Name	Description
--help	print usage and optional parameters
--trim	enable adapter sequence trimming
--recalibration	perform quality score recalibration (GATK)
--alt	enable alternative contig handling (for reference genome hg38)
--bwa_option_M	Trigger the -M option in bwa and the corresponding compatibility option in samblaster (marks shorter split hits as secondary)

Usage

To run the pipeline on a series of fastq or BAM files in folder input and a fasta reference file hg19.fasta, one can type:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output

To run the pipeline without singularity just remove "-profile singularity". Alternatively, one can run the pipeline using a docker container (-profile docker) the conda receipe containing all required dependencies (-profile conda).

Use bwa-mem instead of bwa-mem2

To use bwa-mem, one can type:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output --bwa_mem "bwa mem"

Enable adapter trimming

To use the adapter trimming step, you must add the --trim option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output --trim

Enable ALT mode

To use the alternative contigs handling mode, you must provide the path to an ALT aware genome reference (e.g., hg38) AND add the --alt option, as well as satisfy the above-mentionned requirements. For example:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output --postaltjs /user/bin/bwa-0.7.15/bwakit/bwa-postalt.js --alt

Enable base quality score recalibration

To use the base quality score recalibration step, you must provide the path to 2 GATK bundle VCF files with lists of known snps and indels, respectively, AND add the --recalibration option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf -r v1.3 -profile singularity  --input_folder input/ --ref hg19.fasta --output_folder output --snp_vcf GATKbundle/dbsnp.vcf.gz --indel_vcf GATKbundle/Mills_1000G_indels.vcf.gz --recalibration

Output

Type	Description
BAM/	folder with BAM and BAI files of alignments or realignments
QC/BAM/multiqc_qualimap_flagstat_*report.html	multiQC report for qualimap and samtools flagstat (duplicates)
QC/BAM/multiqc_qualimap_flagstat_*report_data	data used for the multiQC report
QC/qualimap/file_BQSRecalibrated.stats.txt	qualimap summary file
QC/qualimap/file_BQSRecalibrated/	qualimap files
QC/BAM/BQSR/	GATK base quality score recalibration outputs (tables and pdf comparing scores before/after recalibration)

Directed Acyclic Graph

FAQ

Why did Indel realignment disappear from version 1.0?

Indel realignment was removed following new GATK best practices for pre-processing.

Contributions

Name	Email	Description
Nicolas Alcala*	AlcalaN@fellows.iarc.fr	Developer to contact for support
Catherine Voegele	VoegeleC@iarc.fr	Tester
Vincent Cahais	CahaisV@iarc.fr	Tester
Alexis Robitaille	RobitailleA@students.iarc.fr	Tester

kubranarci/alignment-nf