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alignment-nf

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

Overview of pipeline workflow

Scheme of alignment/realignment Workflow

workflow

Prerequisites

For the basic fastq files alignment without indel realignment, base quality score recalibration, nor alternative contif handling, the script requires the following programs: - *nextflow*. Install with bash curl -fsSL get.nextflow.io | bash And move it to a location in your $PATH (e.g., /usr/local/bin): bash sudo mv nextflow /usr/local/bin - *bwa* - *samblaster* - *sambamba* and the following files: - a fasta reference genome with its index files (.fai, .sa, .bwt, .ann, .amb, .pac; in the same directory) - a folder with fastq files

In addition, for the bam files realignment: - *samtools* - a folder with bam files

For the adapter sequence trimming: - *AdapterRemoval*

For the ALT contigs handling, additional softwares and scripts are required: - the k8 javascript execution shell (e.g., available in the *bwakit* archive) - javascript bwa-postalt.js and the additional fasta reference .alt file from *bwakit* must be in the same directory as the reference genome file.

For the indel realignment: - GATK *GenomeAnalysisTK.jar* - GATK bundle VCF files with lists of indels (recommended: 1000 genomes and Mills gold standard VCFs)

For the base quality score recalibration: - GATK *GenomeAnalysisTK.jar* - GATK bundle VCF files with lists of indels and SNVs (recommended: 1000 genomes indels, Mills gold standard indels VCFs, dbsnp VCF) - bed file with intervals to be considered

Usage

nextflow run iarcbioinfo/alignment-nf --input_folder input --fasta_ref hg19.fasta --out_folder output

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 --input_folder input --fasta_ref reference/hs38DH.fa -out_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 requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf --input_folder input --fasta_ref reference/hs38DH.fa --js /user/bin/k8/k8 --postaltjs /user/bin/bwa-0.7.15/bwakit/bwa-postalt.js -out_folder output --alt

Enable local indel realignment

To use the local indel realignment step, you must provide the path to the GATK jar file, the GATK bundle folder, AND add the *--indel_realignment* option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf --GATK_bundle GATKbundle/hg19 --input_folder input --fasta_ref reference/hg19.fa --GATK_folder /user/bin7GATK-3.6-0 --out_folder output --indel_realignment

Enable base quality score recalibration

To use the base quality score recalibration step, you must provide the path to the GATK jar file, the GATK bundle folder, a bed file, AND add the *--recalibration* option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf --GATK_bundle GATKbundle/hg19 --input_folder input --fasta_ref reference/hg19.fa --GATK_folder /user/bin7GATK-3.6-0 --intervals reference/hg19_intervals.bed --out_folder output --recalibration

All parameters

**PARAMETER* * DEFAULT **DESCRIPTION* *
--help null print usage and optional parameters
--input_fo lder . input folder
--fasta_re f hg19.fasta genome reference
--cpu 8 number of CPUs
--mem 32 memory
--mem_samb amba 1 memory for software sambamba
--RG PL:ILLUMINA sequencing information for aligned (for bwa)
--fastq_ex t 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
--out_fold er . output folder for aligned BAMs
*--intervals *   bed file with interval list
--GATK_bun dle bundle path to GATK bundle files
--GATK_fol der . path to GATK GenomeAnalysi sTK.jar file
--trim false enable adapter sequence trimming
--indel_re alignment false perform local indel realignment (GATK)
--recalibra tion false perform quality score recalibration (GATK)
--js k8 path to javascript interpreter k8
*--postaltjs * bwa-postalt.js" path to postalignment javascript bwa-postalt.j s
--alt false enable alternative contig handling (for reference genome hg38)