TronFlow alignment pipeline

The TronFlow alignment pipeline is part of a collection of computational workflows for tumor-normal pair somatic variant calling.

Find the documentation here

This pipeline aligns paired and single end FASTQ files with BWA aln and mem algorithms and with BWA mem 2. For RNA-seq STAR is also supported. To increase sensitivity of novel junctions use --star_two_pass_mode (recommended for RNAseq variant calling). It also includes an initial step of read trimming using FASTP.

How to run it

Run it from GitHub as follows:

nextflow run tron-bioinformatics/tronflow-alignment -profile conda --input_files $input --output $output --algorithm aln --library paired

Otherwise download the project and run as follows:

nextflow main.nf -profile conda --input_files $input --output $output --algorithm aln --library paired

Find the help as follows:

$ nextflow run tron-bioinformatics/tronflow-alignment  --help
N E X T F L O W  ~  version 19.07.0
Launching `main.nf` [intergalactic_shannon] - revision: e707c77d7b

Usage:
    nextflow main.nf --input_files input_files [--reference reference.fasta]

Input:
    * input_fastq1: the path to a FASTQ file (incompatible with --input_files)
    * input_name: name of the sample (only needed if input_fastq1 is used)
    * input_files: the path to a tab-separated values file containing in each row the sample name and two paired FASTQs (incompatible with --fastq1 and --fastq2)
    when `--library paired`, or a single FASTQ file when `--library single`
    Example input file:
    name1	fastq1.1	fastq1.2
    name2	fastq2.1	fastq2.2
    * reference: path to the indexed FASTA genome reference or the star reference folder in case of using star

Optional input:
    * input_fastq2: the path to a second FASTQ file (incompatible with --input_files, incompatible with --library paired)
    * output: the folder where to publish output (default: output)
    * algorithm: determines the BWA algorithm, either `aln`, `mem`, `mem2` or `star` (default `aln`)
    * library: determines whether the sequencing library is paired or single end, either `paired` or `single` (default `paired`)
    * cpus: determines the number of CPUs for each job, with the exception of bwa sampe and samse steps which are not parallelized (default: 8)
    * memory: determines the memory required by each job (default: 32g)
    * inception: if enabled it uses an inception, only valid for BWA aln, it requires a fast file system such as flash (default: false)
    * skip_trimming: skips the read trimming step
    * star_two_pass_mode: activates STAR two-pass mode, increasing sensitivity of novel junction discovery, recommended for RNA variant calling (default: false)
    * additional_args: additional alignment arguments, only effective in BWA mem, BWA mem 2 and STAR (default: none) 

Output:
    * A BAM file \${name}.bam and its index
    * FASTP read trimming stats report in HTML format \${name.fastp_stats.html}
    * FASTP read trimming stats report in JSON format \${name.fastp_stats.json}

Input tables

The table with FASTQ files expects two tab-separated columns without a header

Sample name	FASTQ 1	FASTQ 2
sample_1	/path/to/sample_1.1.fastq	/path/to/sample_1.2.fastq
sample_2	/path/to/sample_2.1.fastq	/path/to/sample_2.2.fastq

Reference genome

The reference genome has to be provided in FASTA format and it requires two set of indexes:

• FAI index. Create with samtools faidx your.fasta
• BWA indexes. Create with bwa index your.fasta

For bwa-mem2 a specific index is needed:

bwa-mem2 index your.fasta

For star a reference folder prepared with star has to be provided. In order to prepare it will need the reference genome in FASTA format and the gene annotations in GTF format. Run a command as follows:

STAR --runMode genomeGenerate --genomeDir $YOUR_FOLDER --genomeFastaFiles $YOUR_FASTA --sjdbGTFfile $YOUR_GTF

References

• Li H. and Durbin R. (2010) Fast and accurate long-read alignment with Burrows-Wheeler Transform. Bioinformatics, Epub. https://doi.org/10.1093/bioinformatics/btp698
• Shifu Chen, Yanqing Zhou, Yaru Chen, Jia Gu; fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics, Volume 34, Issue 17, 1 September 2018, Pages i884–i890, https://doi.org/10.1093/bioinformatics/bty560
• Vasimuddin Md, Sanchit Misra, Heng Li, Srinivas Aluru. Efficient Architecture-Aware Acceleration of BWA-MEM for Multicore Systems. IEEE Parallel and Distributed Processing Symposium (IPDPS), 2019.
• Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013 Jan 1;29(1):15-21. doi: 10.1093/bioinformatics/bts635. Epub 2012 Oct 25. PMID: 23104886; PMCID: PMC3530905.