https://hydra-genetics-alignment.readthedocs.io

hydra-genetics/alignment

Snakemake module containing processing steps that should be performed during sequence alignment.

💬 Introduction

The module consists of alignment processing steps, such as alignment of .fastq-files. and duplicates marking .bam-files.

❗ Dependencies

In order to use this module, the following dependencies are required:

Note! Releases of prealignment <= v0.4.0 needs tabulate<0.9.0 added in requirements.txt

🎒 Preparations

Sample and unit data

Input data should be added to samples.tsv and units.tsv. The following information need to be added to these files:

Column Id	Description
samples.tsv
sample	unique sample/patient id, one per row
units.tsv
sample	same sample/patient id as in samples.tsv
type	data type identifier (one letter), can be one of Tumor, Normal, RNA
platform	type of sequencing platform, e.g. NovaSeq
machine	specific machine id, e.g. NovaSeq instruments have @Axxxxx
flowcell	identifier of flowcell used
lane	flowcell lane number
barcode	sequence library barcode/index, connect forward and reverse indices by +, e.g. ATGC+ATGC
fastq1/2	absolute path to forward and reverse reads
adapter	adapter sequences to be trimmed, separated by comma

Reference data

You need have a indexed reference genome: e.g. reference.fna

For bwa the files are generated by bwa index. Dict files is generated using picard CreateSequenceDictionary. fai is generated using samtools index

File	Description	Generated by
reference.dict	dictionary file	picard CreateSequenceDictionary
reference.fna.amb	record appearance of N (or other non-ATGC) in the ref fasta	bwa index
reference.fna.ann	record ref sequences, name, length, etc	bwa index
reference.fna.bwt	the Burrows-Wheeler transformed sequence	bwa index
reference.fna.fai	index file	samtools index
reference.fna.pac	packaged sequence (four base pairs encode one byte)	bwa index
reference.fna.sa	suffix array index	bwa index

For the RNA-aligner Star a star index is needed. This can be generated based on the reference genome. Example command:

STAR --runThreadN 8 --runMode genomeGenerate --genomeDir star_index --genomeFastaFiles reference.fna

✅ Testing

The workflow repository contains a small test dataset .tests/integration which can be run like so:

$ cd .tests/integration
$ snakemake -s ../../Snakefile -j1 --configfile config.yaml --use-singularity

🚀 Usage

To use this module in your workflow, follow the description in the snakemake docs. Add the module to your Snakefile like so:

module alignment:
    snakefile:
        github(
            "hydra-genetics/alignment",
            path="workflow/Snakefile",
            tag="v0.4.0",
        )
    config:
        config


use rule * from alignment as alignment_*

Compatibility

Latest:

• prealignment:v1.2.0

See COMPATIBLITY.md file for a complete list of module compatibility.

Input files

File	Description
hydra-genetics/prealignment data
prealignment/fastp_pe/{sample}_{type}_{flowcell}_{lane}_fastq1.fastq.gz	trimmed forward reads
prealignment/fastp_pe/{sample}_{type}_{flowcell}_{lane}_fastq2.fastq.gz	trimmed reverse reads
original fastq files
PATH/fastq1.fastq.gz	forward reads retrieved from units.tsv
PATH/fastq2.fastq.gz	reverse reads retrieved from units.tsv

Output files

The following output files should be targeted via another rule:

File	Description
alignment/samtools_merge_bam/{sample}_{type}.bam	aligned data which have been duplicate marked
alignment/star/{sample}_{type}.bam	aligned rna data

🧑‍⚖️ Rule Graph

Align and mark duplicates

RNA Alignment