Overview¶
The workflow can be run in two modes: the ‘standard’ workflow (for normal sequencing) data and a ‘PDX’ workflow, which handles host/graft read separation for patient-derived xenograft samples.
Standard workflow¶
The standard (non-PDX) workflow essentially performs the following steps:
- The input reads are trimmed to remove adapters and/or poor quality base calls using cutadapt.
- The trimmed reads are aligned to the reference genome using bwa mem.
- The alignments are sorted and indexed using samtools.
- Bam files from multiple lanes are merged using samtools merge.
- Picard MarkDuplicates is used to remove optical/PCR duplicates.
- The final alignments are indexed using samtools index.
QC statistics are generated using fastqc, samtools stats and picard CollectHSMetrics (to assess bait coverage). The stats are summarized into a single report using multiqc.
This results in the following dependency graph:
PDX workflow¶
The PDX workflow is a slightly modified version of the standard workflow, which aligns the reads to two reference genome (the host and graft reference genomes) and uses disambiguate to remove sequences originating from the host organism. For typical PDX samples, this means removing the mouse (host) reads, leaving the human (graft) reads for further analysis.
The PDX workflow adds the following additional steps:
- The reads are aligned to two references in
bwa_hostandbwa_graft. - The resulting alignments are sorted by queryname using samtools and subsequently ‘disambiguated’ using the disamgibuate tool from AstraZeneca.
- The disambiguated alignments are sorted by coordinate using sambamba.
This results in the following dependency graph:
