Overview of the pipeline

Here is a brief overview of the entire pipeline. For details see subsections and the hydra-genetics documentation.

Essentially, a few extra steps specific to the needs of the geneticists at Clinical Genomics Uppsala are added on the top of the pipeline Poppy (Genomic Medicine Sweden).

Main processing steps from poppy GMS

Depending on the sequencing setup that is used, a preliminary demultiplexing of the samples may be required. We assume in the following steps that the fastq files are demultiplexed.

  1. Input files: directory with fastq files, also used to create the samples.tsv and units.tsv files (see section "Running Poppy Uppsala")
  2. Trimming using fastp and merging
  3. Alignment using BWA-mem
  4. Mark duplicates using Picard
  5. SNV and INDEL
    5.1 Calling using Mutect2 and Vardict
    5.2 Annotation using VEP and hydra-genetics
    5.3 Filtering using bcftools and hydra-genetics
  6. CNV
    6.1 Calling using CNVkit and GATK CNV
    6.2 Merging using SVDB
    6.3 Annotation using SVDB and hydra-genetics
    6.4 Filtering using hydra-genetics
    6.5 CNV html report using hydra-genetics
  7. Pindel for more complex indels
    8.1 Calling using Pindel in chosen restricted regions
    8.2 Annotation using VEP and hydra-genetics
    8.3 Filtering using bcftools and hydra-genetics
  8. QC
    9.1 QC measures from Samtools, Picard, FastQC, GATK
    9.2 MultiQC hmtl report
    9.3 Coverage report from Mosdepth

Additional steps specific to poppy-uppsala

  1. Coverage calculation in chosen exon regions only,
  2. Excel report with filtered and curated variants from different callers (Mutect2, VarDict, Pisces, GATK, Pindel), as well as coverage metrics. The report has been designed to address requests from the geneticists' team in order to facilitate the interpretation of the genetic profile of the patients against the panel of genes "Twist Myeloid".
  3. MultiQC graphical report with reordered samples so that they are displayed in the same order as in the sample sheet used in the wet lab.
  4. Automated screenshot in IGV of the filtered small variants that are located in genes in the panel and that have a VAF > 5%.

Rule graph

rulegraph