Output

This document describes the output produced by the pipeline.

Pipeline overview

The pipeline processes data using the following steps:

• Preprocessing
  • Map to Reference
    • BWA mem
  • Mark Duplicates
    • GATK MarkDuplicates
  • Base (Quality Score) Recalibration
    • GATK BaseRecalibrator
    • GATK ApplyBQSR
  • TSV files
• Variant Calling
  • SNVs and small indels
    • FreeBayes
    • GATK HaplotypeCaller
    • GATK GenotypeGVCFs
    • GATK Mutect2
    • samtools mpileup
    • Strelka2
    • Sentieon DNAseq
    • Sentieon DNAscope
    • Sentieon TNscope
  • Structural Variants
    • Manta
    • TIDDIT
    • Sentieon DNAscope SV
  • Sample heterogeneity, ploidy and CNVs
    • ConvertAlleleCounts
    • ASCAT
    • Control-FREEC
• Variant annotation
  • snpEff
  • VEP
• QC and reporting
  • QC
    • FastQC
    • bamQC
    • MarkDuplicates reports
    • samtools stats
    • bcftools stats
    • VCFtools
    • snpEff reports
    • VEP reports
  • Reporting
    • MultiQC

Preprocessing

Sarek preprocesses raw FastQ files or unmapped BAM files, based on GATK best practices.

BAM files with Recalibration tables can also be used as an input to start with the recalibration of said BAM files, for more information see TSV files output information

Map to Reference

BWA mem

BWA mem is a software package for mapping low-divergent sequences against a large reference genome.

Such files are intermediate and not kept in the final files delivered to users.

Mark Duplicates

GATK MarkDuplicates

GATK MarkDuplicates locates and tags duplicate reads in a BAM or SAM file, where duplicate reads are defined as originating from a single fragment of DNA.

This directory is the location for the BAM files delivered to users. Besides the duplicate marked BAM files, the recalibration tables (*.recal.table) are also stored, and can be used to create base recalibrated files.

For further reading and documentation see the data pre-processing workflow from the GATK best practices.

For all samples: Output directory: results/Preprocessing/[SAMPLE]/DuplicateMarked

• [SAMPLE].md.bam, [SAMPLE].md.bai and [SAMPLE].recal.table
  • BAM file and index with Recalibration Table

Base (Quality Score) Recalibration

GATK BaseRecalibrator

GATK BaseRecalibrator generates a recalibration table based on various covariates.

Such files are intermediate and not kept in the final files delivered to users.

GATK ApplyBQSR

GATK ApplyBQSR recalibrates the base qualities of the input reads based on the recalibration table produced by the BaseRecalibrator tool.

This directory is usually empty, it is the location for the final recalibrated BAM files. Recalibrated BAM files are usually 2-3 times larger than the duplicate marked BAM files. To re-generate recalibrated BAM file you have to apply the recalibration table delivered to the DuplicateMarked directory either within Sarek, or doing this recalibration step yourself.

For further reading and documentation see the data pre-processing workflow from the GATK best practices.

For all samples: Output directory: results/Preprocessing/[SAMPLE]/Recalibrated

• [SAMPLE].recal.bam and [SAMPLE].recal.bam.bai
  • BAM file and index

TSV files

The TSV files are auto-generated and can be used by Sarek for further processing and/or variant calling.

For further reading and documentation see the input documentation.

For all samples: Output directory: results/Preprocessing/TSV

• duplicateMarked.tsv and recalibrated.tsv
  • TSV files to start Sarek from recalibration or variantcalling steps.
• duplicateMarked_[SAMPLE].tsv and recalibrated_[SAMPLE].tsv
  • TSV files to start Sarek from recalibration or variantcalling steps for a specific sample.

⚠️ Only with --sentieon

For all samples: Output directory: results/Preprocessing/TSV

• recalibrated_sentieon.tsv
  • TSV files to start Sarek from variantcalling step.
• recalibrated_sentieon_[SAMPLE].tsv
  • TSV files to start Sarek from variantcalling step for a specific sample.

Variant Calling

All the results regarding Variant Calling are collected in this directory.

Recalibrated BAM files can also be used as an input to start the Variant Calling, for more information see TSV files output information

SNVs and small indels

FreeBayes

FreeBayes is a Bayesian genetic variant detector designed to find small polymorphisms, specifically SNPs, indels, MNPs, and complex events smaller than the length of a short-read sequencing alignment..

For further reading and documentation see the FreeBayes manual.

For a Tumor/Normal pair only: Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/FreeBayes

• FreeBayes_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz and FreeBayes_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz.tbi
  • VCF with Tabix index

GATK HaplotypeCaller

GATK HaplotypeCaller calls germline SNPs and indels via local re-assembly of haplotypes.

Germline calls are provided for all samples, to able comparison of both tumor and normal for possible mixup.

For further reading and documentation see the HaplotypeCaller manual.

For all samples: Output directory: results/VariantCalling/[SAMPLE]/HaploTypeCaller

• HaplotypeCaller_[SAMPLE].vcf.gz and HaplotypeCaller_[SAMPLE].vcf.gz.tbi
  • VCF with Tabix index

GATK GenotypeGVCFs

GATK GenotypeGVCFs performs joint genotyping on one or more samples pre-called with HaplotypeCaller.

Germline calls are provided for all samples, to able comparison of both tumor and normal for possible mixup.

For further reading and documentation see the GenotypeGVCFs manual.

For all samples: Output directory: results/VariantCalling/[SAMPLE]/HaplotypeCallerGVCF

• HaplotypeCaller_[SAMPLE].g.vcf.gz and HaplotypeCaller_[SAMPLE].g.vcf.gz.tbi
  • VCF with Tabix index

GATK Mutect2

GATK Mutect2 calls somatic SNVs and indels via local assembly of haplotypes.

For further reading and documentation see the Mutect2 manual. It is recommended to have panel of normals PON for this version of Mutect2 using at least 40 normal samples, and you can add your PON file to get filtered somatic calls.

For a Tumor/Normal pair only: Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/Mutect2

Files created:

• unfiltered_Mutect2_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz and unfiltered_Mutect2_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz.tbi
  • unfiltered (raw) Mutect2 calls VCF with Tabix index
• filtered_Mutect2_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz and filtered_Mutect2_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz.tbi
  • filtered Mutect2 calls VCF with Tabix index: these entries has a PASS filter, you can get these when supplying a panel of normals using the --pon option
• [TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz.stats
  • a stats file generated during calling raw variants (needed for filtering)
• [TUMORSAMPLE]_contamination.table
  • a text file exported when panel-of-normals provided about sample contamination

samtools mpileup

samtools mpileup generate pileup for a BAM file.

For further reading and documentation see the samtools manual.

For all samples: Output directory: results/VariantCalling/[SAMPLE]/mpileup

• [SAMPLE].pileup.gz
  • The pileup format is a text-based format for summarizing the base calls of aligned reads to a reference sequence. Alignment records are grouped by sample (SM) identifiers in @RG header lines.

Strelka2

Strelka2 is a fast and accurate small variant caller optimized for analysis of germline variation in small cohorts and somatic variation in tumor/normal sample pairs.

For further reading and documentation see the Strelka2 user guide.

For all samples: Output directory: results/VariantCalling/[SAMPLE]/Strelka

• Strelka_Sample_genome.vcf.gz and Strelka_Sample_genome.vcf.gz.tbi
  • VCF with Tabix index
• Strelka_Sample_variants.vcf.gz and Strelka_Sample_variants.vcf.gz.tbi
  • VCF with Tabix index

For a Tumor/Normal pair: Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/Strelka

• Strelka_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_indels.vcf.gz and Strelka_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_indels.vcf.gz.tbi
  • VCF with Tabix index
• Strelka_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_snvs.vcf.gz and Strelka_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_snvs.vcf.gz.tbi
  • VCF with Tabix index

Using Strelka Best Practices with the candidateSmallIndels from Manta: Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/Strelka

• StrelkaBP_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_indels.vcf.gz and StrelkaBP_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_indels.vcf.gz.tbi
  • VCF with Tabix index
• StrelkaBP_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_snvs.vcf.gz and StrelkaBP_[TUMORSAMPLE]_vs_[NORMALSAMPLE]_somatic_snvs.vcf.gz.tbi
  • VCF with Tabix index

Sentieon DNAseq

⚠️ Only with --sentieon

Sentieon DNAseq implements the same mathematics used in the Broad Institute’s BWA-GATK HaplotypeCaller 3.3-4.1 Best Practices Workflow pipeline.

For further reading and documentation see the Sentieon DNAseq user guide.

For all samples: Output directory: results/VariantCalling/[SAMPLE]/SentieonDNAseq

• DNAseq_Sample.vcf.gz and DNAseq_Sample.vcf.gz.tbi
  • VCF with Tabix index

Sentieon DNAscope

⚠️ Only with --sentieon

Sentieon DNAscope calls SNPs and small indels.

For further reading and documentation see the Sentieon DNAscope user guide.

For all samples: Output directory: results/VariantCalling/[SAMPLE]/SentieonDNAscope

• DNAscope_Sample.vcf.gz and DNAscope_Sample.vcf.gz.tbi
  • VCF with Tabix index

Sentieon TNscope

⚠️ Only with --sentieon

Sentieon TNscope calls SNPs and small indels on an Tumor/Normal pair.

For further reading and documentation see the Sentieon TNscope user guide.

For a Tumor/Normal pair: Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/SentieonTNscope

• TNscope_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz and TNscope_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz.tbi
  • VCF with Tabix index

Structural Variants

Manta

Manta calls structural variants (SVs) and indels from mapped paired-end sequencing reads. It is optimized for analysis of germline variation in small sets of individuals and somatic variation in tumor/normal sample pairs. Manta provides a candidate list for small indels also that can be fed to Strelka following Strelka Best Practices.

For further reading and documentation see the Manta user guide.

For all samples: Output directory: results/VariantCalling/[SAMPLE]/Manta

• Manta_[SAMPLE].candidateSmallIndels.vcf.gz and Manta_[SAMPLE].candidateSmallIndels.vcf.gz.tbi
  • VCF with Tabix index
• Manta_[SAMPLE].candidateSV.vcf.gz and Manta_[SAMPLE].candidateSV.vcf.gz.tbi
  • VCF with Tabix index

For Normal sample only:

• Manta_[NORMALSAMPLE].diploidSV.vcf.gz and Manta_[NORMALSAMPLE].diploidSV.vcf.gz.tbi
  • VCF with Tabix index

For a Tumor sample only:

• Manta_[TUMORSAMPLE].tumorSV.vcf.gz and Manta_[TUMORSAMPLE].tumorSV.vcf.gz.tbi
  • VCF with Tabix index

For a Tumor/Normal pair only: Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/Manta

• Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].candidateSmallIndels.vcf.gz and Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].candidateSmallIndels.vcf.gz.tbi
  • VCF with Tabix index
• Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].candidateSV.vcf.gz and Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].candidateSV.vcf.gz.tbi
  • VCF with Tabix index
• Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].diploidSV.vcf.gz and Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].diploidSV.vcf.gz.tbi
  • VCF with Tabix index
• Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].somaticSV.vcf.gz and Manta_[TUMORSAMPLE]_vs_[NORMALSAMPLE].somaticSV.vcf.gz.tbi
  • VCF with Tabix index

TIDDIT

TIDDIT identifies intra and inter-chromosomal translocations, deletions, tandem-duplications and inversions.

Germline calls are provided for all samples, to able comparison of both tumor and normal for possible mixup. Low quality calls are removed internally, to simplify processing of variant calls but they are saved by Sarek.

For further reading and documentation see the TIDDIT manual.

For all samples: Output directory: results/VariantCalling/[SAMPLE]/TIDDIT

• TIDDIT_[SAMPLE].vcf.gz and TIDDIT_[SAMPLE].vcf.gz.tbi
  • VCF with Tabix index
• TIDDIT_[SAMPLE].signals.tab
  • tab file describing coverage across the genome, binned per 50 bp
• TIDDIT_[SAMPLE].ploidy.tab
  • tab file describing the estimated ploïdy and coverage across each contig
• TIDDIT_[SAMPLE].old.vcf
  • VCF including the low qualiy calls
• TIDDIT_[SAMPLE].wig
  • wiggle file containing coverage across the genome, binned per 50 bp
• TIDDIT_[SAMPLE].gc.wig
  • wiggle file containing fraction of gc content, binned per 50 bp

Sentieon DNAscope SV

⚠️ Only with --sentieon

Sentieon DNAscope can perform structural variant calling in addition to calling SNPs and small indels.

For further reading and documentation see the Sentieon DNAscope user guide.

For all samples: Output directory: results/VariantCalling/[SAMPLE]/SentieonDNAscope

• DNAscope_SV_Sample.vcf.gz and DNAscope_SV_Sample.vcf.gz.tbi
  • VCF with Tabix index

Sample heterogeneity, ploidy and CNVs

ConvertAlleleCounts

ConvertAlleleCounts is a R-script for converting output from AlleleCount to BAF and LogR values.

For a Tumor/Normal pair only: Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/ASCAT

• [TUMORSAMPLE].BAF and [NORMALSAMPLE].BAF
  • file with beta allele frequencies
• [TUMORSAMPLE].LogR and [NORMALSAMPLE].LogR
  • file with total copy number on a logarithmic scale

ASCAT

ASCAT is a method to derive copy number profiles of tumor cells, accounting for normal cell admixture and tumor aneuploidy. ASCAT infers tumor purity and ploidy and calculates whole-genome allele-specific copy number profiles.

For further reading and documentation see the Sarek documentation about ASCAT or the ASCAT manual.

For a Tumor/Normal pair only: Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/ASCAT

• [TUMORSAMPLE].aberrationreliability.png
  • Image with information about aberration reliability
• [TUMORSAMPLE].ASCATprofile.png
  • Image with information about ASCAT profile
• [TUMORSAMPLE].ASPCF.png
  • Image with information about ASPCF
• [TUMORSAMPLE].rawprofile.png
  • Image with information about raw profile
• [TUMORSAMPLE].sunrise.png
  • Image with information about sunrise
• [TUMORSAMPLE].tumour.png
  • Image with information about tumor
• [TUMORSAMPLE].cnvs.txt
  • file with information about CNVS
• [TUMORSAMPLE].LogR.PCFed.txt
  • file with information about LogR
• [TUMORSAMPLE].purityploidy.txt
  • file with information about purity ploidy

Control-FREEC

Control-FREEC is a tool for detection of copy-number changes and allelic imbalances (including LOH) using deep-sequencing data. Control-FREEC automatically computes, normalizes, segments copy number and beta allele frequency profiles, then calls copy number alterations and LOH. And also detects subclonal gains and losses and evaluate the likeliest average ploidy of the sample.

For further reading and documentation see the Control-FREEC manual.

For a Tumor/Normal pair only: Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/ControlFREEC

• [TUMORSAMPLE]_vs_[NORMALSAMPLE].config.txt
  • Configuration file used to run Control-FREEC
• [TUMORSAMPLE].pileup.gz_CNVs and [TUMORSAMPLE].pileup.gz_normal_CNVs
  • file with coordinates of predicted copy number alterations
• [TUMORSAMPLE].pileup.gz_ratio.txt and [TUMORSAMPLE].pileup.gz_normal_ratio.txt
  • file with ratios and predicted copy number alterations for each window
• [TUMORSAMPLE].pileup.gz_BAF.txt and [NORMALSAMPLE].pileup.gz_BAF.txt
  • file with beta allele frequencies for each possibly heterozygous SNP position

Variant annotation

This directory contains results from the final annotation steps: two software are used for annotation, snpEff and VEP. Only a subset of the VCF files are annotated, and only variants that have a PASS filter. FreeBayes results are not annotated in the moment yet as we are lacking a decent somatic filter. For HaplotypeCaller the germline variations are annotated for both the tumor and the normal sample.

snpEff

snpeff is a genetic variant annotation and effect prediction toolbox. It annotates and predicts the effects of variants on genes (such as amino acid changes) using multiple databases for annotations. The generated VCF header contains the software version and the used command line.

For further reading and documentation see the snpEff manual

For all samples: Output directory: results/Annotation/[SAMPLE]/snpEff

• VariantCaller_Sample_snpEff.ann.vcf.gz and VariantCaller_Sample_snpEff.ann.vcf.gz.tbi
  • VCF with Tabix index

VEP

VEP (Variant Effect Predictor), based on Ensembl, is a tools to determine the effects of all sorts of variants, including SNPs, indels, structural variants, CNVs. The generated VCF header contains the software version, also the version numbers for additional databases like Clinvar or dbSNP used in the “VEP” line. The format of the consequence annotations is also in the VCF header describing the INFO field. In the moment it contains:

• Consequence: impact of the variation, if there is any
• Codons: the codon change, i.e. cGt/cAt
• Amino_acids: change in amino acids, i.e. R/H if there is any
• Gene: ENSEMBL gene name
• SYMBOL: gene symbol
• Feature: actual transcript name
• EXON: affected exon
• PolyPhen: prediction based on PolyPhen
• SIFT: prediction by SIFT
• Protein_position: Relative position of amino acid in protein
• BIOTYPE: Biotype of transcript or regulatory feature

For further reading and documentation see the VEP manual

For all samples: Output directory: results/Annotation/[SAMPLE]/VEP

• VariantCaller_Sample_VEP.ann.vcf.gz and VariantCaller_Sample_VEP.ann.vcf.gz.tbi
  • VCF with Tabix index

QC and reporting

QC

FastQC

FastQC gives general quality metrics about your reads. It provides information about the quality score distribution across your reads, the per base sequence content (%T/A/G/C). You get information about adapter contamination and other overrepresented sequences.

For further reading and documentation see the FastQC help.

For all samples: Output directory: results/Reports/[SAMPLE]/fastqc

• sample_R1_XXX_fastqc.html and sample_R2_XXX_fastqc.html
  • FastQC report, containing quality metrics for each pair of the raw fastq files
• sample_R1_XXX_fastqc.zip and sample_R2_XXX_fastqc.zip
  • zip file containing the FastQC reports, tab-delimited data files and plot images

bamQC

Qualimap bamqc reports information for the evaluation of the quality of the provided alignment data. In short, the basic statistics of the alignment (number of reads, coverage, GC-content, etc.) are summarized and a number of useful graphs are produced.

Plot will show:

• Stats by non-reference allele frequency, depth distribution, stats by quality and per-sample counts, singleton stats, etc.

For all samples: Output directory: results/Reports/[SAMPLE]/bamQC

• VariantCaller_[SAMPLE].bcf.tools.stats.out
  • RAW statistics used by MultiQC

For more information about how to use Qualimap bamqc reports, see Qualimap bamqc manual

MarkDuplicates reports

GATK MarkDuplicates locates and tags duplicate reads in a BAM or SAM file, where duplicate reads are defined as originating from a single fragment of DNA. Duplicates can arise during sample preparation e.g. library construction using PCR. Duplicate reads can also result from a single amplification cluster, incorrectly detected as multiple clusters by the optical sensor of the sequencing instrument. These duplication artifacts are referred to as optical duplicates.

For all samples: Output directory: results/Reports/[SAMPLE]/MarkDuplicates

• [SAMPLE].bam.metrics
  • RAW statistics used by MultiQC

For further reading and documentation see the MarkDuplicates manual.

samtools stats

samtools stats collects statistics from BAM files and outputs in a text format. Plots will show:

• Alignment metrics.

For all samples: Output directory: results/Reports/[SAMPLE]/SamToolsStats

• [SAMPLE].bam.samtools.stats.out
  • RAW statistics used by MultiQC

For further reading and documentation see the samtools manual

bcftools stats

bcftools is a program for variant calling and manipulating files in the Variant Call Format. Plot will show:

• Stats by non-reference allele frequency, depth distribution, stats by quality and per-sample counts, singleton stats, etc.

For all samples: Output directory: results/Reports/[SAMPLE]/BCFToolsStats

• VariantCaller_[SAMPLE].bcf.tools.stats.out
  • RAW statistics used by MultiQC

For further reading and documentation see the bcftools stats manual

VCFtools

VCFtools is a program package designed for working with VCF files. Plots will show:

• the summary counts of each type of transition to transversion ratio for each FILTER category.
• the transition to transversion ratio as a function of alternative allele count (using only bi-allelic SNPs).
• the transition to transversion ratio as a function of SNP quality threshold (using only bi-allelic SNPs).

For all samples: Output directory: results/Reports/[SAMPLE]/VCFTools

• VariantCaller_[SAMPLE].FILTER.summary
  • RAW statistics used by MultiQC
• VariantCaller_[SAMPLE].TsTv.count
  • RAW statistics used by MultiQC
• VariantCaller_[SAMPLE].TsTv.qual
  • RAW statistics used by MultiQC

For further reading and documentation see the VCFtools manual

snpEff reports

snpeff is a genetic variant annotation and effect prediction toolbox. It annotates and predicts the effects of variants on genes (such as amino acid changes) using multiple databases for annotations.

Plots will shows :

• locations of detected variants in the genome and the number of variants for each location.
• the putative impact of detected variants and the number of variants for each impact.
• the effect of variants at protein level and the number of variants for each effect type.
• the quantity as function of the variant quality score.

For all samples: Output directory: results/Reports/[SAMPLE]/snpEff

• VariantCaller_Sample_snpEff.csv
  • RAW statistics used by MultiQC
• VariantCaller_Sample_snpEff.html
  • Statistics to be visualised with a web browser
• VariantCaller_Sample_snpEff.txt
  • TXT (tab separated) summary counts for variants affecting each transcript and gene

For further reading and documentation see the snpEff manual

VEP reports

VEP (Variant Effect Predictor), based on Ensembl, is a tools to determine the effects of all sorts of variants, including SNPs, indels, structural variants, CNVs.

For all samples: Output directory: results/Reports/[SAMPLE]/VEP

• VariantCaller_Sample_VEP.summary.html
  • Summary of the VEP run to be visualised with a web browser

For further reading and documentation see the VEP manual

Reporting

MultiQC

MultiQC is a visualisation tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available in within the report data directory.

The pipeline has special steps which allow the software versions used to be reported in the MultiQC output for future traceability.

For the whole Sarek run: Output directory: results/Reports/MultiQC

• multiqc_report.html
  • MultiQC report - a standalone HTML file that can be viewed in your web browser
• multiqc_data/
  • Directory containing parsed statistics from the different tools used in the pipeline

For further reading and documentation see the MultiQC website