nf-core/sarek

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Introduction

This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report, which summarises results at the end of the pipeline.

The directories listed below will be created in the results directory after the pipeline has finished. All paths are relative to the top-level results directory.

Pipeline overview

The pipeline is built using Nextflow and processes data using the following steps:

• Preprocessing
  • Map to Reference
    • bwa
    • BWA-mem2
  • Mark Duplicates
    • GATK MarkDuplicates
  • Base (Quality Score) Recalibration
    • GATK BaseRecalibrator
    • GATK ApplyBQSR
  • TSV files
  • TSV files with --skip_markduplicates
  • TSV files with --sentieon
• 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
  • MSI status
    • MSIsensor
• Variant annotation
  • snpEff
  • VEP
• QC and reporting
  • QC
    • FastQC
    • bamQC
    • GATK MarkDuplicates reports
    • samtools stats
    • bcftools stats
    • VCFtools
    • snpEff reports
    • VEP reports
  • Reporting
    • MultiQC
• Pipeline information

Preprocessing

Sarek pre-processes raw FASTQ files or unmapped BAM files, based on GATK best practices.

Map to Reference

bwa

bwa 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.

BWA-mem2

BWA-mem2 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

By default, Sarek will use GATK MarkDuplicatesSpark, Spark implementation of GATK MarkDuplicates, which locates and tags duplicate reads in a BAM or SAM file, where duplicate reads are defined as originating from a single fragment of DNA.

Specify --no_gatk_spark to use GATK MarkDuplicates instead.

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

For all samples:

Output directory: results/Preprocessing/[SAMPLE]/DuplicatesMarked

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

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

Base (Quality Score) Recalibration

GATK BaseRecalibrator

GATK BaseRecalibrator generates a recalibration table based on various co-variates.

For all samples:

Output directory: results/Preprocessing/[SAMPLE]/DuplicatesMarked

• [SAMPLE].recal.table
  • Recalibration table associated to the duplicates-marked BAM file.

GATK ApplyBQSR

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

This directory is the location for the final recalibrated BAM files. Recalibrated BAM files are usually 2-3 times larger than the duplicates-marked BAM files. To re-generate recalibrated BAM file you have to apply the recalibration table delivered to the DuplicatesMarked\ folder either using Sarek ( --step recalibrate ) , or doing this recalibration yourself.

For all samples:

Output directory: results/Preprocessing/[SAMPLE]/Recalibrated

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

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

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 section in the usage documentation.

For all samples:

Output directory: results/Preprocessing/TSV

• duplicates_marked_no_table.tsv, duplicates_marked.tsv and recalibrated.tsv
  • TSV files to start Sarek from prepare_recalibration, recalibrate or variantcalling steps.
• duplicates_marked_no_table_[SAMPLE].tsv, duplicates_marked_[SAMPLE].tsv and recalibrated_[SAMPLE].tsv
  • TSV files to start Sarek from prepare_recalibration, recalibrate or variantcalling steps for a specific sample.

TSV files with --skip_markduplicates

WARNING Only with --skip_markduplicates

For all samples:

Output directory: results/Preprocessing/TSV

• mapped.tsv, mapped_no_duplicates_marked.tsv and recalibrated.tsv
  • TSV files to start Sarek from prepare_recalibration, recalibrate or variantcalling steps.
• mapped_[SAMPLE].tsv, mapped_no_duplicates_marked_[SAMPLE].tsv and recalibrated_[SAMPLE].tsv
  • TSV files to start Sarek from prepare_recalibration, recalibrate or variantcalling steps for a specific sample.

TSV files with --sentieon

WARNING Only with --sentieon

For all samples:

Output directory: results/Preprocessing/TSV

• sentieon_deduped.tsv and recalibrated_sentieon.tsv
  • TSV files to start Sarek from variantcalling step.
• sentieon_deduped_[SAMPLE].tsv and 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. If some results from a variant caller do not appear here, please check out the --tools section in the usage documentation.

Recalibrated BAM files can used as an input to start the Variant Calling.

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 all samples:

Output directory: results/VariantCalling/[SAMPLE]/FreeBayes

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

For further reading and documentation see the FreeBayes manual.

GATK HaplotypeCaller

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

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

For all samples:

Output directory: results/VariantCalling/[SAMPLE]/HaploTypeCaller

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

For further reading and documentation see the HaplotypeCaller manual.

GATK GenotypeGVCFs

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

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

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

For further reading and documentation see the GenotypeGVCFs manual.

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 GATK Mutect2 using at least 40 normal samples. Additionally, you can add your PON file to get filtered somatic calls.

For a Tumor/Normal pair:

Output directory: results/VariantCalling/[TUMOR_vs_NORMAL]/Mutect2

Files created:

• Mutect2_unfiltered_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz and Mutect2_unfiltered_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz.tbi
  • unfiltered (raw) Mutect2 calls VCF with Tabix index
• Mutect2_filtered_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz and Mutect2_filtered_[TUMORSAMPLE]_vs_[NORMALSAMPLE].vcf.gz.tbi
  • filtered Mutect2 calls VCF with Tabix index: these entries have 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 of raw variants (needed for filtering)
• [TUMORSAMPLE]_contamination.table
  • a text file exported when panel-of-normals about sample contamination are provided

samtools mpileup

samtools mpileup generates pileup of a BAM file.

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.

For further reading and documentation see the samtools manual.

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 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

For further reading and documentation see the Strelka2 user guide.

Sentieon DNAseq

WARNING 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 all samples:

Output directory: results/VariantCalling/[SAMPLE]/SentieonDNAseq

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

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

Sentieon DNAscope

WARNING Only with --sentieon

Sentieon DNAscope calls SNPs and small indels.

For all samples:

Output directory: results/VariantCalling/[SAMPLE]/SentieonDNAscope

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

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

Sentieon TNscope

WARNING Only with --sentieon

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

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

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

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 that can be fed to Strelka following Strelka Best Practices.

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:

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

For further reading and documentation see the Manta user guide.

TIDDIT

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

Germline calls are provided for all samples, to enable 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 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 ploidy 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

For further reading and documentation see the TIDDIT manual.

Sentieon DNAscope SV

WARNING Only with --sentieon

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

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

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

Sample heterogeneity, ploidy and CNVs

ConvertAlleleCounts

Running ASCAT on NGS data requires that the BAM files are converted into BAF and LogR values. This can be done using the software AlleleCount followed by the provided ConvertAlleleCounts R-script.

For a Tumor/Normal pair:

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 software for performing allele-specific copy number analysis of tumor samples and for estimating tumor ploidy and purity (normal contamination). It infers tumor purity and ploidy and calculates whole-genome allele-specific copy number profiles. ASCAT is written in R and available here: github.com/Crick-CancerGenomics/ascat. The ASCAT process gives several images as output, described in detail in this book chapter.

For a Tumor/Normal pair:

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

The text file [TUMORSAMPLE].cnvs.txt countains predictions about copy number state for all the segments. The output is a tab delimited text file with the following columns:

• chr: chromosome number
• startpos: start position of the segment
• endpos: end position of the segment
• nMajor: number of copies of one of the allels (for example the chromosome inherited from the father)
• nMinor: number of copies of the other allele (for example the chromosome inherited of the mother)

The file [TUMORSAMPLE].cnvs.txt contains all segments predicted by ASCAT, both those with normal copy number (nMinor = 1 and nMajor =1) and those corresponding to copy number aberrations.

For further reading and documentation see the ASCAT manual.

Control-FREEC

Control-FREEC is a tool for detection of copy-number changes and allelic imbalances (including loss of heterozygoity (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 most likely average ploidy of the sample.

For a Tumor/Normal pair:

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

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

MSI status

Microsatellite instability is a genetic condition associated to deficiencies in the mismatch repair (MMR) system which causes a tendency to accumulate a high number of mutations (SNVs and indels). An altered distribution of microsatellite length is associated to a missed replication slippage which would be corrected under normal MMR conditions.

MSIsensor

MSIsensor is a tool to detect the MSI status of a tumor scanning the length of the microsatellite regions. It requires a normal sample for each tumour to differentiate the somatic and germline cases.

For a Tumor/Normal pair:

Output directory: results/VariantCalling/[TUMORSAMPLE]_vs_[NORMALSAMPLE]/MSIsensor

• [TUMORSAMPLE]_vs_[NORMALSAMPLE]_msisensor
  • MSI score output, contains information about the number of somatic sites.
• [TUMORSAMPLE]_vs_[NORMALSAMPLE]_msisensor_dis
  • The normal and tumor length distribution for each microsatellite position.
• [TUMORSAMPLE]_vs_[NORMALSAMPLE]_msisensor_germline
  • Somatic sites detected.
• [TUMORSAMPLE]_vs_[NORMALSAMPLE]_msisensor_somatic
  • Germline sites detected.

For further reading see the MSIsensor paper.

Variant annotation

This directory contains results from the final annotation steps: two tools are used for annotation, snpEff and VEP. Only a subset of the VCF files are annotated, and only variants that have a PASS filter. Currently, FreeBayes results are not annotated as we are lacking a decent somatic filter.

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 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

For further reading and documentation see the snpEff manual

VEP

VEP (Variant Effect Predictor), based on Ensembl, is a tool 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. Currently, 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 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

For further reading and documentation see the VEP manual

QC and reporting

QC

FastQC

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

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 your untrimmed raw FASTQ files
• sample_R1_XXX_fastqc.zip and sample_R2_XXX_fastqc.zip
  • Zip archive containing the FastQC report, tab-delimited data file and plot images

NB: The FastQC plots displayed in the MultiQC report shows untrimmed reads. They may contain adapter sequence and potentially regions with low quality.

For further reading and documentation see the FastQC help pages.

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 further reading and documentation see the Qualimap bamqc manual

GATK MarkDuplicates reports

More information in the GATK MarkDuplicates section

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 VCF files.

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.genes.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 visualization tool that generates a single HTML report summarizing all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available in the report data directory.

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

Output files:

• multiqc/
  • multiqc_report.html
    • 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
  • multiqc_plots/
    • Directory containing static images from the report in various formats

For more information about how to use MultiQC reports, see https://multiqc.info.

Pipeline information

Nextflow provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to troubleshoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage.

Output files:

• pipeline_info/
  • Reports generated by Nextflow: execution_report.html, execution_timeline.html, execution_trace.txt and pipeline_dag.dot/pipeline_dag.svg.
  • Reports generated by the pipeline: pipeline_report.html, pipeline_report.txt and software_versions.csv.
  • Documentation for interpretation of results in HTML format: results_description.html.