Introduction Usage Parameters Output Releases

Introduction

This document describes the output produced by 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.

output/
│  
├── <group_id-1>/
│   ├── alignments/
│   ├── amber/
│   ├── bamtools/
│   ├── chord/
│   ├── cobalt/
│   ├── cuppa/
│   ├── esvee/
│   ├── isofox/
│   ├── lilac/
│   ├── linx/
│   ├── orange/
│   ├── pave/
│   ├── purple/
│   ├── sage/
│   ├── sigs/
│   ├── virusbreakend/
│   └── virusinterpreter/
│  
├── <group_id-2>/
│   └── ...
│  
...
│  
└── pipeline_info/

Pipeline overview

Read alignment

Alignment functionality in oncoanalyser is simple and rigid, and exists only to meet the exact requirements of the WiGiTS workflow.

BWA-MEM2

BWA-MEM2 is a short-read mapping tool used to align reads to a large reference sequences. In oncoanalyser, BWA-MEM2 is used to align DNA reads to the human genome.

No outputs are published directly from bwa-mem2, see REDUX for the fully processed alignment outputs

STAR

STAR is a specialised mapping tool used to align RNA reads to a reference transcriptome.

No outputs are published directly from STAR, see Picard MarkDuplicates for the fully processed alignment outputs

Alignment post-processing

REDUX

Output files
  • <group_id>/alignments/dna/
    • <tumor_dna_id>.jitter_params.tsv: Tumor DNA sample microsatellite jitter model parameters.
    • <tumor_dna_id>.ms_table.tsv.gz: Tumor DNA sample aggregated repeat units and repeat counts.
    • <tumor_dna_id>.redux.bam: Tumor DNA sample read alignments.
    • <tumor_dna_id>.redux.bam.bai: Tumor DNA sample read alignments index.
    • <tumor_dna_id>.redux.duplicate_freq.tsv: Tumor DNA sample duplicate read frequencies.
    • <tumor_dna_id>.repeat.tsv.gz: Tumor DNA sample repeat units and repeat counts per site.
    • <normal_dna_id>.jitter_params.tsv: Normal DNA sample microsatellite jitter model parameters.
    • <normal_dna_id>.ms_table.tsv.gz: Normal DNA sample aggregated repeat units and repeat counts.
    • <normal_dna_id>.redux.bam: Normal DNA sample: Read alignments.
    • <normal_dna_id>.redux.bam.bai: Normal DNA sample read alignments index.
    • <normal_dna_id>.redux.duplicate_freq.tsv: Normal DNA sample duplicate read frequencies.
    • <normal_dna_id>.repeat.tsv.gz: Normal DNA sample repeat units and repeat counts per site.

REDUX applies various alignment post-processing routines such as duplicate marking and unmapping of problematic regions for DNA reads. It can also handle UMIs when configured to do so.

REDUX is only run on DNA alignments

Picard MarkDuplicates

Output files
  • <group_id>/alignments/rna/
    • <tumor_rna_id>.md.bam: Tumor RNA sample read alignments.
    • <tumor_rna_id>.md.bam.bai: Tumor RNA sample read alignments index.
    • <tumor_rna_id>.md.metrics: Tumor RNA sample read duplicate marking metrics.

Picard MarkDuplicates used to mark duplicate reads following alignment.

Picard MarkDuplicates is only run on RNA alignments

SNV, MNV, INDEL calling

SAGE

Output files

  • <group_id>/sage/append/

    • <tumor_dna_id>.sage.append.vcf.gz: Tumor DNA sample small variant VCF with RNA data appended.
    • <normal_dna_id>.sage.append.vcf.gz: Normal DNA sample small variant VCF with RNA data appended.

  • <group_id>/sage/somatic/

    • <normal_dna_id>.sage.bqr.png: Normal DNA sample base quality recalibration metrics plot.
    • <normal_dna_id>.sage.bqr.tsv: Normal DNA sample base quality recalibration metrics.
    • <tumor_dna_id>.sage.bqr.png: Tumor DNA sample base quality recalibration metrics plot.
    • <tumor_dna_id>.sage.bqr.tsv: Tumor DNA sample base quality recalibration metrics.
    • <tumor_dna_id>.sage.exon.medians.tsv: Tumor DNA sample exon median depths.
    • <tumor_dna_id>.sage.gene.coverage.tsv: Tumor DNA sample gene coverages.
    • <tumor_dna_id>.sage.somatic.vcf.gz.tbi: Tumor DNA sample small variant calls index.
    • <tumor_dna_id>.sage.somatic.vcf.gz: Tumor DNA sample small variant calls.

  • <group_id>/sage/germline/

    • <normal_dna_id>.sage.bqr.png: Normal DNA sample base quality recalibration metrics plot.
    • <normal_dna_id>.sage.bqr.tsv: Normal DNA sample base quality recalibration metrics.
    • <normal_dna_id>.sage.exon.medians.tsv: Normal DNA sample exon median depths.
    • <normal_dna_id>.sage.gene.coverage.tsv: Normal DNA sample gene coverages.
    • <tumor_dna_id>.sage.bqr.png: Normal DNA sample base quality recalibration metrics plot.
    • <tumor_dna_id>.sage.bqr.tsv: Normal DNA sample base quality recalibration metrics.
    • <tumor_dna_id>.sage.germline.vcf.gz.tbi: Normal DNA sample small variant calls index.
    • <tumor_dna_id>.sage.germline.vcf.gz: Normal DNA sample small variant calls.

SAGE is a precise and highly sensitive somatic SNV, MNV and small INDEL caller. It has dynamically scaling sensitivity based on the depth of the provided tumor and germline BAMs, but performs best if both BAMs have at least 30x typical depth.

PAVE

Output files
  • <group_id>/pave/
    • <tumor_dna_id>.sage.germline.pave.vcf.gz.tbi: Annotated SAGE germline small variants index.
    • <tumor_dna_id>.sage.germline.pave.vcf.gz: Annotated SAGE germline small variants.
    • <tumor_dna_id>.sage.somatic.pave.vcf.gz.tbi: Annotated SAGE somatic small variants index.
    • <tumor_dna_id>.sage.somatic.pave.vcf.gz: Annotated SAGE somatic small variants.

PAVE annotates variants called by SAGE with impact information with regards to transcript and coding effects.

SV calling

ESVEE

Output files

  • <group_id>/esvee/prep/

    • <tumor_dna_id>.esvee.prep.bam: Tumor DNA sample BAM with candidate SV reads.
    • <tumor_dna_id>.esvee.prep.bam.bai: Tumor DNA sample BAM index.
    • <tumor_dna_id>.esvee.prep.disc_stats.tsv: Tumor DNA sample discordant reads stats.
    • <tumor_dna_id>.esvee.prep.fragment_length.tsv: Tumor DNA sample fragment length stats.
    • <tumor_dna_id>.esvee.prep.junction.tsv: Tumor DNA sample candidate junctions.
    • <normal_dna_id>.esvee.prep.bam: Tumor DNA sample BAM with candidate SV reads.
    • <normal_dna_id>.esvee.prep.bam.bai: Tumor DNA sample BAM index.

  • <group_id>/esvee/assemble/

    • <tumor_dna_id>.esvee.assembly.tsv: Tumor DNA sample breakend assemblies.
    • <tumor_dna_id>.esvee.alignment.tsv: Tumor DNA sample assemblies realigned to the reference genome.
    • <tumor_dna_id>.esvee.breakend.tsv: Tumor DNA sample breakends.
    • <tumor_dna_id>.esvee.phased_assembly.tsv: Tumor DNA sample phased assemblies.
    • <tumor_dna_id>.esvee.raw.vcf.gz: Tumor DNA sample VCF with candidate breakends.
    • <tumor_dna_id>.esvee.raw.vcf.gz.tbi: Tumor DNA sample VCF with candidate breakends.

  • <group_id>/esvee/depth_annotation/

    • <tumor_dna_id>.esvee.ref_depth.vcf.gz: Tumor DNA sample VCF annotated with normal sample read depths.
    • <tumor_dna_id>.esvee.ref_depth.vcf.gz.tbi: Tumor DNA sample VCF index.

  • <group_id>/esvee/caller/

    • <tumor_dna_id>.esvee.germline.vcf.gz: Tumor DNA sample VCF with germline breakends.
    • <tumor_dna_id>.esvee.germline.vcf.gz.tbi: Tumor DNA sample VCF index.
    • <tumor_dna_id>.esvee.somatic.vcf.gz: Tumor DNA sample VCF with somatic breakends.
    • <tumor_dna_id>.esvee.somatic.vcf.gz.tbi: Tumor DNA sample VCF index.
    • <tumor_dna_id>.esvee.unfiltered.vcf.gz: Tumor DNA sample VCF with unfiltered breakends.
    • <tumor_dna_id>.esvee.unfiltered.vcf.gz.tbi: Tumor DNA sample VCF index.

ESVEE is a SV caller that uses both read support and local breakend/breakpoint assemblies to call variants.

CNV calling

AMBER

Output files
  • <group_id>/amber/
    • amber.version: AMBER version file.
    • <tumor_dna_id>.amber.baf.pcf: Tumor DNA sample piecewise constant fit.
    • <tumor_dna_id>.amber.baf.tsv.gz: Tumor DNA sample β-allele frequencies.
    • <tumor_dna_id>.amber.contamination.tsv: Tumor DNA sample contamination TSV.
    • <tumor_dna_id>.amber.contamination.vcf.gz: Tumor DNA sample contamination sites.
    • <tumor_dna_id>.amber.contamination.vcf.gz.tbi: Tumor DNA sample contamination sites index.
    • <tumor_dna_id>.amber.qc: AMBER QC file.
    • <normal_dna_id>.amber.homozygousregion.tsv: Normal DNA sample regions of homozygosity.
    • <normal_dna_id>.amber.snp.vcf.gz: Normal DNA sample SNP sites.
    • <normal_dna_id>.amber.snp.vcf.gz.tbi: Normal DNA sample SNP sites index.

AMBER generates β-allele frequencies in tumor samples for CNV calling in PURPLE.

COBALT

Output files
  • <group_id>/cobalt/
    • cobalt.version: COBALT version file.
    • <tumor_dna_id>.cobalt.gc.median.tsv: Tumor DNA sample GC median read depths.
    • <tumor_dna_id>.cobalt.ratio.pcf: Tumor DNA sample piecewise constant fit.
    • <tumor_dna_id>.cobalt.ratio.tsv.gz: Tumor DNA sample read counts and ratios (with reference or supposed diploid regions).
    • <normal_dna_id>.cobalt.gc.median.tsv: Normal DNA sample GC median read depths.
    • <normal_dna_id>.cobalt.ratio.median.tsv: Normal DNA sample chromosome median ratios.
    • <normal_dna_id>.cobalt.ratio.pcf: Normal DNA sample piecewise constant fit.

COBALT generates read depth ratios (or an estimation for tumor-only) for CNV calling in PURPLE.

PURPLE

Output files
  • <group_id>/purple/
    • circos/: Circos plot data.
    • <tumor_dna_id>.purple.cnv.gene.tsv: Somatic gene copy number.
    • <tumor_dna_id>.purple.cnv.somatic.tsv: Copy number variant segments.
    • <tumor_dna_id>.purple.driver.catalog.germline.tsv: Normal DNA sample driver catalogue.
    • <tumor_dna_id>.purple.driver.catalog.somatic.tsv: Tumor DNA sample driver catalogue.
    • <tumor_dna_id>.purple.germline.deletion.tsv: Normal DNA deletions.
    • <tumor_dna_id>.purple.germline.vcf.gz: Normal DNA SAGE small variants with PURPLE annotations.
    • <tumor_dna_id>.purple.germline.vcf.gz.tbi: Normal DNA SAGE small variants with PURPLE annotations index.
    • <tumor_dna_id>.purple.purity.range.tsv: Purity/ploid model fit scores across a range of purity values.
    • <tumor_dna_id>.purple.purity.tsv: Purity/ploidy summary.
    • <tumor_dna_id>.purple.qc: PURPLE QC file.
    • <tumor_dna_id>.purple.segment.tsv: Genomic copy number segments.
    • <tumor_dna_id>.purple.somatic.clonality.tsv: Clonality peak model data.
    • <tumor_dna_id>.purple.somatic.hist.tsv: Somatic variants histogram data.
    • <tumor_dna_id>.purple.somatic.vcf.gz: Tumor DNA sample small variants with PURPLE annotations.
    • <tumor_dna_id>.purple.somatic.vcf.gz.tbi: Tumor DNA sample small variants with PURPLE annotations index.
    • <tumor_dna_id>.purple.sv.germline.vcf.gz: Germline structural variants with PURPLE annotations.
    • <tumor_dna_id>.purple.sv.germline.vcf.gz.tbi: Germline structural variants with PURPLE annotations index.
    • <tumor_dna_id>.purple.sv.vcf.gz: Somatic structural variants with PURPLE annotations.
    • <tumor_dna_id>.purple.sv.vcf.gz.tbi: Somatic structural variants with PURPLE annotations.
    • plot/: PURPLE plots.
    • purple.version: PURPLE version file.

PURPLE is a CNV caller that also infers tumor purity/ploidy and annotates both small and structural variant calls with copy-number information.

SV event interpretation

LINX

Output files

  • <group_id>/linx/germline_annotations/

    • linx.version: LINX version file.
    • <tumor_dna_id>.linx.germline.breakend.tsv: Normal DNA sample breakend data.
    • <tumor_dna_id>.linx.germline.clusters.tsv: Normal DNA sample clustered events.
    • <tumor_dna_id>.linx.germline.disruption.tsv: Normal DNA sample breakend data.
    • <tumor_dna_id>.linx.germline.driver.catalog.tsv: Normal DNA sample driver catalogue.
    • <tumor_dna_id>.linx.germline.links.tsv: Normal DNA sample cluster links.
    • <tumor_dna_id>.linx.germline.svs.tsv: Normal DNA sample structural variants.

  • <group_id>/linx/somatic_annotations/

    • linx.version: LINX version file.
    • <tumor_dna_id>.linx.breakend.tsv: Tumor DNA sample breakend data.
    • <tumor_dna_id>.linx.clusters.tsv: Tumor DNA sample clustered events.
    • <tumor_dna_id>.linx.driver.catalog.tsv: Tumor DNA sample driver catalogue.
    • <tumor_dna_id>.linx.drivers.tsv: Tumor DNA sample LINX drivers.
    • <tumor_dna_id>.linx.fusion.tsv: Tumor DNA sample fusions.
    • <tumor_dna_id>.linx.links.tsv: Tumor DNA sample cluster links.
    • <tumor_dna_id>.linx.neoepitope.tsv: Tumor DNA sample neoepitopes.
    • <tumor_dna_id>.linx.svs.tsv: Tumor DNA sample structural variants.
    • <tumor_dna_id>.linx.vis_*: Tumor DNA sample visualisation data.

  • <group_id>/linx/somatic_plots/

    • all/*png: All available tumor DNA sample cluster plots.
    • reportable/*png: Driver-only tumor DNA sample cluster plots.

LINX clusters PURPLE-annotated SVs into high-order events and classifies these events within a biological context. Following clustering and interpretation, events are visualised as LINX plots.

Transcript analysis

ISOFOX

Output files
  • <group_id>/isofox/
    • <tumor_rna_id>.isf.alt_splice_junc.csv: Tumor RNA sample alternative splice junctions.
    • <tumor_rna_id>.isf.fusions.csv: Tumor RNA sample fusions, unfiltered.
    • <tumor_rna_id>.isf.gene_collection.csv: Tumor RNA sample gene-collection fragment counts.
    • <tumor_rna_id>.isf.gene_data.csv: Tumor RNA sample gene fragment counts.
    • <tumor_rna_id>.isf.pass_fusions.csv: Tumor RNA sample fusions, filtered.
    • <tumor_rna_id>.isf.retained_intron.csv: Tumor RNA sample retained introns.
    • <tumor_rna_id>.isf.summary.csv: Tumor RNA sample analysis summary file.
    • <tumor_rna_id>.isf.transcript_data.csv: Tumor RNA sample transcript fragment counts.

ISOFOX analyses RNA alignment data to quantify transcripts, identify novel splice junctions, and caller fusions.

Oncoviral detection

VIRUSBreakend

Output files
  • <group_id>/virusbreakend/
    • <tumor_dna_id>.virusbreakend.vcf: Tumor DNA sample viral integratino sites.
    • <tumor_dna_id>.virusbreakend.vcf.summary.tsv: Tumor DNA sample analysis summary file.

VIRUSBreakend detects the presence of oncoviruses and intergration sites in tumor samples.

VirusInterpreter

Output files
  • <group_id>/virusinterpreter/
    • <tumor_dna_id>.virus.annotated.tsv: Processed oncoviral call/annotation data.

VirusInterpreter performs post-processing for VIRUSBreakend calls to provide higher-level interpretation of data.

Immune analysis

LILAC

Output files
  • <group_id>/lilac/
    • <tumor_dna_id>.lilac.candidates.coverage.tsv: Coverage of high scoring candidates.
    • <tumor_dna_id>.lilac.qc.tsv: LILAC QC file.
    • <tumor_dna_id>.lilac.tsv: Analysis summary.

LILAC calls HLA Class I and characterises allelic status (copy-number alterations, somatic mutations) in the tumor sample. Analysis can also incorporate RNA data as an indirect measurement of allele expression.

NEO

Output files

  • <group_id>/neo/finder/

    • <tumor_dna_id>.neo_data.tsv: Tumor sample neoepitope candidates.

  • <group_id>/neo/annotated_fusions/

    • <tumor_dna_id>.isf.neoepitope.tsv: Tumor sample annotated Isofox fusions.

  • <group_id>/neo/scorer/

    • <tumor_dna_id>.neo.peptide_scores.tsv: Tumor sample peptide scores.
    • <tumor_dna_id>.neo.neoepitope.tsv: Tumor sample neoepitope predictions.

NEO identifies neoepitopes from point mutations, small indels and gene fusions, as well as calculating allele specific neoepitope binding and presentation likelihoods.

HRD status prediction

CHORD

Output files
  • <group_id>/chord/
    • <tumor_dna_id>.chord.prediction.tsv: Tumor DNA sample analysis summary file.
    • <tumor_dna_id>.chord.mutation_contexts.tsv: Tumor DNA sample counts of mutation types.

CHORD predicts the HRD status of a tumor using statistical inference on the basis of relative somatic mutation counts.

Mutational signature fitting

SIGS

Output files
  • <group_id>/sigs/
    • <tumor_dna_id>.sig.allocation.tsv: Tumor DNA sample signature allocations.
    • <tumor_dna_id>.sig.snv_counts.csv: Tumor DNA sample variant counts contributing to signatures.

Sigs fits defined COSMIC trinucleotide mutational signatures to tumor sample data.

Tissue of origin prediction

CUPPA

Output files
  • <group_id>/cuppa/
    • <tumor_dna_id>.cuppa_data.tsv.gz: Tumor sample input features
    • <tumor_dna_id>.cuppa.pred_summ.tsv: Tumor sample prediction summary
    • <tumor_dna_id>.cuppa.vis_data.tsv: Tumor sample predication visualisation data
    • <tumor_dna_id>.cuppa.vis.png: Tumor sample prediction visualisation

CUPPA predicts tissue of origin for a given tumor sample using DNA and/or RNA features generated by upstream WiGiTS components.

Report generation

ORANGE

Output files
  • <group_id>/orange/
    • <tumor_dna_id>.orange.json: Aggregated report data.
    • <tumor_dna_id>.orange.pdf: Static report PDF.

ORANGE summaries and integrates key results from hmftool components into a single static PDF report.

linxreport

Output files
  • <group_id>/linx/
    • <tumor_dna_id>_linx.html: Interactive HTML report.

linxreport generates an interactive report containing LINX annotations and plots.

Pipeline information

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.yml. The pipeline_report* files will only be present if the --email / --email_on_fail parameter’s are used when running the pipeline.
    • Reformatted samplesheet files used as input to the pipeline: samplesheet.valid.csv.
    • Parameters used by the pipeline run: params.json.

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.
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