Output files

Output files

PCGR generates multiple output files with annotations of molecular aberrations, including an interactive report, an Excel workbook, and pure text-based annotation files (TSV).

HTML report - quarto-based

An interactive and structured HTML report that shows the most relevant findings in the query cancer genome has the following naming convention:

• <sample_id>.pcgr.<genome_assembly>.html
  • The sample_id is provided as input by the user, and reflects a unique identifier of the tumor-normal sample pair to be analyzed.

The report is structured in various sections, pending upon the input provided by the user. The following sections may be included in the report:

1. Settings
   • Lists key configurations for the analysis, including the genome assembly, type of sequencing assay (WES/WGS/TARGETED), the cancer type (as provided by the user), and the tumor purity and ploidy.
2. Somatic SNVs/InDels
   • Provides an overview of the somatic SNVs and InDels detected in the tumor sample
   • Includes a global distribution of allelic support, statistics with respect to variant types and consequences
   • Variants are classified with respect to predicted oncogenicity (ClinGen/CGC/VICC standard operating procedures)
     • permits also exploration of somatic mutations through interactive filtering according to several dimensions (variant sequencing depth/support, variant consequence etc.)
   • Variants are classified with respect to clinical actionability (AMP/ASCO/CAP guidelines)
     • individual evidence items linked to actionable variants can be explored, indicating strength of evidence, tumor type and therapeutic context, and clinical significance
3. Somatic CNAs
   • Aberrations are classified with respect to clinical actionability (AMP/ASCO/CAP guidelines)
     • individual evidence items linked to actionable variants can be explored, indicating strength of evidence, tumor type and therapeutic context, and clinical significance
   • Other potentially oncogenic aberrations are listed: proto-oncogenes subject to copy number amplifications, and tumor suppressor genes subject to homozygous deletions
   • Two-hit events: tumor suppressor genes may be subject to biallelic inactivation through a combination of:
     • Loss of heterozygosity (LOH) — a copy number segment affecting one allele of the gene
     • A somatic loss-of-function variant (e.g. frameshift, nonsense, splice-site) in the tumor
     • A germline loss-of-function variant (as classified by CPSR, if provided as input)
4. MSI status

• Indicates predicted microsatellite stability from the somatic mutation profile and supporting evidence (details of the underlying MSI statistical classifier can be found here)
• The MSI classifier was trained on TCGA exome samples.

5. Tumor mutational burden (TMB)
   • given a coding target region size specified by the user (ideally the callable target size), an estimate of the mutational burden is provided
   • The estimated TMB is shown in the context of TMB distributions from different primary sites in TCGA
6. Mutational signatures

• Estimation of relative contribution of known mutational signatures in tumor sample (using MutationalPatterns as the underlying framework)
• Datatable with signatures found and proposed underlying etiologies

7. Kataegis events

• Kataegis events are clusters of particular SNV types. The report includes a datatable with all detected kataegis events.

8. RNA fusions

• Fusion partners are annotated with respect to oncogene status and overlap in the Mitelman database of chromosomal aberrations in cancer
• Clinical actionability evidence linked to fusion events is shown where available (AMP/ASCO/CAP guidelines)

9. RNA expression analysis

• Datatable with expression outliers - as compared to distribution in reference cohorts
• Datatable with correlation between gene expression in query sample and samples in other reference cohorts (TCGA, Treehouse Childhood Cancer Initiative, DepMap)
• Immune contexture profiling

10. Documentation

• Annotation resources - databases with version and licensing information
  • Report contents - brief description of the main sections in the report
• References - supporting scientific literature (key report elements)

Example reports

SNVs/InDels

1. Variant call format - VCF

A VCF file containing annotated, somatic calls (single nucleotide variants and insertion/deletions) is generated with the following naming convention:

• <sample_id>.pcgr.<genome_assembly>.vcf.gz
  • The sample_id is provided as input by the user, and reflects a unique identifier of the tumor-normal sample pair to be analyzed. Following common standards, the annotated VCF file is compressed with bgzip and indexed with tabix. Below follows a description of all annotations/tags present in the VCF INFO column after processing with the PCGR annotation pipeline:

VEP consequence annotations

Tag	Description
CSQ	Complete consequence annotations from VEP. Format (separated by a |): Allele, Consequence, IMPACT, SYMBOL, Gene, Feature_type, Feature, BIOTYPE, EXON, INTRON, HGVSc, HGVSp, cDNA_position, CDS_position, Protein_position, Amino_acids, Codons, Existing_variation, ALLELE_NUM, DISTANCE, STRAND, FLAGS, PICK, VARIANT_CLASS, SYMBOL_SOURCE, HGNC_ID, CANONICAL, MANE_SELECT, MANE_PLUS_CLINICAL, TSL, APPRIS, CCDS, ENSP, SWISSPROT, TREMBL, UNIPARC, UNIPROT_ISOFORM, RefSeq, DOMAINS, HGVS_OFFSET, AF, AFR_AF, AMR_AF, EAS_AF, EUR_AF, SAS_AF, gnomAD_AF, gnomAD_AFR_AF, gnomAD_AMR_AF, gnomAD_ASJ_AF, gnomAD_EAS_AF, gnomAD_FIN_AF, gnomAD_NFE_AF, gnomAD_OTH_AF, gnomAD_SAS_AF, CLIN_SIG, SOMATIC, PHENO, CHECK_REF, MOTIF_NAME, MOTIF_POS, HIGH_INF_POS, MOTIF_SCORE_CHANGE, TRANSCRIPTION_FACTORS, NearestExonJB
Consequence	Impact modifier for the consequence type (picked by VEP’s --flag_pick_allele option)
Gene	Ensembl stable ID of affected gene (picked by VEP’s --flag_pick_allele option)
Feature_type	Type of feature. Currently one of Transcript, RegulatoryFeature, MotifFeature (picked by VEP’s --flag_pick_allele option)
Feature	Ensembl stable ID of feature (picked by VEP’s --flag_pick_allele option)
cDNA_position	Relative position of base pair in cDNA sequence (picked by VEP’s --flag_pick_allele option)
CDS_position	Relative position of base pair in coding sequence (picked by VEP’s --flag_pick_allele option)
CDS_RELATIVE_POSITION	Ratio of variant coding position to length of coding sequence
CDS_CHANGE	Coding, transcript-specific sequence annotation (picked by VEP’s --flag_pick_allele option)
ALTERATION	HGVSp/HGVSc identifier
AMINO_ACID_START	Protein position indicating absolute start of amino acid altered (fetched from Protein_position)
AMINO_ACID_END	Protein position indicating absolute end of amino acid altered (fetched from Protein_position)
Protein_position	Relative position of amino acid in protein (picked by VEP’s --flag_pick_allele option)
Amino_acids	Only given if the variant affects the protein-coding sequence (picked by VEP’s --flag_pick_allele option)
GRANTHAM_DISTANCE	Grantham distance between the reference and variant amino acids
Codons	The alternative codons with the variant base in upper case (picked by VEP’s --flag_pick_allele option)
IMPACT	Impact modifier for the consequence type (picked by VEP’s --flag_pick_allele option)
VARIANT_CLASS	Sequence Ontology variant class (picked by VEP’s --flag_pick_allele option)
SYMBOL	Gene symbol (picked by VEP’s --flag_pick_allele option)
SYMBOL_SOURCE	The source of the gene symbol (picked by VEP’s --flag_pick_allele option)
STRAND	The DNA strand (1 or -1) on which the transcript/feature lies (picked by VEP’s --flag_pick_allele option)
ENSP	The Ensembl protein identifier of the affected transcript (picked by VEP’s --flag_pick_allele option)
FLAGS	Transcript quality flags: cds_start_NF: CDS 5’, incomplete cds_end_NF: CDS 3’ incomplete (picked by VEP’s --flag_pick_allele option)
SWISSPROT	Best match UniProtKB/Swiss-Prot accession of protein product (picked by VEP’s --flag_pick_allele option)
TREMBL	Best match UniProtKB/TrEMBL accession of protein product (picked by VEP’s --flag_pick_allele option)
UNIPARC	Best match UniParc accession of protein product (picked by VEP’s --flag_pick_allele option)
HGVSc	The HGVS coding sequence name (picked by VEP’s --flag_pick_allele option)
HGVSc_RefSeq	The HGVSc coding sequence name using RefSeq transcript identifiers (MANE select) - picked by VEP’s --flag_pick_allele option)
HGVSp	The HGVS protein sequence name (picked by VEP’s --flag_pick_allele option)
HGVSp_short	The HGVS protein sequence name, short version (picked by VEP’s --flag_pick_allele option)
HGVS_OFFSET	Indicates by how many bases the HGVS notations for this variant have been shifted (picked by VEP’s --flag_pick_allele option)
NearestExonJB	VEP plugin that finds nearest exon junction for a coding sequence variant. Format: Ensembl exon identifier+distanceto exon boundary+boundary type(start/end)+exon length
MOTIF_NAME	The source and identifier of a transcription factor binding profile aligned at this position (picked by VEP’s --flag_pick_allele option)
MOTIF_POS	The relative position of the variation in the aligned TFBP (picked by VEP’s --flag_pick_allele option)
HIGH_INF_POS	A flag indicating if the variant falls in a high information position of a transcription factor binding profile (TFBP) (picked by VEP’s --flag_pick_allele option)
MOTIF_SCORE_CHANGE	The difference in motif score of the reference and variant sequences for the TFBP (picked by VEP’s --flag_pick_allele option)
CELL_TYPE	List of cell types and classifications for regulatory feature (picked by VEP’s --flag_pick_allele option)
CANONICAL	A flag indicating if the transcript is denoted as the canonical transcript for this gene (picked by VEP’s --flag_pick_allele option)
CCDS	The CCDS identifier for this transcript, where applicable (picked by VEP’s --flag_pick_allele option)
INTRON	The intron number (out of total number) (picked by VEP’s --flag_pick_allele option)
EXON	The exon number (out of total number) (picked by VEP’s --flag_pick_allele option)
EXON_AFFECTED	The exon affected by the variant (picked by VEP’s --flag_pick_allele option)
LAST_EXON	Logical indicator for last exon of transcript (picked by VEP’s --flag_pick_allele option)
LAST_INTRON	Logical indicator for last intron of transcript (picked by VEP’s --flag_pick_allele option)
INTRON_POSITION	Relative position of intron variant to nearest exon/intron junction (NearestExonJB VEP plugin)
EXON_POSITION	Relative position of exon variant to nearest intron/exon junction (NearestExonJB VEP plugin)
DISTANCE	Shortest distance from variant to transcript (picked by VEP’s --flag_pick_allele option)
BIOTYPE	Biotype of transcript or regulatory feature (picked by VEP’s --flag_pick_allele option)
TSL	Transcript support level (picked by VEP’s --flag_pick_allele option)
PUBMED	PubMed ID(s) of publications that cite existing variant - VEP
PHENO	Indicates if existing variant is associated with a phenotype, disease or trait - VEP
GENE_PHENO	Indicates if overlapped gene is associated with a phenotype, disease or trait - VEP
ALLELE_NUM	Allele number from input; 0 is reference, 1 is first alternate etc - VEP
REFSEQ_MATCH	The RefSeq transcript match status; contains a number of flags indicating whether this RefSeq transcript matches the underlying reference sequence and/or an Ensembl transcript (picked by VEP’s --flag_pick_allele option)
PICK	Indicates if this block of consequence data was picked by VEP’s --flag_pick_allele option
VEP_ALL_CSQ	All transcript consequences (Consequence:SYMBOL:Feature_type:Feature:BIOTYPE) - VEP
EXONIC_STATUS	Indicates if variant consequence type is ‘exonic’ or ‘nonexonic’. We here define ‘exonic’ as any variant with either of the following consequences: stop_gained / stop_lost, start_lost, frameshift_variant, missense_variant, splice_donor_variant, splice_acceptor_variant, inframe_insertion / inframe_deletion, synonymous_variant, start_retained, stop_retained, protein_altering
CODING_STATUS	Indicates if primary variant consequence type is ‘coding’ or ‘noncoding’ (wrt. protein-alteration). ‘coding’ variants are here defined as those with an ‘exonic’ status, with the exception of synonymous variants
EXONIC_STATUS	Indicates if variant consequence type is ‘exonic’ or ‘nonexonic’. We define ‘exonic’ as any variants with the following consequence types: stop_gained / stop_lost, start_lost, frameshift_variant, missense_variant, splice_donor_variant, splice_acceptor_variant, inframe_insertion / inframe_deletion, synonymous_variant, protein_altering
CODING_STATUS	Indicates if primary variant consequence type is ‘coding’ or ‘noncoding’. ‘coding’ variants are here defined as those consequence types with an ‘exonic’ status, with the exception of synonymous variants. All other consequence types are considered ‘noncoding’
NULL_VARIANT	Primary variant consequence type is frameshift or stop_gained
LOSS_OF_FUNCTION	Loss-of-function variant - primary variant consequence being either stop_gained / stop_lost, start_lost, frameshift_variant, splice_donor_variant, or splice_acceptor_variant
LOF_FILTER	Loss-of-function filter - exceptions to putative LOF variants - GC to GT at splice donor sites or truncations within the last 5% of coding sequence
SPLICE_DONOR_RELEVANT	Logical indicating if variant is located at a particular location near the splice donor site (+3A/G, +4A or +5G)
REGULATORY_ANNOTATION	Comma-separated list of all variant annotations of Feature_type, RegulatoryFeature, and MotifFeature. Format (separated by a |): <Consequence>, <Feature_type>, <Feature>, <BIOTYPE>, <MOTIF_NAME>, <MOTIF_POS>, <HIGH_INF_POS>, <MOTIF_SCORE_CHANGE>, <TRANSCRIPTION_FACTORS>

Gene information

Tag	Description
ENTREZGENE	Entrez gene identifier
APPRIS	Principal isoform flags according to the APPRIS principal isoform database
MANE_SELECT	Indicating if the transcript is the MANE Select for the gene (picked by VEP’s --flag_pick_allele_gene option)
MANE_PLUS_CLINICAL	Indicating if the transcript is MANE Plus Clinical, as required for clinical variant reporting (picked by VEP’s --flag_pick_allele_gene option)
UNIPROT_ID	UniProt identifier
UNIPROT_ACC	UniProt accession(s)
ENSEMBL_GENE_ID	Ensembl gene identifier for VEP’s picked transcript (ENSGXXXXXXX)
ENSEMBL_TRANSCRIPT_ID	Ensembl transcript identifier for VEP’s picked transcript (ENSTXXXXXX)
ENSEMBL_PROTEIN_ID	Ensembl corresponding protein identifier for VEP’s picked transcript (ENSPXXXXXX)
REFSEQ_TRANSCRIPT_ID	Corresponding RefSeq transcript(s) identifier for VEP’s picked transcript (NM_XXXXX)
MANE_SELECT2	MANE select transcript identifer: one high-quality representative transcript per protein-coding gene that is well-supported by experimental data and represents the biology of the gene - provided through BioMart
MANE_PLUS_CLINICAL2	transcripts chosen to supplement MANE Select when needed for clinical variant reporting - provided through BioMart
GENCODE_TAG	tag for gencode transcript (basic etc)
GENCODE_TRANSCRIPT_TYPE	type of transcript (protein-coding etc.)
TSG	Flag indicating whether gene is predicted as a tumor suppressor gene, from Cancer Gene Census, Network of Cancer Genes (NCG) & the CancerMine text-mining resource
TSG_SUPPORT	Underlying evidence for gene being a tumor suppressor. Format: NCG&CancerMine:num_citations
ONCOGENE	Flag indicating whether gene is predicted as an oncogene, from Cancer Gene Census, Network of Cancer Genes (NCG) & the CancerMine text-mining resource.
ONCOGENE_SUPPORT	Underlying evidence for gene being an oncogene. Format: NCG&CancerMine:num_citations
INTOGEN_DRIVER	Gene is predicted as a cancer driver in the IntoGen Cancer Drivers Database
TCGA_DRIVER	Gene is predicted as a cancer driver in the TCGA pan-cancer analysis of cancer driver genes and mutations
PROB_EXAC_LOF_INTOLERANT	dbNSFP_gene: the probability of being loss-of-function intolerant (intolerant of both heterozygous and homozygous lof variants) based on ExAC r0.3 data
PROB_EXAC_LOF_INTOLERANT_HOM	dbNSFP_gene: the probability of being intolerant of homozygous, but not heterozygous lof variants based on ExAC r0.3 data
PROB_EXAC_LOF_TOLERANT_NULL	dbNSFP_gene: the probability of being tolerant of both heterozygous and homozygous lof variants based on ExAC r0.3 data
PROB_EXAC_NONTCGA_LOF_INTOLERANT	dbNSFP_gene: the probability of being loss-of-function intolerant (intolerant of both heterozygous and homozygous lof variants) based on ExAC r0.3 nonTCGA subset
PROB_EXAC_NONTCGA_LOF_INTOLERANT_HOM	dbNSFP_gene: the probability of being intolerant of homozygous, but not heterozygous lof variants based on ExAC r0.3 nonTCGA subset
PROB_EXAC_NONTCGA_LOF_TOLERANT_NULL	dbNSFP_gene: the probability of being tolerant of both heterozygous and homozygous lof variants based on ExAC r0.3 nonTCGA subset
PROB_GNOMAD_LOF_INTOLERANT	dbNSFP_gene: the probability of being loss-of-function intolerant (intolerant of both heterozygous and homozygous lof variants based on gnomAD 2.1 data
PROB_GNOMAD_LOF_INTOLERANT_HOM	dbNSFP_gene: the probability of being intolerant of homozygous, but not heterozygous lof variants based on gnomAD 2.1 data
PROB_GNOMAD_LOF_TOLERANT_NULL	dbNSFP_gene: the probability of being tolerant of both heterozygous and homozygous lof variants based on gnomAD 2.1 data
PROB_HAPLOINSUFFICIENCY	dbNSFP_gene: Estimated probability of haploinsufficiency of the gene (from http://dx.doi.org/10.1371/journal.pgen.1001154)
ESSENTIAL_GENE_CRISPR	dbNSFP_gene: Essential (E) or Non-essential phenotype-changing (N) based on large scale CRISPR experiments. from http://dx.doi.org/10.1126/science.aac7041
ESSENTIAL_GENE_CRISPR2	dbNSFP_gene: Essential (E), context-Specific essential (S), or Non-essential phenotype-changing (N) based on large scale CRISPR experiments. from http://dx.doi.org/10.1016/j.cell.2015.11.015

Variant effect and protein-coding information

Tag	Description
MUTATION_HOTSPOT	mutation hotspot codon in cancerhotspots.org. Format: gene_symbol | codon | q-value
MUTATION_HOTSPOT_TRANSCRIPT	hotspot-associated transcripts (Ensembl transcript ID)
MUTATION_HOTSPOT_CANCERTYPE	hotspot-associated cancer types (from cancerhotspots.org)
PFAM_DOMAIN	Pfam domain identifier (from VEP)
INTOGEN_DRIVER_MUT	Indicates if existing variant is predicted as driver mutation from IntoGen Catalog of Driver Mutations
EFFECT_PREDICTIONS	In silico predictions of variant effect on protein function and pre-mRNA splicing from database of non-synonymous functional predictions - dbNSFP v5.0. Predicted effects are provided by different sources/algorithms (separated by &), T = Tolerated, N = Neutral, D = Damaging
SPLICE_EFFECT	Effect of splicing, from MutSpliceDB and/or MaxEntScan. Format:
MES
DBNSFP_BAYESDEL_ADDAF	predicted effect from BayesDel (dbNSFP)
DBNSFP_LIST_S2	predicted effect from LIST-S2 (dbNSFP)
DBNSFP_SIFT	predicted effect from SIFT (dbNSFP)
DBNSFP_POLYPHEN2_HVAR	predicted effect from PolyPhen2 (dbNSFP)
DBNSFP_PROVEAN	predicted effect from PROVEAN (dbNSFP)
DBNSFP_MUTATIONTASTER	predicted effect from MUTATIONTASTER (dbNSFP)
DBNSFP_MUTATIONASSESSOR	predicted effect from MUTATIONASSESSOR (dbNSFP)
DBNSFP_M_CAP	predicted effect from M-CAP (dbNSFP)
DBNSFP_MUTPRED	score from MUTPRED (dbNSFP)
DBNSFP_CLINPRED	predicted effect from ClinPred (dbNSFP)
DBNSFP_FATHMM	predicted effect from FATHMM-XF (dbNSFP)
DBNSFP_PRIMATEAI	predicted effect from PRIMATEAI (dbNSFP)
DBNSFP_DEOGEN2	predicted effect from DEOGEN2 (dbNSFP)
DBNSFP_PHACTBOOST	predicted effect from PHACTboost (dbNSFP)
DBNSFP_ALPHA_MISSENSE	predicted effect from AlphaMissense (dbNSFP)
DBNSFP_MUTFORMER	predicted effect from MutFormer (dbNSFP)
DBNSFP_ESM1B	predicted effect from ESM1b (dbNSFP)
DBNSFP_GERP	evolutionary constraint measure from GERP (dbNSFP)
DBNSFP_CADD	Combined Annotation Dependent Depletion (CADD) score (dbNSFP)
DBNSFP_VEST4	VEST4 score (dbNSFP)
DBNSFP_FATHMM_XF	predicted effect from FATHMM-XF (dbNSFP)
DBNSFP_META_RNN	predicted effect from ensemble prediction (deep learning - dbNSFP)
DBNSFP_SPLICE_SITE_RF	predicted effect of splice site disruption, using random forest (dbscSNV)
DBNSFP_SPLICE_SITE_ADA	predicted effect of splice site disruption, using boosting (dbscSNV)

Variant frequencies/annotations in germline/somatic databases

Tag	Description
gnomADe_AFR_AF	African/American germline allele frequency - exome set (gnomAD release 4.1)
gnomADe_AMR_AF	Latino/Admixed American germline allele frequency - exome set (gnomAD release 4.1)
gnomADe_AF	Adjusted global germline allele frequency - exome set (gnomAD release 4.1)
gnomADe_SAS_AF	South Asian germline allele frequency - exome set (gnomAD release 4.1)
gnomADe_EAS_AF	East Asian germline allele frequency - exome set (gnomAD release 4.1)
gnomADe_FIN_AF	Finnish germline allele frequency - exome set (gnomAD release 4.1)
gnomADe_NFE_AF	Non-Finnish European germline allele frequency - exome set (gnomAD release 4.1)
gnomADe_OTH_AF	Other germline allele frequency - exome set (gnomAD release 4.1)
gnomADe_ASJ_AF	Ashkenazi Jewish allele frequency - exome set (gnomAD release 4.1)
gnomADg_AFR_AF	African/American germline allele frequency - genome set (gnomAD release 4.1)
gnomADg_AMR_AF	Latino/Admixed American germline allele frequency - genome set (gnomAD release 4.1)
gnomADg_AF	Adjusted global germline allele frequency - genome set (gnomAD release 4.1)
gnomADg_SAS_AF	South Asian germline allele frequency - genome set (gnomAD release 4.1)
gnomADg_EAS_AF	East Asian germline allele frequency - genome set (gnomAD release 4.1)
gnomADg_FIN_AF	Finnish germline allele frequency - genome set (gnomAD release 4.1)
gnomADg_NFE_AF	Non-Finnish European germline allele frequency - genome set (gnomAD release 4.1)
gnomADg_MID_AF	Middle Eastern germline allele frequency - genome set (gnomAD release 4.1)
gnomADg_OTH_AF	Other germline allele frequency - genome set (gnomAD release 4.1)
gnomADg_ASJ_AF	Ashkenazi Jewish allele frequency - genome set (gnomAD release 4.1)
DBSNP_RSID	dbSNP reference ID, as provided by VEP
COSMIC_MUTATION_ID	Mutation identifier in Catalog of somatic mutations in cancer database, as provided by VEP
TCGA_PANCANCER_COUNT	Raw variant count across all TCGA tumor types
TCGA_FREQUENCY	Frequency of variant across TCGA tumor types. Format: tumortype| percent affected|affected cases|total cases

Clinical associations

Tag	Description
CLINVAR_MSID	ClinVar Measure Set/Variant ID
CLINVAR_ALLELE_ID	ClinVar allele ID
CLINVAR_PMID	Associated Pubmed IDs for variant in ClinVar - germline state-of-origin
CLINVAR_HGVSP	Protein variant expression using HGVS nomenclature
CLINVAR_PMID_SOMATIC	Associated Pubmed IDs for variant in ClinVar - somatic state-of-origin
CLINVAR_CLNSIG	Clinical significance for variant in ClinVar - germline state-of-origin
CLINVAR_CLNSIG_SOMATIC	Clinical significance for variant in ClinVar - somatic state-of-origin
CLINVAR_MEDGEN_CUI	Associated MedGen concept identifiers (CUIs) - germline state-of-origin
CLINVAR_MEDGEN_CUI_SOMATIC	Associated MedGen concept identifiers (CUIs) - somatic state-of-origin
CLINVAR_VARIANT_ORIGIN	Origin of variant (somatic, germline, de novo etc.) for variant in ClinVar
CLINVAR_REVIEW_STATUS_STARS	Rating of the ClinVar variant (0-4 stars) with respect to level of review
KNOWN_ONCOGENIC	Variant matches with known oncogenic variants in ClinVar, through ClinGen/CGC/VICC SOP. Format:
KNOWN_ONCOGENIC_SITE	Variant matches with known oncogenic site/codon in ClinVar, through ClinGen/CGC/VICC SOP. The variant must have approximately the same Grantham distance as the one recorded for the oncogenic site. Format:

Other

Tag	Description
BIOMARKER_MATCH	Variant matches with biomarker evidence in CIViC/CGI. Format: ||::::|. Multiple evidence items are separated by ‘&’. Example: civic
ONCOGENICITY	Oncogenicity annotation - ClinGen/CGC/VICC SOP implementation
ONCOGENICITY_CODE	Variant-matching oncogenicity code(s) - ClinGen/CGC/VICC SOP implementation
ONCOGENICITY_SCORE	Variant oncogenicity score - ClinGen/CGC/VICC SOP implementation

2. Tab-separated values (TSV)

We provide a tab-separated values file with most important annotations for SNVs/InDels. The file has the following naming convention:

• <sample_id>.pcgr.<genome_assembly>.snv_indel_ann.tsv.gz

The following variables are included in the TSV file (VCF tags issued by the user (--retained_info_tags) will be appended at the end). Columns suffixed with _OKB are only present when OncoKB annotation is enabled via --oncokb_api_token.

Variable	Description
1. SAMPLE_ID	Sample identifier
2. VARIANT_CLASS	Variant type, e.g. SNV/insertion/deletion/indel
3. VAR_ID	Variant identifier. Format: <chrom>_<position>_<ref_allele>_<alt_allele>
4. GENOMIC_CHANGE	Identifier for variant at the genome (VCF) level, e.g. 1:g.152382569A>G. Format: <chrom>:g.<position><ref_allele><alt_allele>
5. GENOME_VERSION	Assembly version, e.g. GRCh38
6. SYMBOL	Gene symbol
7. ENTREZGENE	Entrez gene identifier
8. ENSEMBL_GENE_ID	Ensembl gene identifier
9. GENENAME	Gene name
10. ALTERATION	Combined HGVSp/HGVSc annotation
11. CDS_CHANGE	Coding sequence change
12. HGVSc	HGVS coding sequence name
13. HGVSc_RefSeq	HGVS coding sequence name (RefSeq/MANE select)
14. HGVSp	HGVS protein sequence name
15. HGVSP	Protein change in one-letter amino-acid HGVS notation (HGVSp_short)
16. SPLICE_EFFECT	Splice effect annotations from MutSpliceDB and MaxEntScan (see details above)
17. MAXENTSCAN	MaxEntScan splice site impact summary. Format: MaxEntScan|<stratum>|<evidence_tier>
18. EFFECT_PREDICTIONS	Variant effect predictions - from dbNSFP
19. EXON	Exon number (out of total number of exons)
20. CONSEQUENCE	Variant consequence - from VEP
21. PFAM_DOMAIN_NAME	Pfam domain name
22. LOSS_OF_FUNCTION	Loss of function flag
23. LOF_FILTER	Loss of function filter
24. CODING_STATUS	Coding status - flag indicating if consequence is protein-altering/affecting splice sites
25. EXONIC_STATUS	Exonic status - flag indicating if consequence is silent/protein-altering/affecting splice sites
26. DP_TUMOR	Depth of coverage at variant position in tumor sample
27. VAF_TUMOR	Variant allele fraction at variant position in tumor sample
28. AD_TUMOR	Allelic depth (number of reads supporting alt allele) in tumor sample
29. CALL_CONFIDENCE	Call confidence
30. DP_CONTROL	Depth of coverage at variant position in control sample
31. VAF_CONTROL	Variant allele fraction at variant position in control sample
32. AD_CONTROL	Allelic depth (number of reads supporting alt allele) in control sample
33. MUTATION_HOTSPOT	Mutation hotspot annotation
34. MUTATION_HOTSPOT_CANCERTYPE	Mutation hotspot-associated cancer types (from cancerhotspots.org)
35. ACTIONABILITY_TIER	Variant clinical actionability tier - AMP/ASCO/CAP implementation
36. ACTIONABILITY	Variant clinical actionability significance - AMP/ASCO/CAP implementation
37. ONCOGENICITY	Oncogenicity annotation - ClinGen/CGC/VICC SOP implementation
38. ONCOGENICITY_CODE	Variant-matching oncogenicity code(s) - ClinGen/CGC/VICC SOP implementation
39. ONCOGENICITY_SCORE	Variant oncogenicity score - ClinGen/CGC/VICC SOP implementation
40. MUTATION_EFFECT_OKB	(OncoKB only) OncoKB mutation effect annotation (e.g. Loss-of-function, Gain-of-function)
41. MUTATION_EFFECT_CITATIONS_OKB	(OncoKB only) OncoKB mutation effect citations
42. MUTATION_EFFECT_DESCRIPTION_OKB	(OncoKB only) OncoKB mutation effect description
43. ONCOGENICITY_OKB	(OncoKB only) OncoKB oncogenicity annotation (e.g. Oncogenic, Likely Oncogenic, Likely Neutral etc.)
44. TUMOR_TYPE_SUMMARY_OKB	(OncoKB only) OncoKB tumor type summary
45. VARIANT_SUMMARY_OKB	(OncoKB only) OncoKB variant summary
46. HOTSPOT_OKB	(OncoKB only) OncoKB hotspot flag - indicates if variant overlaps a known OncoKB hotspot
47. VUS_OKB	(OncoKB only) OncoKB variant of uncertain significance flag
48. CANONICAL	Flag indicating if transcript is canonical
49. CCDS	CCDS identifier
50. UNIPROT_ACC	UniProt accession
51. ENSEMBL_TRANSCRIPT_ID	Ensembl transcript identifier
52. ENSEMBL_PROTEIN_ID	Ensembl protein identifier
53. REFSEQ_TRANSCRIPT_ID	RefSeq transcript identifier
54. REFSEQ_PROTEIN_ID	RefSeq protein identifier
55. MANE_SELECT	MANE transcript select
56. MANE_PLUS_CLINICAL	MANE transcript plus clinical
57. ONCOGENE	Flag indicating if gene is oncogene (CancerMine/NCG)
58. ONCOGENE_SUPPORT	Oncogene annotation support (CancerMine/NCG)
59. TUMOR_SUPPRESSOR	Flag indicating if gene is tumor suppressor (CancerMine/NCG)
60. TUMOR_SUPPRESSOR_SUPPORT	Tumor suppressor annotation support (CancerMine/NCG)
61. TARGETED_INHIBITORS2	Targeted inhibitors
62. REGULATORY_ANNOTATION	Regulatory annotation
63. VEP_ALL_CSQ	VEP consequence - all transcripts
64. gnomADe_AF	gnomAD exomes allele frequency - globally
65. gnomADg_AF	gnomAD genomes allele frequency - globally
66. DBSNP_RSID	dbSNP identifier
67. COSMIC_ID	COSMIC identifier
68. TCGA_FREQUENCY	Frequency of variant across TCGA tumor types. Format: tumortype | percent affected | affected cases | total cases
69. TCGA_PANCANCER_COUNT	Raw variant count across all TCGA tumor types
70. CLINVAR_MSID	ClinVar measureset identifier
71. CLINVAR_CLASSIFICATION	ClinVar variant classification
72. CLINVAR_VARIANT_ORIGIN	ClinVar variant origin
73. CLINVAR_NUM_SUBMITTERS	ClinVar number of submitters
74. CLINVAR_GOLD_STARS	ClinVar review status gold stars (0-4)
75. CLINVAR_CONFLICTED	ClinVar variant classification is conflicted

Tumor-only runs

For tumor-only runs, we provide a similarly formatted tab-separated values file that include both filtered (i.e. likely germline events) and unfiltered (deemed somatic) variants. The file has the following naming convention:

• <sample_id>.pcgr.<genome_assembly>.snv_indel_unfiltered.ann.tsv.gz

This file shares most columns with the standard/filtered TSV above, with the following differences:

• Added SOMATIC_CLASSIFICATION (column 5, after VARIANT_CLASS): indicates whether a variant is classified as somatic or germline. Value is SOMATIC (not caught by any filter), or one or more of the following germline tags: GERMLINE_GNOMAD (always on), GERMLINE_CLINVAR, GERMLINE_DBSNP, GERMLINE_HET, GERMLINE_HOM, GERMLINE_PON.
• Added per-population gnomAD columns: gnomADg_AFR_AF, gnomADg_AMR_AF, gnomADg_ASJ_AF, gnomADg_EAS_AF, gnomADg_FIN_AF, gnomADg_NFE_AF, gnomADg_OTH_AF, gnomADg_SAS_AF (genome set) and gnomADe_AFR_AF, gnomADe_AMR_AF, gnomADe_EAS_AF, gnomADe_FIN_AF, gnomADe_NFE_AF, gnomADe_SAS_AF (exome set).
• Omitted compared to the filtered TSV: DP_CONTROL, VAF_CONTROL, AD_CONTROL (not applicable in tumor-only runs)

Tumor mutational burden (TSV)

We provide a tab-separated values (TSV) file with information about mutational burden detected in the tumor sample. The file has the following naming convention:

• <sample_id>.pcgr.<genome_assembly>.tmb.tsv

The format of the TSV file is the following:

Variable	Description
1. SAMPLE_ID	sample identifier
2. N_SOMATIC_VARIANTS	number of somatic variants in total for sample
3. TMB_MEASURE	TMB measure - type of variants included
4. TMB_CSQ_REGEX	VEP consequence regex for variants included in TMB calculation
5. TMB_TARGET_SIZE_MB	target size in megabases
6. TMB_DP_MIN	minimum depth of coverage for variant to be included in TMB calculation
7. TMB_AF_MIN	minimum allele frequency for variant to be included in TMB calculation
8. TMB_N_VARIANTS	number of variants included in TMB calculation
9. TMB_ESTIMATE	TMB estimate
10. TMB_UNIT	TMB unit (i.e. mutations/Mb)

Mutational signature contributions (TSV)

We provide a tab-separated values (TSV) file with information about mutational signatures detected in the tumor sample. The file has the following naming convention:

• <sample_id>.pcgr.<genome_assembly>.msigs.tsv.gz

The format of the TSV file is the following:

Variable	Description
1. SAMPLE_ID	sample identifier
2. SIGNATURE_ID	identifier for signature
3. N_BS_ITERATIONS	number of bootstrap iterations
4. PROP_SIGNATURE	relative contribution of mutational signature
5. PROP_SIGNATURE_CI_LOWER	lower bound of confidence interval for relative contribution of mutational signature
6. PROP_SIGNATURE_CI_UPPER	upper bound of confidence interval for relative contribution of mutational signature
7. AETIOLOGY	underlying aetiology of mutational signature
8. COMMENTS	additional comments regarding aetiology
9. GROUP	keyword for signature aetiology
10. ALL_REFERENCE_SIGNATURES	logical indicating if all reference signatures were used for reconstruction/inference
11. TUMOR_TYPE	tumor type (used for retrieval of reference signatures)
12. REFERENCE_COLLECTION	collection used for reference signatures
13. REFERENCE_SIGNATURES	signatures present in reference collection
14. FITTING_ACCURACY	accuracy of mutational signature fitting

Copy number aberrations

1. Tab-separated values (TSV)

Copy number segments are intersected with the genomic coordinates of all transcripts from GENCODE’s basic gene annotation. In addition, PCGR attaches cancer-relevant annotations for the affected transcripts. The naming convention of the compressed TSV files are as follows:

• <sample_id>.pcgr.<genome_assembly>.cna_segment.tsv.gz
  • segment level information only
• <sample_id>.pcgr.<genome_assembly>.cna_gene_ann.tsv.gz
  • This file is organized according to the affected transcripts (i.e. one line/record per affected transcript, segments not overlapping with any transcripts will thus not be included in this files).

The format of the compressed cna_gene_ann.tsv.gz is the following. Columns suffixed with _OKB are only present when OncoKB annotation is enabled via --oncokb_api_token. Note that HOTSPOT_OKB and VUS_OKB are not included for CNA output.

Variable	Description
1. SAMPLE_ID	Sample identifier
2. VAR_ID	Variant identifier. Format: <chromosome>:<segment_start>-<segment_end>:<major_cn>:<minor_cn>
3. GENOME_VERSION	Assembly version, e.g. GRCh38
4. CN_MAJOR	Major copy number
5. CN_MINOR	Minor copy number
6. LOH	Loss of heterozygosity flag
7. TWOHIT_CANDIDATE_SOMATIC	Comma-separated somatic loss-of-function variants overlapping the segment (TSG with allele-specific LOH: deletion or copy-neutral). Each entry has the format VAR_ID;CONSEQUENCE;VAF_FLAG;ALTERATION;VAF_TUMOR;DP_TUMOR;ONCOGENICITY, where VAF_FLAG is one of: VAF_CONSISTENT (observed VAF consistent with variant on retained allele given tumor purity), VAF_LOW (VAF below purity-adjusted expectation — variant may reside on the deleted allele or be subclonal to the LOH event), or VAF_UNKNOWN (tumor purity not provided); ALTERATION is the protein-level consequence (or . if absent); VAF_TUMOR is the allele fraction in the tumor; DP_TUMOR is the read depth; and ONCOGENICITY is the OncoKB classification (or . if OncoKB is not enabled).
8. TWOHIT_CANDIDATE_GERMLINE	Comma-separated germline pathogenic/likely-pathogenic loss-of-function variants matching by gene symbol (TSG with allele-specific LOH: deletion or copy-neutral; requires CPSR input). Each entry has the format VAR_ID;CONSEQUENCE.
9. FOLD_CHANGE	Fold change of segment copy number relative to tumor ploidy
10. TUMOR_PLOIDY	Tumor ploidy estimate
11. TUMOR_PLOIDY_SOURCE	Source of tumor ploidy estimate
12. TUMOR_PURITY	Tumor purity estimate
13. SEGMENT_LENGTH_MB	Length of segment in Mb
14. CYTOBAND	Associated cytoband
15. EVENT_TYPE	Focal or broad (covering more than 25% of chromosome arm)
16. VARIANT_CLASS	amplification: total copy number >= user-defined threshold; homdel: total copy number equal to zero; hetdel: total copy number equal to one; gain: copy number gain below amplification threshold; undefined: other copy number states
17. VARIANT_CLASS_DISPLAY	Display label for variant class
18. SYMBOL	Gene symbol
19. ENTREZGENE	Entrez gene identifier
20. GENENAME	Gene name
21. ENSEMBL_GENE_ID	Ensembl gene identifier
22. TUMOR_SUPPRESSOR	Flag indicating if gene is tumor suppressor (CancerMine/NCG)
23. TUMOR_SUPPRESSOR_SUPPORT	Tumor suppressor annotation support (CancerMine/NCG)
24. ONCOGENE	Flag indicating if gene is oncogene (CancerMine/NCG)
25. ONCOGENE_SUPPORT	Oncogene annotation support (CancerMine/NCG)
26. MUTATION_EFFECT_OKB	(OncoKB only) OncoKB mutation effect annotation (e.g. Loss-of-function, Gain-of-function)
27. MUTATION_EFFECT_CITATIONS_OKB	(OncoKB only) OncoKB mutation effect citations
28. MUTATION_EFFECT_DESCRIPTION_OKB	(OncoKB only) OncoKB mutation effect description
29. ONCOGENICITY_OKB	(OncoKB only) OncoKB oncogenicity annotation (e.g. Oncogenic, Likely Oncogenic)
30. TUMOR_TYPE_SUMMARY_OKB	(OncoKB only) OncoKB tumor type summary
31. VARIANT_SUMMARY_OKB	(OncoKB only) OncoKB variant summary
32. TRANSCRIPT_OVERLAP	Comma-separated list of associated transcripts, including percentage of transcript overlap
33. TRANSCRIPT_OVERLAP_PERCENT	Percentage overlap between copy number segment and transcript
34. ACTIONABILITY_TIER	Variant actionability tier - AMP/ASCO/CAP
35. ACTIONABILITY	Variant clinical actionability significance - AMP/ASCO/CAP
36. BIOMARKER_MATCH	Biomarker match
37. TARGETED_INHIBITORS_ALL2	Molecularly targeted inhibitors - indicated for any tumor type

RNA fusions

Tab-separated values (TSV)

Detected RNA fusions are annotated with gene-level cancer relevance, Mitelman database evidence, and clinical actionability. The output file has the following naming convention:

• <sample_id>.pcgr.<genome_assembly>.fusion_ann.tsv.gz

Columns suffixed with _OKB are only present when OncoKB annotation is enabled via --oncokb_api_token. Note that HOTSPOT_OKB and VUS_OKB are not included for fusion output.

Variable	Description
1. SAMPLE_ID	Sample identifier
2. VARIANT_CLASS	Variant type: fusion
3. VAR_ID	Variant identifier. Format: <gene1>--<gene2>:<breakpoint_5P>:<breakpoint_3P>
4. ENTREZGENE	Entrez gene identifiers for both fusion partners. Format: <entrezgene_5P>::<entrezgene_3P>
5. FUSION_GENE	Fusion gene name. Format: <gene1>--<gene2>
6. FUSION_GENE2	Alternative fusion gene notation used for Mitelman database variant matching
7. SPLIT_READS	Number of split reads supporting the fusion
8. FUSION_GENE_5P	Gene symbol of the 5’ fusion partner
9. FUSION_GENE_3P	Gene symbol of the 3’ fusion partner
10. BREAKPOINT_5P	Chromosomal breakpoint position of the 5’ partner
11. BREAKPOINT_3P	Chromosomal breakpoint position of the 3’ partner
12. GENENAME_5P	Full gene name of the 5’ partner
13. ONCOGENE_5P	Flag indicating if the 5’ partner gene is an oncogene (CancerMine/NCG)
14. ENSEMBL_TRANSCRIPT_ID_5P	Ensembl transcript identifier covering the 5’ breakpoint
15. GENENAME_3P	Full gene name of the 3’ partner
16. ONCOGENE_3P	Flag indicating if the 3’ partner gene is an oncogene (CancerMine/NCG)
17. ENSEMBL_TRANSCRIPT_ID_3P	Ensembl transcript identifier covering the 3’ breakpoint
18. SAMPLE_ALTERATION	Alteration description for the sample. Format: <FUSION_GENE> fusion
19. MITDB_NUM_EVIDENCE	Number of evidence records matching the fusion in the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer
20. MITDB_EVIDENCE	Evidence records for the fusion from the Mitelman database (cancer type associations)
21. ACTIONABILITY_TIER	Variant clinical actionability tier - AMP/ASCO/CAP implementation
22. ACTIONABILITY	Variant clinical actionability significance - AMP/ASCO/CAP implementation
23. MUTATION_EFFECT_OKB	(OncoKB only) OncoKB mutation effect annotation (e.g. Loss-of-function, Gain-of-function)
24. MUTATION_EFFECT_CITATIONS_OKB	(OncoKB only) OncoKB mutation effect citations
25. MUTATION_EFFECT_DESCRIPTION_OKB	(OncoKB only) OncoKB mutation effect description
26. ONCOGENICITY_OKB	(OncoKB only) OncoKB oncogenicity annotation (e.g. Oncogenic, Likely Oncogenic)
27. TUMOR_TYPE_SUMMARY_OKB	(OncoKB only) OncoKB tumor type summary
28. VARIANT_SUMMARY_OKB	(OncoKB only) OncoKB variant summary

Gene expression data

If users provide bulk RNA-seq expression data as input, PCGR will attach basic gene annotations for the affected transcripts, and perform similarity analysis and outlier detection if configured by the user. The naming convention of the compressed TSV files are as follows:

Expression data - basic

• Filename: <sample_id>.pcgr.<genome_assembly>.expression.tsv.gz

One row per affected transcript. Contains TPM expression values with basic gene/transcript annotations.

Variable	Description
1. SAMPLE_ID	Sample identifier
2. ID	Input transcript/gene identifier as provided by the user
3. ID_TYPE	Type of identifier (e.g. ensembl_transcript_id)
4. AMBIGUOUS_ID	Flag indicating whether the identifier maps to multiple genes
5. TPM	Expression level in transcripts per million (TPM)
6. ENSEMBL_TRANSCRIPT_ID	Ensembl transcript identifier
7. ENSEMBL_GENE_ID	Ensembl gene identifier
8. SYMBOL	Gene symbol
9. ENTREZGENE	Entrez gene identifier
10. GENENAME	Full gene name
11. BIOTYPE	Transcript biotype (e.g. protein_coding, lncRNA)

Expression outliers

• Filename: <sample_id>.pcgr.<genome_assembly>.expression_outliers.tsv.gz

One row per gene. Contains outlier statistics comparing the sample’s gene-level expression against a tumor-type-matched reference cohort.

Variable	Description
1. SAMPLE_ID	Sample identifier
2. REF_COHORT	Reference cohort used for comparison (e.g. tcga_skcm)
3. REF_COHORT_SIZE	Number of samples in the reference cohort
4. ENSEMBL_GENE_ID	Ensembl gene identifier
5. TPM_LOG2_GENE	Log2-transformed gene-level TPM for the query sample
6. MEAN	Mean log2 TPM across the reference cohort
7. STD	Standard deviation of log2 TPM across the reference cohort
8. Z_SCORE	Z-score of the query sample relative to the reference cohort
9. Q1	First quartile (25th percentile) of log2 TPM in the reference cohort
10. Q2	Median (50th percentile) of log2 TPM in the reference cohort
11. Q3	Third quartile (75th percentile) of log2 TPM in the reference cohort
12. IQR	Interquartile range (Q3 − Q1) in the reference cohort
13. PERCENTILE	Percentile rank of the query sample within the reference cohort

Sample similarity analysis

• Filename: <sample_id>.pcgr.<genome_assembly>.expression_similarity.tsv.gz

One row per external reference sample. Contains transcriptome-wide (protein-coding only) correlation scores between the query sample and samples from reference expression cohorts.

Variable	Description
1. SAMPLE_ID	Sample identifier
2. EXT_SAMPLE_ID	Identifier of the external reference sample
3. EXT_DB	Source database of the external reference sample (e.g. tcga)
4. CORR	Pearson correlation coefficient between the query sample and the external reference sample
5. PROTEIN_CODING_ONLY	Flag indicating whether the correlation was computed using protein-coding genes only
6. EXT_SAMPLE_ID2	Secondary identifier for the external reference sample
7. EXT_SAMPLE_NAME	Display name of the external reference sample
8. EXT_SAMPLE_TYPE	Sample type of the external reference sample (e.g. tumor subtype)
9. EXT_AGE_AT_DX	Age at diagnosis of the external reference sample donor
10. EXT_AGE_CATEGORY	Age category of the external reference sample donor
11. EXT_GENDER	Gender of the external reference sample donor
12. EXT_LINK	URL link to the external reference sample record
13. EXT_PRIMARY_DIAGNOSIS	Primary diagnosis of the external reference sample
14. EXT_PRIMARY_SITE	Primary tumor site of the external reference sample

Excel workbook (XLSX)

The Excel workbook has the following naming convention:

• <sample_id>.pcgr.<genome_assembly>.xlsx

It contains up to 16 sheets, each populated conditionally on the type of input data provided and the analysis performed. Sheets with no data are omitted entirely. Any sheet exceeding 30,000 rows is truncated to the first 30,000 rows (a warning is emitted in that case; the full data is always available in the corresponding TSV file).

The table below describes each sheet, when it is included, and any filtering or content restrictions applied to keep the workbook manageable.

Sheet	Included when	Content and filtering
SETTINGS	Always	Key parameters used for the analysis, including filtering thresholds
DATA_VERSIONS	Always	Version information for all databases used in the annotation and analysis
SAMPLE_ASSAY	Always	Sample and assay properties: tumor site, sex, purity, ploidy, assay type (WGS/WES/TARGETED), tumor-normal/tumor-only mode, and effective target size
SOMATIC_SNV_INDEL	--input_vcf provided	Somatic SNV/InDel annotations using the same columns as the TSV output, minus BIOMARKER_MATCH and VEP_ALL_CSQ. Filtered to exonic variants (EXONIC_STATUS == "exonic") plus any non-exonic variant with an actionability tier ≤ 3. User-retained VCF INFO tags (--retained_info_tags) are appended as additional columns. HGVSP is renamed to HGVSp_short to avoid column name conflicts
SOMATIC_SNV_INDEL_BIOMARKER	--input_vcf provided and biomarker hits exist	One row per biomarker evidence item matched to a somatic SNV/InDel. A TIER column encodes evidence category and actionability tier (e.g. T1/T2/T3 = therapeutic sensitivity, R1/R2/R3 = therapeutic resistance, PP1–PP2 = poor prognosis, PB1–PB2 = better prognosis, D1–D2 = diagnostic positive). Diagnostic-negative evidence is excluded
SOMATIC_CNA	--input_cna provided	CNA gene-level annotations using the same columns as the TSV output, minus BIOMARKER_MATCH. Filtered to genes with a non-missing ACTIONABILITY_TIER
SOMATIC_CNA_BIOMARKER	--input_cna provided and biomarker hits exist	One row per biomarker evidence item matched to a somatic CNA, using the same TIER encoding and sorting as SOMATIC_SNV_INDEL_BIOMARKER
RNA_FUSION	--input_rna_fusion provided	RNA fusion annotations using the same columns as the TSV output, minus BIOMARKER_MATCH. Filtered to fusions with a non-missing ACTIONABILITY_TIER
RNA_FUSION_BIOMARKER	--input_rna_fusion provided and biomarker hits exist	One row per biomarker evidence item matched to an RNA fusion, using the same TIER encoding and sorting as SOMATIC_SNV_INDEL_BIOMARKER
TMB	--input_vcf provided	Tumor mutational burden estimates (same content as the .tmb.tsv file)
MSI	--input_vcf provided and MSI estimation enabled	MSI classifier statistics and predicted stability status
MUTATIONAL_SIGNATURE	--input_vcf provided and signature estimation enabled	Mutational signature contributions (same content as the .msigs.tsv.gz file)
KATAEGIS_EVENTS	--input_vcf provided and kataegis events detected	Kataegis loci identified from the somatic mutation profile
RNA_EXPRESSION_OUTLIERS	--input_rna_expression provided and outlier detection enabled	Expression outlier genes/transcripts compared to reference cohorts. A subset of display-only columns (GENENAME, CANCERGENE_EVIDENCE, TARGETED_INHIBITORS_ALL, ENSEMBL_GENE_ID) are removed to reduce width
RNA_IMMUNE_CONTEXTURE	--input_rna_expression provided	Immune cell type deconvolution results from bulk RNA-seq (quanTIseq)

Biomarker evidence in the HTML report vs. the Excel workbook

The _BIOMARKER sheets are more comprehensive than what is displayed in the HTML report. The HTML report shows only the tier-defining evidence items — those that directly determined the variant’s actionability tier assignment. The Excel _BIOMARKER sheets include all matched evidence items, each annotated with a BM_ACTIONABILITY_SUPPORT column that records its relationship to the tier assignment.

The possible values of BM_ACTIONABILITY_SUPPORT are:

• tier-defining — the evidence item was the primary basis for the variant’s actionability tier. Only these items appear in the HTML report.
• additional — the evidence item is relevant (correct evidence type, overlapping molecular profile) but was not sufficient on its own to define the tier, either because the tumor type does not match the query or the evidence is weaker than what the tier requires.