- Pair ID - DNA sample ID (if DNA is run) - RNA sample ID (if RNA is run)

- Library Prep Kit - Output date - Output time - Module version - Pipeline version (Docker image version #)

- Run name - Run date - DNA sample index ID (if DNA is run) - RNA sample index ID (if RNA is run) - [HRD] Sample feature - Instrument ID - Instrument control software version - Instrument type

- RTA version - Reagent cartridge lot number

LowQ

Splice variant score < passing quality score threshold value of 1.

PASS

Splice variant score ≥ passing quality score threshold value of 1.

LowUniqueAlignments

All splice junction supporting reads map to a unique genomic interval near at least one of the two splice sites.

DOUBLE_BROKEN_EXON

Confidence filter

If both breakpoints are distant from annotated exon boundaries, the number of supporting reads do not satisfy a high threshold requirement (≥ 10 supporting reads).

LOW_MAPQ

Confidence filter

All fusion supporting read alignments at either of the breakpoints have MAPQ < 20.

LOW_UNIQUE_ALIGNMENTS

Confidence filter

All fusion supporting read alignments map to a unique genomic interval at either of the breakpoints.

LOW_SCORE

Confidence filter

The fusion candidate has probabilistic score as determined by the features of the candidate.

MIN_SUPPORT

Confidence filter

The fusion candidate has very few fusion supporting reads (< 5 supporting read pairs).

READ_THROUGH

Confidence filter

The breakpoints are cis neighbors (< 200 kbp) on the reference genome.

ANCHOR_SUPPORT

Information only

Read alignments of fusion supporting reads are not long enough (12 bp) at either of the two breakpoints.

HOMOLOGOUS

Information only

The candidate is likely a false candidate generated because the two genes involved have high gene homology.

LOW_ALT_TO_REF

Information only

The number of fusion supporting reads is < 1% of the number of reads supporting the reference transcript at either of the two breakpoints.

LOW_GENE_COVERAGE

Information only

Each breakpoint in an enriched gene has fewer than 125 bp with nonzero read coverage.

NO_COMPLETE_SPLIT_READS

Confidence filter

For every fusion-supporting split read, the total number of aligned bases across two breakpoints is less 60% of the read length.

UNENRICHED_GENE

Confidence filter

Neither of the two parent genes is in the enrichment panel.

Gene A

The gene associated with the A side of the fusion. A semicolon delimited list is used for multiple genes.

Gene B

The gene associated with the B side of the fusion. A semicolon delimited list is used for multiple genes.

Gene A Breakpoint

[Information only] The chromosome and offset of the Gene A side of the fusion.

Gene A Location

Location of the breakpoint within Gene A: - IntactExon—Matches exon boundary - BrokenExon—Inside an exon - Intronic—Within an intron - Intergenic—No gene overlap (currently excluded) If multiple genes are in Gene A, then semicolon separated list of locations. This column is used internally to identify genes to report when a breakpoint occurs in a region overlapping multiple genes. Occasionally, additional values are listed for genes that were excluded from the GeneA list.

Gene A Sense

Boolean indicating whether left/right breakpoint order suggests fusion transcript is in the same sense of Gene A. If multiple genes are in Gene A, then semicolon separated list of bools.

Gene A Strand

Strand of Gene A, + for forward, - for reverse.

Gene B Breakpoint

[Information only] The chromosome and offset of the Gene B side of the fusion.

Gene B Location

Location of the breakpoint within Gene B: - IntactExon—Matches exon boundary - BrokenExon—Inside an exon - Intronic—Within an intron - Intergenic—No gene overlap (currently excluded) If multiple genes in Gene B, then semicolon separated list of locations. This column is used internally to identify genes to report when a breakpoint occurs in a region overlapping multiple genes. Occasionally, additional values are listed for genes that were excluded from the GeneB list.

Gene B Sense

Boolean indicating whether left/right breakpoint order suggests fusion transcript is in the same sense of Gene B. If multiple genes are in Gene B, then semicolon separated list of bools.

Gene B Strand

Strand of Gene B, + for forward, - for reverse.

Score

The quality of fusion as determined by DRAGEN server.

Filter

The filter associated with the fusion as determined by the respective caller. Results from different callers are not equivalent.

Ref A Dedup

Gene A uniquely mapping reads paired across or split by the junction. Does not support fusion. Duplicate reads are not included.

Ref B Dedup

Gene B uniquely mapping reads paired across or split by the junction. Does not support fusion. Duplicate reads are not included.

Alt Split Dedup

Uniquely mapping reads split by the junction. Supports fusion. Duplicate reads are not included.

Alt Pair Dedup

Uniquely mapping reads paired across junction. Supports fusion. Duplicate reads are not included.

KeepFusion

The determination whether the fusion should be kept or dropped from the list of fusions.

Fusion Directionality Known

Whether fusion directionality is known and indicated by gene order.

TOTAL_PF_READS (count)

Total number of non-supplementary, non-secondary, and passing QC reads after alignment to the whole genome sequence.

Primarily driven by data output of sequencer, quality of library and balancing of library in library pool. If TOTAL_PF_READS is in line with other samples, but coverage metrics are more may suggest non-specific enrichment.

Low values for all samples indicate a poor quality run with possible low cluster numbers or low numbers of Q30 and PF%.

A low value for an individual sample indicates poor pooling of this library into the final pool.

MEAN_FAMILY_SIZE (count)

A UMI Family is a group of reads that all have the same UMI barcode. The family size is the number of reads in family. MEAN_FAMILY_SIZE is the mean of the entire population of reads assembled into UMI families.

The mean UMI family size decreases with increased unique read numbers, and more input DNA leads to more unique reads. Conversely over sequencing of a fixed population of unique DNA molecules leads to increased family size.

As a guide, for a good run with optimal cluster density, passing specs, even sample pooling, and good quality DNA we usually observe values <10.

UMI family size = 1 is not ideal as it is harder to correct for errors.

UMI family size of 2 to 5 enables efficient error correction without wasting sequencing capacity on high percentages of duplicate reads.

MEDIAN_TARGET_COVERAGE (count)

Median depth across all the unique loci occurring in all regions of the manifest file.

Lower median target coverage may be due to poor sample input/quality, library preparation issues or low sequencing output.

PCT_CHIMERIC_READS (%)

Chimeric reads occur when one sequencing read aligns to two distinct portions of the genome with little or no overlap. Metric is proportion of total number of non-supplementary, non-secondary, and passing QC reads after alignment to the whole genome sequence.

While this can be indicative of large-scale structural rearrangement of the genome, values that are elevated above the usual baseline may indicate enrichment probe contamination during library preparation. A suggested metric USL is 8% (those that are higher might see decrease performance in small variant and tmb scores).

PCT_EXON_100X (%)

Percentage of exon bases with 100X fragment coverage. Calculated against all regions in manifest containing _exon in name.

Can be used in combination with other PCT_EXON metrics to understand under or over coverage of exons.

PCT_READ_ENRICHMENT (%)

Percentage of reads that have overlapping sequence with the target regions defined in the sample manifest.

Indicative of general enrichment performance. Reduced proportions of enriched reads may indicate issues with the enrichment proportion of the library preparation.

PCT_USABLE_UMI_READS (%)

Percentage of reads that have valid UMI sequences associated with them.

As UMI reads are sequenced at the start of each read, loss of valid UMI sequence may be cause by sequencing issues impacting the quality of base calling in this portion of the sequencing read.

MEAN_TARGET_COVERAGE (count)

Mean depth across all the unique loci defined in the manifest file.

Lower mean target coverage may be due to poor sample input/quality, library preparation issues or low sequencing output. Large differences between the median and mean target coverage values may indicated a skewed distribution of target coverage.

PCT_ALIGNED_READS (%)

Proportion of aligned reads that are non-supplementary, non-secondary and pass QC versus aligned reads that are non-supplementary, non-secondary, mapped and pass QC.

PCT_CONTAMINATION_EST (%)

This metric should only be evaluated if the CONTAMINATION_SCORE metric exceed the USL. This metric estimates the amount of contamination in a sample. The contamination level is computed by taking 2.0* the average of the adjusted allele frequencies of all variants that were selected. The adjusted alllele frequency is either the actual allele frequency of the variant if it is less than 0.5, or 1 -allele frequency if it is greater than or equal to 0.5.

If the sample does not fail the CONTAMINATION_SCORE this metric has no intended meaning as it will be driven by statistical noise (e.g. the few variants that naturally fall outside an expected interval around 0.5 due to random chance)

High contamination estimates may be due to any of the following:

Inter-sample contamination caused by mixing of samples during extraction or library preparation.

Intra-sample contamination, due to mixing of clonally different cell populations during extraction. Large scale genomic rearrangements that cause unexpected VAFs for large numbers of variants.

PCT_TARGET_0.4X_MEAN (%)

Parentage of target (all locations in manifest) reads that have a coverage depth of greater the 0.4x the mean target coverage depth (see definition above).

Provides an indication of uniformity of coverage of the target regions in the manifest file. When trended over time reductions in this metric may indicate an issue with the enrichment process resulting in coverage bias.

PCT_TARGET_50X (%)

Percentage of target bases with 50X fragment coverage. Calculated against all regions in manifest file.

Can be used in combination with other PCT_TARGET metrics to understand under or over coverage of targets.

PCT_TARGET_100X (%)

Percentage of target bases with 100X fragment coverage. Calculated against all regions in manifest file.

Can be used in combination with other PCT_TARGET metrics to understand under or over coverage of targets.

PCT_TARGET_250X (%)

Percentage of target bases with 250X fragment coverage. Calculated against all regions in manifest file.

Can be used in combination with other PCT_TARGET metrics to understand under or over coverage of targets.

PCT_SOFT_CLIPPED_BASES (%)

percentage of based that were not used for alignment but retained as part of the alignment file

Soft clipped reads are used as a part of the downstream analysis for small variants calling. A higher-than-expected number could indicate a low-quality enrichment step.

PCT_Q30_BASES (%)

Average percentage of bases ≥ Q30. A prediction of the probability of an incorrect base call (Q‑score).

An indicator of sequencing run quality, low Q30 across all samples on a run could be the result of run overclustering.

ALLELE DOSAGE_RATIO (with HRD add-on)

Proprietary Myriad Genetics estimate of b-allele dosage based on b-allele noise/signal ratio. B-Allele noise is correlated with coverage; lower coverage samples will have higher noise. B-allele signal is also correlated with tumor fraction; a higher tumor fraction produces a higher signal for b-allele sites. Samples with lower tumor fraction and higher amount of noise (or lower coverage) will have higher Allele Dosage Ratio. The upper limit of the score is 50, therefore any sample with 50 Allele Dosage Ratio can be assumed to have tumor fraction close to zero and typically has a GIS = 0.

MEDIAN TARGET HRD (with HRD add-on)

Median target fragment coverage across all target positions in the genome. Coverage is the total number of non-duplicate pair alignments that overlap.

GIS Score*

Proprietary Genomic Instability Score (GIS) indicating level of genomic instability in sample genome. Combination of Loss of Heterozygosity (LOH), Telomeric allelic imbalance and Large-scale State Transitions (LST) scores. The GIS scores provided by TruSight Oncology 500 HRD show good correlation (R2= 0.98) with Myriad Genetics GIS however they are not identical (Refer to TruSight Oncology 500 HRD Product Data Sheet Doc# M-GL-00748 for more details). GIS from alternative HRD assays should be not be considered equivalent to Illumina/Myriad GIS.

PCT_TARGET_HRD_50X

Percent of HRD probe SNP panel covered by at least 50X coverage

DNA Library QC Metrics for GIS

EXCESSIVE_TF

EXCESSIVE TF indicates if there is excessive tumor content in sample. Troubleshooting: Samples with pure tumor fraction >90% are outside the design for GIS estimation (this includes pure tumor cell lines)

DNA Library QC Metrics for GIS

ALLELE_DOSAGE_RATIO

Proprietary Myriad Genetics estimate of b-allele dosage based on b-allele noise/signal ratio. B-Allele noise is correlated with coverage; lower coverage samples will have higher noise. B-allele signal is also correlated with tumor fraction; a higher tumor fraction produces a higher signal for b-allele sites. Samples with lower tumor fraction and higher amount of noise (or lower coverage) will have higher Allele Dosage Ratio. The upper limit of the score is 50, therefore any sample with 50 Allele Dosage Ratio can be assumed to have tumor fraction close to zero and typically has a GIS = 0.

DNA Expanded Metrics

MEDIAN_TARGET_HRD_COVERAGE

Median target fragment coverage across all target positions in the genome. Coverage is the total number of non-duplicate pair alignments that overlap.

DNA Expanded Metrics

PCT_CHIMERIC_READS

Percentage of reads that are aligned as two segments which map to nonconsecutive regions in the genome.

%

PCT_ON_TARGET_READS

Percentage of reads that cross any part of the target region versus total reads. A read that partially maps to a target region is counted as on target.

%

SCALED_MEDIAN_GENE_COVERAGE

Median of median base coverage of genes scaled by length. An indication of median coverage depth of genes in the panel.

Count

TOTAL_PF_READS

Total number of reads passing filter.

Count

GENE_MEDIAN_COVERAGE

The median coverage depth of all genes in the panel.

Count

GENE_ABOVE_MEDIAN_CUTOFF

Number of genes above the median coverage cutoff.

Count

PER_GENE_MEDIAN_COVERAGE

Median deduped coverage across each gene (available in Logs_Intermediates only)

Count

PCT_SOFT_CLIPPED_BASES

percentage of based that were not used for alignment but retained as part of the alignment file

%

RNA_PCT_030_BASES

Average percentage of bases ≥ Q30. A prediction of the probability of an incorrect base call (Q‑score). Troubleshooting: An indicator of sequencing run quality, low Q30 across all samples on a run could be the result of run overclustering.

%

PASS

PASS variants.

base_quality

Site filtered because median base quality of alt reads at this locus does not meet threshold.

filtered_reads

Site filtered because the fraction of reads is too large.

fragment_length

Site filtered because absolute difference between the median fragment length of alt reads and median fragment length of ref reads at this locus exceeds threshold.

low_depth

Site filtered because the read depth is too low.

low_frac_info_reads

Site filtered because the fraction of informative reads is below threshold.

long_indel

Site filtered because the indel length is too long.

mapping_quality

Site filtered because median mapping quality of alt reads at this locus does not meet threshold.

multiallelic

Site filtered because more than two alt alleles pass tumor LOD.

no_reliable_supporting_read

Site filtered because no reliable supporting somatic read exists.

read_position

Site filtered because median of distances between start/end of read and this locus is below threshold.

str_contraction

Site filtered due to suspected PCR error where the alt allele is one repeat unit less than the reference.

too_few_supporting_reads

Site filtered because there are too few supporting reads in the tumor sample.

weak_evidence

Somatic variant score (SQ) does not meet threshold.

systematic_noise

Site filtered based on evidence of systematic noise in normal sample.

excluded_regions

Site overlaps with VC excluded regions bed.

Chromosome

Chromosome

Position

Position of variant

RefCall

Reference base

AltCall

Alternate base

VAF

Variant allele frequency

Depth

Coverage of position

CytoBand

Cytoband of variant

GeneName

Name of gene if applicable. A semicolon delimited list is used for multiple genes.

VariantType

Type of the variant: SNV, insertion, deletion, MNV

CosmicIDs

Cosmic IDs, if multiple concatenated by “;”

MaxCosmicCount

Maximum Cosmic study count

AlleleCountsGnomadExome

Variant allele count in gnomAD exome database

AlleleCountsGnomadGenome

Variant allele count in gnomAD genome database

AlleleCounts1000Genomes

Variant allele count in 1000 genomes database

MaxDatabaseAlleleCounts

Maximum variant allele count over the three databases

GermlineFilterDatabase

TRUE if variant was filtered by the database filter

GermlineFilterProxi

TRUE if variant was filtered by the proxi filter

CodingVariant

TRUE if variant is in the coding region

Nonsynonymous

TRUE if variant has any transcript annotations with nonsynonymous consequences

IncludedinTMBNumberator

TRUE if variant is used in the TMB calculation