Invitae Common Hereditary Cancers Panel

{0}------------------------------------------------ Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue. # EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR Invitae Common Hereditary Cancers Panel DECISION SUMMARY #### I Background Information: - A De Novo Number DEN210011 ## B Applicant Invitae Corporation ### C Proprietary and Established Names Invitae Common Hereditary Cancers Panel ### D Regulatory Information | Product<br>Code(s) | Classification | Regulation<br>Section | Panel | |--------------------|----------------|-------------------------------------------------------------------------------------------------------------------------|-----------| | QVU | Class II | 21 CFR 866.6095— High<br>throughput DNA<br>sequencing for hereditary<br>cancer predisposition<br>assessment test system | Pathology | #### II Submission/Device Overview: # A Purpose for Submission: De Novo request for evaluation of automatic class III designation for the Invitae Common Hereditary Cancers Panel ### B Measurand: Germline substitutions, small insertion and deletion alterations and copy number variants (CNV) in a panel of 47 targeted genes # C Type of Test: Next generation sequencing based cancer-related germline mutation profiling {1}------------------------------------------------ #### Indications for Use: III ## A Indication(s) for Use: The Invitae Common Hereditary Cancers Panel is a qualitative high-throughput sequencing based in vitro diagnostic test system intended for analysis of germline human genomic DNA extracted from whole blood for detection of substitutions, small insertion and deletion alterations and copy number variants (CNV) in a panel of targeted genes. This test system is intended to provide information for use by qualified health care professionals, in accordance with professional guidelines, for hereditary cancer predisposition assessment and to aid in identifying hereditary genetic variants potentially associated with a diagnosed cancer. The test is not intended for cancer screening or prenatal testing. Results are intended to be interpreted within the context of additional laboratory results, family history, and clinical findings. The test is a single-site assay performed at Invitae Corporation. ### B Special Conditions for Use Statement(s): For Prescription Use Only For in vitro diagnostic use ### C Special Instrument Requirements: Illumina NovaSeq 6000 system (qualified by Invitae) #### Device/System Characteristics: IV #### A Device Description: The Invitae Common Hereditary Cancers Panel uses hybridization-based capture, nextgeneration sequencing (NGS), and a custom-built bioinformatics pipeline to compare all positions in targeted regions of 47 genes to a reference sequence and identify variants, including single nucleotide variants (SNVs), insertions and deletions (Indels), and copy number variants (CNVs). Sequence analysis covers clinically important regions of each gene, including coding exons and 10 to 20 base pairs of adjacent intronic sequence on either side of the coding exons in the transcript listed in Table 1. Genes of "high clinical significance" are defined as those for which the test result(s) may lead to prophylactic screening, confirmatory procedures, or treatment that may incur morbidity or mortality to the patient and are shown in bold text. In addition, the {2}------------------------------------------------ analysis covers the select non-coding variants specifically defined in the table. Any variants that fall outside these regions are not analyzed. | Gene* | SNV/Indel<br>Analysis | CNV<br>Analysis | Notes | |---------|-----------------------|-----------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| | APC | YES | YES | The 1B promoter region is covered by both SNV/Indel and CNV<br>analysis. The 1A promoter region is covered by CNV analysis.<br>SNV/Indel analysis for exon 5 is limited to cds +/-10 bp. | | ATM | YES | YES | SNV/Indel analysis for exons 6, 24, 43 includes only cds +/- 10 bp. | | AXIN2 | YES | YES | | | BARDI | YES | YES | | | BMPR1A | YES | YES | CNV analysis covers the promoter region. | | BRCA1 | YES | YES | SNV/Indel analysis includes +/- 20 base pairs of adjacent intronic<br>sequence. | | BRCA2 | YES | YES | SNV/Indel analysis includes +/- 20 base pairs of adjacent intronic<br>sequence. | | BRIP1 | YES | YES | | | CDH1 | YES | YES | | | CDK4 | YES | YES | | | CDKN2A | YES | YES | | | CHEK2 | YES | YES | | | CTNNA1 | YES | YES | | | DICER1 | YES | YES | SNV/Indel analysis for exon 22 includes only cds +/- 10 bp. | | EPCAM | NO | YES | SNV/Indel analysis is not offered for this gene (CNV analysis only). | | GREMI | NO | YES | Promoter region duplication testing only. | | HOXB13 | YES | YES | | | KIT | YES | YES | | | MENI | YES | YES | SNV/Indel analysis for exon 2 is limited to cds +/-10 bp. | | MLH1 | YES | YES | CNV analysis covers the promoter region. SNV/Indel analysis for<br>exon 12 is limited to cds +/-10 bp. | | MSH2 | YES | YES | Analysis includes the exon 1-7 inversion (Boland mutation).<br>SNV/Indel analysis for exons 2, 5 includes only cds +/- 10 bp. | | MSH3 | YES | YES | SNV/Indel analysis of the repeat region of exon 1 (5:79950697-<br>79950765) is not offered | | Gene* | SNV/Indel<br>Analysis | CNV<br>Analysis | Notes | | MSH6 | YES | YES | SNV/Indel analysis for exons 7, 10 includes only cds +/- 10 bp. | | MUTYH | YES | YES | | | NBN | YES | YES | | | NF1 | YES | YES | SNV/Indel analysis for exons 2, 7, 25, 41, 48 includes only cds +/-<br>10 bp. | | NTHLI | YES | YES | | | PALB2 | YES | YES | | | PDGFRA | YES | YES | | | PMS2 | YES | YES | SNV/Indel analysis for exon 7 includes only cds +/- 10 bp. | | POLDI | YES | YES | SNV/Indel analysis for exon 22 includes only cds +/- 10 bp. | | POLE | YES | YES | | | PTEN | YES | NO | CNV analysis is not offered for this gene. SNV/Indel analysis for<br>exons 8 includes only cds +/- 10 bp. | | RAD50 | YES | YES | | | RAD51C | YES | YES | | | RAD51D | YES | YES | | | SDHA | YES | NO | CNV analysis is not offered for this gene. SNV/Indel analysis is not<br>offered for exon 14. SNV/Indel analysis for exons 6-8 includes only<br>cds +/- 10 bp. | | SDHB | YES | YES | | | SDHC | YES | NO | CNV analysis is not offered for this gene. SNV/Indel analysis for<br>exons 2, 6 includes only cds +/- 10 bp. | | SDHD | YES | YES | | | SMAD4 | YES | YES | | | SMARCA4 | YES | YES | | | STK11 | YES | YES | | | TP53 | YES | YES | CNV analysis covers the promoter region. | | TSC1 | YES | YES | SNV/Indel analysis for exon 21 includes only cds +/- 10 bp. | | TSC2 | YES | YES | | | VHL | YES | YES | | Table 1. Variant Type Reporting per Gene {3}------------------------------------------------ {4}------------------------------------------------ *Genes of high clinical significance are defined as those for which the test result(s) may lead to prophylactic screening, confirmatory procedures or treatment that may incur mortality to the patient and are shown in bold text. Identified variants are assessed by clinical professionals using currently available literature and data from public genetic variant databases. Variants are assigned a score, calculated according to an algorithm that weights the available clinical evidence. Possible outcomes include the following, which are based on joint ACMG/AMP Committee guidelines: - . Benign (not reported) - strong evidence of not being disease or risk causing - . Likely benign (not reported) - some evidence of not being disease or risk causing - . Likely pathogenic – some evidence in favor of causing disease or increasing risk - . Pathogenic - strong evidence of causing disease or increasing disease risk - Variant of Uncertain Significance insufficient information to classify in one of the other . four categories Variants are reported using HGVS nomenclature and the human reference genome GRCh37. # B Principle of Operation ## Materials and Equipment The reagents, consumables, and instruments needed to perform the Invitae Common Hereditary Cancers Panel test are used exclusively at, and qualified by the Invitae Corporation clinical laboratory (1400 16th Street, San Francisco, CA 94103). Instrumentation includes integrated, automated systems that incorporate liquid handling instruments, incubators, thermal cvclers, sonicators, and centrifuges, as well as Illumina NovaSeq 6000 sequencers. Reagents include extraction reagents, custom-made molecular barcode (TAFT) plates, buffers, PCR Cleanup Mag Beads, ER/AT Mix, Ligase Mix, PCR Master Mix, custom baits and blockers, PCR primer mix, streptavidin beads, and sequencing reagent kits. ### Specimen Collection and Preparation The test includes a specimen collection kit, which is sent to ordering physicians. The shipping kit contains the following components: - Plastic whole blood collection tube with K2EDTA . - Patient information card with IB code sticker - Test requisition form - Biohazard bag and absorbent pouch, for submission of specimen - . Handling instructions for shipping the specimen The healthcare provider is directed to collect a 3 mL whole blood specimen, label the tube in the space provided with the patient's full name and at least one additional unique identifier, then ship the specimen to the Sponsor's laboratory at room temperature. When the specimen is received by the Sponsor, the accessioning operators inspect it for the following acceptance criteria: - Minimum testing volume of 1.5 mL ● - No evidence of clotting ● {5}------------------------------------------------ - No evidence of leaks ● - Specimen collection date <90 days from receipt of the kit - Paperwork matching tube label ● Samples are processed as soon as they are received. ## DNA Extraction, Normalization and Shearing Genomic DNA is extracted from whole blood specimens per protocol using automated extraction and extraction kit qualified by Invitae. Unique molecular tags (TAFTs, Tagged Amplicons For Tracking) are appended to a region of non-clinical significance and processed with the samples from the time of extraction throughout the wet lab process. This allows sample identification to be maintained when samples are processed multiplexed. Extracted DNA is quantified and normalized to 23ng/uL to create batches for assay processing. The DNA is then sonically sheared into consistently sized pieces. Following DNA shearing, the DNA goes through a series of small enzyme-mediated steps that repair the sheared ends of the DNA sample and attach specific oligonucleotides to the ends of the sample. These oligonucleotides are specific for primers that are used in subsequent steps to anneal to them. This includes end-repair, A-tailing, adapter ligation, and dual-indexing of each sample. ## DNA Processing and Library Preparation The specific fragments that contain targeted regions for the assay (i.e., the exonic regions for all 47 genes that are sequenced in this test) can be isolated from the remaining gDNA in the sample. Library production is fully automated end-to-end with integrated robotic systems; the system can create approximately 1,600 libraries every 8 hours with no human intervention. Libraries are pooled by row to create 8 pools of 12 libraries each. # Hybridization Following library preparation, the barcoded fragments are mixed, and the relevant DNA segments annealed to biotinylated probes for magnetic bead capture. The probes hybridize to the patient DNA samples and these fragments are separated from the rest of the genomic DNA using a magnet that interacts with ferromagnetic beads attached to avidin bound to biotin, which enables all non-bound DNA to be washed away. The beads are manufactured by a third-party vendor per the Sponsor's specification. Both ends of the fragments are sequenced, the sequence of the links is deconvoluted using the molecular barcode to identify which sample each fragment was derived from, and the sequence is aligned to the reference. ### Sequencing The system uses Illumina's NovaSeq, a high throughput sequencing system that employs Sequencing-by-Synthesis chemistry, to perform paired-end reads of 150 bases in length and dual-index barcode reads of 8-nucleotides on the adapters. Equimolar mixing of hybridization reactions forms sequencing pools that allow each library to be sequenced with a minimum of {6}------------------------------------------------ 4.000.000 clusters on the Illumina flow cell. The system's average read depth (i.e., coverage) is 450x; any region with coverage below 50x is flagged as "low" and manually reviewed in the bioinformatics pipeline. Any region below 20x coverage is flagged as "very low," and the sample is failed and either re-run or a new sample is requested. # Sequence Alignment/Mapping and Variant Calling Following sequencing, raw BCL files are demultiplexed, producing individual fastg files for each sample, then mapped to the reference sequence, producing BAM files. The Invitae bioinformatics software then identifies "Active Regions", i.e., regions that may contain a putative variant, and different variant types are then called by the software. # Variant Interpretation and Review Variant calls are annotated based on evidence from published literature, public databases, prediction programs, and an internal curated variants database. Regions with low read depth, split reads, or low CNV quality scores are flagged for manual review. All other variants are processed through a combination of automated processes and manual review. For all variants that have been seen previously, the most recent interpretation and associated evidence is automatically placed in the report. For novel variants, variants that have not been seen recently, and variants that have new clinical evidence available, the bioinformatics software has tools to aid the genetics professional in variant interpretation/classification. Variants are assigned one of the following classifications, which are based on joint ACMG/AMP Committee guidelines: - Pathogenic (Odds >99:1 in favor of causing disease or increasing disease risk) ● - Likely Pathogenic (Odds >9:1 but <99:1 in favor of causing disease or increasing ● risk) - Variant of Uncertain (Unknown) Significance (VUS) - Insufficient information to classify in one of the other four categories - Likely Benign (Odds >9:1 but <99:1 in favor of not being disease or risk causing) ● - Benign (Odds >99:1 in favor of not being disease or risk causing) ● Refer to Section VI. C for more information on the interpretation and curation process. # Controls - a) No Template Control (NTC): A NTC is processed as a negative control through the DNA extraction process and post-extraction DNA quantification process. NTC is checked for position and post-extraction DNA quantification, to ensure the plate is being processed in the correct orientation and that the plate is not contaminated. The NTC is not included in the test steps post DNA quantification. - b) Positive control: A reference cell line sample is processed as a positive control from DNA extraction through sequencing. The positive control is checked for quality metrics such as library concentration, sequence coverage, and gap rates. Failure of the positive control to meet the pre-defined quality metrics will result in a plate failure. {7}------------------------------------------------ ## Result Reporting The Invitae Common Hereditary Cancers Panel test results are for professional use only; the final clinical reports generated from the assay summarize the clinical findings for the ordering physician. Test reports are generated and reviewed by PhD level scientists, genetic counselors, as well as licensed, board-certified clinical molecular geneticists or licensed, board-certified molecular pathologists before signing out. # C Instrument Description Information - 1. Instrument Name: Illumina NovaSeq 6000 system (qualified by Invitae) - 2. Specimen Identification: Whole blood ## 3. Specimen Sampling and Handling: Refer to Section IV. B, Specimen Collection and Preparation - 4. Calibration: Not applicable - 5. Quality Control: Quality metrics are evaluated throughout the bench workflow, including library preparation, hybridization, and sequencing, to verify that samples have appropriate DNA concentrations at key points and that the sequencing run generates reads with adequate quality and depth. Failure to meet the prespecified quality thresholds results in manual review and possible resequencing. Post-sequencing quality metrics are assessed at the sample and batch level to determine sample and batch quality (Table 2). Samples are held for review whenever the value for a metric exceeds the thresholds in the QC Service. Batch quality is determined by reviewing the number of samples that failed for an individual given metric and a batch is held when the number of individual failures exceeds a predetermined threshold for number of failures. Samples and/or batches with holds are reviewed by trained professionals for quality according to Standard Operating Procedure (SOP) documents. {8}------------------------------------------------ | Metric | Description | Sample<br>Threshold | Batch<br>Threshold<br>(# failed<br>samples) | |----------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------|---------------------------------------------| | Mean target coverage | The mean depth of coverage (i.e., number of reads)<br>over a target region | ≥300x | ≤21 | | Very low coverage | Number of targeted bases (assay-wide) with <20x<br>depth of coverage | ≤400 | ≤42 | | Percent Selected Bases | The fraction of bases in aligned reads that map<br>directly to or within a fixed interval containing the<br>baited region. | ≥68% | ≤21 | | Pass Filter and High Quality<br>aligned read base calling error rate | The fraction of bases that mismatch the genomic<br>reference sequence in reads that pass quality filters<br>and align with mapping quality Phred score of 20<br>or higher. | ≤0.01 | ≤21 | | AT dropout (bias) | A measure of how undercovered regions with<br><50% GC content are relative to the mean. A<br>higher score implies that a higher percentage of<br>total reads that should have mapped to regions<br>with ≤50% GC content mapped elsewhere. | ≤6% | ≤10 | | GC dropout (bias) | A measure of how undercovered regions with<br>≥50% GC content are relative to the mean. A<br>higher score implies that a higher percentage of<br>total reads that should have mapped to regions<br>with ≥50% content mapped elsewhere | ≤15% | ≤21 | | Median insert size | The median size of sections of DNA being<br>sequenced | 200 to 320 bp | ≤21 | | Percent duplication | Percent of paired and unpaired reads marked as<br>duplicates | ≤0.2% | ≤21 | | Contamination estimate | A maximum likelihood estimate of the percentage<br>of the DNA in a sample that is from a foreign<br>source. | *** | *** | | Consensus variant rate | The fraction of processed loci that are classified as<br>variant loci. | 0.0006 to<br>0.0029 | ≤21 | | Consensus<br>heterozygous/homozygous ratio | The ratio of heterozygous to homozygous variant<br>loci. | 1 to 5.8 | ≤21 | | Consensus heterozygous rate | The fraction of processed loci that are classified as<br>heterozygous variant loci. | ≤0.0015 | ≤21 | | Sample Goodness-of-fit to a<br>Baseline sample set | A per-sample measure of how well the observed<br>data at each CNV target are described by the mean<br>model of predicted sample read counts as a | *** | *** | | Metric | Description | Sample<br>Threshold | Batch<br>Threshold<br>(# failed<br>samples) | | | function of the read counts for a set of baseline<br>samples. | | | | CNVitae Q-score <35 | Called copy-number error probability p-value<br>represented as a Phred-scaled quality score. | *** | *** | | TAFT (molecular barcode) -<br>minimum expected counts | The number of reads that align to the molecular<br>barcode sequence | *** | *** | | TAFT - proportion of classified | The proportion of reads that align to the correct<br>molecular barcode sequence | *** | *** | | CNVitae_XY_<br>ploidy_match | Whether the X and Y chromosome count observed<br>through sequencing match the biological sex<br>reported by the provider or patient. | *** | *** | # Table 2. Post-Sequencing Sample and Batch QC Metrics {9}------------------------------------------------ *** Pre-specified threshold values provided. Data not shown In addition, based on per variant QC metrics, any variant whose data does not reflect a minimum level of confidence that the variant was correctly called is assigned a "FILTER" flag. The criteria for SNV and Indel variants and CNVs are detailed in Table 3 and Table 4 below, respectively. | Table 3. FILTER Flag Criteria for SNVs/Indels | | | | |-----------------------------------------------|--|--|--| | Metric | Description | Acceptance<br>Criteria | |-----------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------| | QUAL (quality) score | Phred-scaled quality score measures the error<br>probability of the called variant. | < 30 | | QD (quality by depth) score if variant<br>length is ≤3 bp | The QD is the QUAL score normalized by allele<br>depth (AD) for a variant. | < 2.0 | | Allele balance if variant length is ≤7 bp | In a location where a variant has been detected, the<br>proportion of reads that indicate a variant | < 0.15 | | Repeat Unit Wobble Filter | The detected variant is in a region with a high<br>amount of variability between reads with respect to<br>the number of short tandem repeats (STRs - short<br>repeated sequences of DNA) | True | | Variant is on curated "block list" of known<br>artifacts | The detected variant has been repeatedly identified<br>in the past as being a result of the sequencing<br>process or something other than an actual variant. | True | | Variant was removed following manual<br>review of data | Used in specific cases which are selected using<br>additional criteria | True | {10}------------------------------------------------ | Metric | Description | Acceptance Criteria | |----------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------| | Excluded Target | Targets are excluded due to very low counts, as<br>well as inability to confidently call copy number<br>(at that target) in too many of the baseline<br>samples. Baseline samples are used to establish<br>what the expected profile of a sample with a<br>normal number of copies of a target region<br>looks like. | *** | | BaselineCNV | High proportion of copy number variants were<br>called in the baseline OR there was a failure to<br>confidently call copy number in the baseline at<br>this target. | *** | | CNVLowQual: Quality score | The quality score is derived from the likelihood<br>that the call is normal for copy number normal<br>events and at least part of the called segment is<br>a deletion/duplication for deletion/duplication<br>events. Failing values indicate that there<br>remains significant uncertainty in the call. | *** | | Variant is on curated "block list" of<br>known artifacts | The detected variant has been repeatedly<br>identified in the past as being a result of the<br>sequencing process or something other than an<br>actual variant. | True | | Variant was removed following<br>manual review of data | Used in specific cases which are selected using<br>additional criteria | True | Table 4. FILTER Flag Criteria for CNVs *** Pre-specified threshold values provided. Data not shown Variants with a FILTER flag are later discarded. All other variants are assigned one of three flags (WARN, LIGHTWARN or CONFIDENT), which are later used to guide whether manual inspection may be needed. #### V Standards/Guidance Documents Referenced: - . Considerations for Design, Development, and Analytical Validation of Next Generation Sequencing (NGS)-Based In Vitro Diagnostics (IVDs) Intended to Aid in the Diagnosis of Suspected Germline Diseases - Guidance for the Content of Premarket Submissions for Software Contained in Medical ● Devices - General Principles of Software Validation . - . Recommended Content Format of Non-Clinical Bench Performance Testing Information in Premarket Submissions - Content of Premarket Submissions for Management of Cybersecurity in Medical Devices ● - Statistical Guidance on Reporting Results from Studies ● {11}------------------------------------------------ - CLSI standard EP05-A3 Evaluation of Precision of Quantitative Measurement ● Procedures (3rd ed) - CLSI standard EP07 Interference Testing in Clinical Chemistry (3rd ed) - CLSI standard EP37 Supplemental Tables for Interference Testing in Clinical Chemistry ● #### VI Performance Characteristics: # A Analytical Performance: # 1. Precision/Reproducibility: The precision of the Invitae Common Hereditary Cancers Panel was evaluated with two sets of clinical samples. The first set included 25 samples, with a total of 2,047 SNVs across 40 genes (including 11 genes of high clinical significance), 33 Insertions across 7 genes (including 2 genes of high clinical significance), 38 Deletions across 5 genes (including 1 gene of high clinical significance), 5 CNV duplications across 4 genes (including 1 gene of high clinical significance). and 16 CNV deletions across 12 genes (including 5 genes of high clinical significance). The second set included 18 samples enriched for Indels and CNVs, with a total of 19 Insertions across 4 genes (including 1 genes of high clinical significance), 12 Deletions across 7 genes (including 1 gene of high clinical significance), 7 CNV duplications across 6 genes (including 4 genes of high clinical significance) and 16 CNV deletions across 6 genes (including 5 genes of high clinical significance). Each sample was tested with 14 replicates across 3 different operators, 3 different sequencers, and 3 different sequencing reagent lots. The assay runs were performed on 3 different, non-consecutive days. The repeatability of the assay was assessed by analyzing the concordance between sample replicates within the same run. The reproducibility of the assay was assessed by analyzing the concordance between sample replicates across runs, overall and across sequencing reagent lots, sequencing operators, and sequencers. Results of the precision study are summarized in Table 11. The overall PPA is 99.95% (95% CI 99.92-99.97%) for SNVs, 99.57% (95% CI 99.07-99.80%) for Indels and 99.67% (95% CI 98.80-99.91%) for CNVs. The overall NPA is >99.99% for all variant types (95% CI >99.99-100%) (Table 5). | Type | # Total Positive<br>Variants* | # Detected<br>Positive<br>Variants** | # Total Negative<br>Variants* | # Detected<br>Negative<br>Variants** | PPA<br>(95% CI) | NPA<br>(95% CI) | |------------|-------------------------------|--------------------------------------|-------------------------------|--------------------------------------|---------------------------|---------------------------| | SNVs | 28633 | 28619 | 35645541 | 35645506 | 99.95%<br>(99.92-99.97%) | >99.99% (>99.99-<br>100%) | | Indels | 1405 | 1399 | 14854187 | 14854180 | 99.57% (99.07-<br>99.80%) | >99.99% (>99.99-<br>100%) | | Insertions | 728 | 728 | 3973866 | 3973866 | 100%<br>(99.48-100%) | 100%<br>(>99.99-100%) | | Deletions | 677 | 671 | 10880321 | 10880314 | 99.11%<br>(98.08-99.59%) | >99.99% (>99.99-<br>100%) | Table 5. Overall Precision by Variant Type {12}------------------------------------------------ | CNVs | 602 | 600 | 467326 | 467323 | 99.67% (98.80-<br>99.91%) | >99.99% (>99.99-<br>100%) | |---------------------|-----|-----|--------|--------|---------------------------|---------------------------| | CNV<br>duplications | 157 | 157 | 467326 | 467326 | 100%<br>(97.61-100%) | 100%<br>(>99.99-100%) | | CNV<br>deletions | 445 | 443 | 467326 | 467323 | 99.55%<br>(98.38-99.88%) | >99.99% (>99.99-<br>100%) | ** # Detected positive variants: the total number of positive/negative variants that were detected across replicates of the study samples. The PPAs are >99% across different zygosity (for SNVs), variant sizes (for Indels and CNVs) or genomic contexts, except for deletions of 1-5bp, for which the PPA is 97.53% (95% CI 94.72-98.86%) (Table 6 and Table 7). This was due to 6 false negative results for the same variant on the SDHA gene, which were in a low mappability/complexity region. | | Variant Type<br>Stratified by Zygosity and<br>Size | # Total<br>Positive<br>Variants* | PPA | 95% CI | # Total<br>Negative<br>Variants* | NPA | 95% CI | |---------------------|----------------------------------------------------|----------------------------------|--------|--------------|----------------------------------|---------|-------------| | SNVs | Homo | 10248 | 100% | 99.96-100% | N/A | N/A | N/A | | | Hetero | 17181 | 99.92% | 99.86-99.95% | N/A | N/A | N/A | | | Unknown<br>zygosity | 1204 | 100% | 99.68-100% | N/A | N/A | N/A | | Insertions | 1-5 bp | 588 | 100% | 99.35-100% | 3525432 | 100% | >99.99-100% | | | 6-10 bp | 112 | 100% | 96.68-100% | 315417 | 100% | >99.99-100% | | | 11-20 bp | 14 | 100% | 78.47-100% | 131247 | 100% | >99.99-100% | | | 21+ bp | 14 | 100% | 78.47-100% | 1770 | 100% | >99.99-100% | | Deletions | 1-5 bp | 243 | 97.53% | 94.72-98.86% | 9706966 | >99.99% | >99.99-100% | | | 6-10 bp | 196 | 100% | 98.08-100% | 813027 | 100% | >99.99-100% | | | 11-20 bp | 196 | 100% | 98.08-100% | 359433 | 100% | >99.99-100% | | | 21+ bp | 42 | 100% | 91.62-100% | 888 | 100% | >99.99-100% | | CNV<br>Duplications | ≤ Single Exon | 40 | 100% | 91.24-100% | N/A | N/A | N/A | | | 2-5 Exons | 70 | 100% | 94.80-100% | N/A | N/A | N/A | | | 6-9 Exons | 12 | 100% | 75.75-100% | N/A | N/A | N/A | | | 10+ Exons | 14 | 100% | 78.47-100% | N/A | N/A | N/A | Table 6. Precision by Zygosity and Variant Size {13}------------------------------------------------ | Variant Type<br>Stratified by Zygosity and<br>Size | | # Total<br>Positive<br>Variants* | PPA | 95% CI | # Total<br>Negative<br>Variants* | NPA | 95% CI | |----------------------------------------------------|-----------------------------------------------------|----------------------------------|--------|--------------|----------------------------------|-----|--------| | | Entire Coding<br>Sequence | 21 | 100% | 84.54-100% | N/A | N/A | N/A | | | Other<br>(intronic, non-<br>coding,<br>combination) | 0 | N/A | N/A | N/A | N/A | N/A | | CNV<br>Deletions | ≤ Single Exon | 235 | 99.15% | 96.95-99.77% | N/A | N/A | N/A | | | 2-5 Exons | 116 | 100% | 96.79-100% | N/A | N/A | N/A | | | 6-9 Exons | 61 | 100% | 94.08-100% | N/A | N/A | N/A | | | 10+ Exons | 33 | 100% | 89.57-100% | N/A | N/A | N/A | | | Entire Coding<br>Sequence | 0 | N/A | N/A | N/A | N/A | N/A | | | Other<br>(intronic, non-<br>coding,<br>combination) | 0 | N/A | N/A | N/A | N/A | N/A | ** N/A: data not available or not calculated. ## Table 7. Precision by Genomic Context | Variant Type<br>Stratified by Genomic<br>Context | | # Total Positive<br>Variants* | PPA | 95% CI of<br>PPA | # Total<br>Negative<br>Variants* | NPA | 95% CI of<br>NPA | |--------------------------------------------------|---------------------|-------------------------------|-------|------------------|----------------------------------|------|------------------| | High GC<br>Content<br>(>70%) | SNVs | 504 | 100% | 99.24-100% | 118395 | 100% | >99.99-100% | | | Insertions | 0 | N/A** | N/A | 12297 | 100% | 99.97-100% | | | Deletions | 182 | 100% | 97.93-100% | 12951 | 100% | 99.97-100% | | | CNV<br>duplications | 56 | 100% | 93.58-100% | 25223 | 100% | 99.98-100% | | | CNV<br>deletions | 126 | 100% | 97.04-100% | 25223 | 100% | 99.98-100% | | Low GC Content<br>(<30%) | SNVs | 5320 | 100% | 99.93-100% | 7191969 | 100% | >99.99-100% | | | Insertions | 14 | 100% | 78.47-100% | 946545 | 100% | >99.99-100% | | | Deletions | 168 | 100% | 97.76-100% | 3087513 | 100% | >99.99-100% | {14}------------------------------------------------ | Variant Type<br>Stratified by Genomic<br>Context | | # Total Positive<br>Variants* | PPA | 95% CI of<br>PPA | # Total<br>Negative<br>Variants* | NPA | 95% CI of<br>NPA | |--------------------------------------------------|---------------------|-------------------------------|--------|------------------|----------------------------------|---------|------------------| | | CNV<br>duplications | 76 | 100% | 95.19-100% | 129043 | 100% | >99.99-100% | | | CNV<br>deletions | 209 | 100% | 98.17-100% | 129043 | 100% | >99.99-100% | | Low<br>Mappability/<br>Low Complexity | SNVs | 9075 | 99.84% | 99.74-99.91% | 9738339 | >99.99% | >99.99-100% | | | Insertions | 238 | 100% | 98.41-100% | 1163808 | 100% | >99.99-100% | | | Deletions | 607 | 99.01% | 97.86-99.55% | 3817896 | >99.99% | >99.99-100% | | | CNV<br>duplications | 43 | 100% | 91.80-100% | 28710 | 100% | 99.99-100% | | | CNV<br>deletions | 104 | 100% | 96.44-100% | 28710 | 100% | 99.99-100% | **N/A: data not available or not calculated. #### Table 8. Precision by Sources of Variance and Variant Type | Variable | Average Positive Agreement (95% CI) | | | | | |-----------------------------------------------|-------------------------------------|-----------------------|--------------------------|--------------------------|--------------------------| | | SNVs | Insertions | Deletions | CNV duplications | CNV deletions | | Repeatability | 99.91%<br>(99.88-99.94%) | 100%<br>(>99.99-100%) | 98.08%<br>(97.48-99.52%) | 99.83%<br>(99.83-100%) | 99.43%<br>(98.29-99.82) | | Reproducibility –<br>Instrument-to-Instrument | 99.93%<br>(99.91-99.96%) | 100%<br>(>99.99-100%) | 97.72%<br>(96.59-99.05%) | 96.69%<br>(94.17-98.80%) | 98.99%<br>(98.06-99.68%) | | Reproducibility - Lot-to-Lot | 99.87%<br>(99.83%-99.90%) | 100%<br>(>99.99-100%) | 99.24%<br>(99.16-99.85%) | 98.37%<br>(95.14-99.16%) | >99.99%<br>99.13-100% | | Reproducibility -<br>Operator-to-Operator | 99.89%<br>(99.85-99.92%) | 100%<br>(>99.99-100%) | 99.85%<br>(98.90-99.93%) | 98.72%<br>(96.82-100%) | 99.85%<br>(99.16-100%) | | Reproducibility - Run-to-Run/Day-to-Day | 99.83%<br>(99.74-99.89%) | 100%<br>(>99.99-100%) | 95.17%<br>(90.55-96.49%) | 100%<br>(95.63-100%) | 100%<br>(>99.99-100%) | ### Table 9. No Call Rate | No call rate* | Repeatability | Reproducibility<br>- Instrument-<br>to-Instrument | Reproducibility<br>- Lot-to-Lot | Reproducibility<br>- Operator-to-<br>Operator | Reproducibility -<br>Run-to-Run/Day-<br>to-Day | |---------------|---------------|---------------------------------------------------|---------------------------------|-----------------------------------------------|------------------------------------------------| | SNVs/Indels | 0.05% | 0.05% | 0.05% | 0.05% | 0.18% | | CNVs | 1.78% | 1.99% | 1.99% | 1.95% | 1.82% | *For SNVs and Indels, a "no call" is an area that could not be called due to a sample or region-specific limitation like an unfilled coverage gap. The no call (NC) rate for SNVs and Indels is calculated as the proportion of variants with a no call result in the total number of SNV/Indel called variants, assessed across all sample comparisons. {15}------------------------------------------------ For CNVs, a "no call" is a region that could not be called due to a sample or region-specific limitation like an unfilled coverage gap. The NC rate for CNVs is calculated as the proportion of CNV target regions with a no call result in the total number of target regions, assessed across all sample comparisons. | Specimen Stratified by Variant Type | # Total Positive Variants* | # Detected Positive Variants* | PPA | 95% CI of PPA | # Total Negative Variants* | # Detected Negative Variants* | NPA | 95% CI of NPA | | |-------------------------------------------|----------------------------------|------------------------------------------|------------------------------------------|------------------|----------------------------------|------------------------------------------|------------------------------------------|------------------|------------------| | Study 1 | | | | | | | | | | | Specimen 1 SNVs | 1204 | 1204 | 100% | 99.68-100% | 776217 | 776217 | 100% | >99.99-100% | | | Specimen 1 Indels | 56 | 56 | 100% | 93.58-100% | 197977 | 197977 | 100% | >99.99-100% | | | Specimen 1 CNVs | 14 | 14 | 100% | 78.47-100% | 10874 | 10874 | 100% | 99.96-100% | | | Specimen 2 SNVs | 1106 | 1106 | 100% | 99.65-100% | 776217 | 776217 | 100% | >99.99-100% | | | Specimen 2 Indels | 42 | 42 | 100% | 91.62-100% | 198016 | 198016 | 100% | >99.99-100% | | | Specimen 2 CNVs | 14 | 14 | 100% | 78.47-100% | 10878 | 10878 | 100% | 99.96-100% | | | Specimen 3 SNVs | 1512 | 1512 | 100% | 99.75-100% | 776035 | 776035 | 100% | >99.99-100% | | | Specimen 3 Indels | 56 | 56 | 100% | 93.58-100% | 198003 | 198003 | 100% | >99.99-100% | | | Specimen 3 CNVs | 28 | 28 | 100% | 87.94-100% | 10892 | 10892 | 100% | 99.96-100% | | | Specimen 4 SNVs | 1008 | 1008 | 100% | 99.62-100% | 776308 | 776308 | 100% | >99.99-100% | | | Specimen 4 Indels | 28 | 28 | 100% | 87.94-100% | 198029 | 198029 | 100% | >99.99-100% | | | Specimen 4 CNVs | 0 | 0 | N/A*** | N/A | 10888 | 10888 | 100% | 99.96-100% | | | Specimen 5 SNVs | 882 | 882 | 100% | 99.57-100% | 776295 | 776295 | 100% | >99.99-100% | | | Specimen 5 Indels | 70 | 70 | 100% | 94.80-100% | 198003 | 198003 | 100% | >99.99-100% | | | Specimen 5 CNVs | 21 | 21 | 100% | 84.54-100% | 10810 | 10808 | 99.98% | 99.93-99.99% | | | Specimen 6 SNVs | 997 | 994 | 99.70% | 99.12-99.90% | 776228 | 776222 | >99.99% | >99.99-100% | | | Specimen<br>Stratified by<br>Variant Type | | # Total<br>Positive<br>Variants* | # Detected<br>Positive<br>Variants*<br>* | PPA | 95% CI<br>of PPA | # Total<br>Negative<br>Variants* | # Detected<br>Negative<br>Variants*<br>* | NPA | 95% CI<br>of NPA | | | Indels | 30 | 28 | 93.33% | 78.68-98.15% | 197972 | 197969 | >99.99% | >99.99-100% | | | CNVs | 22 | 22 | 100% | 85.13-100% | 10805 | 10805 | 100% | 99.96-100% | | | SNVs | 1190 | 1190 | 100% | 99.68-100% | 776152 | 776152 | 100% | >99.99-100% | | Specimen 7 | Indels | 14 | 14 | 100% | 78.47-100% | 198042 | 198042 | 100% | >99.99-100% | | | CNVs | 14 | 14 | 100% | 78.47-100% | 10850 | 10850 | 100% | 99.96-100% | | | SNVs | 1036 | 1036 | 100% | 99.63-100% | 776295 | 776295 | 100% | >99.99-100% | | Specimen 8 | Indels | 56 | 56 | 100% | 93.58-100% | 198042 | 198042 | 100% | >99.99-100% | | | CNVs | 14 | 14 | 100% | 78.47-100% | 10877 | 10877 | 100% | 99.96-100% | | | SNVs | 1120 | 1120 | 100% | 99.66-100% | 776139 | 776139 | 100% | >99.99-100% | | Specimen 9 | Indels | 14 | 14 | 100% | 78.47-100% | 198068 | 198068 | 100% | >99.99-100% | | | CNVs | 0 | 0 | N/A | N/A | 10888 | 10888 | 100% | 99.96-100% | | | SNVs | 1206 | 1205 | 99.92% | 99.53-99.99% | 776128 | 776125 | >99.99% | >99.99-100% | | Specimen<br>10 | Indels | 14 | 14 | 100% | 78.47-100% | 198055 | 198055 | 100% | >99.99-100% | | | CNVs | 14 | 14 | 100% | 78.47-100% | 10874 | 10874 | 100% | 99.96-100% | | | SNVs | 1050 | 1050 | 100% | 99.64-100% | 776334 | 776330 | >99.99% | >99.99-100% | | Specimen<br>11 | Indels | 63 | 63 | 100% | 94.25-100% | 197994 | 197990 | >99.99% | >99.99-100% | | | CNVs | 14 | 14 | 100% | 78.47-100% | 10864 | 10864 | 100% | 99.96-100% | | Specimen<br>12 | SNVs | 1456 | 1456 | 100% | 99.74-100% | 776063 | 776057 | >99.99% | >99.99-100% | | | Indels | 70 | 70 | 100% | 94.80-100% | 198003 | 198003 | 100% | >99.99-100% | | Specimen<br>Stratified by<br>Variant Type | # Total<br>Positive<br>Variants* | # Detected<br>Positive<br>Variants* | PPA | 95% CI<br>of PPA | # Total<br>Negative<br>Variants* | # Detected<br>Negative<br>Variants* | NPA | 95% CI<br>of NPA | | | CNVs | 14 | 14 | 100% | 78.47-100% | 10878 | 10878 | 100% | 99.96-100% | | | SNVs | 882 | 882 | 100% | 99.57-100% | 776438 | 776438 | 100% | >99.99-100% | | | Specimen<br>13 | Indels | 14 | 14 | 100% | 78.47-100% | 198042 | 198042 | 100% | >99.99-100% | | CNVs | 14 | 14 | 100% | 78.47-100% | 10869 | 10869 | 100% | 99.96-100% | | | SNVs | 1106 | 1106 | 100% | 99.65-100% | 776256 | 776256 | 100% | >99.99-100% | | | Specimen<br>14 | Indels | 28 | 28 | 100% | 87.94-100% | 198016 | 198016 | 100% | >99.99-100% | | | CNVs | 14 | 14 | 100% | 78.47-100% | 10864 | 10864 | 100% | 99.96-100% | | SNVs | 1080 | 1074 | 99.44% | 98.79-99.75% | 776244 | 776238 | >99.99% | >99.99-100% | | | Specimen<br>15 | Indels | 14 | 14 | 100% | 78.47-100% | 198029 | 198029 | 100% | >99.99-100% | | CNVs | 0 | 0 | N/A | N/A | 10892 | 10892 | 100% | 99.96-100% | | | SNVs | 952 | 952 | 100% | 99.60-100% | 776373 | 776373 | 100% | >99.99-100% | | | Specimen<br>16 | Indels | 56 | 56 | 100% | 93.58-100% | 198003 | 198003 | 100% | >99.99-100% | | CNVs | 0 | 0 | N/A | N/A | 10892 | 10892 | 100% | 99.96-100% | | | SNVs | 1302 | 1302 | 100% | 99.71-100% | 776113 | 776113 | 100% | >99.99-100% | | | Specimen<br>17 | Indels | 14 | 14 | 100% | 78.47-100% | 198016 | 198016 | 100% | >99.99-100% | | CNVs | 0 | 0 | N/A | N/A | 10892 | 10892 | 100% | 99.96-100% | | | SNVs | 1694 | 1694 | 100% | 99.77-100% | 775788 | 775788 | 100% | >99.99-100% | | | Specimen<br>18 | Indels | 84 | 84 | 100% | 95.63-100% | 197977 | 197977 | 100% | >99.99-100% | | CNVs | 14 | 14 | 100% | 78.47-100% | 10892 | 10892 | 100% | 99.96-100% | | | Specimen<br>Stratified by<br>Variant Type | # Total<br>Positive<br>Variants* | # Detected<br>Positive<br>Variants* | PPA | 95% CI<br>of PPA | # Total<br>Negative<br>Variants* | # Detected<br>Negative<br>Variants* | NPA | 95% CI<br>of NPA | | | Specimen<br>19 | SNVs | 924 | 924 | 100% | 99.59-100% | 776334 | 776334 | 100% | >99.99-100% | | | Indels | 14 | 14 | 100% | 78.47-100% | 198016 | 198016 | 100% | >99.99-100% | | | CNVs | 0 | 0 | N/A | N/A | 10891 | 10891…