Predicate Devices

Not Found

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

Unknown
The device description mentions a "custom-built bioinformatics pipeline" and an "algorithm that weights the available clinical evidence" for variant assessment. While these could potentially incorporate AI/ML, the summary does not explicitly state the use of AI, ML, DNN, or provide details about training or test sets in a way that confirms AI/ML usage. The performance studies focus on analytical and clinical validation metrics typical of genetic testing, not specific AI/ML performance metrics.

Therapeutic

No
This device is an in vitro diagnostic test intended for genetic analysis to assess hereditary cancer predisposition and aid in identifying variants associated with diagnosed cancer. It provides information for healthcare professionals but does not directly treat or prevent a disease.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the Invitae Common Hereditary Cancers Panel is an "in vitro diagnostic test system." This indicates its purpose is to diagnose specific conditions or genetic predispositions.

SaMD (Software as a Medical Device)

No

The device description explicitly states it uses "hybridization-based capture, next-generation sequencing (NGS), and a custom-built bioinformatics pipeline". Hybridization-based capture and NGS are hardware-dependent laboratory processes, not solely software. The bioinformatics pipeline is software, but it is part of a larger system that includes physical components and processes for DNA analysis.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The "Intended Use / Indications for Use" section explicitly states: "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..."

This statement clearly identifies the device as an in vitro diagnostic test.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications 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.

Product codes (comma separated list FDA assigned to the subject device)

QVU

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 analysis covers the select non-coding variants specifically defined in the table. Any variants that fall outside these regions are not analyzed.

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.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Germline human genomic DNA extracted from whole blood. The genes analyzed are associated with various cancers affecting breast, ovarian, pancreatic, prostate, colorectal, stomach, brain, thyroid, fallopian tube, peritoneal, neural system, bladder, biliary tract, urinary tract, small bowel, kidney, skin, lung, and more.

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Qualified health care professionals; single-site assay performed at Invitae Corporation.

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Precision/Reproducibility:
- Study type: Analytical precision and reproducibility study.
- Sample size: Two sets of clinical samples: 25 samples in the first set, 18 samples in the second set. Each sample was tested with 14 replicates across 3 different operators, 3 different sequencers, and 3 different sequencing reagent lots over 3 non-consecutive days.
- Key results: Overall PPA: 99.95% for SNVs, 99.57% for Indels, 99.67% for CNVs. Overall NPA: >99.99% for all variant types. PPAs were >99% across different zygosity (for SNVs), variant sizes (for Indels and CNVs), or genomic contexts, except for deletions of 1-5bp (PPA 97.53%) due to 6 false negative results for the same variant on the SDHA gene in a low mappability/complexity region. On the gene level, PPA for SDHA SNVs was 99.15%, for Indels 68.42%, and for NF1 CNVs 97.30%.
DNA input:
- Study type: Evaluation of impact of different DNA input levels on test performance.
- Sample size: Eight whole blood clinical specimens tested at 1, 5, 10, 23 (standard), and 46 ng/uL, each in triplicate (total 120 samples).
- Key results: At all DNA input levels (compared to 23 ng/uL), overall concordance, PPA, and NPA were >99%. Five samples failed QC at 1 ng/uL, and six at 46 ng/uL. The minimum DNA input for the Invitae Common Hereditary Cancers Panel was determined to be 5 ng/uL. CNV deletions PPA was slightly below 95% at 5 ng/uL (95.1%) due to 2 false negatives in the NF1 gene in an AT rich region.
Analytical Specificity/Interference:
- Study type: Evaluation of impact of endogenous and exogenous interfering substances.
- Sample size: Two studies. Study 1: Bilirubin, Wash buffer, TAFTs, K2EDTA, Amplicon, Hemoglobin, Triglycerides (5 samples each, 3 replicates); Donor Blood (10 samples, 2 replicates). Study 2 (enriched for Indels and CNVs): Bilirubin, Wash buffer, TAFTs, K2EDTA, Amplicon, Hemoglobin, Triglycerides (5 or 6 samples each, 2 replicates).
- Key results: Bilirubin, TAFTs, hemoglobin, and triglycerides did not affect assay performance. Donor blood caused assay failure at all contamination levels. False negative CNVs were reported with K2EDTA and wash buffer. Inappropriate PMS2 amplicon addition caused QC metric failure for CNV detection and decreased PPA for Indel detection.
Carry-Over and Cross-Contamination:
- Study type: Retrospective analysis of clinical samples.
- Sample size: All clinical samples run over one year (118,471 samples).
- Key results: 0.29% of samples had evidence of contamination above the QC threshold (≥2.5% alien DNA).
DNA Integrity:
- Study type: Evaluation of impact of DNA degradation.
- Sample size: 5 individual gDNA samples sheared to three ranges of DNA Integrity Number (>7, 3-7, 99% at all timepoints for all conditions, supporting specimen stability.
Guardbanding:
- Study type: Robustness by assessing impact of varying critical input parameters (DNA input concentration at library prep, hybridization, sequencing).
- Sample size: Eight DNA samples run in duplicate.
- Key results: Assay was robust when input concentrations were varied up to +/-10%, with PPA >99.9% and NPA >99.99% for most conditions.
Accuracy – Comparison to Orthogonal Method(s):
- Study type: Comparison to Genome in a Bottle (GIAB) samples and supplemental cell lines (non-clinical samples).
- Sample size: 5 GIAB samples (total 778,829 data points, 389 SNVs, 7 Indels). 92 supplemental cell line samples (24 SNVs, 101 Indels, 10 CNVs).
- Key results: Percentage of no calls/invalid calls was 0%. PPA, NPA, and TPPV were 100% across all samples for all variant types.
Comparison Study using Clinical Specimens:
- Study type: Accuracy evaluation against validated orthogonal methods.
- Sample size: 6014 samples for SNVs/Indels (2181 SNVs, 3914 Indels). 3542 samples for CNVs (3601 CNVs). Additional 106 clinical specimens for CNV TNPV evaluation.
- Key results: Overall TPPV: 99.9% for SNVs, 100% for Indels, 99.5% for CNVs. Overall TNPV: 100% for SNVs, 100% for Indels, 99.7% for CNVs. Gene-level accuracy: All genes showed 100% TPPV for SNVs/Indels except SDHA (99.0%). For CNVs, 32 genes showed 100% TPPV; SMAD4 (84.6%) and TSC2 (88.9%) were lower. TPPV for CNV duplications with single exon or less was 95.5%. TPPV for CNV deletions with GC content 25-30% was 98.6%. TPPV for CNV duplications with GC content 30-55% was 98.5% and >55% was 95.5%.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Precision (Overall PPA):
- SNVs: 99.95% (95% CI 99.92-99.97%)
- Indels: 99.57% (95% CI 99.07-99.80%)
- CNVs: 99.67% (95% CI 98.80-99.91%)
Precision (Overall NPA):
- All variant types: >99.99% (95% CI >99.99-100%)
Accuracy (Clinical Specimens):
- TPPV:
  - SNVs: 99.9% (95% CI 99.7->99.9%)
  - Indels: 100% (95% CI 99.9- 100%)
  - CNVs: 99.5% (95% CI 99.2- 99.7%)
- TNPV:
  - SNVs: 100% (95% CI >99.9%- 100%)
  - Indels: 100% (95% CI >99.9- 100%)
  - CNVs: 99.7% (95% CI 99.6- 99.7%)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Not Found

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

N/A

Summary

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
Code(s) | Classification | Regulation
Section | Panel |
|--------------------|----------------|-------------------------------------------------------------------------------------------------------------------------|-----------|
| QVU | Class II | 21 CFR 866.6095— High
throughput DNA
sequencing for hereditary
cancer predisposition
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
Analysis | CNV
Analysis | Notes |---------|-----------------------|---- | APC | YES | YES analysis. The 1A promoter region SNV/Indel analysis for exon 5 | ATM | YES | YES | AXIN2 | YES | YES | BARDI | YES | YES | BMPR1A | YES | YES | BRCA1 | YES | YES sequence. | BRCA2 | YES | YES sequence. | BRIP1 | YES | YES | CDH1 | YES | YES | CDK4 | YES | YES | CDKN2A | YES | YES | CHEK2 | YES | YES | CTNNA1 | YES | YES | DICER1 | YES | YES | EPCAM | NO | YES | GREMI | NO | YES | HOXB13 | YES | YES | KIT | YES | YES | MENI | YES | YES | MLH1 | YES | YES exon 12 is limited to cds +/-10 bp. | MSH2 | YES | YES SNV/Indel analysis for exons 2, | MSH3 | YES | YES 79950765) is not offered | Gene* | SNV/Indel
Analysis | CNV
Analysis | Notes | MSH6 | YES | YES | MUTYH | YES | YES | NBN | YES | YES | NF1 | YES | YES 10 bp. | NTHLI | YES | YES | PALB2 | YES | YES | PDGFRA | YES | YES | PMS2 | YES | YES | POLDI | YES | YES | POLE | YES | YES | PTEN | YES | NO exons 8 includes only cds +/- 10 bp. | RAD50 | YES | YES | RAD51C | YES | YES | RAD51D | YES | YES | SDHA | YES | NO offered for exon 14. SNV/Indel cds +/- 10 bp. | SDHB | YES | YES | SDHC | YES | NO exons 2, 6 includes only cds +/- 10 bp. | SDHD | YES | YES | SMAD4 | YES | YES | SMARCA4 | YES | YES | STK11 | YES | YES | TP53 | YES | YES | TSC1 | YES | YES | TSC2 | YES | YES | VHL | YES | YES |
-------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| The 1B promoter region is covered by both SNV/Indel and CNV
is covered by CNV analysis.
is limited to cds +/-10 bp. |
| SNV/Indel analysis for exons 6, 24, 43 includes only cds +/- 10 bp. |
| |
| |
| CNV analysis covers the promoter region. |
| SNV/Indel analysis includes +/- 20 base pairs of adjacent intronic
|
| SNV/Indel analysis includes +/- 20 base pairs of adjacent intronic
|
| |
| |
| |
| |
| |
| |
| SNV/Indel analysis for exon 22 includes only cds +/- 10 bp. |
| SNV/Indel analysis is not offered for this gene (CNV analysis only). |
| Promoter region duplication testing only. |
| |
| |
| SNV/Indel analysis for exon 2 is limited to cds +/-10 bp. |
| CNV analysis covers the promoter region. SNV/Indel analysis for
|
| Analysis includes the exon 1-7 inversion (Boland mutation).
5 includes only cds +/- 10 bp. |
| SNV/Indel analysis of the repeat region of exon 1 (5:79950697-
|
|
| SNV/Indel analysis for exons 7, 10 includes only cds +/- 10 bp. |
| |
| |
| SNV/Indel analysis for exons 2, 7, 25, 41, 48 includes only cds +/-
|
| |
| |
| |
| SNV/Indel analysis for exon 7 includes only cds +/- 10 bp. |
| SNV/Indel analysis for exon 22 includes only cds +/- 10 bp. |
| |
| CNV analysis is not offered for this gene. SNV/Indel analysis for
|
| |
| |
| |
| CNV analysis is not offered for this gene. SNV/Indel analysis is not
analysis for exons 6-8 includes only
|
| |
| CNV analysis is not offered for this gene. SNV/Indel analysis for
|
| |
| |
| |
| |
| CNV analysis covers the promoter region. |
| SNV/Indel analysis for exon 21 includes only cds +/- 10 bp. |
| |
| |

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 99:1 in favor of causing disease or increasing disease risk) ●
Likely Pathogenic (Odds >9:1 but 9:1 but 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)
1. Specimen Identification:
  Whole blood

3. Specimen Sampling and Handling:

Refer to Section IV. B, Specimen Collection and Preparation

1. Calibration:
  Not applicable
1. 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
Threshold | Batch
Threshold
(# failed
samples) |
|----------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------|---------------------------------------------|
| Mean target coverage | The mean depth of coverage (i.e., number of reads)
over a target region | ≥300x | ≤21 |
| Very low coverage | Number of targeted bases (assay-wide) with 99.99% for all variant types (95% CI >99.99-100%) (Table 5).

| Type | # Total Positive
Variants* | # Detected
Positive
Variants** | # Total Negative
Variants* | # Detected
Negative
Variants** | PPA
(95% CI) | NPA
(95% CI) |
|------------|-------------------------------|--------------------------------------|-------------------------------|--------------------------------------|---------------------------|---------------------------|
| SNVs | 28633 | 28619 | 35645541 | 35645506 | 99.95%
(99.92-99.97%) | >99.99% (>99.99-
100%) |
| Indels | 1405 | 1399 | 14854187 | 14854180 | 99.57% (99.07-
99.80%) | >99.99% (>99.99-
100%) |
| Insertions | 728 | 728 | 3973866 | 3973866 | 100%
(99.48-100%) | 100%
(>99.99-100%) |
| Deletions | 677 | 671 | 10880321 | 10880314 | 99.11%
(98.08-99.59%) | >99.99% (>99.99-
100%) |

Table 5. Overall Precision by Variant Type

12

| CNVs | 602 | 600 | 467326 | 467323 | 99.67% (98.80-
99.91%) | >99.99% (>99.99-
100%) |
|---------------------|-----|-----|--------|--------|---------------------------|---------------------------|
| CNV
duplications | 157 | 157 | 467326 | 467326 | 100%
(97.61-100%) | 100%
(>99.99-100%) |
| CNV
deletions | 445 | 443 | 467326 | 467323 | 99.55%
(98.38-99.88%) | >99.99% (>99.99-
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
Stratified by Zygosity and
Size | # Total
Positive
Variants* | PPA | 95% CI | # Total
Negative
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
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
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
Stratified by Zygosity and
Size | | # Total
Positive
Variants* | PPA | 95% CI | # Total
Negative
Variants* | NPA | 95% CI |
|----------------------------------------------------|-----------------------------------------------------|----------------------------------|--------|--------------|----------------------------------|-----|--------|
| | Entire Coding
Sequence | 21 | 100% | 84.54-100% | N/A | N/A | N/A |
| | Other
(intronic, non-
coding,
combination) | 0 | N/A | N/A | N/A | N/A | N/A |
| CNV
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
Sequence | 0 | N/A | N/A | N/A | N/A | N/A |
| | Other
(intronic, non-
coding,
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
Stratified by Genomic
Context | | # Total Positive
Variants* | PPA | 95% CI of
PPA | # Total
Negative
Variants* | NPA | 95% CI of
NPA |
|--------------------------------------------------|---------------------|-------------------------------|-------|------------------|----------------------------------|------|------------------|
| High GC
Content
(>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
duplications | 56 | 100% | 93.58-100% | 25223 | 100% | 99.98-100% |
| | CNV
deletions | 126 | 100% | 97.04-100% | 25223 | 100% | 99.98-100% |
| Low GC Content
(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
Stratified by Genomic
Context | | # Total Positive
Variants* | PPA | 95% CI of
PPA | # Total
Negative
Variants* | NPA | 95% CI of
NPA |
|--------------------------------------------------|---------------------|-------------------------------|--------|------------------|----------------------------------|---------|------------------|
| | CNV
duplications | 76 | 100% | 95.19-100% | 129043 | 100% | >99.99-100% |
| | CNV
deletions | 209 | 100% | 98.17-100% | 129043 | 100% | >99.99-100% |
| Low
Mappability/
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
duplications | 43 | 100% | 91.80-100% | 28710 | 100% | 99.99-100% |
| | CNV
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%
(99.88-99.94%)	100%
(>99.99-100%)	98.08%
(97.48-99.52%)	99.83%
(99.83-100%)	99.43%
(98.29-99.82)
Reproducibility –
Instrument-to-Instrument	99.93%
(99.91-99.96%)	100%
(>99.99-100%)	97.72%
(96.59-99.05%)	96.69%
(94.17-98.80%)	98.99%
(98.06-99.68%)
Reproducibility - Lot-to-Lot	99.87%
(99.83%-99.90%)	100%
(>99.99-100%)	99.24%
(99.16-99.85%)	98.37%
(95.14-99.16%)	>99.99%
99.13-100%
Reproducibility -
Operator-to-Operator	99.89%
(99.85-99.92%)	100%
(>99.99-100%)	99.85%
(98.90-99.93%)	98.72%
(96.82-100%)	99.85%
(99.16-100%)
Reproducibility - Run-to-Run/Day-to-Day	99.83%
(99.74-99.89%)	100%
(>99.99-100%)	95.17%
(90.55-96.49%)	100%
(95.63-100%)	100%
(>99.99-100%)

Table 9. No Call Rate

| No call rate* | Repeatability | Reproducibility

Instrument-
to-Instrument | Reproducibility
Lot-to-Lot | Reproducibility
Operator-to-
Operator | Reproducibility -
Run-to-Run/Day-
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
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 |
| | 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
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
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
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
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 | |
| 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
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
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
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
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
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
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
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 | |
| Specimen
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 | 100% | 99.96-100% |
| Specimen
20 | SNVs | 1120 | 1120 | 100% | 99.66-100% | 776308 | 776308 | 100% | >99.99-100% |
| | Indels | 28 | 28 | 100% | 87.94-100% | 198016 | 198016 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 10878 | 10878 | 100% | 99.96-100% |
| Specimen
21 | SNVs | 1540 | 1540 | 100% | 99.75-100% | 775892 | 775892 | 100% | >99.99-100% |
| | Indels | 42 | 42 | 100% | 91.62-100% | 198003 | 198003 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 10878 | 10878 | 100% | 99.96-100% |
| Specimen
22 | SNVs | 1162 | 1162 | 100% | 99.67-100% | 776165 | 776165 | 100% | >99.99-100% |
| | Indels | 28 | 28 | 100% | 87.94-100% | 198016 | 198016 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 10884 | 10884 | 100% | 99.96-100% |
| Specimen
23 | SNVs | 1032 | 1028 | 99.61% | 99.01-99.85% | 776283 | 776273 | >99.99% | >99.99-100% |
| | Indels | 56 | 56 | 100% | 93.58-100% | 198003 | 198003 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 10878 | 10878 | 100% | 99.96-100% |
| Specimen
24 | SNVs | 924 | 924 | 100% | 99.59-100% | 776399 | 776399 | 100% | >99.99-100% |
| | Indels | 28 | 28 | 100% | 87.94-100% | 198029 | 198029 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 10864 | 10864 | 100% | 99.96-100% |
| | SNVs | 1148 | 1148 | 100% | 99.67-100% | 776217 | 776217 | 100% | >99.99-100% |
| 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 |
| Specimen
25 | Indels | 56 | 56 | 100% | 93.58-100% | 197990 | 197990 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 10889 | 10889 | 100% | 99.96-100% |
| Study 2 | | | | | | | | | |
| Specimen 1 | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| | Indels | 28 | 28 | 100% | 87.94-100% | 198107 | 198107 | 100% | >99.99-100% |
| | CNVs | 28 | 28 | 100% | 87.94-100% | 10907 | 10907 | 100% | 99.96-100% |
| Specimen 2 | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| | Indels | 28 | 28 | 100% | 87.94-100% | 198120 | 198120 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 10959 | 10959 | 100% | 99.96-100% |
| Specimen 3 | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| | Indels | 28 | 28 | 100% | 87.94-100% | 198120 | 198120 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 10988 | 10988 | 100% | 99.96-100% |
| Specimen 4 | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| | Indels | 14 | 14 | 100% | 78.47-100% | 198146 | 198146 | 100% | >99.99-100% |
| | CNVs | 22 | 22 | 100% | 85.13-100% | 11001 | 11001 | 100% | 99.96-100% |
| Specimen 5 | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| | Indels | 28 | 28 | 100% | 87.94-100% | 198133 | 198133 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 10975 | 10975 | 100% | 99.96-100% |
| Specimen 6 | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| 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 |
| | Indels | 42 | 42 | 100% | 91.62-100% | 198094 | 198094 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 10792 | 10792 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Specimen 7 | Indels | 14 | 14 | 100% | 78.47-100% | 198133 | 198133 | 100% | >99.99-100% |
| | CNVs | 20 | 20 | 100% | 83.89-100% | 10783 | 10783 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Specimen 8 | Indels | 14 | 14 | 100% | 78.47-100% | 198159 | 198159 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 10994 | 10994 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Specimen 9 | Indels | 14 | 14 | 100% | 78.47-100% | 198146 | 198146 | 100% | >99.99-100% |
| | CNVs | 17 | 17 | 100% | 81.57-100% | 10991 | 10991 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Specimen
10 | Indels | 28 | 28 | 100% | 87.94-100% | 198120 | 198120 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 10998 | 10998 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Specimen
11 | Indels | 28 | 28 | 100% | 87.94-100% | 198146 | 198146 | 100% | >99.99-100% |
| | CNVs | 22 | 22 | 100% | 85.13-100% | 10990 | 10990 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Specimen
12 | Indels | 0 | 0 | N/A | N/A | 198133 | 198133 | 100% | >99.99-100% |
| 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 | |
| Specimen
13 | CNVs | 14 | 14 | 100% | 78.47-100% | 10963 | 10963 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Indels | 28 | 28 | 100% | 87.94-100% | 198146 | 198146 | 100% | >99.99-100% | |
| Specimen
14 | CNVs | 60 | 58 | 96.67% | 88.64-99.08% | 10229 | 10228 | 99.99% | 99.94-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Indels | 14 | 14 | 100% | 78.47-100% | 198159 | 198159 | 100% | >99.99-100% | |
| Specimen
15 | CNVs | 14 | 14 | 100% | 78.47-100% | 10969 | 10969 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Indels | 42 | 42 | 100% | 91.62-100% | 198107 | 198107 | 100% | >99.99-100% | |
| Specimen
16 | CNVs | 28 | 28 | 100% | 87.94-100% | 10938 | 10938 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Indels | 10 | 6 | 60.00% | 31.27-83.18% | 167662 | 167662 | 100% | >99.99-100% | |
| Specimen
17 | CNVs | 0 | 0 | 0 | N/A | 11026 | 11026 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Indels | 28 | 28 | 100% | 87.94-100% | 198133 | 198133 | 100% | >99.99-100% | |
| Specimen
18 | CNVs | 14 | 14 | 100% | 78.47-100% | 10958 | 10958 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Indels | 42 | 42 | 100% | 91.62-100% | 198068 | 198068 | 100% | >99.99-100% | |
| CNVs | 26 | 26 | 100% | 87.13-100% | 10824 | 10824 | 100% | 99.96-100% | |

Table 10. Precision per specimen

16

17

18

19

20

21

22

** # Detected positive variants: the total number of positive/negative variants that were detected across replicates of the study samples.

*** N/A: values not calculated.

On the gene level, for SNVs, all the genes tested in the study show PPA of 100%, except SDHA (99.15% with 95% CI 98.59-99.50%); for Indels, all the genes tested in the study show PPA of 100%, except SDHA (68.42% with 95% CI 46.01-84.64%); and for CNVs, all the genes tested in the study show PPA of 100%, except NF1 (97.30% with 95% CI 90.67-99.26%) (Table 11).

	Table 11. Precision per Gene

| Gene | Type | # Total
Positive
Variants* | # Detected
Positive
Variants* | PPA | 95% CI
of PPA | # Total
Negative
Variants* | # Detected
Negative
Variants* | NPA | 95% CI
of NPA |
|----------------|--------|----------------------------------|-------------------------------------|--------|------------------|----------------------------------|-------------------------------------|---------|------------------|
| APC | SNVs | 2562 | 2562 | 100% | 99.85-100% | 2448957 | 2448957 | 100% | >99.99-100% |
| APC | Indels | 56 | 56 | 100% | 93.58-100% | 838419 | 838419 | 100% | >99.99-100% |
| APC | CNVs | 0 | 0 | N/A | N/A | 10234 | 10234 | 100% | 99.96-100% |
| ATM | SNVs | 770 | 770 | 100% | 99.50-100% | 2767848 | 2767848 | 100% | >99.99-100% |
| ATM | Indels | 168 | 168 | 100% | 97.76-100% | 1086495 | 1086495 | 100% | >99.99-100% |
| ATM | CNVs | 28 | 28 | 100% | 87.94-100% | 37266 | 37266 | 100% | 99.99-100% |
| AXIN2 | SNVs | 1008 | 1008 | 100% | 99.62-100% | 626001 | 626001 | 100% | >99.99-100% |
| AXIN2 | Indels | 14 | 14 | 100% | 78.47-100% | 79332 | 79332 | 100% | >99.99-100% |
| AXIN2 | CNVs | 0 | 0 | N/A | N/A | 6018 | 6018 | 100% | 99.94-100% |
| BARD1 | SNVs | 812 | 812 | 100% | 99.53-100% | 708975 | 708975 | 100% | >99.99-100% |
| BARD1 | Indels | 420 | 420 | 100% | 99.09-100% | 182457 | 182457 | 100% | >99.99-100% |
| BARD1 | CNVs | 0 | 0 | N/A | N/A | 6621 | 6621 | 100% | 99.94-100% |
| BMPR1A | SNVs | 210 | 210 | 100% | 98.20-100% | 403335 | 403335 | 100% | >99.99-100% |
| Gene | Type | # Total
Positive
Variants* | # Detected
Positive
Variants* | PPA | 95% CI
of PPA | # Total
Negative
Variants* | # Detected
Negative
Variants* | NPA | 95% CI
of NPA |
| | Indels | 0 | 0 | N/A | N/A | 92028 | 92028 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 8764 | 8764 | 100% | 99.96-100% |
| | SNVs | 1330 | 1330 | 100% | 99.71-100% | 1364151 | 1364151 | 100% | >99.99-100% |
| BRCA1 | Indels | 0 | 0 | N/A | N/A | 1726671 | 1726671 | 100% | >99.99-100% |
| | CNVs | 82 | 82 | 100% | 95.52-100% | 11956 | 11956 | 100% | 99.97-100% |
| | SNVs | 2338 | 2338 | 100% | 99.84-100% | 2694108 | 2694108 | 100% | >99.99-100% |
| BRCA2 | Indels | 14 | 14 | 100% | 78.47-100% | 2326866 | 2326866 | 100% | >99.99-100% |
| | CNVs | 42 | 42 | 100% | 91.62-100% | 16183 | 16183 | 100% | 99.98-100% |
| | SNVs | 952 | 952 | 100% | 99.60-100% | 1005642 | 1005642 | 100% | >99.99-100% |
| BRIP1 | Indels | 0 | 0 | N/A | N/A | 304578 | 304578 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 11436 | 11436 | 100% | 99.97-100% |
| | SNVs | 882 | 882 | 100% | 99.57-100% | 745317 | 745317 | 100% | >99.99-100% |
| CDH1 | Indels | 0 | 0 | N/A | N/A | 173172 | 173172 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 9632 | 9632 | 100% | 99.96-100% |
| | SNVs | 0 | 0 | N/A | N/A | 175518 | 175518 | 100% | >99.99-100% |
| CDK4 | Indels | 0 | 0 | N/A | N/A | 25350 | 25350 | 100% | 99.98-100% |
| | CNVs | 0 | 0 | N/A | N/A | 4214 | 4214 | 100% | 99.91-100% |
| CDKN2A | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| | Indels | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| Gene | Type | # Total
Positive
Variants* | # Detected
Positive
Variants* | PPA | 95% CI
of PPA | # Total
Negative
Variants* | # Detected
Negative
Variants* | NPA | 95% CI
of NPA |
| CHEK2 | CNVs | 0 | 0 | N/A | N/A | 2310 | 2310 | 100% | 99.83-100% |
| | SNVs | 14 | 14 | 100% | 78.47-100% | 541458 | 541458 | 100% | >99.99-100% |
| | Indels | 0 | 0 | N/A | N/A | 303225 | 303225 | 100% | >99.99-100% |
| CTNNA1 | CNVs | 14 | 14 | 100% | 78.47-100% | 8391 | 8391 | 100% | 99.95-100% |
| | SNVs | 350 | 350 | 100% | 98.91-100% | 453696 | 453696 | 100% | >99.99-100% |
| | Indels | 14 | 14 | 100% | 78.47-100% | 64707 | 64707 | 100% | 99.99-100% |
| DICER1 | CNVs | 0 | 0 | N/A | N/A | 10234 | 10234 | 100% | 99.96-100% |
| | SNVs | 420 | 420 | 100% | 99.09-100% | 1110225 | 1110225 | 100% | >99.99-100% |
| | Indels | 14 | 14 | 100% | 78.47-100% | 276732 | 276732 | 100% | >99.99-100% |
| EPCAM | CNVs | 0 | 0 | N/A | N/A | 15652 | 15652 | 100% | 99.98-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| | Indels | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| GREM1 | CNVs | 0 | 0 | N/A | N/A | 5418 | 5418 | 100% | 99.93-100% |
| | SNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| | Indels | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| HOXB13 | CNVs | 21 | 21 | 100% | 84.54-100% | 3530 | 3528 | 99.94% | 99.79-99.98% |
| | SNVs | 294 | 294 | 100% | 98.71-100% | 261696 | 261696 | 100% | >99.99-100% |
| | Indels | 0 | 0 | N/A | N/A | 181350 | 181350 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 1204 | 1204 | 100% | 99.68-100% |
| Gene | Type | # Total
Positive
Variants* | # Detected
Positive
Variants* | PPA | 95% CI
of PPA | # Total
Negative
Variants* | # Detected
Negative
Variants* | NPA | 95% CI
of NPA |
| KIT | SNVs | 266 | 266 | 100% | 98.58-100% | 522657 | 522657 | 100% | >99.99-100% |
| KIT | Indels | 0 | 0 | N/A | N/A | 43278 | 43278 | 100% | 99.99-100% |
| KIT | CNVs | 0 | 0 | N/A | N/A | 12642 | 12642 | 100% | 99.97-100% |
| MEN1 | SNVs | 994 | 994 | 100% | 99.62-100% | 379650 | 379650 | 100% | >99.99-100% |
| MEN1 | Indels | 0 | 0 | N/A | N/A | 174147 | 174147 | 100% | >99.99-100% |
| MEN1 | CNVs | 0 | 0 | N/A | N/A | 6020 | 6020 | 100% | 99.94-100% |
| MLH1 | SNVs | 224 | 224 | 100% | 98.31-100% | 683862 | 683862 | 100% | >99.99-100% |
| MLH1 | Indels | 0 | 0 | N/A | N/A | 457191 | 457191 | 100% | >99.99-100% |
| MLH1 | CNVs | 14 | 14 | 100% | 78.47-100% | 11984 | 11984 | 100% | 99.97-100% |
| MSH2 | SNVs | 532 | 532 | 100% | 99.28-100% | 904152 | 904152 | 100% | >99.99-100% |
| MSH2 | Indels | 0 | 0 | N/A | N/A | 536250 | 536250 | 100% | >99.99-100% |
| MSH2 | CNVs | 83 | 83 | 100% | 95.58-100% | 8096 | 8096 | 100% | 99.95-100% |
| MSH3 | SNVs | 1330 | 1330 | 100% | 99.71-100% | 771639 | 771639 | 100% | >99.99-100% |
| MSH3 | Indels | 518 | 518 | 100% | 99.26-100% | 141462 | 141462 | 100% | >99.99-100% |
| MSH3 | CNVs | 14 | 14 | 100% | 78.47-100% | 14419 | 14419 | 100% | 99.97-100% |
| MSH6 | SNVs | 602 | 602 | 100% | 99.37-100% | 1285641 | 1285641 | 100% | >99.99-100% |
| MSH6 | Indels | 28 | 28 | 100% | 87.94-100% | 628074 | 628074 | 100% | >99.99-100% |
| MSH6 | CNVs | 28 | 28 | 100% | 87.94-100% | 5978 | 5978 | 100% | 99.94-100% |
| MUTYH | SNVs | 336 | 336 | 100% | 98.87-100% | 511395 | 511395 | 100% | >99.99-100% |
| Gene | Type | # Total Positive Variants* | # Detected Positive Variants* | PPA | 95% CI of PPA | # Total Negative Variants* | # Detected Negative Variants* | NPA | 95% CI of NPA |
| | Indels | 0 | 0 | N/A | N/A | 104325 | 104325 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 9632 | 9632 | 100% | 99.96-100% |
| NBN | SNVs | 1260 | 1260 | 100% | 99.70-100% | 652761 | 652761 | 100% | >99.99-100% |
| NBN | Indels | 0 | 0 | N/A | N/A | 222897 | 222897 | 100% | >99.99-100% |
| NBN | CNVs | 20 | 20 | 100% | 83.89-100% | 9408 | 9408 | 100% | 99.96-100% |
| NF1 | SNVs | 560 | 560 | 100% | 99.32-100% | 1968074 | 1968074 | 100% | >99.99-100% |
| NF1 | Indels | 0 | 0 | N/A | N/A | 1360956 | 1360956 | 100% | >99.99-100% |
| NF1 | CNVs | 74 | 72 | 97.30% | 90.67-99.26% | 32719 | 32718 | >99.99% | 99.98-100% |
| NTHL1 | SNVs | 0 | 0 | N/A | N/A | 306858 | 306858 | 100% | >99.99-100% |
| NTHL1 | Indels | 0 | 0 | N/A | N/A | 51675 | 51675 | 100% | 99.99-100% |
| NTHL1 | CNVs | 0 | 0 | N/A | N/A | 3603 | 3603 | 100% | 99.89-100% |
| PALB2 | SNVs | 252 | 252 | 100% | 98.50-100% | 993345 | 993345 | 100% | >99.99-100% |
| PALB2 | Indels | 0 | 0 | N/A | N/A | 505269 | 505269 | 100% | >99.99-100% |
| PALB2 | CNVs | 14 | 14 | 100% | 78.47-100% | 7812 | 7812 | 100% | 99.95-100% |
| PDGFRA | SNVs | 1330 | 1330 | 100% | 99.71-100% | 585288 | 585288 | 100% | >99.99-100% |
| PDGFRA | Indels | 0 | 0 | N/A | N/A | 53625 | 53625 | 100% | 99.99-100% |
| PDGFRA | CNVs | 0 | 0 | N/A | N/A | 13242 | 13242 | 100% | 99.97-100% |
| PMS2 | SNVs | 2338 | 2338 | 100% | 99.84-100% | 835626 | 835626 | 100% | >99.99-100% |
| PMS2 | Indels | 0 | 0 | N/A | N/A | 244884 | 244884 | 100% | >99.99-100% |
| Gene | Type | # Total
Positive
Variants* | # Detected
Positive
Variants* | PPA | 95% CI
of PPA | # Total
Negative
Variants* | # Detected
Negative
Variants* | NPA | 95% CI
of NPA |
| POLDI | CNVs | 112 | 112 | 100% | 96.68-100% | 2279 | 2279 | 100% | 99.83-100% |
| | SNVs | 630 | 630 | 100% | 99.39-100% | 834690 | 834690 | 100% | >99.99-100% |
| | Indels | 28 | 28 | 100% | 87.94-100% | 144075 | 144075 | 100% | >99.99-100% |
| POLE | CNVs | 0 | 0 | N/A | N/A | 15652 | 15652 | 100% | 99.98-100% |
| | SNVs | 1750 | 1750 | 100% | 99.78-100% | 1696266 | 1696266 | 100% | >99.99-100% |
| | Indels | 14 | 14 | 100% | 78.47-100% | 293832 | 293832 | 100% | >99.99-100% |
| | CNVs | 28 | 28 | 100% | 87.94-100% | 29256 | 29256 | 100% | 99.99-100% |
| PTEN | SNVs | 0 | 0 | N/A | N/A | 316410 | 316410 | 100% | >99.99-100% |
| | Indels | 0 | 0 | N/A | N/A | 268125 | 268125 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| RAD50 | SNVs | 70 | 70 | 100% | 94.80-100% | 896586 | 896586 | 100% | >99.99-100% |
| | Indels | 0 | 0 | N/A | N/A | 211575 | 211575 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 15050 | 15050 | 100% | 99.97-100% |
| RAD51C | SNVs | 14 | 14 | 100% | 78.47-100% | 353403 | 353403 | 100% | >99.99-100% |
| | Indels | 0 | 0 | N/A | N/A | 94953 | 94953 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 5418 | 5418 | 100% | 99.93-100% |
| RAD51D | SNVs | 336 | 336 | 100% | 98.87-100% | 314091 | 314091 | 100% | >99.99-100% |
| | Indels | 0 | 0 | N/A | N/A | 85203 | 85203 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 6020 | 6020 | 100% | 99.94-100% |
| Gene | Type | # Total
Positive
Variants* | # Detected
Positive
Variants* | PPA | 95% CI
of PPA | # Total
Negative
Variants* | # Detected
Negative
Variants* | NPA | 95% CI
of NPA |
| SDHA | SNVs | 1655 | 1641 | 99.15% | 98.59-99.50% | 523186 | 523151 | 99.99% | 99.99-100% |
| SDHA | Indels | 19 | 13 | 68.42% | 46.01-84.64% | 87287 | 87280 | 99.99% | 99.98-100% |
| SDHA | CNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| SDHB | SNVs | 378 | 378 | 100% | 98.99-100% | 234600 | 234600 | 100% | >99.99-100% |
| SDHB | Indels | 0 | 0 | N/A | N/A | 73965 | 73965 | 100% | 99.99-100% |
| SDHB | CNVs | 0 | 0 | N/A | N/A | 4813 | 4813 | 100% | 99.92-100% |
| SDHC | SNVs | 0 | 0 | N/A | N/A | 121191 | 121191 | 100% | >99.99-100% |
| SDHC | Indels | 84 | 84 | 100% | 95.63-100% | 23274 | 23274 | 100% | 99.98-100% |
| SDHC | CNVs | 0 | 0 | N/A | N/A | 0 | 0 | N/A | N/A |
| SDHD | SNVs | 70 | 70 | 100% | 94.80-100% | 127032 | 127032 | 100% | >99.99-100% |
| SDHD | Indels | 0 | 0 | N/A | N/A | 43875 | 43875 | 100% | 99.99-100% |
| SDHD | CNVs | 0 | 0 | N/A | N/A | 2408 | 2408 | 100% | 99.84-100% |
| SMAD4 | SNVs | 14 | 14 | 100% | 78.47-100% | 335493 | 335493 | 100% | >99.99-100% |
| SMAD4 | Indels | 0 | 0 | N/A | N/A | 91650 | 91650 | 100% | >99.99-100% |
| SMAD4 | CNVs | 0 | 0 | N/A | N/A | 7224 | 7224 | 100% | 99.95-100% |
| SMARCA4 | SNVs | 378 | 378 | 100% | 98.99-100% | 924498 | 924498 | 100% | >99.99-100% |
| SMARCA4 | Indels | 0 | 0 | N/A | N/A | 107847 | 107847 | 100% | >99.99-100% |
| SMARCA4 | CNVs | 0 | 0 | N/A | N/A | 21070 | 21070 | 100% | 99.98-100% |
| STK11 | SNVs | 154 | 154 | 100% | 97.57-100% | 390417 | 390417 | 100% | >99.99-100% |
| Gene | Type | # Total
Positive
Variants* | # Detected
Positive
Variants* | PPA | 95% CI
of PPA | # Total
Negative
Variants* | # Detected
Negative
Variants* | NPA | 95% CI
of NPA |
| | Indels | 0 | 0 | N/A | N/A | 129675 | 129675 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 5418 | 5418 | 100% | 99.93-100% |
| | SNVs | 378 | 378 | 100% | 98.99-100% | 346773 | 346773 | 100% | >99.99-100% |
| TP53 | Indels | 0 | 0 | N/A | N/A | 281292 | 281292 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 7224 | 7224 | 100% | 99.95-100% |
| | SNVs | 294 | 294 | 100% | 98.71-100% | 722523 | 722523 | 100% | >99.99-100% |
| TSC1 | Indels | 0 | 0 | N/A | N/A | 233940 | 233940 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 13830 | 13830 | 100% | 99.97-100% |
| | SNVs | 490 | 490 | 100% | 99.22-100% | 1499727 | 1499727 | 100% | >99.99-100% |
| TSC2 | Indels | 14 | 14 | 100% | 78.47-100% | 390954 | 390954 | 100% | >99.99-100% |
| | CNVs | 0 | 0 | N/A | N/A | 25282 | 25282 | 100% | 99.98-100% |
| | SNVs | 56 | 56 | 100% | 93.58-100% | 300780 | 300780 | 100% | >99.99-100% |
| VHL | Indels | 0 | 0 | N/A | N/A | 107250 | 107250 | 100% | >99.99-100% |
| | CNVs | 14 | 14 | 100% | 78.47-100% | 1764 | 1764 | 100% | 99.78-100% |

23

24

25

26

27

28

29

** # Detected positive/negative variants: the total number of positive/negative variants that were detected across replicates of the study samples.

*** N/A: values not calculated.

Linearity:

Not applicable.

1. DNA input:

30

During the test procedure, extracted DNA is quantified and normalized to 23ng/uL to create batches for assay processing. To evaluate the impact of different levels of DNA input on the Invitae Common Hereditary Cancers Panel test performance, eight whole blood clinical specimens with representative variants were tested across five input levels spanning 10 fold below and 2 fold above the standard input level of 23ng/uL. These samples include a total of 659 SNVs across 36 genes, 12 Indels across 6 genes, and 13 CNV across 4 genes in different genomic contexts (Table 12). The samples were tested at 1, 5, 10, 23 (standard concentration), and 46 ng/uL, each in triplicate, for a total of 120 samples (8 unique samples x 5 input levels x 3 replicates/ input level = 120 samples). Number of failed samples, number of samples with low coverage, overall concordance, PPA, and NPA compared to the 23ng/uL input were evaluated at each input level.

Results of the study are summarized in Table 13 to Table 15. At all DNA input levels, the overall concordance, PPA and NPA are >99% compared to the standard input. However, five sample failed QC metrics at 1ng/uL, and six samples failed QC metrics at 46ng/uL. Therefore, the minimum DNA input for the Invitae Common Hereditary Cancers Panel was determined to be 5 ng/uL. DNA input higher than 23ng/uL may lead to sample failure and should be normalized to 23ng/uL for assay processing. One variant type - CNV deletions - fell slightly below 95% PPA at 5 ng/uL. This was due to 2 false negative results for the same variant on the NF1 gene, which was in an AT rich region.

Genomic Context	SNVs	Insertions	Deletions	CNVs - Dup	CNVs - Del
Low mappability/complexity	212	2	4	2	7
GC rich regions	11	0	0	0	3
AT rich regions	115	0	3	1	6
All other	370	5	1	0	1

Table 12. Variant Distribution by Genomic Context

Table 13. DNA input Study Results Summary

| DNA Conc | # Samples with
Low Coverage* | # Failed
Samples | PPA
(95% CI) | NPA
(95% CI) | Overall Concordance
(95% CI) | No call
rate** |
|----------|---------------------------------|---------------------|--------------------------|---------------------------|---------------------------------|-------------------|
| 1 ng/uL | 0 | 5 | 99.95%
(99.72-99.99%) | >99.99%
(>99.99%-100%) | >99.99%
(>99.99%-100%) | 0.32% |
| 5 ng/uL | 0 | 0 | 99.90%
(99.65-99.97%) | >99.99%
(>99.99%-100%) | >99.99%
(>99.99%-100%) | 0.05% |
| 10 ng/uL | 0 | 0 | 99.95%
(99.72-99.99%) | >99.99%
(>99.99%-100%) | >99.99%
(>99.99%-100%) | 0.03% |
| 46 ng/uL | 0 | 6 | 99.71%
(99.36-99.87%) | >99.99%
(>99.99%-100%) | >99.99%
(>99.99%-100%) | 0.02% |

Samples with >400 bp (SNV and Indel analysis) or 138 target regions (CNV analysis) at 99.99% | >99.99-
100% | 364387 | 2 | >99.99% | >99.99-
100% | 15174 | 0 | 100% | >99.99-
100% |
| 5 ng/uL | 1451671 | 2 | >99.99% | >99.99-
100% | 364386 | 3 | >99.99% | >99.99-
100% | 19271 | 0 | 100% | >99.99-
100% |
| 10
ng/uL | 1451673 | 1 | >99.99% | >99.99-
100% | 364387 | 2 | >99.99% | >99.99-
100% | 19271 | 0 | 100% | >99.99-
100% |
| 46
ng/uL | 1451669 | 4 | >99.99% | >99.99-
100% | 364386 | 3 | >99.99% | >99.99-
100% | 14404 | 0 | 100% | >99.99-
100% |

TP: true positive; FN: false negative

4. Analytical Specificity/Interference:

Two studies were performed to evaluate the impact on assay performance of endogenous and exogenous substances that may be present in test samples due to carry-through from patient samples or due to processing conditions. In each study, seven potential interfering substances, including bilirubin, K2EDTA, wash buffer, TAFT, hemoglobin, and triglycerides, were each spiked at varying levels into specimens sourced from a blood bank. Blood from another donor was also tested in the first study to mimic a stem cell or bone marrow transplant patient. In the first study, samples were tested in three replicates at each level; in the second study, samples, enriched for Indels and CNVs, were tested in two replicates at each level (Table 16). The variants tested included those in challenging genomic contexts such as AT rich, GC rich, and low complexity regions. Results were analyzed for number of failed samples, number of samples with low coverage, overall concordance, PPA, and NPA compared to the un-spiked condition. Results of the interfering substances study are summarized in Table 20. Bilirubin, TAFTs, hemoglobin, and triglycerides were determined not to affect assay performance with the tested levels. Blood from a second donor caused the assay to fail at all levels of contamination as expected. False negative CNVs were reported in the samples with K2EDTA and wash buffer

32

added. In both cases, the replicates of the control sample were discordant with each other for the same variants. Inappropriate addition of PMS2 amplicon caused all samples to fail QC metrics for CNV detection, as well as decreased PPA for Indel detection. This is because the CNV calling algorithm cannot form a baseline when there is an unexpected read count due to the presence of the inappropriately introduced large amplicons. Therefore, amplicon presence should be stringently controlled in laboratory workflows, through use of filtered tips, automation and other good laboratory practices.

Study	Substance	# Samples	# Replicates	# SNVs	# Indels	# CNVs
1	Bilirubin	5	3	332	15	0
	Wash Buffer	5	3	383	11	0
	TAFTS	5	3	393	18	0
	K2EDTA	5	3	375	12	0
	Amplicon	5	3	395	0	0
	Hemoglobin	5	3	355	9	0
	Triglycerides	5	3	375	12	0
	Donor Blood	10	2	N/A*	N/A	N/A
2	Bilirubin	5	2	0	10	5
	Wash Buffer	5	2	0	11	9
	TAFTS	5	2	0	8	4
	K2EDTA	5	2	0	9	4
	Amplicon	5	2	0	8	0
	Hemoglobin	6	2	0	8	8
	Triglycerides	5	2	0	8	5

Table 16. Samples and Variants Tested in the Interference Substances Studies

*N/A: values not calculated because all samples failed QC metrics

Table 17. Interfering Substances Study Results Summary for SNVs (Study 1)

| Substance | Level | # Samples with
Low Coverage* | # Failed
Samples | PPA (95% CI) | NPA (95% CI) | Concordance (95% CI) |
|--------------------------------|------------|---------------------------------|---------------------|--------------------------|-------------------------|-------------------------|
| Bilirubin | 0 | 0 | 2 | N/A** | N/A | N/A |
| | 137 mmol/L | 0 | 1 | 100%
(98.9- 100%) | >99.9%
(>99.9- 100%) | >99.9%
(>99.9- 100%) |
| | 684 mmol/L | 0 | 1 | 100%
(98.9- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Wash buffer | 0 | 0 | 0 | N/A | N/A | N/A |
| | 15% v/v | 0 | 0 | 99.7%
(98.54- 99.95%) | 100%
(>99.9- 100%) | >99.9%
(>99.9- 100%) |
| TAFT
(molecular
barcode) | 0 | 0 | 0 | N/A | N/A | N/A |
| | 5% | 0 | 0 | 100%
(99.0- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |

33

| Substance | Level | # Samples with
Low Coverage* | # Failed
Samples | PPA (95% CI) | NPA (95% CI) | Concordance (95% CI) |
|-----------------------------------------|-----------|---------------------------------|---------------------|------------------------|-------------------------|-------------------------|
| | 15% | 0 | 0 | 100%
(99.0- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| | 30% | 0 | 0 | 100%
(99.0- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| K2EDTA | 0 | 0 | 0 | N/A | N/A | N/A |
| | 2.8 mg/mL | 0 | 0 | 100%
(99.0- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| | 7 mg/mL | 0 | 0 | 100%
(99.0- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Post-PCR
Amplicon | 0 | 0 | 0 | N/A | N/A | N/A |
| | 5% | 0 | 0 | 96.6%
(94.1- 98.0%) | >99.9%
(>99.9- 100%) | >99.9%
(>99.9- 100%) |
| | 15% | 0 | 0 | 96.6%
(94.1- 98.0%) | >99.9%
(>99.9- 100%) | >99.9%
(>99.9- 100%) |
| | 30% | 0 | 0 | 96.6%
(94.1- 98.0%) | >99.9%
(>99.9- 100%) | >99.9%
(>99.9- 100%) |
| Hemoglobin | 0 | 0 | 1 | N/A | N/A | N/A |
| | 1 mg/mL | 0 | 0 | 100%
(98.9- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| | 2 mg/mL | 0 | 2 | 100%
(98.9- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Triglycerides | 0 | 0 | 0 | N/A | N/A | N/A |
| | 100 mg/dL | 0 | 0 | 99.7%
(98.5- 100%) | 100%
(>99.9- 100%) | >99.9%
(>99.9- 100%) |
| | 250 mg/dL | 0 | 1 | 99.7%
(98.5- 100%) | 100%
(>99.9- 100%) | >99.9%
(>99.9- 100%) |
| Bone marrow/
stem cell
transplant | 0% | N/A | 20 | N/A | N/A | N/A |
| | 5% | N/A | 20 | N/A | N/A | N/A |
| | 10% | N/A | 20 | N/A | N/A | N/A |
| | 25% | N/A | 20 | N/A | N/A | N/A |
| | 75% | N/A | 20 | N/A | N/A | N/A |
| | 90% | N/A | 20 | N/A | N/A | N/A |
| | 95% | N/A | 20 | N/A | N/A | N/A |

Samples with >400 bp (SNV and Indel analysis) or 138 target regions (CNV analysis) at 99.9- 100%) | 100%
(>99.9- 100%) |
| Wash buffer | 0 | 0 | 0 | N/A | N/A | N/A |
| Wash buffer | 15% v/v | 0 | 0 | 100%
(74.1- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| TAFT
(molecular
barcode) | 0 | 0 | 0 | N/A | N/A | N/A |
| TAFT
(molecular
barcode) | 5% | 0 | 0 | 100%
(82.4- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| TAFT
(molecular
barcode) | 15% | 0 | 0 | 100%
(82.4- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| TAFT
(molecular
barcode) | 30% | 0 | 0 | 100%
(82.4- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| K2EDTA | 0 | 0 | 0 | N/A | N/A | N/A |
| K2EDTA | 2.8 mg/mL | 0 | 0 | 100%
(75.8- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| K2EDTA | 7 mg/mL | 0 | 0 | 100%
(75.8- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Post-PCR
Amplicon | 0 | 0 | 0 | N/A | N/A | N/A |
| Post-PCR
Amplicon | 5% | 0 | 0 | 100%
(75.8- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Post-PCR
Amplicon | 15% | 0 | 0 | 100%
(75.8- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Post-PCR
Amplicon | 30% | 0 | 0 | 100%
(75.8- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Hemoglobin | 0 | 0 | 1 | N/A | N/A | N/A |
| Hemoglobin | 1 mg/mL | 0 | 0 | 100%
(81.6- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Hemoglobin | 2 mg/mL | 0 | 2 | 100%
(81.6- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Triglycerides | 0 | 0 | 0 | N/A | N/A | N/A |
| Triglycerides | 100 mg/dL | 0 | 0 | 100%
(82.4- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Triglycerides | 250 mg/dL | 0 | 1 | 100%
(82.4- 100%) | 100%
(>99.9- 100%) | 100%
(>99.9- 100%) |
| Bone marrow/
stem cell
transplant | 0% | N/A | 20 | N/A | N/A | N/A |
| Bone marrow/
stem cell
transplant | 5% | N/A | 20 | N/A | N/A | N/A |
| Bone marrow/
stem cell
transplant | 10% | N/A | 20 | N/A | N/A | N/A |
| Bone marrow/
stem cell
transplant | 25% | N/A | 20 | N/A | N/A | N/A |
| Bone marrow/
stem cell
transplant | 75% | N/A | 20 | N/A | N/A | N/A |
| Substance | Level | Samples with
Low Coverage* | Failed
Samples | PPA (95% CI) | NPA (95% CI) | Concordance (95% CI) |
| | 90% | N/A | 20 | N/A | N/A | N/A |
| | તે તે જેની જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી ત | N/A | 20 | N/A | N/A | N/A |

34

35

Samples with >400 bp (SNV and Indel analysis) or 138 target regions (CNV analysis) at 99.99- 100%) |
| | 684 mmol/L | 0 | 0 | 100%
(83.89- 100%) | 100%
(99.95-100%) | 100%
(>99.9- 100%) |
| | 0 | 0 | 0 | N/A | N/A | N/A |
| Wash buffer | 15% v/v | 0 | 0 | 100%
(85.13- 100%) | 100%
(>99.99- 100%) | 100%
(>99.99- 100%) |
| | 0 | 0 | 0 | N/A | N/A | N/A |
| TAFT
(molecular
barcode) | 5% | 0 | 0 | 100%
(80.64- 100%) | 100%
(>99.99- 100%) | 100%
(>99.99- 100%) |
| | 15% | 0 | 1 | 100%
(78.47- 100%) | 100%
(>99.99- 100%) | 100%
(>99.99- 100%) |
| | 30% | 0 | 0 | 100%
(80.64- 100%) | 100%
(>99.99- 100%) | 100%
(>99.99- 100%) |
| | 0 | 0 | 0 | N/A | N/A | N/A |
| K2EDTA | 2.8 mg/mL | 0 | 0 | 100%
(82.41- 100%) | 100%
(>99.99- 100%) | 100%
(>99.99- 100%) |
| | 7 mg/mL | 0 | 0 | 100%
(82.41- 100%) | 100%
(>99.99- 100%) | 100%
(>99.99- 100%) |
| | 0 | 0 | 0 | N/A | N/A | N/A |
| Post-PCR
Amplicon | 5% | 0 | 0 | 93.75%
(71.67- 98.89%) | 100%
(>99.99- 100%) | >99.99%
(99.99- 100%) |
| | 15% | 0 | 0 | 87.50%
(63.98- 96.50%) | 100%
(>99.99- 100%) | >99.99%
(99.99- 100%) |
| | 30% | 0 | 0 | 93.75%
(71.67- 98.89%) | 100%
(>99.99- 100%) | >99.99%
(99.99- 100%) |
| Hemoglobin | 0 | 0 | 0 | N/A | N/A | N/A |
| | 1 mg/mL | 0 | 2 | 100%
(80.64- 100%) | 100%
(>99.99- 100%) | 100%
(99.9- 100%) |
| | 2 mg/mL | 0 | 1 | 100%
(80.64- 100%) | 100%
(>99.99- 100%) | 100%
(99.9- 100%) |
| | 0 | 0 | 0 | N/A | N/A | N/A |
| Triglycerides | 100 mg/dL | 0 | 0 | 100%
(80.64- 100%) | 100%
(>99.99- 100%) | 100%
(99.9- 100%) |

36

| Substance | Level | Samples with
Low Coverage* | Failed
Samples | PPA (95% CI) | NPA (95% CI) | Concordance (95% CI) |
|-----------|-----------|-------------------------------|-------------------|-----------------------|------------------------|----------------------|
| | 250 mg/dL | 0 | 0 | 100%
(80.64- 100%) | 100%
(>99.99- 100%) | 100%
(99.9- 100%) |

Samples with >400 bp (SNV and Indel analysis) or 138 target regions (CNV analysis) at 99.99%
(99.99-100%) |
| | 7 mg/mL | 0 | 0 | 87.5%
(52.91 - 97.6%) | 99.99%
(99.93-100%) | >99.99%
(99.99-100%) |
| | 0 | 0 | 10 | N/A | N/A | N/A |
| Post-PCR
Amplicon | 5% | 0 | 10 | N/A | N/A | N/A |
| | 15% | 0 | 10 | N/A | N/A | N/A |
| | 30% | 0 | 10 | N/A | N/A | N/A |
| | 0 | 0 | 0 | N/A | N/A | N/A |
| Hemoglobin | 1 mg/mL | 0 | 2 | 100%
(78.47- 100%) | 100%
(99.95-100%) | 100%
(99.9- 100%) |
| | 2 mg/mL | 0 | 1 | 100%
(79.61- 100%) | 100%
(99.95-100%) | 100%
(99.9- 100%) |
| | 0 | 0 | 0 | N/A | N/A | N/A |
| Triglycerides | 100 mg/dL | 0 | 0 | 100%
(72.25- 100%) | 100%
(99.95-100%) | 100%
(99.9- 100%) |
| | 250 mg/dL | 0 | 0 | 100%
(72.25- 100%) | 100%
(99.95-100%) | 100%
(99.9- 100%) |

37

Samples with >400 bp (SNV and Indel analysis) or 138 target regions (CNV analysis) at 7), medium molecular weight (3-7), and low molecular weight (7 | 0 | 0 | N/A** | N/A | N/A |
| 3-7 | 0 | 0 | 100%
(99.0-100%) | 100%
(>99.9-100%) | 100%
(100%-100%) |

Table 22. DNA Integrity Study Results Summary

38

| 99.9- 100%) | (100%-100%) |
| 0 | | | |

Samples with >400 bp (SNV and Indel analysis) or 138 target regions (CNV analysis) at 99% at all timepoints for all conditions, supporting the stability of the specimens for the assay performance with the Invitae Common Hereditary Cancers Panel.

| Condition | Time
Point | PPA | | | | NPA | | | | Overall concordance | |
|--------------|---------------|------|----|-------|---------------|--------|----|--------|----------------|----------------------|--|
| | | TP | FN | PPA | 95%
CI | TN | FP | NPA | 95% CI | Concordance (95% CI) | |
| Room
Temp | 7 days | 670 | 0 | 100% | 99.4-
100% | 680802 | 1 | >99.9% | >99.9-
100% | >99.9% (>99.9-100%) | |
| | 30
days | 670 | 0 | 100% | 99.4-
100% | 679563 | 0 | 100% | >99.9-
100% | 100% (>99.9-100%) | |
| | 60
days | 670 | 0 | 100% | 99.4-
100% | 680815 | 0 | 100% | >99.9-
100% | >99.9% (>99.9-100%) | |
| | 90
days | 587* | 0 | 100% | 99.4-
100% | 605178 | 0 | 100% | >99.9-
100% | >99.9% (>99.9-100%) | |
| 4°C | 7 days | 807 | 2 | 99.8% | 99.3-
100% | 756443 | 1 | >99.9% | >99.9-
100% | >99.9% (>99.9-100%) | |

Table 23. Specimen Stability Study Results Summary

39

| Condition | Time
Point | PPA | | | | NPA | | | | Overall concordance |
|-----------------|---------------|-------------|----|--------|----------------|---------|----|--------|----------------|----------------------|
| | | TP | FN | PPA | 95%
CI | TN | FP | NPA | 95% CI | Concordance (95% CI) |
| | 30
days | 412** | 0 | 100% | 99.1 -
100% | 452488 | 0 | 100% | >99.9-
100% | 100% (>99.9-100%) |
| | 60
days | 807 | 1 | 99.9% | 99.3-
100% | 756471 | 1 | >99.9% | >99.9-
100% | >99.9% (>99.9-100%) |
| | 90
days | 807 | 1 | 99.9% | 99.3-
100% | 756469 | 1 | >99.9% | >99.9-
100% | >99.9% (>99.9-100%) |
| 40 °C | 3 hr | 2612 | 0 | 100% | 99.9-
100% | 2190407 | 0 | 100% | >99.9-
100% | 100% (>99.9-100%) |
| | 12 hr | 2421*
** | 0 | 100% | 99.9-
100% | 2039346 | 2 | >99.9% | >99.9-
100% | >99.9% (>99.9-100%) |
| | 7 days | 2612 | 0 | 100% | 99.9-
100% | 2190405 | 0 | 100% | >99.9-
100% | 100% (>99.9-100%) |
| Freeze/
Thaw | 1
cycle | 2612 | 0 | 100% | 99.9-
100% | 2190407 | 1 | >99.9% | >99.9-
100% | >99.9% (>99.9-100%) |
| | 3
cycles | 2611 | 1 | >99.9% | 99.9-
100% | 2190402 | 0 | >99.9% | >99.9-
100% | >99.9% (>99.9-100%) |

9 samples were included in the analysis at this time point

** 4 samples failed due to an instrument-related failure, unrelated to the study condition.

*** 2 samples failed due to an instrument-related failure, unrelated to the study condition.

b) Reagent Stability

Studies were performed to establish the stability of the master plates of TAFTs (molecular barcodes), the diluted TAFT plates that are stamped and later used in production, and the baits. For master stock TAFT plates, five stamped plates from each of 3 stock plate designs from each of three manufactured plate lots, representing 303 ± 94 samples per stock plate were analyzed for the percentage of samples that passed the TAFT QC metrics. For diluted TAFT plates, all available plates older than 1 year at time of use, for each of 3 selected plate designs from each of three manufactured plate lots, representing 450±48 samples run were analyzed for the percentage of samples that passed the TAFT QC metrics. For the baits, control sample data for the genomic regions targeted by the bait components were analyzed for gaps in coverage and concordance with the reference sequence. The results of the study are summarized in Table 24. All reagents analyzed met the acceptance criteria, establishing stability as follows:

Master stock plates stored frozen are stable for at least 36 months ●
Diluted plates are stable for at least 12 months ●
Baits are stable for at least 33 months ●

Table 24 Summary of Reagent Stability

Reagent	Passed Number of Reads	Passed Proportion Classified
Master molecular barcode plates	95.3% - 99.2%	99.0% - 100%
Diluted molecular barcode plates	96.4% - 99.0%	96.3% - 100%

40

Reagent	Gaps in Coverage	Concordance
Baits	0	100%

9. Multiplexing:

The Invitae Common Hereditary Cancers Panel test is multiplexed at two steps:

At library prep: Libraries are pooled by row to create 8 pools of 12 libraries each. .
At sequencing: Equimolar mixing of hybridization reactions forms sequencing pools that . allow each library to be sequenced with a minimum of 4,000,000 clusters on the Illumina flow cell. The number of samples in a pool depends on what other assays are being run at the same time.

Unique molecular barcodes are added to individual samples during extraction to ensure that sample identification is maintained throughout the process, even when multiplexed. QC checks built into the test system verify that the molecular barcodes have performed as expected. As demonstrated in the interfering substances study, the molecular barcodes - referred to as TAFTs - spiked at 5%. 15%, and 30% above the standard operational levels did not impact performance of the test. An additional analysis was performed using data from the analytical validation studies. In these studies, each sample has, on average, over 60 variant calls distributed across the 47 loci. Thus, each sample has a nearly unique sequencing footprint. This provides high confidence that the performance of the molecular barcodes for preserving sample identity fidelity can be confirmed using the concordance analysis of the sequencing results. All of the studies performed across all validations were multiplexed with post hybridization multiplexing levels (pool sizes) between 49 to 1485. Pooling sizes and configurations are designed so that read depth can be maintained at a level that meets quality thresholds. Read depth are maintained at a level that meets quality thresholds for all post hybridization multiplexing levels. No trends were observed that suggested that the pool size negatively impacted the read depth.

10. Guardbanding:

A guardbanding study was conducted to establish the robustness of the Invitae Common Hereditary Cancers Panel test by assessing the impact of varying critical input parameters - the DNA input concentration at the library preparation, hybridization, and sequencing steps - related to test run quality metrics. To assess the impact of varying these parameters, eight DNA samples from clinical specimens were run in duplicate with a series of manual manipulations during runs, wherein the input concentrations were varied by +/-5% and +/-10% at each step. The samples contained representative variants across variant types and different genomic context, including 753 SNVs across 39 genes, 14 insertions/deletions across 7 genes and 8 CNVs across 6 genes. Assay results were analyzed for number of failed samples, number of samples with low coverage, overall concordance, PPA, and NPA compared to the standard condition. Results of the study are summarized in Table 25. The assay was demonstrated to be robust when input concentrations for the library prep, hybridization, and sequencing steps were varied up to 10%.

41

| DNA Input
Concentration | | Samples with
Low
coverage* | Failed
Samples | PPA
(95% CI) | NPA
(95% CI) | Concordance
(95% CI) |
|----------------------------|------|----------------------------------|-------------------|-------------------------|--------------------------|--------------------------|
| Library Prep | -10% | 1 | 0 | 99.9%
(99.53-99.96%) | 100%
(100-100%) | >99.99%
(>99.99-100%) |
| | -5% | 0 | 0 | 99.9%
(99.53-99.96%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| | +5% | 0 | 0 | 99.9%
(99.43-99.93%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| | +10% | 0 | 0 | 99.9%
(99.43-99.93%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| Hybridization | -10% | 0 | 0 | 99.9%
(99.53-99.96%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| | -5% | 0 | 0 | 99.9%
(99.53-99.96%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| | +5% | 0 | 0 | 100%
(99.75-100%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| | +10% | 0 | 0 | 99.9%
(99.53-99.96%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| Sequencing | -10% | 1 | 0 | 99.9%
(99.53-99.96%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| | -5% | 0 | 0 | 99.9%
(99.53-99.96%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| | +5% | 0 | 0 | 100%
(99.75-100%) | >99.99%
(>99.99-100%) | >99.99%
(>99.99-100%) |
| | +10% | 0 | 0 | 100%
(99.75-100%) | 100% (100-
100%) | 100% (100-
100%) |

Table 25. Guard banding Study Results Summary

Samples with >400 bp (SNV and Indel analysis) or 138 target regions (CNV analysis) at 99.9%) for SNVs, 100% (95% CI 99.9- 100%) for Indels and 99.5% (95% CI 99.2- 99.7%) for CNVs. The overall TNPV is 100% for SNVs (95% CI >99.9%- 100%), 100% for Indels (95% CI >99.9- 100%), and 99.7% for CNVs (95% CI 99.6- 99.7%).

4 For methods of calculation, refer to Section B, Test Performance Characteristics in the Guidance Document 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 Stakeholders and Food and Drug Administration Staff.

44

| Gene | #
Samples | #
Variants | TP | TN | FP | FN | TPPV
(95% CI) | TNPV
(95% CI) |
|-------------------------|--------------|---------------|------|---------|----|-----|------------------------|---------------------------|
| SNVs | 2151 | 2181 | 2180 | 413004 | 1 | 0 | 99.9%
(99.7->99.9%) | 100%
(>99.9%- 100%) |
| Indels | 3900 | 3914 | 3914 | 1127320 | 0 | 0 | 100%
(99.9- 100%) | 100%
(>99.9- 100%) |
| Insertion
s | 1240 | 1240 | 1240 | 781671 | 0 | 0 | 100%
(99.7-100%) | 100%
(>99.9-100%) |
| Deletions | 2671 | 2674 | 2674 | 345649 | 0 | 0 | 100%
(99.9-100%) | 100%
(>99.9-100%) |
| CNVs | 3648 | 3601 | 3582 | 95965 | 19 | 317 | 99.5%
(99.2- 99.7%) | 99.7%
(99.6- 99.7%) |
| CNV
deletions | 2983 | 3110 | 3103 | 95965 | 7 | 292 | 99.8%
(99.5-99.9%) | 99.7% (99.6-
99.7%) |
| CNV
duplicati
ons | 484 | 491 | 479 | 95965 | 12 | 25 | 97.6%
(95.8-98.6%) | 99.97% (99.96-
99.98%) |

Table 29. Summary of Accuracy Study with Clinical Specimens

TP: True positive; TN: True negative; FP: False positive; FN: False negative

Accuracy result on the gene level is shown in Table 30 and Table 31. For SNVs and Indels (Table 30), all genes tested show TPPV of 100%, except SDHA (99.0% with 95% CI 94.4-99.8%). For CNVs (Table 31), 32 genes show TPPV of 100%, 10 genes show TPPV ranging between 90% to 100%, and 2 genes show TPPV between 80% to 90%, including SMAD4 (84.6% with 95% CI 57.8-95.7%) and TSC2 (88.9% with 95% CI 56.5-98.0%).

| Gene | #
Samples | # Variants | | TP | TN | FP | FN | TPPV
(95% CI) | TNPV
(95% CI) |
|----------------|--------------|---------------|--------|-------|--------|-----|-----------------------|-------------------------|------------------------|
| | | SNVs | Indels | | | | | | |
| APC | 165 | 40 | 125 | 165 | 51987 | 0 | 0 | 100%
(97.7-100.0%) | 100%
(>99.9-100.0%) |
| ATM | 629 | 255 | 386 | 641 | 108628 | 0 | 0 | 100%
(99.1-100.0%) | 100%
(>99.9-100.0%) |
| AXIN2 | 32 | 28 | 4 | 32 | 7151 | 0 | 0 | 100%
(89.3-100.0%) | 100%
(>99.9-100.0%) |
| BARDI | 89 | 17 | 72 | 89 | 14267 | 0 | 0 | 100%
(95.9-100.0%) | 100%
(>99.9-100.0%) |
| BMPR1A | 10 | 4 | 6 | 10 | 1503 | 0 | 0 | 100%
(72.2-100.0%) | 100%
(99.8-100.0%) |
| BRCA1 | 606 | 154 | 452 | 606 | 177627 | 0 | 0 | 100%
(99.4-100.0%) | 100%
(>99.9-100.0%) |
| BRCA2 | 1093 | 97 | 996 | 1093 | 480847 | 0 | 0 | 100%
(99.8-100.0%) | 100%
(>99.9-100.0%) |
| BRIP1 | 260 | 121 | 139 | 260 | 50700 | 0 | 0 | 100%
(98.5-100.0%) | 100%
(>99.9-100.0%) |
| Gene | # Samples | # Variants | | TP | TN | FP | FN | TPPV (95% CI) | TNPV (95% CI) |
| | | SNVs | Indels | | | | | | |
| CDH1 | 39 | 7 | 32 | 39 | 8539 | 0 | 0 | 100%
(91.0-100.0%) | 100%
(>99.9-100.0%) |
| CDK4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | N/A | N/A |
| CDKN2A | 128 | 62 | 66 | 128 | 26877 | 0 | 0 | 100%
(97.1-100.0%) | 100%
(>99.9-100.0%) |
| CHEK2 | 354 | 170 | 185 | 355 | 48503 | 0 | 0 | 100%
(98.4-100.0%) | 100%
(>99.9-100.0%) |
| CTNNA1 | 7 | 6 | 1 | 7 | 1379 | 0 | 0 | 100%
(64.6-100.0%) | 100%
(99.7-100.0%) |
| DICER1 | 24 | 6 | 18 | 24 | 8360 | 0 | 0 | 100%
(86.2-100.0%) | 100%
(>99.9-100.0%) |
| EPCAM | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
| GREM1 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
| HOXB13 | 2 | 2 | 0 | 2 | 607 | 0 | 0 | 100%
(34.2-100.0%) | 100%
(99.4-100.0%) |
| KIT | 6 | 5 | 1 | 6 | 1038 | 0 | 0 | 100%
(61.0-100.0%) | 100%
(99.6-100.0%) |
| MEN1 | 77 | 22 | 55 | 77 | 28100 | 0 | 0 | 100%
(95.2-100.0%) | 100%
(>99.9-100.0%) |
| MLH1 | 102 | 14 | 88 | 102 | 16999 | 0 | 0 | 100%
(96.4-100.0%) | 100%
(>99.9-100.0%) |
| MSH2 | 154 | 105 | 49 | 154 | 27897 | 0 | 0 | 100%
(97.6-100.0%) | 100%
(>99.9-100.0%) |
| MSH3 | 74 | 35 | 39 | 74 | 10489 | 0 | 0 | 100%
(95.1-100.0%) | 100%
(>99.9-100.0%) |
| MSH6 | 365 | 30 | 335 | 365 | 104841 | 0 | 0 | 100%
(99.0-100.0%) | 100%
(>99.9-100.0%) |
| MUTYH | 85 | 52 | 35 | 87 | 14587 | 0 | 0 | 100%
(95.8-100.0%) | 100%
(>99.9-100.0%) |
| NBN | 93 | 15 | 78 | 93 | 17339 | 0 | 0 | 100%
(96.0-100.0%) | 100%
(>99.9-100.0%) |
| NF1 | 396 | 241 | 156 | 397 | 71058 | 0 | 0 | 100%
(99.0-100.0%) | 100%
(>99.9-100.0%) |
| NTHL1 | 43 | 37 | 6 | 43 | 9795 | 0 | 0 | 100%
(91.8-100.0%) | 100%
(>99.9-100.0%) |
| PALB2 | 167 | 50 | 117 | 167 | 50788 | 0 | 0 | 100%
(97.8-100.0%) | 100%
(>99.9-100.0%) |
| PDGFRA | 6 | 6 | 0 | 6 | 1191 | 0 | 0 | 100%
(61.0-100.0%) | 100%
(99.7-100.0%) |
| PMS2 | 293 | 123 | 171 | 294 | 65417 | 0 | 0 | 100% | 100% |
| Gene | # Samples | # Variants | | TP | TN | FP | FN | TPPV
(95% CI) | TNPV
(95% CI) |
| | | SNVs | Indels | | | | | | |
| POLDI | 15 | 13 | 2 | 15 | 2621 | 0 | 0 | 100%
(79.6-100.0%) | 100%
(>99.9-100.0%) |
| POLE | 22 | 16 | 6 | 22 | 3899 | 0 | 0 | 100%
(85.1-100.0%) | 100%
(99.9-100.0%) |
| PTEN | 21 | 12 | 8 | 20 | 3877 | 0 | 0 | 100%
(77.3-99.2%) | 100%
(99.9-100.0%) |
| RAD50 | 143 | 17 | 127 | 144 | 30893 | 0 | 0 | 100%
(97.4-100.0%) | 100%
(>99.9-100.0%) |
| RAD51C | 17 | 3 | 14 | 17 | 1521 | 0 | 0 | 100%
(81.6-100.0%) | 100%
(99.8-100.0%) |
| RAD51D | 18 | 9 | 9 | 18 | 2175 | 0 | 0 | 100%
(82.4-100.0%) | 100%
(99.8-100.0%) |
| SDHA | 97 | 79 | 18 | 96 | 12367 | 1 | 0 | 99.0%
(94.4-99.8%) | 100%
(>99.9-100.0%) |
| SDHB | 47 | 15 | 32 | 47 | 5752 | 0 | 0 | 100%
(92.4-100.0%) | 100%
(>99.9-100.0%) |
| SDHC | 5 | 2 | 3 | 5 | 470 | 0 | 0 | 100%
(56.6-100.0%) | 100%
(99.2-100.0%) |
| SDHD | 11 | 2 | 9 | 11 | 1554 | 0 | 0 | 100%
(74.1-100.0%) | 100%
(99.8-100.0%) |
| SMAD4 | 18 | 4 | 14 | 18 | 2822 | 0 | 0 | 100%
(82.4-100.0%) | 100%
(99.9-100.0%) |
| SMARCA4 | 19 | 19 | 0 | 19 | 3770 | 0 | 0 | 100%
(83.2-100.0%) | 100%
(99.9-100.0%) |
| STK11 | 15 | 6 | 9 | 15 | 2576 | 0 | 0 | 100%
(79.6-100.0%) | 100%
(99.9-100.0%) |
| TP53 | 289 | 260 | 32 | 294 | 50845 | 0 | 0 | 100%
(97.6-99.8%) | 100%
(>99.9-100.0%) |
| TSC1 | 9 | 5 | 4 | 9 | 2516 | 0 | 0 | 100%
(70.1-100.0%) | 100%
(99.9-100.0%) |
| TSC2 | 26 | 11 | 15 | 26 | 5342 | 0 | 0 | 100%
(87.1-100.0%) | 100%
(99.9-100.0%) |
| VHL | 4 | 4 | 0 | 4 | 810 | 0 | 0 | 100%
(51.0-100.0%) | 100%
(99.5-100.0%) |
| Gene | #
Samples | #
Variants | TP | TN | FP | FN | TPPV* | TNPV | |
| APC | 113 | 117 | 114 | 3149 | 3 | 15 | 97.4%
(92.7-99.1%) | 99.5%
(99.2-99.7%) | |
| ATM | 177 | 183 | 183 | 7745 | 0 | 37 | 100%
(97.9-100%) | 99.5%
(99.4-99.7%) | |
| AXIN2 | 1 | 1 | 1 | N/A | 0 | N/A | 100%
(20.7-100%) | N/A ** | |
| BARDI | 66 | 67 | 67 | N/A | 0 | N/A | 100%
(94.6-100%) | N/A | |
| BMPR1A | 26 | 26 | 26 | 1995 | 0 | 4 | 100%
(87.1-100%) | 99.8%
(99.5-99.9%) | |
| BRCA1 | 577 | 580 | 577 | 3208 | 3 | 3 | 99.5%
(98.5-99.8%) | 99.91%
(99.7->99.9%) | |
| BRCA2 | 181 | 183 | 181 | 6895 | 2 | 25 | 98.9%
(96.1-99.7%) | 99.64%
(99.5-99.8%) | |
| BRIP1 | 63 | 69 | 69 | 1995 | 0 | 7 | 100%
(94.7-100%) | 99.65%
(99.3-99.8%) | |
| CDH1 | 59 | 59 | 59 | 2396 | 0 | 6 | 100%
(93.9-100%) | 99.75%
(99.5-99.9%) | |
| CDK4 | 2 | 2 | 2 | N/A | 0 | N/A | 100%
(34.2-100%) | N/A | |
| CDKN2A | 12 | 12 | 12 | 1886 | 0 | 8 | 100%
(75.8-100%) | 99.6%
(99.2-99.8%) | |
| CHEK2 | 263 | 266 | 266 | 1717 | 0 | 8 | 100%
(98.6-100%) | 99.5%
(99.1-99.8%) | |
| CTNNA1 | 12 | 12 | 12 | N/A | 0 | N/A | 100%
(75.8-100%) | N/A | |
| DICERI | 10 | 10 | 10 | N/A | 0 | N/A | 100%
(72.3-100%) | N/A | |
| EPCAM | 113 | 114 | 113 | 523 | 1 | 5 | 99.1%
(95.2-99.8%) | 99.1%
(97.8-99.6%) | |
| GREM1 | 23 | 24 | 23 | N/A | 1 | N/A | 95.8%
(79.8-99.3%) | N/A | |
| HOXB13 | 2 | 2 | 2 | N/A | 0 | N/A | 100%
(34.2-100%) | N/A | |
| KIT | 1 | 1 | 1 | N/A | 0 | N/A | 100%
(20.7-100%) | N/A | |
| MENI | 13 | 13 | 13 | 2730 | 0 | 5 | 100%
(77.2-100%) | 99.8%
(99.6-99.9%) | |
| MLH1 | 134 | 135 | 135 | 4505 | 0 | 18 | 100%
(97.2-100%) | 99.6%
(99.4-99.75%) | |
| MSH2 | 285 | 297 | 297 | 3147 | 0 | 7 | 100%
(98.7-100%) | 99.8%
(99.5-99.9%) | |
| MSH3 | 45 | 45 | 44 | N/A | 1 | N/A | 97.8%
(88.4-99.6) | N/A | |
| Gene | #
Samples | #
Variants | TP | TN | FP | FN | TPPV* | TNPV | |
| MSH6 | 60 | 63 | 63 | 2306 | 0 | 4 | 100%
(94.3-100%) | 99.8%
(99.6-99.9%) | |
| MUTYH | 20 | 21 | 21 | 5879 | 0 | 4 | 100%
(84.5-100%) | 99.9%
(99.8-99.97%) | |
| NBN | 67 | 68 | 68 | 1787 | 0 | 9 | 100%
(94.7-100%) | 99.5%
(99.1-99.7%) | |
| NF1 | 104 | 108 | 107 | 11296 | 1 | 39 | 99.1%
(94.9-99.8%) | 99.7%
(99.5-99.8%) | |
| NTHL1 | 7 | 7 | 7 | 1048 | 0 | 7 | 100%
(64.6-100%) | 99.3%
(98.6-99.68%) | |
| PALB2 | 255 | 255 | 255 | 1674 | 0 | 7 | 100%
(98.5-100%) | 99.6%
(99.1-99.8%) | |
| PDGFRA | 1 | 1 | 1 | N/A | 0 | N/A | 100%
(20.7-100%) | N/A | |
| PMS2 | 414 | 420 | 418 | 3428 | 2 | 26 | 99.5%
(98.3-99.9%) | 99.3%
(98.9-99.5%) | |
| POLD1 | 3 | 3 | 3 | N/A | 0 | N/A | 100%
(43.9-100%) | N/A | |
| POLE | 10 | 10 | 10 | N/A | 0 | N/A | 100%
(72.3-100%) | N/A | |
| PTEN | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
| RAD50 | 33 | 33 | 33 | 735 | 0 | 2 | 100%
(89.6-100%) | 99.7%
(99.0-99.9%) | |
| RAD51C | 116 | 116 | 116 | 1050 | 0 | 6 | 100%
(96.8-100%) | 99.4%
(98.8-99.7%) | |
| RAD51D | 43 | 44 | 44 | N/A | 0 | N/A | 100%
(92.0-100%) | N/A | |
| SDHA | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
| SDHB | 35 | 35 | 35 | N/A | 0 | N/A | 100%
(90.1-100%) | N/A | |
| SDHC | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | |
| SDHD | 17 | 18 | 17 | N/A | 1 | N/A | 94.4%
(74.2-99.0%) | N/A | |
| SMAD4 | 12 | 13 | 11 | 1888 | 2 | 3 | 84.6%
(57.8-95.7%) | 99.8%
(99.5-99.95%) | |
| SMARCA
4 | 4 | 4 | 4 | N/A | 0 | N/A | 100%
(51.0-100%) | N/A | |
| STK11 | 50 | 50 | 50 | 1302 | 0 | 12 | 100%
(92.9-100%) | 99.1%
(98.4-99.5%) | |
| TP53 | 33 | 33 | 32 | 3045 | 1 | 10 | 97.0%
(84.7-99.5%) | 99.7%
(99.4-99.8%) | |
| TSC1 | 17 | 17 | 17 | 3450 | 0 | 6 | 100%
(81.6-100%) | 99.8%
(99.6-99.9%) | |
| TSC2 | 9 | 9 | 8 | 14258 | 1 | 32 | 88.9%
(56.5-98.0%) | 99.78%
(99.7-99.8%) | |
| VHL | 55 | 55 | 55 | 928 | 0 | 2 | 100% | 99.78% | |
| Gene | #
Samples | #
Variants | TP | TN | FP | FN | TPPV* | TNPV | |
| | | | | | | | (93.5-100%) | (99.2-99.9%) | |

Table 30. Accuracy by Stratified by Gene - SNVs and Indels

45

46

TP: True positive; TN: True negative; FP: False positive; FN: False negative

*N/A: values not calculated (for CDK4) or not evaluated as the test does not offer SNV/Indel detection (for EPCAM and GREM1)

47

Table 31 Accuracy Stratified by Gene – CNVs

48

49

TP: True positive; TN: True negative; FP: False positive; FN: False negative

*Genes in bold are those that did not meet the 99% performance expectation for CNVs. The data showed that the false positives were due to those positives called based on one exon.

**N/A: not evaluated as the test does not offer CNV detection for the genes or data not available for calculation.

Accuracy stratified by indel size is shown in Table 32. TPPV is 100% across all insertion and deletion sizes.

Variant Type	Size	TP	TPPV (95% CI)
Insertions	1-5 bp	974	100% (99.6-100%)
	6-10 bp	92	100% (96.0-100%)
	11-20 bp	87	100% (95.8-100%)
	21+ bp*	87	100% (95.8-100%)
Deletions	1-5 bp	2351	100% (99.8-100%)
	6-10 bp	157	100% (97.6-100%)
	11-20 bp	114	100% (96.7-100%)
	21+ bp*	52	100% (93.1-100%)

Table 32. Accuracy Stratified by Indel Size

TP: True positive; FP: False positive

*The largest size tested are 338bp for insertions and 313bp for deletions

Accuracy stratified by CNV size is shown in Table 33. TPPV is >99% across all CNV deletion and duplication sizes except that lower TPPV was observed for CNV duplications with single exon or less (95.5% with 95% CI 92.4-97.4%).

Table 33 Accuracy Stratified by CNV Size

CNV type	CNV Type	TP	FP	TPPV (95% CI)
CNV Deletions	≤ Single Exon	1093	6	99.5% (98.8-99.8%)
	2-5 Exons	1075	0	100% (99.6-100%)
	6-9 Exons	345	0	100% (98.9-100%)
	10+ Exons	226	1	99.6% (97.6-99.9%)
	Entire Coding Sequence	308	0	100% (98.8-100%)
	Other (intronic, non-coding, combination)	56	0	100% (93.6-100%)

50

CNV type	CNV Type	TP	FP	TPPV (95% CI)
CNV Duplications	≤ Single Exon	257	12	95.5% (92.4-97.4%)
	2-5 Exons	108	0	100% (96.6-100%)
	6-9 Exons	28	0	100% (87.9-100%)
	10+ Exons	41	0	100% (91.4-100%)
	Entire Coding Sequence	40	0	100% (91.2-100%)
	Other (intronic, non-
coding, combination)	5	0	100% (56.6-100%)

TP: True positive; FP: False positive

Accuracy stratified by GC content is shown in Table 34. TPPV is >99% across all GC content ranges except for CNV deletions with GC content between 25% to 30% (98.6% with 95% CI 92.3-99.8%), CNV duplications with GC content between 30% to 55% (98.5% with 95% CI 96.6-99.4%), and GC content >55% (95.5% with 95% CI 91.0-97.8%).

| Variant

Type	Stratification	# of Variants	TP	FP	TPPV (95% CI)
SNVs	GC content 0-15	0	0	0	-
	GC content 15- 20	1	1	0	100%
(20.7-100%)
	GC content 20- 25	236	236	0	100%
(98.4-100%)
	GC content 25- 30	216	216	0	100%
(98.3-100%)
	GC content 30- 55	1123	1123	0	100%
(99.7-100%)
	GC content >55	605	604	1	99.8%
(99.1->99.9%)
Insertions	GC content 0-15	0	0	0	-
	GC content 15- 20	0	0	0	-
	GC content 20- 25	14	14	0	100%
(78.5-100%)
	GC content 25- 30	183	183	0	100%
(97.9-100%)
	GC content 30- 55	837	837	0	100%
(99.5-100%)
	GC content >55	125	125	0	100%
(97.0-100%)
Deletions	GC content 0-15	0	0	0	-
	GC content 15- 20	1	1	0	100%
Variant
Type	Stratification	# of Variants	TP	FP	TPPV (95% CI)
	GC content 20-25	74	74	0	100%
(95.1-100%)
	GC content 25-30	408	408	0	100%
(99.1-100%)
	GC content 30-55	2049	2049	0	100%
(99.8-100)
	GC content >55	142	142	0	100%
(97.4-100%)
	GC content 0-15	0	0	0	-
	GC content 15-20	0	0	0	-
Delins*	GC content 20-25	1	1	0	100%
(20.7-100%)
	GC content 25-30	2	2	0	100%
(34.24-100%)
	GC content 30-55	66	66	0	100%
(94.5-100%)
	GC content >55	12	12	0	100%
(75.8-100%)
	GC content 0-15	0	0	0	-
	GC content 15-20	0	0	0	-
	GC content 20-25	18	18	0	100%
(82.4-100%)
CNV
Deletions	GC content 25-30	70	69	1	98.6%
(92.3-99.8%)
	GC content 30-55	2596	2591	5	99.8%
(99.6-99.9%)
	GC content >55	426	425	1	99.8%
(98.7-100%)
	GC content 0-15	0	0	0	-
	GC content 15-20	0	0	0	-
	GC content 20-25	0	0	0	-
CNV
Duplications	GC content 25-30	0	0	0	-
	GC content 30-55	335	330	5	98.5%
(96.6-99.4%)
	GC content >55	156	149	7	95.5%
(91.0-97.8%)
	GC content 0-15	0	0	0	-

Table 34. Accuracy Stratified by Variant Type and GC content

51

TP: True positive; FP: False positive

*A delins refers to a sequence change where, compared to a reference sequence, one or more nucleotides are replaced by one or more other nucleotides and which is not a substitution, inversion or conversion.

2. Matrix Comparison:

52

Not applicable

C Clinical Studies:

1. Clinical Sensitivity:
  Refer to Section VI.B, Comparison Study using Clinical Specimens

2. Clinical Specificity:

Refer to Section VI.B, Comparison Study using Clinical Specimens

3. Other Clinical Supportive Data: Evaluation of Variant Classification, Interpretation, and Reporting

a) Genotype-phenotype associations
Genotype-phenotype associations for the 47 panel genes are summarized in Table 35.

Gene*	Syndrome / Cancer
APC	AD familial adenomatous polyposis (FAP), attenuated FAP (AFAP), gastric adenocarcinoma and
proximal polyposis of the stomach (GAPPS), colorectal, small bowel, gastric, liver, brain, pancreatic,
and thyroid cancers.
ATM	AR ataxia-telangiectasia, AD predisposition to breast, ovarian, pancreatic and prostate cancer.
AXIN2	AD oligodontia-colorectal cancer syndrome
BARDI	AD breast cancer
BMPR1A	AD juvenile polyposis syndrome (JPS), colorectal, small bowel, gastric and pancreatic cancers
BRCA1	AR Fanconi anemia, AD hereditary breast and ovarian cancer (HBOC) syndrome, breast, ovarian,
fallopian tube, peritoneal, pancreatic, and prostate cancers, and affected individuals may be at
increased risk for melanoma
BRCA2	AR Fanconi anemia, AD hereditary breast and ovarian cancer (HBOC) syndrome, breast, ovarian,
fallopian tube, peritoneal, pancreatic, and prostate cancers.
BRIP1	AD predisposition to ovarian cancer and AR Fanconi anemia.
CDH1	AD hereditary diffuse gastric cancer (HDGC) syndrome, stomach and breast cancers.
CDK4	AD melanoma
CDKN2A	AD hereditary melanoma-pancreatic cancer syndrome, melanoma, pancreatic cancer and neural system
tumors
CHEK2	AD breast, colon, thyroid and prostate cancers.
Gene*	Syndrome / Cancer
CTNNA1	AD butterfly-shaped pigmentary macular dystrophy, hereditary diffuse gastric cancer
DICER1	AD pleuropulmonary blastoma familial tumor predisposition syndrome, ovarian sex cord-stromal
tumors, rhabdomyosarcoma, renal sarcoma, cystic nephroma, Wilms tumor, thyroid cancer
EPCAM	AR congenital tufting enteropathy, AR constitutional mismatch repair deficiency syndrome (CMMR-
D), AD Lynch syndrome, colorectal, uterine, endometrial, ovarian, gastric, bladder, biliary tract,
urinary tract, prostate, brain, and small bowel cancers.
GREM1	AD hereditary mixed polyposis syndrome (HMPS), colorectal polyps.
HOXB13	AD prostate cancer
KIT	AD piebaldism, AD gastrointestinal stromal tumors, and AD familial mastocytosis.
MEN1	AD familial isolated hyperparathyroidism, multiple endocrine neoplasia type 1 (MEN1) syndrome,
parathyroid, pituitary, thymus, gastric, small bowel (duodenal endocrine cells), pancreatic and adrenal
gland tumors
MLH1	AR constitutional mismatch repair deficiency syndrome (CMMR-D), AD Lynch syndrome, colorectal,
uterine, endometrial, ovarian, gastric, bladder, biliary tract, prostate, brain, and small
bowel cancers.
MSH2	AR constitutional mismatch repair deficiency syndrome (CMMR-D), AD Lynch syndrome, colorectal,
uterine, endometrial, ovarian, gastric, bladder, biliary tract, urinary tract, prostate, brain, and small
bowel cancers.
MSH3	AR MSH3-associated polyposis, colorectal cancer.
MSH6	AR constitutional mismatch repair deficiency syndrome (CMMR-D), AD Lynch syndrome, colorectal,
uterine, endometrial, ovarian, gastric, bladder, biliary tract, urinary tract, prostate, brain, and small
bowel cancers.
MUTYH	AR MUTYH-associated polyposis (MAP), colorectal cancer, duodenal adenomas, ovarian, bladder,
breast, endometrial, skin and thyroid cancers
NBN	AR Nijmegen breakage syndrome (NBS), lymphoma, medulloblastoma, glioma, and
rhabdomyosarcoma.
NF1	AD Neurofibromatosis type 1 (NF1), AD neurofibromatosis-Noonan syndrome, AD Watson syndrome,
central nervous system neoplasms, breast cancer, gastrointestinal stromal tumors, pheochromocytoma,
and sarcomas.
NTHL1	AR NTHL1-associated polyposis, colorectal cancer.
PALB2	AR Fanconi anemia, AD predisposition to breast, pancreatic, ovarian and male breast cancer
PDGFRA	AD GIST-plus syndrome, gastrointestinal stromal tumors.
PMS2	AR constitutional mismatch repair deficiency syndrome (CMMR-D), AD Lynch syndrome, colorectal,
uterine, endometrial, ovarian, gastric, bladder, biliary tract, urinary tract, prostate, brain, and small
bowel cancers.
POLD1	AD MDPL syndrome (mandibular hypoplasia, deafness, progeroid features, and lipodystrophy), AD
colorectal cancer.
Gene*	Syndrome / Cancer
POLE	AR facial dysmorphism, immunodeficiency, livedo, and short stature (FILS) syndrome, AD
predisposition colon cancer.
PTEN	AD PTEN hamartoma tumor syndrome (PHTS), melanoma, breast, thyroid, renal, endometrial, and
colorectal cancer.
RAD50	AR Nijmegen breakage syndrome-like disorder
RAD51C	AD breast and ovarian cancer
RAD51D	AD ovarian and breast cancer
SDHA	AR mitochondrial complex II deficiency, AD hereditary paraganglioma-pheochromocytoma (PGL-
PCC) syndrome, gastrointestinal stromal tumors (GIST), renal cancer.
SDHB	AR mitochondrial complex II deficiency, AD hereditary paraganglioma-pheochromocytoma (PGL-
PCC) syndrome, gastrointestinal stromal tumors (GIST), renal cancer.
SDHC	AD hereditary paraganglioma-pheochromocytoma (PGL-PCC) syndrome, gastrointestinal stromal
tumors (GIST), renal cancer.
SDHD	AD hereditary paraganglioma-pheochromocytoma (PGL-PCC) syndrome, gastrointestinal stromal
tumors (GIST), renal cancer. AR mitochondrial complex II deficiency
SMAD4	AD juvenile polyposis syndrome (JPS), colorectal, gastric and pancreatic cancer
SMARCA4	AD Coffin-Siris syndrome, AD rhabdoid tumor predisposition syndrome, ovarian cancer.
STK11	AD Peutz-Jeghers syndrome (PJS), breast, ovarian, non-epithelial ovarian cancer (sex cord tumors with
annular tubules), testicular, uterine, cervical, colorectal, small bowel, pancreatic, gastric and lung
cancers
TP53	AD Li-Fraumeni syndrome (LFS), osteosarcoma, brain, breast, adrenocortical, leukemia, lymphoma,
head and neck, renal, lung, laryngeal, skin, ovarian, pancreatic, prostate, testicular and thyroid cancers.
TSC1	AD tuberous sclerosis complex (TSC), hamartomas, facial angiofibromas, ungual fibromas, cortical
tubers, subependymal giant cell astrocytomas, cardiac rhabdomyomas, renal angiomyolipomas, retinal
nodular hamartomas, lymphangioleiomyomas and pancreatic neuroendocrine tumors.
TSC2	AD tuberous sclerosis complex (TSC), hamartomas, facial angiofibromas, ungual fibromas, cortical
tubers, subependymal giant cell astrocytomas, cardiac rhabdomyomas, renal angiomyolipomas, retinal
nodular hamartomas, lymphangioleiomyomas and pancreatic neuroendocrine tumors.
VHL	AR familial erythrocytosis, AD von Hippel-Lindau (VHL), hemangioblastomas, paragangliomas,
pheochromocytomas, endolymphatic sac tumors, epididymal cystadenomas, pancreatic neuroendocrine
tumors, and clear cell renal cell carcinoma

Table 35. Gene/Disease Associations

53

54

AD = autosomal dominant; AR = autosomal recessive

*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.

b) Database

The Invitae Common Hereditary Cancers Panel test system employs multiple databases that store variant information, including one within the VDB software and another within the CROP software.

55

VDB contains normalized representations of variants, as well as both left- and right-aligned representations of variants. These variants are annotated. As new variants are encountered in patient samples, they are added to the database and go through the annotation process using various annotation sources. Variants that have been encountered before are verified to ensure that annotations are present and up to date, with any missing annotations flagged for manual review.

CROP contains variant classifications (i.e., pathogenic, likely pathogenic, uncertain significance, benign, likely benign) and the supporting evidence supporting the classifications, which has been curated by qualified Invitae staff. Gene-disease relationship curation and variant interpretation is performed according to controlled SOPs by trained individuals who have passed a competency assessment. A variety of external databases are consulted. When a novel variant is encountered, CROP pulls in relevant clinical evidence for evaluation. Variant interpreters review the PMIDs and website resources associated with the variant's HGVS nomenclature and assign an evidence type to each individual piece of evidence. Based on this evidence, a classification is calculated, assigned, reviewed, and finalized. The final interpretation is then stored in CROP. When the variant is encountered again, CROP automatically checks how recently the variant was last seen and triggers a new search for and evaluation of any new evidence.

c) Interpretation Agreement

To evaluate the performance of variant classification, interpretation and reporting, a study was performed to compare result interpretations made by the Invitae Common Hereditary Cancers Panel and independently generated prior clinical laboratory testing results. In this study, results from a total of 975 patients, representing 1874 unique BRCA1/BRCA2 variants, were examined. These patients were referred for hereditary breast/ovarian cancer (HBOC) counseling, or have known familial mutations, or have personal or familial HBOC high risk factors. Patients had prior BRCA1/2 results using an orthogonal method, as well as results obtained from a 29-gene panel, which is a subset of the Invitae Common Hereditary Cancers Panel that included BRCA1 and BRCA2, and utilizes consistent variant annotation framework as the Invitae Common Hereditary Cancers Panel. Variant interpretation was compared between the Invitae results and the prior clinical laboratory testing. Across the total of 975 patients, there was 98.9% concordance (964 patients, 1765 variants) between the Invitae interpretation and the prior results from an orthogonal laboratory.

A second analysis was performed to evaluate the concordance between Invitae variant classifications and ClinVar classifications. At the time of the analysis, 9 of the 47 target genes (BRCA1, BRCA2, CDH1, MLH1, MSH2, MSH6, PMS2, PTEN and TP53) have Expert Panel submissions in ClinVar. All available variants (total of 3190, including 3102 SNVs, 17 Indels and 71 CNVs) were evaluated across these 9 genes, and 97% of Invitae classifications were concordant with ClinVar. Results of the analysis are summarized in Table 36. The concordance between Invitae variant classifications and ClinVar classifications is evaluated annually to ensure that the rule sets and the professional application of those rule sets continues to agree with the clinical interpretations issued by other clinical and medical groups.

56

Interpretation		ClinVar
		Positive (P, LP)	Uncertain	Negative (B, LB)
Invitae	Positive (P, LP)	1783	7	0
	Uncertain	26	52	63
	Negative (B, LB)	0	6	1253

Table 36 Invitae-ClinVar Variant Classification Concordance

In addition, to evaluate the performance of the Invitae bioinformatics pipeline in appropriately linking variant classification to report level clinical results, a total of 95 cases were evaluated, for which clinical results were determined and reviewed manually by professionals, representing every possible combination, comparison was made between the software output and manual evaluation. 100% concordance of clinical results were observed between manual evaluation and software output, based on variant classification inputs.

D Clinical Cut-Off:

Not applicable

Expected Values/Reference Range: E

Not applicable

Other Supportive Performance Characteristics Data: F

Not applicable

VII Proposed Labeling:

The labeling supports the decision to grant the De Novo request for this device.

Identified Risks and Mitigations: VIII

Risks to Health	Mitigation Measures
False positive, false negative, or failure to provide a
result.	Certain design verification and validation
including certain analytical and clinical studies, and
mutation annotation and clinical interpretation rules
identified in special control (1).
	Certain labeling information including
limitations, device descriptions, methodology and
protocols, and performance information identified in
special controls (2).
Incorrect interpretation of variants/alterations by the lab.	Certain labeling information including

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Risks to Health	Mitigation Measures
	limitations, device descriptions, methodology and
protocols, and performance information identified in
special controls (2).
	Certain design verification and validation
including certain analytical and clinical studies, and
mutation annotation and clinical interpretation rules
identified in special control (1).
Incorrect interpretation of test results by healthcare
provider.	Certain labeling information including
limitations, device descriptions, methodology and
protocols, and performance information identified in
special controls (2).
	Certain design verification and validation
including certain analytical and clinical studies, and
mutation annotation and clinical interpretation rules
identified in special control (1).

IX Benefit/Risk Assessment:

Summary of the Assessment of Benefit: A

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.

There are probable benefits to the population(s) for whom the Invitae Common Hereditary Cancers Panel test system is intended, including individuals with a diagnosed cancer, and individuals with a family history of developing a certain type or types of cancer. The benefit includes detection of cancer predisposition variants and is supported by the extensive analytical validation and clinical validation of the device, which indicates that use of this device, may aid in appropriate medical management for patients with identified variants. Overall, the Invitae Common Hereditary Cancers Panel test would inform qualified health care professionals, to act in accordance with professional guidelines, for hereditary cancer predisposition assessment and to aid in identifying hereditary genetic variants potentially associated with a diagnosed cancer. This would provide benefit to patients and other family members, in receiving the appropriate medical management for the mutation/alteration identified. The performance of the analytical accuracy study in particular, supported the probable benefit of this device. A total of 6014 samples with SNVs or Indels and 3648 samples with CNVs were included in the analytical accuracy study. The overall TPPV is 99.9% for SNVs, 100% for Indels and 99.5% for CNVs.

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The overall TNPV is 100% for SNVs. 100% for Indels, and 99.7% for CNVs. The totality of the analytical and clinical data provided support a probable benefit of this device, for the uses indicated.

B Summary of the Assessment of Risk:

The probable risk associated with the use of this device are mainly due to 1) analytical false positive, false negatives, or failure to provide a result and 2) incorrect interpretation of variants/alterations by the lab and 3) incorrect interpretation and use of test results by the enduser.

False negatives may lead to patients not being able to avail of appropriate surveillance or management that could benefit the patients. False positives may lead to patients being offered surveillance or management options that are inappropriate for the patients, and that can be associated with other clinical sequelae. Erroneous device results could adversely influence clinical interpretation and consultation for patients. However, this test is not conclusive or prescriptive for the use of any specific therapeutic product or therapeutic pathway and the interpretive statements regarding the clinical implications of a given mutation should not be viewed as a formal treatment or management recommendation.

There is a degree of probable risk of mismanagement of patient care, in accordance with professional guidelines, based on false test results from this test, or incorrect interpretation of test results. These risks are mitigated by the analytical performance of this device, clinical validation and labeling of this device.

Patient Perspectives: C

This submission did not include specific information on patient perspectives for this device.

D Summary of the Assessment of Benefit-Risk:

In conclusion, given the available information above, for the following indications for use statement:

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.

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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.

The probable benefits outweigh the probable risks for the Invitae Common Hereditary Cancers Panel, considering the mitigations of the risks provided in the special controls as well as general controls.

Conclusion: X

The De Novo request is granted and the device is classified under the following and subject to the special controls identified in the letter granting the De Novo request:

Product Code(s): QVU Device Type: High throughput DNA sequencing for hereditary cancer predisposition assessment test system Class: II Regulation: 21 CFR 866.6095