AncestryDNA Factor V Leiden Genetic Health Risk Test

• K Number: K192944
• Device Name: AncestryDNA Factor V Leiden Genetic Health Risk Test
• Manufacturer: Ancestry Genomics, Inc.
• Date Cleared: 2020-08-13 ( 300 days)
• Product Code: PTA
• Regulation Number: 866.5950

Intended Use

The AncestryDNA Factor V Leiden Genetic Health Risk Test for Hereditary Thrombophilia uses qualitative genotyping to detect clinically relevant variants in genomic DNA isolated from human saliva collected from individuals 18 years and older with the AncestryDNA Saliva Collection Kit for the purpose of reporting and interpreting Genetic Health Risks (GHR). The AncestryDNA Factor V Leiden Genetic Health Risk Report for Hereditary Thrombophilia is indicated for reporting of the Factor V Leiden variant in the F5 gene. This report describes if a person has variants associated with a higher risk of developing harmful blood clots, but it does not describe a person's overall risk of developing harmful blood clots. This test is most relevant for people of European descent.

Device Description

A user's saliva is self-collected using the AncestryDNA Saliva Collection Kit, which consists of a sealable collection tube containing a stabilizing buffer solution. Once the sample is collected, it is shipped to one of two Clinical Laboratory Improvement Amendments (CLIA) certified laboratories for processing. DNA is isolated from the saliva, quantified, and tested in a multiplex assay using a customized genotyping chip and instrumentation manufactured by Illumina. The multiplex assay simultaneously tests for more than 500,000 variants, including those for the indication proposed herein. The raw data is generated using Illumina GenomeStudio software, and then sent to Ancestry Genomics (the Manufacturer). The data are analyzed using the Manufacturer's proprietary GHR software, and a genotype is determined for each tested variant. The results for the Factor V Leiden variant are used to generate personalized reports for users that provide information about the disease associated with the detected variant. Personalized reports are generated for each user that provide results of the testing performed. These reports tell the user which variant(s) has/have been detected in their sample and provide information on the risk of disease associated with the variant(s). If no variant was detected, that information is also provided. The personalized reports are designed to present scientific concepts to users in an easy-to-understand format. The reports provide scientifically valid information about the risks associated with the presence of a particular variant. The reports are designed to help users understand the meaning of their results and any appropriate actions that may be taken based on their results.

Predicate Devices

DEN160026

Reference Devices

DEN140044, K192947, K141410

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes standard genetic testing procedures, data analysis using proprietary software, and report generation. There is no mention of AI or ML being used in the genotyping, analysis, or interpretation process. The focus is on qualitative genotyping and comparison to known variants.

Therapeutic

No
This device is a genetic test that provides information about genetic health risks; it does not treat or prevent disease.

Diagnostic

Yes

The device uses qualitative genotyping to detect clinically relevant variants in genomic DNA from human saliva for the purpose of reporting and interpreting Genetic Health Risks (GHR) related to Factor V Leiden. This involves identifying a condition or predisposition, which falls under the definition of a diagnostic device.

SaMD (Software as a Medical Device)

No

The device description explicitly mentions hardware components like the AncestryDNA Saliva Collection Kit, Illumina genotyping chip and instrumentation, and the Illumina iScan optical imaging system. While software is used for data analysis and report generation, the overall system includes significant hardware components for sample collection, processing, and data acquisition.

IVD (In Vitro Diagnostic)

Based on the provided information, yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

• Intended Use: The device is intended for "qualitative genotyping to detect clinically relevant variants in genomic DNA isolated from human saliva... for the purpose of reporting and interpreting Genetic Health Risks (GHR)." This clearly describes a test performed in vitro (outside the body) on a biological sample (saliva) to provide information about a person's health risk.
• Sample Type: It uses "genomic DNA isolated from human saliva," which is a biological specimen.
• Testing Method: It employs "qualitative genotyping" using a "customized genotyping chip and instrumentation." This is a laboratory-based testing method.
• Purpose: The purpose is to report and interpret "Genetic Health Risks (GHR)," specifically for "Hereditary Thrombophilia" related to the Factor V Leiden variant. This is a diagnostic purpose, even though it's a risk assessment rather than a definitive diagnosis.
• Regulatory Context: The device is being submitted for regulatory review (implied by the structure of the document, which resembles a regulatory submission). The comparison to a predicate device (23andMe Personal Genome Service Test, DEN160026) further indicates it's being treated as a medical device subject to regulatory oversight, which is typical for IVDs.
• Performance Studies: The document details analytical and clinical performance studies, including accuracy, reproducibility, sensitivity, and user comprehension. These types of studies are required for IVD devices to demonstrate their reliability and validity.

The fact that it's an Over The Counter (OTC) test and provides risk information rather than a definitive diagnosis does not preclude it from being an IVD. Many IVDs are designed for OTC use and provide information about risk factors or predispositions.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The AncestryDNA Factor V Leiden Genetic Health Risk Test for Hereditary Thrombophilia uses qualitative genotyping to detect clinically relevant variants in genomic DNA isolated from human saliva collected from individuals 18 years and older with the AncestryDNA Saliva Collection Kit for the purpose of reporting and interpreting Genetic Health Risks (GHR).

The AncestryDNA Factor V Leiden Genetic Health Risk Report for Hereditary Thrombophilia is indicated for reporting of the Factor V Leiden variant in the F5 gene. This report describes if a person has variants associated with a higher risk of developing harmful blood clots, but it does not describe a person's overall risk of developing harmful blood clots. This test is most relevant for people of European descent.

Product codes

PTA

Device Description

A user's saliva is self-collected using the AncestryDNA Saliva Collection Kit, which consists of a sealable collection tube containing a stabilizing buffer solution. Once the sample is collected, it is shipped to one of two Clinical Laboratory Improvement Amendments (CLIA) certified laboratories for processing.

DNA is isolated from the saliva, quantified, and tested in a multiplex assay using a customized genotyping chip and instrumentation manufactured by Illumina. The multiplex assay simultaneously tests for more than 500,000 variants, including those for the indication proposed herein.

The raw data is generated using Illumina GenomeStudio software, and then sent to Ancestry Genomics (the Manufacturer). The data are analyzed using the Manufacturer's proprietary GHR software, and a genotype is determined for each tested variant. The results for the Factor V Leiden variant are used to generate personalized reports for users that provide information about the disease associated with the detected variant.

Personalized reports are generated for each user that provide results of the testing performed. These reports tell the user which variant(s) has/have been detected in their sample and provide information on the risk of disease associated with the variant(s). If no variant was detected, that information is also provided. The personalized reports are designed to present scientific concepts to users in an easy-to-understand format. The reports provide scientifically valid information about the risks associated with the presence of a particular variant. The reports are designed to help users understand the meaning of their results and any appropriate actions that may be taken based on their results.

Mentions image processing

Yes

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

individuals 18 years and older

Intended User / Care Setting

For over-the-counter (OTC) use.

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

A total of 378 individuals were enrolled as participants into each of four study arms, one for each of the GHR report types: 0 Variants Identified, 1 Variant Identified, 2 Variants Identified, Result Not Determined.

A total of 213 individuals were enrolled as participants into each of two study arms, one for each of the following two GHR report types: 1 Variant Identified, Result Not Determined.

Participants were recruited to match the demographics (education, age, sex/gender, and race/ethnicity) of the adult U.S. population as of the most recent estimates released by the U.S. Census Bureau. Geographic diversity was addressed through participant recruitment and comprehension testing from each of the four U.S. Census geographic regions: Northeast, Midwest, South, and West.

In Study 1, which was conducted in-person, participants were observed discretely, and each interview session was administered by a trained interviewer/moderator using a series of predefined questions. Participants were given a time limit from start of report review to completion of the comprehension survey. There was a 100% response rate for all questions for all participants included in the data analysis. Failing to respond to numerous items in the questionnaire was one of the criteria used to identify participants for exclusion from data analysis.

In Study 2, which was conducted via live televideo interviews, participants were observed via live video, and each interview session was administered online by a trained interviewer/moderator using a series of predefined questions. Participants were first administered a pre-test questionnaire prior to being shown any of the representative samples. Participants were then given a time limit from start of report review to completion of the post-test questionnaire. There was a 100% response rate for all questions for all participants included in the data analysis. Failing to respond to numerous items in the questionnaire was one of the criteria used to identify participants for exclusion from data analysis. All pre-test questions were repeated again in the posttest questionnaire to assess pre-test vs post-test improvement.

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

Analytical Performance - Reproducibility/Precision:
Study Purpose: Determine precision and reproducibility of the AncestryDNA Factor V Leiden GHR Test at multiple sites, on multiple days, using multiple operator teams, instrument combinations, and assay critical reagent lots tested with samples collected using multiple lots of the AncestryDNA Saliva Collection Kit (SCK).
Sample Size: Saliva samples were collected from nine donors with known Factor V Leiden genotypes (three each with homozygous common, heterozygous, and homozygous rare). Each donor provided 19 saliva samples into three lots of AncestryDNA SCKs.
Methodology: Genotyping with the AncestryDNA Factor V Leiden GHR Test was conducted over a minimum of six non-consecutive starting days at Lab 1 and two non-consecutive days at Lab 2. For inter-laboratory reproducibility, two saliva samples from each donor were extracted at Lab 1 and plated for processing, tested in triplicate at Lab 1 and Lab 2.
Results:

• Lab 1 within-laboratory testing: 1617 concordant calls out of 1620 total replicates, 3 failures (FQC 0.19%).
• Lab 1 within-run repeatability: 45 concordant calls out of 45 total replicates, 0 failures (FQC 0.00%).
• Lab 2 within-laboratory testing: 540 concordant calls out of 540 total replicates, 0 failures (FQC 0.00%).
• Inter-Laboratory Reproducibility: For both Lab 1 and Lab 2, 81 concordant calls out of 81 total replicates, 0 failures (FQC 0.00%).
• Overall Percent Agreement (OPA) for Repeatability and Genotyping: All OPA point estimates for repeatability and each genotype exceeded the 99% predefined protocol acceptance criteria. Overall precision point estimate was > 99%.

Analytical Performance - Analytical Sensitivity:
Study Purpose: Determine Limit of Blank (LoB) and Limit of Detection (LoD).
Methodology:

• Saliva samples study: Samples from 15 donors with known genotypes were used. Pooled DNA created two-fold dilution series. Six replicates of each dilution series were genotyped, two per critical reagent lot (n=3).
• Cell line samples study: Four cell lines with known genotypes were used. DNA from each cell line diluted to generate 8 samples in each series. 27 replicates of each cell line dilution series were genotyped using the AncestryDNA Factor V Leiden GHR Assay; three replicates per critical reagent lot (n=3) on three testing days.
  Results:
• LoB = 1.004 ng/uL
• LoD = 1.53 ng/uL
• Upper limit of concentration = 50 ng/uL
• All genotyping attempts on samples containing the measurand with call rates ≥ 98% and concentrations between 1.53 ng/uL and 50 ng/uL produced genotypes concordant with bidirectional sequencing.

Analytical Performance - Interfering Substances (Endogenous Interference):
Study Purpose: Evaluate four potential common endogenous interferents (Salivary α-amylase, Hemoglobin, IgA, Total Protein) on test performance.
Sample Size: Ten saliva donors.
Methodology: Endogenous substances were individually spiked into saliva samples (collected using Oragene® Dx and AncestryDNA SCK) prior to DNA extraction and genotyping. Saliva with PBS served as control. Each sample genotyped in triplicate (450 total initial genotyping attempts).
Results: Concordance for all interfering substances was 100% for all samples where control samples passed QC, meeting the acceptance criterion of ≥ 95% agreement with true variant status.

Analytical Performance - Interfering Substances (Exogenous Interference):
Study Purpose: Evaluate six potential exogenous interferents (eating chicken, drinking alcohol, using mouthwash, eating beef, chewing gum, smoking).
Sample Size: 10 non-smokers and 10 smokers.
Methodology: Donors performed activities, then provided saliva samples (before, immediately after, and 30-minutes after).
Results: For all samples where control samples and replicates containing interfering substances passed QC, the concordance for all interfering substances was 100%. A higher rate of QC failures was observed for samples collected immediately after eating chicken and beef activities.

Analytical Performance - Interfering Substances (Microbial Interference):
Study Purpose: Evaluate five different species of microbial DNA on test performance.
Sample Size: Six human cell lines with known Factor V Leiden genotypes.
Methodology: DNA from human cell lines spiked with two concentrations (low/normal and high) of microbial DNA. Human cell line DNA spiked with buffer functioned as a spike-in control. Resulting DNA mixtures genotyped in replicates of 6.
Results: The assay produced concordant genotypes with bidirectional sequencing in all genotyping events. Overall percent agreement was 100% for all microbial interferents at both low/normal and high concentrations, meeting the acceptance criteria of > 95% agreement.

Analytical Performance - Interfering Substances (Mutational Interference):
Study Purpose: Evaluate nucleotide mutations for interference.
Methodology: In silico analysis to identify mutations within one probe length (50 bp) downstream of the Factor V Leiden mutation using gnomAD database.
Results: Three potentially interfering mutations (rs770011773, rs773367113, rs143663052) were identified. Impact on assay performance not empirically evaluated. Two mutations from predicate (DEN160026) will not interfere as they are >50 bp away.

Comparison Studies - Method Comparison with the Predicate:
Study Purpose: Establish accuracy by comparing results to true variant status determined by bi-directional sequencing.
Sample Size: 209 donors (73 homozygous common, 69 heterozygous, 67 homozygous rare).
Methodology: Saliva samples collected using Oragene Dx Ogd-500.001 (OGD) for bi-directional sequencing and AncestryDNA SCK for GHR Test. Samples regenotyped if they failed a first-pass QC.
Results:

• Of 209 samples, 198 passed SCR quality control (95%).
• Zero (0) 'no-call' events observed in any samples that passed quality control.
• Overall percent agreement and the percent agreement for each genotype with bi-directional sequencing genotypes was 100% for all samples that passed quality control (198/198).
• The result met the predefined protocol acceptance criterion of ≥ 99% agreement with true variant determination overall and per genotype tested.

Clinical Studies - Clinical Performance (Odds ratios and Likelihood ratios):
Study Type: Meta-analyses from published literature (Simone et al., 2013).
Results:

• Odds ratio for one copy, heterozygote: 4.22 (3.35 - 5.32)
• Odds ratio for two copies, homozygote: 5.45 (6.79 - 19.29)
• Likelihood ratios and post-test risk were calculated from the meta-analyses data. For 0 variants, LR=0.87, Post-test risk=10%. For 1 variant, LR=2.82, Post-test risk=26%. For 2 variants, LR=9.69, Post-test risk=54%.
• Likelihood ratio lower bounds for heterozygotes and homozygotes are greater than one, indicating post-test risk significantly greater than pre-test risk.

Clinical Studies - User Comprehension Studies:
Study Type: Two user comprehension studies (Study 1 and Study 2).
Study Purpose: Assess user comprehension of the AncestryDNA Factor V Leiden GHR Test's Genetic Health Risk (GHR) reports.
Study 1:

• Sample Size: 378 individuals (96 for 0 Variant, 86 for 1 Variant, 106 for 2 Variant, 90 for Result Not Determined reports).
• Methodology: Post-test administered questionnaire, in-person interviews.
• Results: Average comprehension rates per core concept ranged from 90.7% to 97.4%. Overall comprehension rate was 93.2%.
  Study 2:
• Sample Size: 213 individuals (103 for 1 Variant, 110 for Result Not Determined reports).
• Methodology: Pre-test and post-test administered questionnaire, live televideo interviews.
• Results: Average comprehension rates per core concept ranged from 90.9% to 99.1%. Overall comprehension rate was 96%.
• Improvement in comprehension between pre-test and post-test shown for Purpose, Other Risk Factors, and Limitations concepts.
• Acceptance criteria (≥ 90% comprehension score for each domain) met for both studies.

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

Analytical Sensitivity / Limit of Detection:

• LoB = 1.004 ng/uL
• LoD = 1.53 ng/uL
• Upper limit of concentration = 50 ng/uL

Reproducibility/Precision:

• Overall Percent Agreement:
  • All operator teams: 100.00% (99.84 - 100.00)
  • All instrument combinations: 100.00% (99.84 - 100.00)
  • All reagent lot combinations: 100.00% (99.84 - 100.00)
  • Within-run precision at Lab 1: 100.00% (99.77 - 100.00)
  • Within-run precision at Lab 2: 100.00% (99.32 - 100.00)
  • Within-run repeatability: 100.00% (92.13 - 100.00)
  • Inter-lab data at Lab 1: 100.00% (95.55 - 100.00)
  • Inter-lab data at Lab 2: 100.00% (95.55 - 100.00)
  • All "GG": 100.00% (99.53 - 100.00)
  • All "GA": 100.00% (99.53 - 100.00)
  • All "AA": 100.00% (99.53 - 100.00)

Accuracy (Comparison with Bi-directional Sequencing):

• Percent Agreement and Confidence Intervals for AncestryDNA Factor V Leiden GHR Test Genotypes:
  • GG: 100% (94.8–100%)
  • GA: 100% (94.5–100%)
  • AA: 100% (94.4–100%)
  • All genotypes: 100% (98.2–100%)
• Estimated Rate of No-Call Events: 0% (0 – 1.8%)
• Overall Percent Agreement and Confidence Interval for Genotypes Obtained Using AncestryDNA SCK Compared to OGD: 100% (98.2 - 100%)

Interfering Substances (Overall Percent Agreement - OPA):

• Endogenous Interferents: All 100%
• Exogenous Interferents: All 100% (at T0 and T30 time points)
• Microbial Interferents: All 100% (at Low/Normal and High Concentrations)

Clinical Performance (Odds Ratios and Likelihood Ratios):

• Odds ratio (95% confidence interval) for Factor V Leiden one copy, heterozygote: 4.22 (3.35 - 5.32)
• Odds ratio (95% confidence interval) for Factor V Leiden two copies, homozygote: 5.45 (6.79 - 19.29)
• Likelihood Ratios (LR)
  • 0 variants: 0.87 (0.86 – 0.88), Post-test risk: 10%
  • 1 variant: 2.82 (2.64 – 3.02), Post-test risk: 26%
  • 2 variants: 9.69 (6.19 – 15.16), Post-test risk: 54%

User Comprehension Studies:

• Study 1 - Overall Comprehension Rate: 93.2%
• Study 2 - Overall Comprehension Rate: 96%
• Study 2 - % Improvement (Pre-test vs Post-test):
  • Purpose: 10.1%
  • Other Risk Factors: 4.7%
  • Limitations: 8.5%

Predicate Device(s):

DEN160026

Reference Device(s):

DEN140044, K192947, K141410

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.5950 Genetic health risk assessment system.

(a)
Identification. A genetic health risk assessment system is a qualitative in vitro molecular diagnostic system used for detecting variants in genomic deoxyribonucleic acid (DNA) isolated from human specimens that will provide information to users about their genetic risk of developing a disease to inform lifestyle choices and/or conversations with a health care professional. This assessment system is for over-the-counter use. This device does not determine the person's overall risk of developing a disease.(b)
Classification. Class II (special controls). The genetic health risk assessment system device, when it has previously received a first-time FDA marketing authorization (e.g., 510(k) clearance) for the genetic health risk assessment system (a “one-time FDA reviewed genetic health risk assessment system”), is exempt from the premarket notification procedures in part 807, subpart E, of this chapter subject to the limitations in § 866.9. The device must comply with the following special controls:(1) The 21 CFR 809.10 compliant labeling and any prepurchase page and test report generated, unless otherwise specified, must include:
(i) A section addressed to users with the following information:
(A) The limiting statement explaining that this test provides genetic risk information based on assessment of specific genetic variants but does not report on a user's entire genetic profile. This test [does not/may not, as appropriate] detect all genetic variants related to a given disease, and the absence of a variant tested does not rule out the presence of other genetic variants that may be related to the disease.
(B) The limiting statement explaining that other companies offering a genetic risk test may be detecting different genetic variants for the same disease, so the user may get different results using a test from a different company.
(C) The limiting statement explaining that other factors such as environmental and lifestyle risk factors may affect the risk of developing a given disease.
(D) The limiting statement explaining that some people may feel anxious about getting genetic test health results. This is normal. If the potential user feels very anxious, such user should speak to his or her doctor or other health care professional prior to collection of a sample for testing. This test is not a substitute for visits to a doctor or other health care professional. Users should consult with their doctor or other health care professional if they have any questions or concerns about the results of their test or their current state of health.
(E) Information about how to obtain access to a genetic counselor, board-certified clinical molecular geneticist, or equivalent health care professional about the results of a user's test.
(F) The limiting statement explaining that this test is not intended to diagnose a disease, tell you anything about your current state of health, or be used to make medical decisions, including whether or not you should take a medication or how much of a medication you should take.
(G) A limiting statement explaining that the laboratory may not be able to process a sample, and a description of the next steps to be taken by the manufacturer and/or the customer, as applicable.
(ii) A section in your 21 CFR 809.10 labeling and any test report generated that is for health care professionals who may receive the test results from their patients with the following information:
(A) The limiting statement explaining that this test is not intended to diagnose a disease, determine medical treatment, or tell the user anything about their current state of health.
(B) The limiting statement explaining that this test is intended to provide users with their genetic information to inform lifestyle decisions and conversations with their doctor or other health care professional.
(C) The limiting statement explaining that any diagnostic or treatment decisions should be based on testing and/or other information that you determine to be appropriate for your patient.
(2) The genetic test must use a sample collection device that is FDA-cleared, -approved, or -classified as 510(k) exempt, with an indication for in vitro diagnostic use in over-the-counter DNA testing.
(3) The device's labeling must include a hyperlink to the manufacturer's public Web site where the manufacturer shall make the information identified in paragraph (b)(3) of this section publicly available. The manufacturer's home page, as well as the primary part of the manufacturer's Web site that discusses the device, must provide a hyperlink to the Web page containing this information and must allow unrestricted viewing access. If the device can be purchased from the Web site or testing using the device can be ordered from the Web site, the same information must be found on the Web page for ordering the device or provided in a publicly accessible hyperlink on the Web page for ordering the device. Any changes to the device that could significantly affect safety or effectiveness would require new data or information in support of such changes, which would also have to be posted on the manufacturer's Web site. The information must include:
(i) An index of the material being provided to meet the requirements in paragraph (b)(3) of this section and its location.
(ii) A section that highlights summary information that allows the user to understand how the test works and how to interpret the results of the test. This section must, at a minimum, be written in plain language understandable to a lay user and include:
(A) Consistent explanations of the risk of disease associated with all variants included in the test. If there are different categories of risk, the manufacturer must provide literature references that support the different risk categories. If there will be multiple test reports and multiple variants, the risk categories must be defined similarly among them. For example, “increased risk” must be defined similarly between different test reports and different variant combinations.
(B) Clear context for the user to understand the context in which the cited clinical performance data support the risk reported. This includes, but is not limited to, any risks that are influenced by ethnicity, age, gender, environment, and lifestyle choices.
(C) Materials that explain the main concepts and terminology used in the test that include:
(
1 )Definitions: Scientific terms that are used in the test reports.(
2 )Prepurchase page: This page must contain information that informs the user about what information the test will provide. This includes, but is not limited to, variant information, the condition or disease associated with the variant(s), professional guideline recommendations for general genetic risk testing, the limitations associated with the test (e.g., test does not detect all variants related to the disease) and any precautionary information about the test the user should be aware of before purchase. When the test reports the risk of a life-threatening or irreversibly debilitating disease or condition for which there are few or no options to prevent, treat, or cure the disease, a user opt-in section must be provided. This opt-in page must be provided for each disease that falls into this category and must provide specific information relevant to each test result. The opt-in page must include:(
i ) An option to accept or decline to receive this specific test result;(
ii ) Specification of the risk involved if the user is found to have the specific genetic test result;(
iii ) Professional guidelines that recommend when genetic testing for the associated target condition is or is not recommended; and(
iv ) A recommendation to speak with a health care professional, genetic counselor, or equivalent professional before getting the results of the test.(
3 )Frequently asked questions (FAQ) page: This page must provide information that is specific for each variant/disease pair that is reported. Information provided in this section must be scientifically valid and supported by corresponding publications. The FAQ page must explain the health condition/disease being tested, the purpose of the test, the information the test will and will not provide, the relevance of race and ethnicity to the test results, information about the population to which the variants in the test is most applicable, the meaning of the result(s), other risk factors that contribute to disease, appropriate followup procedures, how the results of the test may affect the user's family, including children, and links to resources that provide additional information.(iii) A technical information section containing the following information:
(A) Gene(s) and variant(s) the test detects using standardized nomenclature, Human Genome Organization nomenclature and coordinates as well as Single Nucleotide Polymorphism Database (dbSNP) reference SNP numbers (rs#).
(B) Scientifically established disease-risk association of each variant detected and reported by the test. This risk association information must include:
(
1 ) Genotype-phenotype information for the reported variants.(
2 ) Table of expected frequency and risks of developing the disease in relevant ethnic populations and the general population.(
3 ) A statement about the current professional guidelines for testing these specific gene(s) and variant(s).(
i ) If professional guidelines are available, provide the recommendations in the professional guideline for the gene, variant, and disease, for when genetic testing should or should not be performed, and cautionary information that should be communicated when a particular gene and variant is detected.(
ii ) If professional guidelines are not available, provide a statement that the professional guidelines are not available for these specific gene(s) and variant(s).(C) The specimen type (
e.g., saliva, capillary whole blood).(D) Assay steps and technology used.
(E) Specification of required ancillary reagents, instrumentation, and equipment.
(F) Specification of the specimen collection, processing, storage, and preparation methods.
(G) Specification of risk mitigation elements and description of all additional procedures, methods, and practices incorporated into the directions for use that mitigate risks associated with testing.
(H) Information pertaining to the probability of test failure (
i.e., percentage of tests that failed quality control) based on data from clinical samples, a description of scenarios in which a test can fail (i.e., low sample volume, low DNA concentration, etc.), how users will be notified of a test failure, and the nature of followup actions on a failed test to be taken by the user and the manufacturer.(I) Specification of the criteria for test result interpretation and reporting.
(J) Information that demonstrates the performance characteristics of the test, including:
(
1 ) Accuracy of study results for each claimed specimen type.(
i ) Accuracy of the test shall be evaluated with fresh clinical specimens collected and processed in a manner consistent with the test's instructions for use. If this is impractical, fresh clinical samples may be substituted or supplemented with archived clinical samples. Archived samples shall have been collected previously in accordance with the instructions for use, stored appropriately, and randomly selected. In some limited circumstances, use of contrived samples or human cell line samples may also be appropriate and used as an acceptable alternative. The contrived or human cell line samples shall mimic clinical specimens as much as is feasible and provide an unbiased evaluation of the device accuracy.(
ii ) Accuracy must be evaluated by comparison to bidirectional Sanger sequencing or other methods identified as appropriate by FDA. Performance criteria for both the comparator method and the device must be predefined and appropriate to the device's intended use. Detailed study protocols must be provided.(
iii ) Test specimens must include all genotypes that will be included in the tests and reports. The number of samples tested in the accuracy study for each variant reported must be based on the variant frequency using either the minimum numbers of samples identified in this paragraph or, when determined appropriate and identified by FDA, a minimum number of samples determined using an alternative method. When appropriate, the same samples may be used in testing to demonstrate the accuracy of testing for multiple genotypes by generating sequence information at multiple relevant genetic locations. At least 20 unique samples representing the wild-type genotype must be tested. To test samples that are heterozygous for the reported variant(s), common variants (>0.1 percent variant frequency in the relevant population) must be tested with at least 20 unique samples. Rare variants (≤0.1 percent variant frequency in the relevant population) must be tested with at least three unique samples. To test samples that are homozygous for the reported variant(s), variants with ≥2 percent variant frequency in a relevant population must be tested with at least 20 unique samples. Variants with a frequency in the relevant population

Summary

0

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August 13, 2020

Ancestry Genomics, Inc. Raaj Venkatesan Associate Director, Regulatory Affairs 153 Townsend Street. Suite 800 San Francisco, California 94107

Re: K192944

Trade/Device Name: AncestryDNA Factor V Leiden Genetic Health Risk Test Regulation Number: 21 CFR 866.5950 Regulation Name: Genetic Health Risk Assessment System Regulatory Class: Class II Product Code: PTA Dated: October 17, 2019 Received: October 18, 2019

Dear Raaj Venkatesan:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part

1

801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4. Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

Takeesha Taylor-Bell Chief Division of Immunology and Hematology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K192944

Device Name

AncestryDNA Factor V Leiden Genetic Health Risk Test

Indications for Use (Describe)

The AncestryDNA Factor V Leiden Genetic Health Risk Test for Hereditary Thrombophilia uses qualitative genotyping to detect clinically relevant variants in genomic DNA isolated from human saliva collected from individuals 18 years and older with the AncestryDNA Saliva Collection Kit for the purpose of reporting and interpreting Genetic Health Risks (GHR).

The AncestryDNA Factor V Leiden Genetic Health Risk Report for Hereditary Thrombophilia is indicated for reporting of the Factor V Leiden variant in the F5 gene. This report describes if a person has variants associated with a higher risk of developing harmful blood clots, but it does not describe a person's overall risk of developing harmful blood clots. This test is most relevant for people of European descent.

Type of Use (Select one or both, as applicable)

Prescription Use (Part 21 CER 801 Subpart D)
Over The Counter Use (21 CER 801 Subpart C)

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510(K) SUMMARY

A. GENERAL INFORMATION

Submitter Information:

| Submitted By: | Ancestry Genomics, Inc.
153 Townsend Street, Suite 800
San Francisco, CA 94107 |
|---------------------------|------------------------------------------------------------------------------------------------------|
| Contact Person: | Raajdeep Venkatesan, MS, RAC, CMQ-OE, CBA, CQE
Vice President of RA/QA
Ancestry Genomics, Inc. |
| Alternate Contact Person: | Julie Wood
Director of Quality
Ancestry Genomics, Inc. |

B. PURPOSE FOR SUBMISSION

To obtain a substantial equivalence determination for the AncestryDNA Factor V Leiden Genetic Health Risk Test.

C. MEASURAND

Factor V Leiden c.1601G>A variant in the F5 gene

D. TYPE OF TEST

Qualitative in vitro molecular diagnostic system

E. APPLICANT

Ancestry Genomics, Inc.

F. PROPRIETARY AND ESTABLISHED NAMES

AncestryDNA Factor V Leiden Genetic Health Risk Test

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G. REGULATORY INFORMATION

Trade Name:	AncestryDNA Factor V Leiden Genetic Health Risk Test
Classification:	Class II
Regulation:	21 CFR 866.5950
Regulation Name:	Genetic health risk assessment system
Product Code:	PTA
Panel:	Immunology

H. INTENDED USE

See Indications for Use below.

I. INDICATIONS FOR USE

1. Indications for Use:

The AncestryDNA Factor V Leiden Genetic Health Risk Test for Hereditary Thrombophilia uses qualitative genotyping to detect clinically relevant variants in genomic DNA isolated from human saliva collected from individuals 18 years and older with the AncestryDNA Saliva Collection Kit for the purpose of reporting and interpreting Genetic Health Risks (GHR).

The AncestryDNA Factor V Leiden Genetic Health Risk Report for Hereditary Thrombophilia is indicated for reporting of the Factor V Leiden variant in the F5 gene. This report describes if a person has variants associated with a higher risk of developing harmful blood clots, but it does not describe a person's overall risk of developing harmful blood clots. This test is most relevant for people of European descent.

2. Special Conditions for Use Statements:

• For over-the-counter (OTC) use. a.
• This test is not a substitute for visits to a healthcare provider. It is recommended that b. you consult with a healthcare provider if you have any questions or concerns about your results.
• The AncestryDNA Factor V Leiden Genetic Health Risk (GHR) Test does not detect C. all genetic variants associated with Hereditary Thrombophilia. The absence of a variant tested does not rule out the presence of other genetic variants that may be disease-related.
• d. The test is intended for users ≥ 18 years old.
• The test does not diagnose any specific health conditions. Results should not be used e. to make medical decisions.
• The laboratory may not be able to process a user's sample. The probability that the f. laboratory cannot process a sample can be up to 3% (This estimate was obtained from samples processed in one lab only). If this happens, we will notify you by e-mail and you may request one free replacement kit to provide us with a new sample.

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• g. A user's race, ethnicity, age, and other lifestyle factors may affect how the genetic test results are interpreted.
• Subject to meeting the limitations contained in the special controls under regulation h. 21 CFR 866.5950.
• 3. Special Instrument Requirements:

The AncestryDNA Factor V Leiden Genetic Health Risk Test is to be performed using the Tecan Evo and Illumina iScan instruments.

GenomeStudio Software is a modular software application that is used to view and analyze genotypic data obtained from the Illumina iScan System and based on the cluster definition file. AncestryDNA GHR software conducts a variety of control checks on the file, resulting in a final genotype profile for each sample. These data are used to generate test reports on a user's genotype and associated risk of disease.

J. DEVICE DESCRIPTION

A user's saliva is self-collected using the AncestryDNA Saliva Collection Kit, which consists of a sealable collection tube containing a stabilizing buffer solution. Once the sample is collected, it is shipped to one of two Clinical Laboratory Improvement Amendments (CLIA) certified laboratories for processing.

DNA is isolated from the saliva, quantified, and tested in a multiplex assay using a customized genotyping chip and instrumentation manufactured by Illumina. The multiplex assay simultaneously tests for more than 500,000 variants, including those for the indication proposed herein.

The raw data is generated using Illumina GenomeStudio software, and then sent to Ancestry Genomics (the Manufacturer). The data are analyzed using the Manufacturer's proprietary GHR software, and a genotype is determined for each tested variant. The results for the Factor V Leiden variant are used to generate personalized reports for users that provide information about the disease associated with the detected variant.

Personalized reports are generated for each user that provide results of the testing performed. These reports tell the user which variant(s) has/have been detected in their sample and provide information on the risk of disease associated with the variant(s). If no variant was detected, that information is also provided. The personalized reports are designed to present scientific concepts to users in an easy-to-understand format. The reports provide scientifically valid information about the risks associated with the presence of a particular variant. The reports are designed to help users understand the meaning of their results and any appropriate actions that may be taken based on their results.

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K. SUBSTANTIAL EQUIVALENCE INFORMATION

• 1. Predicate device name(s): 23andMe Personal Genome Service (PGS) Test
• 2. Predicate 510(k) number(s): DEN160026
• 3. Comparison with predicate:

Table 5-1: Predicate Device Comparison

| | AncestryDNA Factor V Leiden
Genetic Health Risk Test | 23andMe Personal Genome
Service Test
(Predicate Device) |
|--------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| K Number | K192944 | DEN160026/DEN140044 |
| SIMILARITIES | | |
| Intended Use | The AncestryDNA Factor V Leiden
Genetic Health Risk Report for
Hereditary Thrombophilia is
indicated for reporting of the Factor
V Leiden variant in the F5 gene. This
report describes if a person has
variants associated with a higher risk
of developing harmful blood clots,
but it does not describe a person's
overall risk of developing harmful
blood clots. This test is most relevant
for people of European descent. | The 23andMe PGS Genetic Health
Risk Report for Hereditary
Thrombophilia is indicated for
reporting of the Factor V Leiden
variant in the F5 gene, and the
Prothrombin G20210A variant in the
F2 gene. This report describes if a
person has variants associated with a
higher risk of developing harmful
blood clots, but it does not describe a
person's overall risk of developing
harmful blood clots. This test is most
relevant for people of European
descent. |
| Special
Conditions for
Use Statements | a. For over-the-counter (OTC) use.
b. This test is not a substitute for
visits to a healthcare provider. It
is recommended that you consult
with a healthcare provider if you
have any questions or concerns
about your results.
c. The AncestryDNA Factor V
Leiden Genetic Health Risk Test
does not detect all genetic
variants associated with
Hereditary Thrombophilia. The
absence of a variant tested does
not rule out the presence of other
genetic variants that may be
disease-related | a. For over-the-counter (OTC) use.
b. This test is not a substitute for
visits to a healthcare provider. It is
recommended that you consult
with a healthcare provider if you
have any questions or concerns
about your results.
c. The 23andMe PGS Genetic Health
Risk Tests for Hereditary
Thrombophilia do not detect all
genetic variants associated with
the aforementioned diseases. The
absence of a variant tested does
not rule out the presence of other
genetic variants that may be
disease-related |
| | AncestryDNA Factor V Leiden
Genetic Health Risk Test | 23andMe Personal Genome
Service Test
(Predicate Device) |
| | d. The test is intended for users ≥ 18 years old.
e. The test does not diagnose any specific health conditions. Results should not be used to make medical decisions.
f. The laboratory may not be able to process a user's sample. The probability that the laboratory cannot process a sample can be up to 3% (This estimate was obtained from samples processed in one lab only). If this happens, we will notify you by e-mail and you may request one free replacement kit to provide us with a new sample.
g. A user's race, ethnicity, age, and other lifestyle factors may affect how the genetic test results are interpreted.
h. Subject to meeting the limitations contained in the special controls under regulation 21 CFR 866.5950. | d. The test is intended for users ≥ 18 years old.
e. The test does not diagnose any specific health conditions. Results should not be used to make medical decisions.
f. The laboratory may not be able to process a user's sample. The probability that the laboratory cannot process a sample can be up to 7.6%.
g. A user's race, ethnicity, age, and sex may affect how the genetic test results are interpreted.
h. Subject to meeting the limitations contained in the special controls under regulation 21 CFR 866.5950. |
| Special
Instrument
Requirements | The AncestryDNA Factor V Leiden
Genetic Health Risk Test for
Hereditary Thrombophilia is to be
performed using the Tecan Evo and
Illumina iScan instruments.

GenomeStudio is a modular software
application that is used to view and
analyze genotypic data obtained
from the Illumina iScan System and
based on the cluster definition file.
AncestryDNA GHR software
conducts a variety of control checks
on the file, resulting in a final
genotype profile for each sample.
These data are used to generate test
reports on a user's genotype and
associated risk of disease. | The 23andMe PGS Genetic Health
Risk Tests for Hereditary
Thrombophilia is to be performed
using the Tecan Evo and Illumina
iScan instruments.

GenomeStudio is a modular software
application that is used to view and
analyze genotypic data obtained
from the iScan. Coregen software
conducts a variety of control checks
on the file, resulting in a final
genotype profile for each sample.
These data are used to generate test
reports on a user's genotype and
associated risk of disease. |
| | AncestryDNA Factor V Leiden
Genetic Health Risk Test | 23andMe Personal Genome
Service Test
(Predicate Device) |
| Classification | Class II | Same |
| Type of Test | Qualitative in vitro molecular
diagnostic system | Same |
| Measurand | Factor V Leiden c.1601G>A variant
in the F5 gene | Factor V Leiden variant in the F5
gene, and the Prothrombin G20210A
variant in the F2 gene |
| Sample
Preparation
Method | DNA extraction | Same |
| Analytical
Sensitivity | The performance requirement for the
AncestryDNA GHR has been set at a
minimum of 1.53 ng/µL DNA and
maximum of 50 ng/µL DNA. | The performance requirement for the
PGS, has been set at a minimum of
15 ng/µL DNA and maximum of 50
ng/µL DNA. |
| Reproducibility/
Precision | 100% correct genotype calls for all
samples with a valid call across
multiple sites, multiple days, using
multiple operator teams, instrument
combinations, and assay reagent lots
tested with samples collected using
multiple lots of the AncestryDNA
Sample Collection Kit at both
laboratory sites.
Genotyping results produced 99.9%
(2337/2340) replicates that were
called correctly and 0.13% (3/2340)
replicates that did not pass quality
control acceptance criteria. Samples
with failed quality control (FQC) on
the first run are re-tested per
laboratory SOPs. | 100% correct genotype calls for all
samples with a valid call across
multiple days, operator teams,
instruments, and reagent lots at both
laboratory sites.
Genotyping results produced 98.0%
(1852/1890) replicates that were
called correctly and 2.01% (38/1890)
replicates that did not pass quality
control acceptance criteria. Samples
with failed quality control (FQC) on
the first run are re-tested per
laboratory SOPs; an anticipated rate
of samples with 2 FQCs based on the
reproducibility study data of cell line
samples is 0.04% (0.020 x 0.020). |
| Endogenous
Interfering
Substances | N = 4 endogenous agents were tested
in saliva: salivary α-amylase,
hemoglobin, IgA, and total protein.
There was no impact on test
performance with all interferents
tested. | N = 4 endogenous agents were tested
in saliva: salivary α-amylase,
hemoglobin, IgA, and total protein.
There was no impact on test
performance with all interferents
tested. |
| Exogenous
Interfering
Substances | N = 6 exogenous agents were tested
in saliva samples collected after
performing the following actions:
eating food containing beef, eating
food not containing beef, drinking | N = 6 exogenous agents were tested
in saliva samples collected after
performing the following actions:
eating food containing beef, eating
food not containing beef, drinking |
| | AncestryDNA Factor V Leiden
Genetic Health Risk Test | 23andMe Personal Genome
Service Test
(Predicate Device) |
| | alcohol, chewing gum, using
mouthwash, and smoking.
There was no impact on test
performance at the 30 minute
timepoint with all interferents tested. | alcohol, chewing gum, using
mouthwash, and smoking.
There was no impact on test
performance at the 30 minute
timepoint with all interferents tested. |
| Microbial
Interfering
Substances | N = 5 microbial agents were tested in
saliva: Staphylococcus epidermis ,
Streptococcus mutans , Lactobacillus
casei , Actinomyces odontolyticus ,
and Candida albicans . There was no
impact on performance with all
interferents tested. | From 23andMe Personal Genome
Service Carrier Screening Test for
Bloom Syndrome (DEN140044):
N = 5 microbial agents were tested in
saliva: Staphylococcus epidermis ,
Streptococcus mutans , Lactobacillus
casei , A., and Candida albicans .
There was no impact on performance
with all interferents tested. |
| Comparison
with Sanger
Bi-directional
Sequencing | Overall agreement was 100%
(198/198) with bi-directional
sequencing. | Overall agreement was 100%
(203/203) with bi-directional
sequencing. |
| Clinical
Performance | The genotype frequencies for Factor
V Leiden in various US population
were obtained from the 2018 ACMG
reporting standard (Zhang et al.,
2018): “In the United States, Factor
V Leiden heterozygosity is present in
5.1%, 2.0%, and 1.2% of Caucasians,
Hispanics, and African Americans
respectively; the frequencies of
homozygosity for the above
populations are 65, 10, and 4 per
100,000 individuals
correspondingly.” | The minor variant frequency for
Factor V Leiden in individuals of
European descent reported in
published literature is 3%-15%;
technical (analytical) positive
predictive values for 23andMe PGS
test results of CT and TT are ≥
99.5% and ≥ 99.1 %
correspondingly.
The minor variant frequency for
Prothrombin G2021 0A in
individuals of European descent
reported in published literature is 1
%--3%; technical (analytical)
positive predictive values for
23andMe PGS test results of AG and
AA are ≥ 98.6% and ≥ 97.2%
correspondingly. |
| DIFFERENCES | | |
| Sample
Collection
Device | AncestryDNA Saliva Collection Kit
(K192947) | Oragene Dx Ogd-500.001
(K141410) |
| Interfering
Mutations | The potential interfering mutations
include rs770011773, rs773367113, | For Factor V Leiden the potential
interfering mutations include |
| AncestryDNA Factor V Leiden
Genetic Health Risk Test | 23andMe Personal Genome
Service Test
(Predicate Device) | |
| and rs143663052. The impact of
these mutations on the performance
of the assay has not been evaluated. | rs760488939 and rs763859650. The
impact of these mutations on the
performance of the assay has not
been evaluated. | |

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L. STANDARDS/GUIDANCE DOCUMENTS REFERENCED

• . Special Controls for Genetic Health Risk Assessment System, as detailed in 21 CFR 866.5950.
• CLSI Guideline EP07-A3, Interference Testing in Clinical Chemistry; Approved Guideline . – Third Edition.
• CLSI Guideline EP09-A3, Measurement Procedure Comparison and Bias Estimation ● Using Patient Samples; Approved Guideline - Third Edition
• CLSI Guideline EP12-A2, User Protocol for Evaluation of Qualitative Test Performance; . Approved Guideline-Second Edition
• CLSI Guideline EP17-A2, Evaluation of Detection Capability for Clinical Laboratory . Measurement Procedures; Approved Guideline - Second Edition.
• CLSI Guideline EP25-A, Evaluation of Stability of In Vitro Diagnostic Reagents; ● Approved Guideline.
• CLSI Guideline EP37-A1, Supplemental Tables for Interference Testing in Clinical . Chemistry.

M. TEST PRINCIPLE

The AncestryDNA Factor V Leiden GHR Test is performed by CLIA-certified laboratories using the BeadChip v10 assay (Illumina Infinium HumanOmniExpress-24 format chip) on the Illumina Infinium Platform. Samples collected using the AncestryDNA Saliva Collection Kit are delivered to laboratories for testing and analysis. DNA from saliva is quantified and samples with DNA concentrations greater than or equal to the limit of detection (1.53 ng/uL) and less than or equal to the maximum DNA concentration (50 ng/uL) are eligible for further processing. These samples are fragmented and captured on a bead array by hybridization to immobilized variant-specific primers, followed by extension with haptenlabeled nucleotides. The primers hybridize adjacent to the variants and are extended with a single nucleotide corresponding to the variant allele. The incorporated hapten-modified nucleotides are detected by adding fluorescently labeled antibodies in several steps to amplify the signals. The Tecan Evo is used in extraction and processing of the DNA, and the Illumina iScan is used to scan and quantify the results. Genotypes are determined using the GenomeStudio software package and delivered to Ancestry Genomics for analysis using the AncestryDNA GHR software.

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N. PERFORMANCE CHARACTERISTICS

The analytical and clinical studies conducted to support the intended use and substantial equivalence claim to the predicate device are summarized below. Execution of the analytical studies, clinical studies and genotyping using the AncestryDNA Factor V Leiden GHR Test was performed by a CLIA-certified laboratory on the Illumina Infinium array platform. Results were analyzed using the Illumina iScan System and Genome Studio software to generate genotypes and calculate call rates. The data was then delivered to Ancestry Genomics where AncestryDNA performed quality control of genotype results and associated the genotype variants to donor identification.

1. Analytical Performance

a. Reproducibility/Precision

The purpose of this study was to determine the precision and reproducibility of the AncestryDNA Factor V Leiden GHR Test at multiple sites, on multiple days, using multiple operator teams, instrument combinations, and assay critical reagent lots tested with samples collected using multiple lots of the AncestryDNA Saliva Collection Kit (SCK). Execution of the study protocol and genotyping using the AncestryDNA Factor V Leiden GHR Test was performed at two (2) CLIA-certified laboratories (Lab 1 and Lab 2) on the Illumina Infinium array platform by six different operator teams (3 per laboratory) on eight instrument combinations (4 per laboratory).

Saliva samples were collected from nine donors with known Factor V Leiden genotypes as determined using bi-directional sequencing: three donors each with homozygous common, heterozygous, and homozygous rare. Each of the nine (9) donors provided 19 saliva samples into three lots of AncestryDNA SCKs. This study was performed over multiple days with three critical assay reagent lots for the AncestryDNA Factor V Leiden GHR Test evaluated in the Lab 1 and one critical assay reagent lot in the Lab 2 (Multi-Sample Amplification Master Mix (MSM), Fragmentation Solution (FMS), BeadChip, XP1 stain, and Two-Color Extension Master Mix (EML)). Genotyping with the AncestryDNA Factor V Leiden GHR Test was conducted over a minimum of six nonconsecutive starting days at Lab 1 and two non-consecutive days at Lab 2.

Each of the donor collections within a given AncestryDNA SCK lot were pooled and mixed, then returned to the AncestryDNA SCK tubes for double extraction. Replicates that did not pass sample call rate (SCR) QC in the first genotyping run underwent second, and when eligible, third genotyping run.

Single Site Precision and Repeatability

At the Lab 1 testing site, repeatability (within-run) and intermediate precision (within laboratory, across days, operator teams, and lots) was performed. For the intermediate precision, each of three operator teams tested each of the nine donor DNA samples in singlicate 20 times over five non-consecutive dates for each of three critical reagent lots,

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across four different instrument combinations. For the within-run repeatability, each donor DNA sample was genotyped an additional four times on one plate (batch). Testing for this batch was performed by one operator team using one critical reagent lot and one instrument combination within a single day.

| Genotype | Total
Number of
Replicates | Number of
Concordant
Calls | Number
of "No-
Calls" | Number
of Call
Rate QC
Failures | FQC
(%) |
|----------|----------------------------------|----------------------------------|-----------------------------|------------------------------------------|------------|
| GG | 540 | 540 | 0 | 0 | 0.00 |
| GA | 540 | 537 | 0 | 3* | 0.56 |
| AA | 540 | 540 | 0 | 0 | 0.00 |
| Total | 1620 | 1617 | 0 | 3 | 0.19 |

Summary of Lab 1 Within-Laboratory Testing Results

*All three (3) QC failures were from the same donor

Results from the within-run repeatability study are shown in the table below. There were no genotyping repeats.

Within-Run Repeatability Results from Lab 1

| Genotype | Total
number of
replicates | Number of
concordant
calls | Number
of “No-
Calls” | Number of
call rate QC
failures | FQC
(%) |
|----------|----------------------------------|----------------------------------|-----------------------------|---------------------------------------|------------|
| GG | 15 | 15 | 0 | 0 | 0.00 |
| GA | 15 | 15 | 0 | 0 | 0.00 |
| AA | 15 | 15 | 0 | 0 | 0.00 |
| Totals | 45 | 45 | 0 | 0 | 0.00 |

At the Lab 2 testing site, intermediate precision (within laboratory, across days, and operator teams) was performed. Each of three operator teams tested each of the nine donor DNA samples in singlicate 20 times over five non-consecutive dates using one critical reagent lot with four different instrument combinations.

Summary of Lab 2 Within-Laboratory Testing Results

| Genotype | Total
Number of
Replicates | Number of
Concordant
Calls | Number
of “No-
Calls” | Number
of Call
Rate QC
Failures | FQC
(%) |
|----------|----------------------------------|----------------------------------|-----------------------------|------------------------------------------|------------|
| GG | 180 | 180 | 0 | 0 | 0.00 |
| GA | 180 | 180 | 0 | 0 | 0.00 |
| AA | 180 | 180 | 0 | 0 | 0.00 |
| Total | 540 | 540 | 0 | 0 | 0.00 |

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Inter-laboratory Reproducibility

For the inter-laboratory reproducibility study, two saliva samples from each donor were extracted at Lab 1 and plated to DNA plates for processing. DNA samples were tested in triplicate at Lab 1 and at Lab 2. This resulted in 18 additional genotyping events per specimen (9 total replicates at Lab 1 and 9 total replicates at Lab 2).

The final genotyping results for the inter-laboratory reproducibility study (n=2) across two instrument combinations, six operator teams, and two different reagent lot combinations (n=1 per lab) is in the table below. The number of concordant calls includes replicates that pass call rate quality control and also have genotypes concordant with the expected genotype (bi-directional sequencing genotype result).

Inter-Laboratory Reproducibility

| Genotype | Total Number
of Replicates | | Number of
Concordant
Calls | | Number of
"No-Calls" | | Number of Call
Rate QC
Failures | | FQC (%) | |
|----------|-------------------------------|-------|----------------------------------|-------|-------------------------|-------|---------------------------------------|-------|---------|-------|
| | Lab 1 | Lab 2 | Lab 1 | Lab 2 | Lab 1 | Lab 2 | Lab 1 | Lab 2 | Lab 1 | Lab 2 |
| GG | 27 | 27 | 27 | 27 | 0 | 0 | 0 | 0 | 0.00 | 0.00 |
| GA | 27 | 27 | 27 | 27 | 0 | 0 | 0 | 0 | 0.00 | 0.00 |
| AA | 27 | 27 | 27 | 27 | 0 | 0 | 0 | 0 | 0.00 | 0.00 |
| Totals | 81 | 81 | 81 | 81 | 0 | 0 | 0 | 0 | 0.00 | 0.00 |

Results by Site and Operator Team

The final genotyping results by site per operator team per genotype across four reagent lot combinations, eight different instrument configurations, and three AncestryDNA SCK lots is in the table below. The number of concordant calls includes replicates that pass call rate QC and have genotypes concordant with the expected genotype as determined by bi-directional sequencing.

| Site/
Operator
Team | Expected
Genotype | Total
Number of
Replicates | Number of
Concordant
Calls | Number of
"No-Calls" | Number of
Call Rate
QC Failures* | Proportion
of FQC
(%) |
|---------------------------|----------------------|----------------------------------|----------------------------------|-------------------------|----------------------------------------|-----------------------------|
| Lab 1
Team 1 | AA | 195 | 195 | 0 | 0 | 0.00 |
| Lab 1
Team 1 | GA | 195 | 194 | 0 | 1 | 0.51 |
| Lab 1
Team 1 | GG | 195 | 195 | 0 | 0 | 0.00 |
| Total | | 585 | 584 | 0 | 1 | 0.17 |
| Lab 1
Team 2 | AA | 189 | 189 | 0 | 0 | 0.00 |
| Lab 1
Team 2 | GA | 189 | 188 | 0 | 1 | 0.53 |
| Lab 1
Team 2 | GG | 189 | 189 | 0 | 0 | 0.00 |
| Total | | 567 | 566 | 0 | 1 | 0.18 |

Site and Operator Team Results

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| Site/
Operator
Team | Expected
Genotype | Total
Number of
Replicates | Number of
Concordant
Calls | Number of
"No-Calls" | Number of
Call Rate
QC Failures* | Proportion
of FQC
(%) |
|----------------------------------|----------------------|----------------------------------|----------------------------------|-------------------------|----------------------------------------|-----------------------------|
| Lab 1
Team 3 | AA | 189 | 189 | 0 | 0 | 0.00 |
| | GA | 189 | 188 | 0 | 1 | 0.53 |
| | GG | 189 | 189 | 0 | 0 | 0.00 |
| Total | | 567 | 566 | 0 | 1 | 0.18 |
| Lab 2
Team 1 | AA | 69 | 69 | 0 | 0 | 0.00 |
| | GA | 69 | 69 | 0 | 0 | 0.00 |
| | GG | 69 | 69 | 0 | 0 | 0.00 |
| Total | | 207 | 207 | 0 | 0 | 0.00 |
| Lab 2
Team 2 | AA | 69 | 69 | 0 | 0 | 0.00 |
| | GA | 69 | 69 | 0 | 0 | 0.00 |
| | GG | 69 | 69 | 0 | 0 | 0.00 |
| Total | | 207 | 207 | 0 | 0 | 0.00 |
| Lab 2
Team 3 | AA | 69 | 69 | 0 | 0 | 0.00 |
| | GA | 69 | 69 | 0 | 0 | 0.00 |
| | GG | 69 | 69 | 0 | 0 | 0.00 |
| Total | | 207 | 207 | 0 | 0 | 0.00 |
| All teams'
totals
combined | GG, GA,
AA | 2,340 | 2,337 | 0 | 3 | 0.13 |

• The heterozygous donor failures in each of the Lab 1 operator teams are from a single donor.

Results by Site and Instrument Combination

The final genotyping results by site per instrument combination per genotype across four reagent lot combinations, six different operator teams, and three AncestryDNA SCK lots is in the table below. The number of genotype calls includes replicates that pass the QC call rate and have genotypes concordance with the expected genotype as determined by bi-directional sequencing.

| Site/Instrument
Combination | Expected
Genotype | Total
Number of
Replicates | Number of
Concordant
Calls | Number
of “No-
Calls” | Number of
Call Rate
QC Failures | Proportion
of FQC
(%) |
|---------------------------------------|----------------------|----------------------------------|----------------------------------|-----------------------------|---------------------------------------|-----------------------------|
| Lab 1-
Instrument
combination A | AA | 168 | 168 | 0 | 0 | 0.00 |
| | GA | 168 | 168 | 0 | 0 | 0.00 |
| | GG | 168 | 168 | 0 | 0 | 0.00 |
| Total | | 504 | 504 | 0 | 0 | 0.00 |
| Lab 1-
Instrument
combination B | AA | 135 | 135 | 0 | 0 | 0.00 |
| | GA | 135 | 134 | 0 | 1 | 0.74 |
| | GG | 135 | 135 | 0 | 0 | 0.00 |
| Site/Instrument
Combination | Expected
Genotype | Total
Number of
Replicates | Number of
Concordant
Calls | Number
of “No-
Calls” | Number of
Call Rate
QC Failures | Proportion
of FQC
(%) |
| Total | | 405 | 404 | 0 | 1 | 0.25 |
| Lab 1-
Instrument
combination C | AA | 135 | 135 | 0 | 0 | 0.00 |
| | GA | 135 | 133 | 0 | 2 | 1.48 |
| | GG | 135 | 135 | 0 | 0 | 0.00 |
| Total | | 405 | 403 | 0 | 2 | 0.49 |
| Lab 1-
Instrument
combination D | AA | 135 | 135 | 0 | 0 | 0.00 |
| | GA | 135 | 135 | 0 | 0 | 0.00 |
| | GG | 135 | 135 | 0 | 0 | 0.00 |
| Total | | 405 | 405 | 0 | 0 | 0.00 |
| Lab 2
-Instrument
combination A | AA | 72 | 72 | 0 | 0 | 0.00 |
| | GA | 72 | 72 | 0 | 0 | 0.00 |
| | GG | 72 | 72 | 0 | 0 | 0.00 |
| Total | | 216 | 216 | 0 | 0 | 0.00 |
| Lab 2
-Instrument
combination B | AA | 45 | 45 | 0 | 0 | 0.00 |
| | GA | 45 | 45 | 0 | 0 | 0.00 |
| | GG | 45 | 45 | 0 | 0 | 0.00 |
| Total | | 135 | 135 | 0 | 0 | 0.00 |
| Lab 2
-Instrument
combination C | AA | 45 | 45 | 0 | 0 | 0.00 |
| | GA | 45 | 45 | 0 | 0 | 0.00 |
| | GG | 45 | 45 | 0 | 0 | 0.00 |
| Total | | 135 | 135 | 0 | 0 | 0.00 |
| Lab 2
-Instrument
combination D | AA | 45 | 45 | 0 | 0 | 0.00 |
| | GA | 45 | 45 | 0 | 0 | 0.00 |
| | GG | 45 | 45 | 0 | 0 | 0.00 |
| Total | | 135 | 135 | 0 | 0 | 0.00 |
| All instruments'
totals combined | GG, GA,
AA | 2,340 | 2,337 | 0 | 3 | 0.13 |
| Site/Reagent
Lot
Combination | Expected
Genotype | Total
Number of
Replicates | Number of
Concordant
Calls | Number
of “No-
Calls” | Number of
Call Rate
QC Failures | Proportion
of FQC
(%) |
| Lab 1-Lot 1 | AA | 213 | 213 | 0 | 0 | 0.00 |
| Lab 1-Lot 1 | GA | 213 | 211 | 0 | 2 | 0.94 |
| Lab 1-Lot 1 | GG | 213 | 213 | 0 | 0 | 0.00 |
| Total | | 639 | 637 | 0 | 2 | 0.31 |
| Lab 1-Lot 2 | AA | 180 | 180 | 0 | 0 | 0.00 |
| Lab 1-Lot 2 | GA | 180 | 180 | 0 | 0 | 0.00 |
| Lab 1-Lot 2 | GG | 180 | 180 | 0 | 0 | 0.00 |
| Total | | 540 | 540 | 0 | 0 | 0.00 |
| Lab 1-Lot 3 | AA | 180 | 180 | 0 | 0 | 0.00 |
| Lab 1-Lot 3 | GA | 180 | 179 | 0 | 1 | 0.56 |
| Lab 1-Lot 3 | GG | 180 | 180 | 0 | 0 | 0.00 |
| Total | | 540 | 539 | 0 | 1 | 0.19 |
| Lab 2-Lot 1 | AA | 207 | 207 | 0 | 0 | 0.00 |
| Lab 2-Lot 1 | GA | 207 | 207 | 0 | 0 | 0.00 |
| Lab 2-Lot 1 | GG | 207 | 207 | 0 | 0 | 0.00 |
| Total | | 621 | 621 | 0 | 0 | 0.00 |
| All reagent lots'
totals combined | GG, GA,
AA | 2,340 | 2,337 | 0 | 3 | 0.13 |

Site and Instrument Combination Results

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Results by Site and Factor V Leiden Reagent Lot Combination

The final genotyping results by site per critical reagent lot combination per genotype across eight instrument combinations, six different operator teams and three AncestryDNA SCK lots. The number of genotype calls includes replicates that pass the QC call rate and have genotype concordance with the expected genotype as determined by bi-directional sequencing.

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Site and Critical Reagent Lot Combination Results
---------------------------------------------------	--

Overall Percent Agreement (OPA) for Repeatability and Genotyping

The OPA point estimates for repeatability exceeded the 99% predefined protocol acceptance criteria, and the OPA point estimates for each genotype exceeded the 99% predefined protocol acceptance criteria as seen in the table below.

| Attribute | Concordant Replicates
(total QC passing replicates) | Point Estimate Percent
Agreement (%)
(95% confidence interval) |
|--------------------------------|--------------------------------------------------------|----------------------------------------------------------------------|
| Lab 1 laboratory | 1,716 (1,716) | 100.00 (99.79 - 100.00) |
| Lab 2 laboratory | 621 (621) | 100.00 (99.41 - 100.00) |
| Lab 1 operator team 1 | 584 (584) | 100.00 (99.37 - 100.00) |
| Lab 1 operator team 2 | 566 (566) | 100.00 (99.35 - 100.00) |
| Lab 1 operator team 3 | 566 (566) | 100.00 (99.35- 100.00) |
| Lab 2 operator team 1 | 207 (207) | 100.00 (98.23 - 100.00) |
| Lab 2 operator team 2 | 207 (207) | 100.00 (98.23 - 100.00) |
| Lab 2 operator team 3 | 207 (207) | 100.00 (98.23 - 100.00) |
| All operator teams | 2,337 (2,337) | 100.00 (99.84 - 100.00) |
| Lab 1 instrument combination A | 504 (504) | 100.00 (99.27 - 100.00) |

Point Estimates for Overall Percent Agreement for Repeatability and Genotype

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| Attribute | Concordant Replicates
(total QC passing replicates) | Point Estimate Percent
Agreement (%)
(95% confidence interval) |
|--------------------------------|--------------------------------------------------------|----------------------------------------------------------------------|
| Lab 1 instrument combination B | 404 (404) | 100.00 (99.09 - 100.00) |
| Lab 1 instrument combination C | 403 (403) | 100.00 (99.09 - 100.00) |
| Lab 1 instrument combination D | 405 (405) | 100.00 (99.09 - 100.00) |
| Lab 2 instrument combination A | 216 (216) | 100.00 (98.31- 100.00) |
| Lab 2 instrument combination B | 135 (135) | 100.00 (97.30 - 100.00) |
| Lab 2 instrument combination C | 135 (135) | 100.00 (97.30 - 100.00) |
| Lab 2 instrument combination D | 135 (135) | 100.00 (97.30 - 100.00) |
| All instrument combinations | 2,337 (2,337) | 100.00 (99.84 - 100.00) |
| Lab 1 reagent lot 1 | 637 (637) | 100.00 (99.42 - 100.00) |
| Lab 1 reagent lot 2 | 540 (540) | 100.00 (99.32 - 100.00) |
| Lab 1 reagent lot 3 | 539 (539) | 100.00 (99.32 - 100.00) |
| Lab 2 reagent lot 1 | 621 (621) | 100.00 (99.41 - 100.00) |
| All reagent lot combinations | 2,337 (2,337) | 100.00 (99.84 - 100.00) |
| Within-run precision at Lab 1 | 1617 (1617) | 100.00 (99.77 - 100.00) |
| Within-run precision at Lab 2 | 540 (540) | 100.00 (99.32 - 100.00) |
| Within-run repeatability | 45 (45) | 100.00 (92.13 - 100.00) |
| Inter-lab data at Lab 1 | 81 (81) | 100.00 (95.55 - 100.00) |
| Inter-lab data at Lab 2 | 81 (81) | 100.00 (95.55 - 100.00) |
| All "GG" | 780 (780) | 100.00 (99.53 - 100.00) |
| All "GA" | 777 (777) | 100.00 (99.53 - 100.00) |
| All "AA" | 780 (780) | 100.00 (99.53 - 100.00) |

The AncestryDNA Factor V Leiden GHR Test was evaluated at multiple labs, on multiple days, by multiple personnel teams, using multiple instrument combinations, and multiple reagent lots. The results demonstrate that the AncestryDNA Factor V Leiden GHR Test met the acceptance criteria for overall precision > 99% point estimate, and for each genotype ≥ 99% agreement. The AncestryDNA SCK in combination with the AncestryDNA Factor V Leiden GHR Test consistently produced results that were in agreement with the true variant status, which was determined by bidirectional sequencing.

b. Linearity/Assay Reportable Range

Not applicable.

c. Traceability, Stability, Expected Values (controls, calibrators, or methods)

Controls

The AncestryDNA Factor V Leiden Genetic Health Risk Test uses one (1) control material, which serves as both the sample processing control and the reproducibility control. The control material is genotyped on the Illumina BeadChip according to routine SOPs at the laboratory. Each new lot of the control is tested by comparison with reference BeadChip genotype results.

18

The control was tested for shelf life stability when stored at 2 to 8°C. This study is ongoing and performance will be evaluated against the pre-established acceptance criteria at 3-months and 4-months post-manufacturing. The interim data shows that the control is stable for a minimum of 1 month post-manufacturing.

The control was also tested for in-use stability. Data shows that the control has an in-use stability of 30 days from first opening.

Reagent Stability

This study is ongoing, and performance will be evaluated against the pre-established acceptance criteria at 3-months, 6-months, 12-months, and 13-months postmanufacturing. The interim data shows that the critical assay reagents are stable for a minimum of 2 months post-manufacturing.

The storage condition and temperature for the critical reagents (all are single use) are listed in the table below.

Critical Reagent	Storage condition	Storage temperature
MSM	Frozen	-15°C to -25°C
FMS	Frozen	-15°C to -25°C
BeadChip	Refrigerated	2 to 8°C
X-Stain Plate	Frozen	-15°C to -25°C
EML	Frozen	-15°C to -25°C

Storage conditions and temperatures for the critical assay reagents

d. Analytical Sensitivity

Two studies were conducted to determine Analytical Sensitivity, one using saliva samples and one using cell line samples.

The study was designed around the regression (probit/logit) approach from section 5.5 of the CLSI EP17-A2 to determine Limit of Blank (LoB) and Limit of Detection (LoD). Each sample was serially diluted to different DNA concentrations and genotyped using three (3) lots of critical reagents. To confirm the genotype call, each sample was also sequenced by bi-directional sequencing to determine the rates of correct genotype calls at each DNA concentration. The Limit of Detection (LoD) was defined as the lowest DNA concentration at which at least 95% of samples yielded the correct call.

Limit of Blank Test Method

For the study using saliva samples, to establish the Limit of Blank (LoB), blanks consisting of molecular grade water and the standard volume of DNA stabilizing solution from the AncestryDNA SCK were extracted, quantified, and genotyped using three lots of critical reagents.

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For the cell line study, blanks consisting of molecular grade water were quantified and genotyped using three lots of critical reagents.

Limit of Detection Test Method

For the study using saliva, samples were collected from 15 donors with known Factor V Leiden genotypes as determined using bi-directional sequencing: five donors each with homozygous common, heterozygous, and homozygous rare. Samples were collected using the Oragene® Dx Collection Device, model OGD-500.001 (OGD) (n=1) and the AncestryDNA SCK (n=4). DNA extracted from each donor's saliva samples were pooled to create a homogenous solution. The pooled DNA was used to create a two-fold dilution series, including a neat sample and four additional dilutions for a total of five samples per donor per genotype in the series. Six replicates of each dilution series were genotyped, two (2) replicates per critical reagent lot (n=3) using the AncestryDNA Factor V Leiden GHR Test. Testing was performed under the standard protocol for the AncestryDNA GHR Test, except that each donor sample in the series was genotyped in duplicate per critical reagent lot. A total of 450 replicates were tested in the LoD study.

For the study using cell lines, four (4) cell lines with known Factor V Leiden genotypes as determined using bi-directional sequencing were used. DNA from each of the cell lines was diluted to generate one (1) four-fold dilution and six (6) two-fold dilutions. In addition, a neat sample was used for a total of eight (8) samples in each dilution series. The samples and water blanks were quantified by UV-Vis spectroscopy to determine DNA concentration and purity (A260/A280). A total of 27 replicates of each cell line dilution series were genotyped using the AncestryDNA Factor V Leiden GHR Assay; three (3) replicates per critical reagent lot (n=3) on three (3) testing days for a total of 216 data points per cell line. A total of 855 blank replicates were genotyped using the AncestryDNA Factor V Leiden GHR Assay: 95 replicates per critical reagent lot (n=3) on three (3) testing days. Testing was performed by one (1) operator team using one (1) instrument line.

Based on both studies using saliva samples and cell line samples:

• The LoB = 1.004 ng/uL, based on the non-parametric rank method from section ● 5.3.3.1 of EP17-A2 to account for sources of measurement variability in the both UV-Vis spectrophotometry and the bead-based fluorescence assay.
• . The LoD = 1.53 ng/uL, a limit concentration that is statistically distinguishable from blank samples.
• . The upper limit of concentration = 50 ng/uL.

All genotyping attempts on samples containing the measurand with call rates ≥ 98% and concentrations between 1.53 ng/uL and 50 ng/uL produced genotypes concordant with bidirectional sequencing.

20

e. Interfering Substances

The analytical specificity studies were designed using CLSI EP07 - Interference Testing in Clinical Chemistrv: Approved Guideline – Third Edition for determining potential interference with the AncestryDNA Factor V Leiden GHR Test. Endogenous, exogenous, microbial DNA, and mutational interferents were evaluated as part of the analytical specificity study.

Endogenous Interference

Four potential common endogenous interferents were evaluated to determine the effect on the performance of the AncestryDNA Factor V Leiden GHR Test as listed in the table below.

Endogenous Substance	Final Concentration (1x) in Saliva
PBS (reference/control)	N/A
Salivary α-amylase	395 U/mL
Hemoglobin	20 mg/mL
IgA	0.44 mg/mL
Total Protein	0.185 mg/mL Salivary α-amylase
	0.44 mg/mL IgA
	2.05 mg/mL human serum albumin

Endogenous Interferent Concentrations

A total of ten saliva donors with unknown Factor V Leiden genotypes were utilized in the specificity study. A saliva sample from each donor was collected with the Oragene® Dx Collection Device, model OGD-500.001 (OGD) (K141410) and sent to a third party laboratory to determine the true variant status using bi-directional sequencing analysis. Each donor provided saliva samples into five AncestryDNA SCK Saliva Collection Tubes that were shipped to the laboratory. The endogenous substances were individually spiked into saliva prior to DNA extraction and genotyping (see above table). Saliva that was spiked with PBS served as the reference/control. The assay was executed by the same two (2) operators for each genotyping replicate. Two lots of the assay reagents were used during the execution of the study. Each sample was genotyped in triplicate for a total of 30 replicate genotyping attempts (3 replicates for each of 10 donors) per each interferent and 30 control replicate genotyping attempts (450 total initial genotyping attempts).

For each endogenous interferent, the acceptance criteria of > 90% agreement with true variant status for all samples that had passed quality control had been met. For all samples where the control samples passed QC, the concordance for all interfering substances was 100%, meeting the acceptance criterion of ≥ 95% agreement with true variant status for all controls and endogenous substances determined by bi-directional sequencing. The point estimate of overall percent agreement from each of the

21

endogenous interferents is provided in the table below. The results indicate that the performance of the AncestryDNA Factor V Leiden GHR Test when tested from samples collected with the AncestryDNA SCK are not affected by the tested interferents.

Endogenous Interferent	Overall Percent Agreement Point Estimate
PBS (reference/control)	100% (30/30)
Salivary α-amylase	100% (30/30)
Hemoglobin	100% (30/30)
IgA	100% (30/30)
Total Protein	100% (30/30)

Overall Percent Agreement for the Endogenous Interference Study

Exogenous Interference

Six potential exogenous interferents were evaluated to determine their effect on the performance of the AncestryDNA Factor V Leiden GHR Test. The exogenous interference study included samples from non-smokers and smokers. The study was performed with nine lots of assay reagents. Saliva samples were collected from 10 nonsmokers in 15 AncestryDNA SCKs over the course of five (5) days. Each day, the donor performed one (1) of the five (5) activities (eating chicken, drinking alcohol, chewing gum, or using mouthwash). The donors provided three (3) tubes per day as follows: before consuming the exogenous substance (control/baseline), immediately after, and 30-minutes after performing the activity. Saliva samples were also collected from 10 smokers into 3 AncestryDNA SCK per day as follows: before smoking (control/baseline), immediately after smoking, and 30-minutes after smoking. There was a total of 594 data points as summarized in the table below.

Exogenous Activity	Donor Count	Time Point	Replicates	Total
Eating chicken	12	3	3	108
Drinking alcohol	12	3	3	108
Using mouthwash	12	3	3	108
Eating beef	10	3	3	90
Chewing gum	10	3	3	90
Smoking	10	3	3	90
Total	--	--	--	594

Overview of Exogenous Interferent Study Design

For all samples where the control samples and replicates containing the interfering substances passed QC, the concordance for all interfering substances was 100%. This met the acceptance criterion of ≥ 95% agreement with true variant status from bidirectional sequencing for all samples that have passed QC. Results indicate that the performance of the AncestryDNA Factor V Leiden GHR Test when tested from samples collected with the AncestryDNA SCK are not affected by the tested interferents. The table below summarizes the overall percent agreement (OPA) point estimate calculated on genotyping events with control samples that passed QC for each interferent and time point.

22

| Exogenous Substance | OPA Point Estimate
(Concordant Replicates / Total QC Passing Replicates) | |
|---------------------|-----------------------------------------------------------------------------|--------------|
| | T0 | T30 |
| Chicken | 100% (33/33) | 100% (36/36) |
| Alcohol | 100% (36/36) | 100% (36/36) |
| Mouthwash | 100% (36/36) | 100% (36/36) |
| Beef | 100% (27/27) | 100% (30/30) |
| Gum | 100% (30/30) | 100% (30/30) |
| Smoking | 100% (30/30) | 100% (30/30) |

Overall Percent Agreement for Exogenous Interferents
------------------------------------------------------

For the eating chicken and eating beef activities, a higher rate of QC failures for samples collected immediately after completing the activity (T0) when matched donor control samples passed QC was observed. Therefore, the AncestryDNA Saliva Collection Kit will include in the labeling the following sentence as part of the saliva collection warning: "Do NOT eat, drink, smoke or chew gum for 30 minutes before giving your saliva sample".

Microbial Interference

Microbial DNA from five (5) different species (Staphylococcus epidermis, Streptococcus mutans, Lactobacillus casei, Actinomyces odontolyticus, and Candida albicans) were evaluated to determine its impact on the performance of the AncestryDNA Factor V Leiden GHR Test. DNA from six (6) human cell lines was obtained for this study:

• Four cell lines were Factor V Leiden homozygous common (GG), .
• One cell line was Factor V Leiden heterozygous (GA), and
• One cell line was Factor V Leiden homozygous rare (AA). .

All cell lines were subjected to bi-directional sequencing by a third-party laboratory to verify the Factor V Leiden genotype as part of the study. DNA from each of the six human cell lines was spiked with two concentrations (low/normal (2.8 ng/uL) and high (12.5 ng/uL)) of the five different species of microbial DNA. Human cell line DNA spiked with buffer functioned as a spike-in control at both concentrations. Each of the human cell lines was spiked a total of 12 times (5 microbial interferents and a control at 2 levels per cell line). The resulting 72 DNA mixtures were genotyped in replicates of 6 using the AncestryDNA Factor V Leiden GHR Test, for a total of 432 genotyping results (6 cell lines x 6 microbe/control x 2 concentrations x 6 replicates = 432). One lot of reagent was used during the execution of the study.

Each sample and replicate, spiked with two levels of microbial interferent, and unspiked (spiked with PBS) was compared directly to bidirectional sequencing results. The assay produced concordant genotypes with bidirectional sequencing in all genotyping events. The point estimate of overall percent agreement (OPA) with true variant status from each condition is provided below. The Factor V Leiden GHR Test performs to the internal specifications and meets the study acceptance criteria of a > 95% agreement with true

23

variant status. The assay reproduced the true variant status, as determined by bidirectional sequencing, for each replicate that was tested, including all control genotyping replicates and all interferent-spiked genotyping replicates. The results indicate that there is no significant impact of common microbial interferents on the performance of the Factor V Leiden GHR Test in either low/normal or higher-than-average concentrations.

Microbial Interferent	OPA Point Estimate
	Low/Normal Concentration	High Concentration
Buffer (reference/control)	100% (36/36)	100% (36/36)
S. epidermis	100% (36/36)	100% (36/36)
S. mutans	100% (36/36)	100% (36/36)
L. casei	100% (36/36)	100% (36/36)
A. odontolyticus	100% (36/36)	100% (36/36)
C. albicans	100% (36/36)	100% (36/36)

Microbial Interferent Testing Results for Overall Percent Agreement

Mutational Interference

Nucleotide mutations were evaluated to determine the impact on the performance of the AncestryDNA Factor V Leiden GHR Test. An in silico analysis was performed to identify nucleotide mutations that could potentially interfere with the AncestryDNA Factor V Leiden GHR Test within the genomic region that is one probe length (50 base pairs (bp)) downstream of the Factor V Leiden mutation (rs6025; NC 000001.11: g.169549811G>A; NM 000130.4: c.1601G>A). The genome aggregation database (gnomAD database) was utilized to search for mutations in this region. Only mutations that have been observed in more than one individual were evaluated. Three potentially interfering mutations were identified as summarized in below. These mutations are located within one (1) probe length of the Factor V Mutation and have an allele count >1. The impact of these mutations on the performance of the assay has not been evaluated.

| rsID | Chromosome
Position | Transcript
Consequence | Annotation | Allele
Count | Allele
Frequency |
|-------------|------------------------|---------------------------|---------------------|-----------------|---------------------|
| rs770011773 | g.169549812 | c.1600C>T | Stop Gained | 3 | 0.00001062 |
| rs773367113 | g.169549835 | c.1577G>A | Missense
Variant | 2 | 0.000007073 |
| rs143663052 | g.169549848 | c.1564C>G | Missense
Variant | 14 | 0.00004951 |

Summary of Identified Potentially Interfering Mutations

Two (2) mutations that were classified as potentially interfering in the predicate submission (DEN160026), rs760488939 and rs763859650, will not interfere in the AncestryDNA Factor V Leiden GHR Test since their genomic location is greater than 50 bp (one probe length) from rs6025. These potentially interfering mutations cannot be empirically tested due to the absence of independent probes on the array.

24

f. Assay Cut-off

Not Applicable.

g. Specimen Stability

Saliva samples for testing are collected with the AncestryDNA Saliva Collection Kit. See K192947 for sample stability information.

h. Shipping Stability

Saliva samples for testing are shipped in the AncestryDNA Saliva Collection Kit. See K192947 for sample shipping stability information.

2. Comparison Studies

a. Method Comparison with the Predicate

The accuracy of the AncestryDNA Factor V Leiden GHR Test was established by comparing the results of the test to the true variant status as determined by bi-directional sequencing analysis at a third-party laboratory.

Saliva samples were collected from 209 donors with known Factor V Leiden genotypes: 200 initial study donors plus nine alternate study donors. The genotypes of the donors as determined by bi-directional sequencing were 73 homozygous common, 69 heterozygous, and 67 homozygous rare. Samples were collected using Oragene Dx Ogd-500.001 (OGD) and the AncestryDNA SCK.

• Each sample collected using the OGD device was subjected to bi-directional ● sequencing by a third-party laboratory to verify the Factor V Leiden genotype,
• Each donor sample collected using the AncestryDNA SCK was used in the accuracy . study with the AncestryDNA Factor V Leiden GHR Test.

Of the 200 samples initially genotyped, 185 passed the first pass genotyping quality control sample-level call rate (SCR) of > 98% and 15 did not ( A), 2018 update: a technical standard of the American College of Medical Genetics and Genomics (ACMG)." Genetics in Medicine 20.12 (2018): 1489.

• 2. Simone, Benedetto, et al. "Risk of venous thromboembolism associated with single and combined effects of Factor V Leiden, Prothrombin 20210A and Methylenetethraydrofolate reductase C677T: a meta-analysis involving over 11,000 cases and 21,000 controls." (2013): 621-647.

b. User Comprehension Studies

Ancestry Genomics sponsored two studies to assess user comprehension of the AncestryDNA Factor V Leiden GHR Test's Genetic Health Risk (GHR) reports.

The first user comprehension study, Study 1, was designed across all report types using a post- test administered questionnaire. Hence, a second study, Study 2, was designed to assess comprehension across the two most challenging reports and evaluate comprehension using a pre-test and post-test administered questionnaire.

In both studies, a Genetic Risk Education Module was developed to provide eligible participants with a brief overview of genetics in order to prepare them for review of the GHR report and explain the significance of genetic risk reports. Participants were representative of the intended use population: adults aged 18 years and older in the U.S. Participants were recruited to match the demographics (education, age, sex/gender, and race/ethnicity) of the adult U.S. population as of the most recent estimates released by the U.S. Census Bureau. Geographic diversity was addressed through participant recruitment and comprehension testing from each of the four U.S. Census geographic regions: Northeast, Midwest, South, and West.

In Study 1, a total of 378 individuals were enrolled as participants into each of four study arms, one for each of the GHR report types:

• 1. 0 Variants Identified
• 2. 1 Variant Identified
• 3. 2 Variants Identified
• 4. Result Not Determined

In Study 2, a total of 213 individuals were enrolled as participants into each of two study arms, one for each of the following two GHR report types:

• 1. 1 Variant Identified
• 2. Result Not Determined

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Both user comprehension studies were performed on the different types of the GHR reports developed using representative samples of the materials below ("supplemental materials").

Supplemental Materials

• Education Module including definition of terms, .
• Pre-purchase page, ●
• Frequently Asked Questions, and .
• . Technical Details.

The representative samples were developed by Ancestry Genomics based on FDA guidance for medical device patient labeling and designed for readability no higher than 8th grade reading level using a Flesch-Kincaid Readability Test. The representative samples were reviewed by a Certified Genetic Counselor to confirm that the materials tested accomplished the following:

• Defined the target condition being tested and related symptoms, .
• Explained the intended use and limitations of the test,
• Explained the relevant ethnicities in regard to the variant tested, ●
• Explained genetic health risks and relevance to the user's ethnicity, and ●
• Assessed participants' ability to understand the following comprehension concepts: . the test's limitations, purpose, appropriate follow-up action, test results, ethnic relevance and other risk factors that may have an impact on the test results.

In Study 1, which was conducted in-person, participants were observed discretely, and each interview session was administered by a trained interviewer/moderator using a series of predefined questions. Participants were given a time limit from start of report review to completion of the comprehension survey. There was a 100% response rate for all questions for all participants included in the data analysis. Failing to respond to numerous items in the questionnaire was one of the criteria used to identify participants for exclusion from data analysis.

In Study 2, which was conducted via live televideo interviews, participants were observed via live video, and each interview session was administered online by a trained interviewer/moderator using a series of predefined questions. Participants were first administered a pre-test questionnaire prior to being shown any of the representative samples. Participants were then given a time limit from start of report review to completion of the post-test questionnaire. There was a 100% response rate for all questions for all participants included in the data analysis. Failing to respond to numerous items in the questionnaire was one of the criteria used to identify participants for exclusion from data analysis. All pre-test questions were repeated again in the posttest questionnaire to assess pre-test vs post-test improvement.

The table below summarizes comprehension scores in Study 1 for each core concept evaluated by report type and for the overall comprehension rate. The average comprehension rates per core comprehension concept range from 90.7% to 97.4% for all subjects who participated in the study.

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| Core
Concept | Comprehension Rates by GHR Report Type (%) | 0 Variant | 1 Variant | 2 Variant | Result Not
Determined | Overall
Comprehension
Rates |
|---------------------------------|--------------------------------------------|-----------|-----------|-----------|--------------------------|-----------------------------------|
| Appropriate
Follow-Up Action | 95.8 | 97.7 | 100 | 95.6 | 97.4% | |
| Ethnic Relevance | 98.9 | 89.5 | 97.2 | N/A | 95.5% | |
| Other Risk Factors | 89.6 | 94.2 | 92.5 | 92.2 | 92.1% | |
| Limitations of Test | 88.5 | 89.5 | 98.1 | 88.9 | 91.5% | |
| Purpose of Test | 89.0 | 95.3 | 93.4 | 93.3 | 92.7% | |
| Results of Test | 93.8 | 83.7 | 93.4 | 91.1 | 90.7% | |
| Total Number of
Reports | N = 96 | N = 86 | N = 106 | N = 90 | N = 378 | |

Study 1 - Overall User Comprehension Rates for Factor V Leiden GHR Reports
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The table below summarizes comprehension scores in Study 2 for each core concept evaluated by report type and for the overall comprehension rate. The average comprehension rates per core comprehension concept range from 90.9% to 99.1% for all subjects who participated in the study.

| Core
Concept | Comprehension Rates by GHR Report Type (%) | | Overall
Comprehension
Rates |
|---------------------------------|--------------------------------------------|-----------------------|-----------------------------------|
| | 1 Variant | Result Not Determined | |
| Appropriate
Follow-Up Action | 94.2 | 98.6 | 96.5% |
| Ethnic Relevance | 98.1 | 97.3 | 97.7% |
| Other Risk Factors | 98.1 | 93.6 | 95.8% |
| Limitations of Test | 93.7 | 98.6 | 96.2% |
| Purpose of Test | 98.5 | 99.6 | 99.1% |
| Results of Test | 88.8 | 92.7 | 90.9% |
| Total Number of
Reports | N = 103 | N = 110 | N = 213 |

Study 2 - Overall User Comprehension Rates for Factor V Leiden GHR Reports

The acceptance criteria were met for both User Comprehension Studies which stated ≥ 90% comprehension score for each of the domains evaluated: test results, test purpose, test limitations, appropriate follow-up action, ethnic relevance, and other risk factors that may impact the test results. In addition, the overall comprehension score across all GHR reports was 93.2% for Study 1 and 96% for Study 2.

In addition, Study 2 was able to show that there was an improvement in comprehension between pre-test versus post-test. The table below shows the pre-test versus post-test statistical comparison by comprehension concept.

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| Comprehension
Concept | Pre-test (%) | Post Test (%) | %
Improvement | p-value |
|--------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|---------|
| Purpose | 89.0 | 99.1 | 10.1 |