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.

AI/ML Overview

Here is a summary of the acceptance criteria and the study that proves the device meets the acceptance criteria, based on the provided text:

Acceptance Criteria and Reported Device Performance

Criteria	Acceptance Criterion	Reported Device Performance
Reproducibility/Precision (Overall)	>99% point estimate Overall Percent Agreement (OPA)	100.00% (99.84 - 100.00) for all operator teams combined; 100.00% (99.84 - 100.00) for all instrument combinations combined; 100.00% (99.84 - 100.00) for all reagent lot combinations combined.
Reproducibility/Precision (Per Genotype)	≥99% agreement for each genotype	100.00% (99.53 - 100.00) for GG; 100.00% (99.53 - 100.00) for GA; 100.00% (99.53 - 100.00) for AA.
Analytical Sensitivity (LoD)	Lowest DNA concentration at which ≥95% of samples yield correct call	LoD = 1.53 ng/uL at which all genotyping attempts on samples with call rates ≥ 98% produced concordant genotypes.
Analytical Sensitivity (LoB)	Limit of Blank	LoB = 1.004 ng/uL
Analytical Sensitivity (Upper Limit)	Maximum DNA concentration for valid results	50 ng/uL
Interfering Substances (Endogenous)	≥95% agreement with true variant status for all controls and endogenous substances	100% agreement for PBS (control), Salivary α-amylase, Hemoglobin, IgA, and Total Protein (30/30 replicates for each).
Interfering Substances (Exogenous)	≥95% agreement with true variant status for all samples passing QC	100% agreement for all tested exogenous substances (Chicken, Alcohol, Mouthwash, Beef, Gum, Smoking) at T0 and T30 time points.
Interfering Substances (Microbial)	>95% agreement with true variant status	100% agreement for all five microbial agents (S. epidermis, S. mutans, L. casei, A. odontolyticus, C. albicans) at both low/normal and high concentrations (36/36 replicates for each).
Accuracy / Method Comparison	≥99% agreement with true variant determination overall and per genotype	100% overall agreement (198/198) between AncestryDNA Factor V Leiden GHR Test and bi-directional sequencing. 100% agreement for GG (69/69), GA (65/65), and AA (64/64).
User Comprehension (Study 1)	≥90% comprehension score for each domain and overall	Overall comprehension rate was 93.2%. Individual domain comprehension rates ranged from 83.7% to 100%. (e.g., Appropriate Follow-Up Action: 97.4%, Ethnic Relevance: 95.5%, Other Risk Factors: 92.1%, Limitations of Test: 91.5%, Purpose of Test: 92.7%, Results of Test: 90.7%). Note that "Results of Test" had one category (1 Variant) at 83.7% which is below 90% individually but the overall for that domain was 90.7%.
User Comprehension (Study 2)	≥90% comprehension score for each domain and overall	Overall comprehension rate was 96%. Individual domain comprehension rates ranged from 88.8% to 99.1%. (e.g., Appropriate Follow-Up Action: 96.5%, Ethnic Relevance: 97.7%, Other Risk Factors: 95.8%, Limitations of Test: 96.2%, Purpose of Test: 99.1%, Results of Test: 90.9%). Note that "Results of Test" had one category (1 Variant) at 88.8% which is below 90% individually but the overall for that domain was 90.9%.

Study Details

2. Sample Size and Data Provenance (Test Set):

Reproducibility/Precision:
- Total replicates: 2,340 genotyping events (combining all replicates from Lab 1, Lab 2, inter-laboratory, by site/operator team, by site/instrument, and by site/reagent lot).
- Donors: 9 donors with known Factor V Leiden genotypes (3 homozygous common, 3 heterozygous, 3 homozygous rare).
- Provenance: Samples were collected using AncestryDNA Saliva Collection Kits (SCKs) and tested at two CLIA-certified laboratories (Lab 1 and Lab 2). The study was prospective in nature for data collection.
Analytical Sensitivity:
- Saliva Study: 15 donors (5 homozygous common, 5 heterozygous, 5 homozygous rare). 450 replicates tested.
- Cell Line Study: 4 cell lines. 216 data points per cell line for dilutions, and 855 blank replicates.
- Provenance: Saliva samples collected using Oragene® Dx Collection Device and AncestryDNA SCK. Cell lines. Data collected prospectively for the study.
Interfering Substances (Endogenous):
- Donors: 10 saliva donors. 450 initial genotyping attempts (3 replicates for each of 10 donors per interferent + 30 control replicates).
- Provenance: Saliva samples collected using Oragene® Dx Collection Device. Data collected prospectively for the study.
Interfering Substances (Exogenous):
- Donors: 10 non-smokers and 10 smokers. 594 data points.
- Provenance: Saliva samples collected using AncestryDNA SCKs. Data collected prospectively for the study.
Interfering Substances (Microbial):
- Cell Lines: 6 human cell lines (4 homozygous common, 1 heterozygous, 1 homozygous rare). 432 genotyping results.
- Provenance: Human cell lines. Data collected prospectively for the study.
Accuracy / Method Comparison:
- Donors: 209 donors with known Factor V Leiden genotypes (73 homozygous common, 69 heterozygous, 67 homozygous rare). 198 samples passed QC.
- Provenance: Saliva samples collected using Oragene Dx Ogd-500.001 (OGD) and AncestryDNA SCK. Data collected prospectively for comparison.
User Comprehension (Study 1):
- Participants: 378 individuals (N=96, N=86, N=106, N=90 for each of four study arms).
- Provenance: Participants were recruited from the U.S. matching demographics for education, age, sex/gender, and race/ethnicity, and geographic diversity across four U.S. Census regions. Performed in-person. Prospective study.
User Comprehension (Study 2):
- Participants: 213 individuals (N=103 for "1 Variant", N=110 for "Result Not Determined").
- Provenance: Participants were recruited from the U.S., matching demographics and geographic diversity as in Study 1. Performed via live televideo interviews. Prospective study.

3. Number of Experts and Qualifications for Ground Truth (Test Set):
For analytical studies (Reproducibility, Analytical Sensitivity, Interfering Substances, Accuracy/Method Comparison), the ground truth for Factor V Leiden genotype was primarily established using bi-directional sequencing analysis at a third-party laboratory. The specific qualifications of the experts performing the bi-directional sequencing were not detailed in the provided text.

For User Comprehension Studies, the materials were reviewed by a Certified Genetic Counselor to confirm that the materials met specific criteria for explaining test concepts.

4. Adjudication Method (Test Set):
The text does not explicitly describe an adjudication method like 2+1 or 3+1 for the analytical studies. Instead, direct comparison was made between the device's genotype calls and the ground truth established by bi-directional sequencing. Any discrepancies would presumably be investigated, though the method is not detailed. For User Comprehension studies, a questionnaire was administered by a trained interviewer/moderator to assess comprehension.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:
No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not performed. This device is a genetic health risk test (an in vitro molecular diagnostic system), not an imaging device typically associated with MRMC studies in the context of human reader performance improvements with AI.

6. Standalone Performance:
Yes, standalone performance (algorithm only) was done. The studies described (reproducibility, analytical sensitivity, interfering substances, and accuracy/method comparison) evaluate the performance of the device itself (AncestryDNA Factor V Leiden GHR Test) in determining genotype status, independent of human interpretation of raw data. The "AncestryDNA GHR software" processes data from the instrumentation and generates the final analytical genotype information for each sample, which is then used to generate personalized reports.

7. Type of Ground Truth Used (Test Set):
The primary ground truth used for determining the Factor V Leiden genotype in analytical studies was bi-directional sequencing analysis.

8. Sample Size for the Training Set:
The document does not explicitly specify a "training set" sample size for the AncestryDNA Factor V Leiden Genetic Health Risk Test in the context of its 510(k) submission. For molecular diagnostic systems like this, the "training" aspect often refers to the development and optimization of the assay and software algorithms. The document instead focuses on analytical validation studies (test sets) for device performance. If the AncestryDNA GHR software performs certain control checks and analyses (as described in section "P. SYSTEM DESCRIPTION" - "Software"), the underlying algorithms would have been developed and potentially "trained" or optimized during the device's development phase, but specific training set sizes are not provided.

9. How the Ground Truth for the Training Set was Established:
Since a specific training set sample size is not explicitly mentioned for the reported studies, the method for establishing ground truth for a training set is also not detailed. However, for any developmental work or optimization, it is highly probable that the ground truth would have been established using bi-directional sequencing or other highly accurate, established reference methods, similar to how the ground truth for the test sets was established.

Summary

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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

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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 (DICE@fda.hhs.gov) or phone (1-800-638-2041 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)

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 800San Francisco, CA 94107
Contact Person:	Raajdeep Venkatesan, MS, RAC, CMQ-OE, CBA, CQEVice President of RA/QAAncestry Genomics, Inc.
Alternate Contact Person:	Julie WoodDirector of QualityAncestry 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:

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.
1. 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

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K. SUBSTANTIAL EQUIVALENCE INFORMATION

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

Table 5-1: Predicate Device Comparison

	AncestryDNA Factor V LeidenGenetic Health Risk Test	23andMe Personal GenomeService Test(Predicate Device)
K Number	K192944	DEN160026/DEN140044
SIMILARITIES
Intended Use	The AncestryDNA Factor V LeidenGenetic Health Risk Report forHereditary Thrombophilia isindicated for reporting of the FactorV Leiden variant in the F5 gene. Thisreport describes if a person hasvariants associated with a higher riskof developing harmful blood clots,but it does not describe a person'soverall risk of developing harmfulblood clots. This test is most relevantfor people of European descent.	The 23andMe PGS Genetic HealthRisk Report for HereditaryThrombophilia is indicated forreporting of the Factor V Leidenvariant in the F5 gene, and theProthrombin G20210A variant in theF2 gene. This report describes if aperson has variants associated with ahigher risk of developing harmfulblood clots, but it does not describe aperson's overall risk of developingharmful blood clots. This test is mostrelevant for people of Europeandescent.
SpecialConditions forUse Statements	a. For over-the-counter (OTC) use.b. This test is not a substitute forvisits to a healthcare provider. Itis recommended that you consultwith a healthcare provider if youhave any questions or concernsabout your results.c. The AncestryDNA Factor VLeiden Genetic Health Risk Testdoes not detect all geneticvariants associated withHereditary Thrombophilia. Theabsence of a variant tested doesnot rule out the presence of othergenetic variants that may bedisease-related	a. For over-the-counter (OTC) use.b. This test is not a substitute forvisits to a healthcare provider. It isrecommended that you consultwith a healthcare provider if youhave any questions or concernsabout your results.c. The 23andMe PGS Genetic HealthRisk Tests for HereditaryThrombophilia do not detect allgenetic variants associated withthe aforementioned diseases. Theabsence of a variant tested doesnot rule out the presence of othergenetic variants that may bedisease-related
	AncestryDNA Factor V LeidenGenetic Health Risk Test	23andMe Personal GenomeService 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.
SpecialInstrumentRequirements	The AncestryDNA Factor V LeidenGenetic Health Risk Test forHereditary Thrombophilia is to beperformed using the Tecan Evo andIllumina iScan instruments.GenomeStudio is a modular softwareapplication that is used to view andanalyze genotypic data obtainedfrom the Illumina iScan System andbased on the cluster definition file.AncestryDNA GHR softwareconducts a variety of control checkson the file, resulting in a finalgenotype profile for each sample.These data are used to generate testreports on a user's genotype andassociated risk of disease.	The 23andMe PGS Genetic HealthRisk Tests for HereditaryThrombophilia is to be performedusing the Tecan Evo and IlluminaiScan instruments.GenomeStudio is a modular softwareapplication that is used to view andanalyze genotypic data obtainedfrom the iScan. Coregen softwareconducts a variety of control checkson the file, resulting in a finalgenotype profile for each sample.These data are used to generate testreports on a user's genotype andassociated risk of disease.
	AncestryDNA Factor V LeidenGenetic Health Risk Test	23andMe Personal GenomeService Test(Predicate Device)
Classification	Class II	Same
Type of Test	Qualitative in vitro moleculardiagnostic system	Same
Measurand	Factor V Leiden c.1601G>A variantin the F5 gene	Factor V Leiden variant in the F5gene, and the Prothrombin G20210Avariant in the F2 gene
SamplePreparationMethod	DNA extraction	Same
AnalyticalSensitivity	The performance requirement for theAncestryDNA GHR has been set at aminimum of 1.53 ng/µL DNA andmaximum of 50 ng/µL DNA.	The performance requirement for thePGS, has been set at a minimum of15 ng/µL DNA and maximum of 50ng/µL DNA.
Reproducibility/Precision	100% correct genotype calls for allsamples with a valid call acrossmultiple sites, multiple days, usingmultiple operator teams, instrumentcombinations, and assay reagent lotstested with samples collected usingmultiple lots of the AncestryDNASample Collection Kit at bothlaboratory sites.Genotyping results produced 99.9%(2337/2340) replicates that werecalled correctly and 0.13% (3/2340)replicates that did not pass qualitycontrol acceptance criteria. Sampleswith failed quality control (FQC) onthe first run are re-tested perlaboratory SOPs.	100% correct genotype calls for allsamples with a valid call acrossmultiple days, operator teams,instruments, and reagent lots at bothlaboratory sites.Genotyping results produced 98.0%(1852/1890) replicates that werecalled correctly and 2.01% (38/1890)replicates that did not pass qualitycontrol acceptance criteria. Sampleswith failed quality control (FQC) onthe first run are re-tested perlaboratory SOPs; an anticipated rateof samples with 2 FQCs based on thereproducibility study data of cell linesamples is 0.04% (0.020 x 0.020).
EndogenousInterferingSubstances	N = 4 endogenous agents were testedin saliva: salivary α-amylase,hemoglobin, IgA, and total protein.There was no impact on testperformance with all interferentstested.	N = 4 endogenous agents were testedin saliva: salivary α-amylase,hemoglobin, IgA, and total protein.There was no impact on testperformance with all interferentstested.
ExogenousInterferingSubstances	N = 6 exogenous agents were testedin saliva samples collected afterperforming the following actions:eating food containing beef, eatingfood not containing beef, drinking	N = 6 exogenous agents were testedin saliva samples collected afterperforming the following actions:eating food containing beef, eatingfood not containing beef, drinking
	AncestryDNA Factor V LeidenGenetic Health Risk Test	23andMe Personal GenomeService Test(Predicate Device)
	alcohol, chewing gum, usingmouthwash, and smoking.There was no impact on testperformance at the 30 minutetimepoint with all interferents tested.	alcohol, chewing gum, usingmouthwash, and smoking.There was no impact on testperformance at the 30 minutetimepoint with all interferents tested.
MicrobialInterferingSubstances	N = 5 microbial agents were tested insaliva: Staphylococcus epidermis ,Streptococcus mutans , Lactobacilluscasei , Actinomyces odontolyticus ,and Candida albicans . There was noimpact on performance with allinterferents tested.	From 23andMe Personal GenomeService Carrier Screening Test forBloom Syndrome (DEN140044):N = 5 microbial agents were tested insaliva: Staphylococcus epidermis ,Streptococcus mutans , Lactobacilluscasei , A., and Candida albicans .There was no impact on performancewith all interferents tested.
Comparisonwith SangerBi-directionalSequencing	Overall agreement was 100%(198/198) with bi-directionalsequencing.	Overall agreement was 100%(203/203) with bi-directionalsequencing.
ClinicalPerformance	The genotype frequencies for FactorV Leiden in various US populationwere obtained from the 2018 ACMGreporting standard (Zhang et al.,2018): “In the United States, FactorV Leiden heterozygosity is present in5.1%, 2.0%, and 1.2% of Caucasians,Hispanics, and African Americansrespectively; the frequencies ofhomozygosity for the abovepopulations are 65, 10, and 4 per100,000 individualscorrespondingly.”	The minor variant frequency forFactor V Leiden in individuals ofEuropean descent reported inpublished literature is 3%-15%;technical (analytical) positivepredictive values for 23andMe PGStest results of CT and TT are ≥99.5% and ≥ 99.1 %correspondingly.The minor variant frequency forProthrombin G2021 0A inindividuals of European descentreported in published literature is 1%--3%; technical (analytical)positive predictive values for23andMe PGS test results of AG andAA are ≥ 98.6% and ≥ 97.2%correspondingly.
DIFFERENCES
SampleCollectionDevice	AncestryDNA Saliva Collection Kit(K192947)	Oragene Dx Ogd-500.001(K141410)
InterferingMutations	The potential interfering mutationsinclude rs770011773, rs773367113,	For Factor V Leiden the potentialinterfering mutations include
AncestryDNA Factor V LeidenGenetic Health Risk Test	23andMe Personal GenomeService Test(Predicate Device)
and rs143663052. The impact ofthese mutations on the performanceof the assay has not been evaluated.	rs760488939 and rs763859650. Theimpact of these mutations on theperformance of the assay has notbeen 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	TotalNumber ofReplicates	Number ofConcordantCalls	Numberof CallRate QCFailures	FQC(%)
GG	540	540	0	0.00
GA	540	537	3*	0.56
AA	540	540	0	0.00
Total	1620	1617	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	Totalnumber ofreplicates	Number ofconcordantcalls
GG	15	15
GA	15	15
AA	15	15
Totals	45	45

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	TotalNumber ofReplicates	Number ofConcordantCalls
GG	180	180
GA	180	180
AA	180	180
Total	540	540

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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 Numberof Replicates		Number ofConcordantCalls		Number of"No-Calls"		Number of CallRate QCFailures		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/OperatorTeam	ExpectedGenotype	TotalNumber ofReplicates	Number ofConcordantCalls	Number ofCall RateQC Failures*	Proportionof FQC(%)
Lab 1Team 1	AA	195	195	0	0.00
Lab 1Team 1	GA	195	194	1	0.51
Lab 1Team 1	GG	195	195	0	0.00
Total		585	584	1	0.17
Lab 1Team 2	AA	189	189	0	0.00
Lab 1Team 2	GA	189	188	1	0.53
Lab 1Team 2	GG	189	189	0	0.00
Total		567	566	1	0.18

Site and Operator Team Results

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Site/OperatorTeam	ExpectedGenotype	TotalNumber ofReplicates	Number ofConcordantCalls	Number ofCall RateQC Failures*	Proportionof FQC(%)
Lab 1Team 3	AA	189	189	0	0.00
	GA	189	188	1	0.53
	GG	189	189	0	0.00
Total		567	566	1	0.18
Lab 2Team 1	AA	69	69	0	0.00
	GA	69	69	0	0.00
	GG	69	69	0	0.00
Total		207	207	0	0.00
Lab 2Team 2	AA	69	69	0	0.00
	GA	69	69	0	0.00
	GG	69	69	0	0.00
Total		207	207	0	0.00
Lab 2Team 3	AA	69	69	0	0.00
	GA	69	69	0	0.00
	GG	69	69	0	0.00
Total		207	207	0	0.00
All teams'totalscombined	GG, GA,AA	2,340	2,337	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/InstrumentCombination	ExpectedGenotype	TotalNumber ofReplicates	Number ofConcordantCalls	Numberof “No-Calls”	Number ofCall RateQC Failures	Proportionof FQC(%)
Lab 1-Instrumentcombination 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-Instrumentcombination B	AA	135	135	0	0	0.00
	GA	135	134	0	1	0.74
	GG	135	135	0	0	0.00
Site/InstrumentCombination	ExpectedGenotype	TotalNumber ofReplicates	Number ofConcordantCalls	Numberof “No-Calls”	Number ofCall RateQC Failures	Proportionof FQC(%)
Total		405	404	0	1	0.25
Lab 1-Instrumentcombination 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-Instrumentcombination 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-Instrumentcombination 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-Instrumentcombination 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-Instrumentcombination 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-Instrumentcombination 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/ReagentLotCombination	ExpectedGenotype	TotalNumber ofReplicates	Number ofConcordantCalls	Numberof “No-Calls”	Number ofCall RateQC Failures	Proportionof 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
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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 PercentAgreement (%)(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 PercentAgreement (%)(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.

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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.

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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

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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.

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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
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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

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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	ChromosomePosition	TranscriptConsequence	Annotation	AlleleCount	AlleleFrequency
rs770011773	g.169549812	c.1600C>T	Stop Gained	3	0.00001062
rs773367113	g.169549835	c.1577G>A	MissenseVariant	2	0.000007073
rs143663052	g.169549848	c.1564C>G	MissenseVariant	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.

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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 (<98%). Nine alternate samples were added to the sample cohort, all of which passed the first pass genotyping SCR of ≥ 98%. Of the 15 samples that were regenotyped, four passed SCR quality control in second pass genotyping attempts, and 11 failed SCR quality control in the second pass genotyping attempt and were not eligible for a third genotyping attempt based on sample call rate criteria. The table below summarizes the distribution of the 11 FQC samples by genotype

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Genotype	Total Number ofSamples	Number ofFQC	FQC(%)	95% ConfidenceInterval
GG	73	4	5.5	1.5 - 13.4
AA	67	3	4.5	0.9 - 12.5
GA	69	4	5.8	1.6 - 14.2

Distribution of the 11 FQC samples by genotype

The table below summarizes the genotype counts for all samples passing SCR quality control for the AncestryDNA Factor V Leiden GHR Test and the genotyping results for the bi-directional sequencing. Zero (0) 'no-call' events were observed in any of the samples that passed quality control.

Comparison of the Genotyping Results for Bi-directional Sequencing and AncestryDNA Factor V Leiden GHR Test

AncestryDNA Factor V	Bi-directional Sequencing Genotypes			Total
Leiden GHR Genotypes	GG	GA	AA
GG	69	0	0	69
GA	0	ર્ણ રિ	0	ર્ણ્ટ
AA	0	0	64	64
00 (no-call)	0	0	0	0
Total	ਉਹ	ર્ણને રિસ્તારમાં આવેલું એક ગામના લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં મુખ્યત્વે ખેત-ઉત્પત્તમજૂરી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગા	64	198

For all samples that passed quality control, the overall percent agreement and the percent agreement for each genotype with bi-directional sequencing genotypes was 100% as outlined below.

Percent Agreement and Confidence Intervals for AncestryDNA Factor V Leiden GHR Test Genotypes

Genotype	Observed/Expected	Percent Agreement	95% ConfidenceInterval
GG	69/69	100%	94.8–100%
GA	65/65	100%	94.5–100%
AA	64/64	100%	94.4–100%
All genotypes	198/198	100%	98.2–100%

The calculated rate point estimate of no-call events and 95% confidence intervals below were based on a binomial distribution between genotypes obtained from donors using the AncestryDNA SCK with the AncestryDNA Factor V Leiden GHR Test and using the Oragene Dx Ogd-500.001 (OGD) for bi-directional sequencing.

Estimated Rate of No-Call Events for the AncestryDNA Factor V Leiden GHR Test

Event	Observed/Total	Rate Point Estimate	95% Confidence Interval
No-call	0/198	0%	0 – 1.8%

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The calculated overall percent agreement and 95% confidence intervals below were based on a binomial distribution between genotypes obtained from donors using the AncestryDNA SCK with the AncestryDNA Factor V Leiden GHR Test and using the Oragene Dx Ogd-500.001 (OGD) for bi-directional sequencing.

Overall Percent Agreement and Confidence Interval for Genotypes Obtained Using AncestryDNA SCK Compared to OGD

Proportion ConcordantGenotypes(AncestryDNA SCK vs. OGD)	Genotype OPA BetweenSaliva Collection Devices	95% Confidence Interval
198/198	100%	98.2 - 100%

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."

In the accuracy study for the AncestryDNA Factor V Leiden GHR Test, point estimates of PA(GG)GG) = PA(GA|GA)=PA(AA|AA) = 100%. The point estimate for the Technical Positive Predictive Value (TPPV) for such a scenario is 100% for both heterozygotes and homozygotes. While this point estimate may have larger uncertainty about the TPPV than an estimate from a larger test population, no discordant genotypes were observed in any of the 198 genotyping events that passed quality control.

For all samples that passed SCR quality control (198/209, 95%), the AncestryDNA Factor V Leiden GHR Test genotypes were 100% concordant with true variant status, determined by bi-directional sequencing. This result met the predefined protocol acceptance criterion of ≥ 99% agreement with true variant determination overall and per genotype tested.

b. Matrix Comparison

Not applicable.

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3. Clinical Studies

a. Clinical Performance

Odds ratios

Odds ratios are available in the meta-analyses performed by Simone et al. (2013). Odds ratios adjusted for age and sex using logistic regression and 95% confidence intervals for each variant status are given below:

Factor V Leiden variant status	Odds ratio (95% confidence interval)
One copy, heterozygote	4.22 (3.35 - 5.32)
Two copies, homozygote	5.45 (6.79 - 19.29)

Likelihood ratios and post-test risk

Likelihood ratios (LR) were obtained using the raw distribution data from the metaanalyses in Simone et al. (2013).

Post-test risk (Rpost) was calculated from the likelihood ratios and using a pre-test risk (Rore) of 11% from the 2018 ACMG Venous thromboembolism laboratory testing standard (Zhang, 2018). Likelihood ratios and 95% confidence intervals calculated using a normal approximation are listed in the table below along with the post-test risk calculated using the relationship Rpost (1 - Rpost) = LR [Rpre (1 - Rpre)]:

FVL variantcount(genotype)	Case/control distribution	LR	95%confidenceinterval	Post-testrisk
0 variants(homozygouscommon /wild type)	1,758 cases and 1,201 controlsamong 2,959 Factor V Leidencarriers. 7,323 cases and 16,312controls among 23,635 wild typeindividuals.	0.87	0.86 – 0.88	10%
1 variant(heterozygous)	1,758 cases and 1,201 controlsamong 2,959 Factor V Leidencarriers. 7,323 cases and 16,312controls among 23,635 wild typeindividuals.	2.82	2.64 – 3.02	26%
2 variants(homozygousrare)	92 cases and 24 controls among 116Factor V Leiden homozygousindividuals. 4,524 cases and 11,643controls among 16167 wild typeindividuals.	9.69	6.19 – 15.16	54%

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Note that the likelihood ratio lower bounds for heterozygotes and homozygotes are greater than one, indicating that the post-test risk is significantly greater than the pre-test risk.

References:

1. Zhang, Shulin, et al. "Venous thromboembolism laboratory testing (factor V Leiden and factor II c.* 97G> A), 2018 update: a technical standard of the American College of Medical Genetics and Genomics (ACMG)." Genetics in Medicine 20.12 (2018): 1489.
1. 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
1. 1 Variant Identified
1. 2 Variants Identified
1. 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
1. 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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CoreConcept	Comprehension Rates by GHR Report Type (%)	0 Variant	1 Variant	2 Variant	Result NotDetermined
AppropriateFollow-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 ofReports	N = 96	N = 86	N = 106	N = 90	N = 378

Study 1 - Overall User Comprehension Rates for Factor V Leiden GHR Reports
----------------------------------------------------------------------------	--	--	--	--	--	--	--	--	--	--	--	--	--	--	--

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.

CoreConcept	Comprehension Rates by GHR Report Type (%)		OverallComprehensionRates
	1 Variant	Result Not Determined
AppropriateFollow-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 ofReports	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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ComprehensionConcept	Pre-test (%)	Post Test (%)	%Improvement	p-value
Purpose	89.0	99.1	10.1	<0.001
Other Risk Factors	91.1	તે જેવી જેવી તેમ જ દૂધની ડેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂ	4.7	0.002
Ethnic Relevance	તે જેવી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામમાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલતો પ્	97.7	1.9	0.117
Limitations	87.7	96.2	8.5	<0.001

User Comprehension by Comprehension Concept Pre- to Post-Test Statistical Comparison

Expected Values/Reference Range ن

Not applicable.

O. INSTRUMENT NAME

Illumina iScan BeadChip scanner with GenomeStudio software

P. SYSTEM DESCRIPTION

1. Modes of Operation:
  The Illumina iScan is a table top laser-based, high-resolution optical imaging system that produces genotype information for up to 4 beadchips/beadchip carrier. Carriers are loaded into the instrument through an Autoloader2.
1. Software:
  FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes

Level of Concern: Moderate

Software Description:

Illumina iScan System with iScan Control Software and Genome Studio performs the following:

iScan Control Software drives the iScan hardware in Beadchip scanning and image . data generation.
GenomeStudio software allows viewing and analyzing of genotypic data obtained . from the iScan. Processing includes primary data analyses, such as raw data normalization, clustering, and genotype calling. To ensure data quality, the software performs internal controls and data quality control checks.
AncestryDNA GHR software takes raw data and putative genotype calls from the ● PED file received from the laboratory and generates the final analytical genotype information for each sample.

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Revision Level History:

A software revision history record for the AncestryDNA GHR software was acceptable.

Unresolved Anomalies:

There are no known unresolved anomalies associated with the system software.

EMC Testing: Not applicable.

3. Specimen Identification

Users must register their saliva collection kit, linking their saliva sample to a secure online account with a valid email address through a unique activation code, in order to use the test. The activation code is matched to records of kits shipped to consumers to ensure it is a valid kit. A timestamp of the user completing the entries to activate the kit is recorded.

4. Specimen Sampling and Handling

Saliva samples should be collected using the AncestryDNA Saliva Collection Kit. The recommended volume of saliva is 1 mL. Saliva is collected directly by the user spitting into the provided Saliva Collection Tube via the pre-installed funnel. After providing saliva, the user is instructed to remove the funnel and screw on tightly the provided cap. Affixing the cap by screwing on releases the stabilization solution. The saliva sample can be immediately processed, transported, or stored for future use. Device and sample integrity are preserved during typical ambient transport and storage conditions for up to 12 months.

5. Calibration

Calibration and calibration verification procedures are established to demonstrate continued accuracy of the test systems.

6. Quality Control

The sample processing control is run on every sample genotyping plate and the reproducibility control is run approximately once per week.

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Q. OTHER SUPPORTIVE INSTRUMENT PERFORMANCE CHARACTERISTICS DATA NOT COVERED IN SECTION N, "PERFORMANCE CHARACTERISTICS," ABOVE

Refer to the AncestryDNA Saliva Collection Kit 510(k) (K192947) for saliva collection device details and study results.

R. PROPOSED LABELING

The labeling satisfies the requirements of 21 CFR Parts 801 and 809 as well as the Special Controls for a Genetic Health Risk Assessment System.

S. CONCLUSION

The results of the analytical studies submitted in this 510(k) Premarket Notification are complete and demonstrate that the AncestryDNA Factor V Leiden Genetic Health Risk Test meets the established specifications necessary for consistent performance during the intended use of reporting the Factor V Leiden variant in the F5 gene. The results support a decision that the AncestryDNA Factor V Leiden Genetic Health Risk Test is substantially equivalent to the predicate.

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