(a) Identification. A whole exome sequencing constituent device is for germline whole exome sequencing of genomic deoxyribonucleic acid (DNA) isolated from human specimens. The DNA sequence generated by this device is intended as input for clinical germline DNA assays that have FDA marketing authorization and are intended for use with this device.

(b) Classification. Class II (special controls). The special controls for this device are:

(1) The intended use on the device's label and labeling required under § 809.10 of this chapter must include:

(i) The indicated variant types for which acceptable, as determined by FDA, validation data has been provided. Distinct variant types are considered as single nucleotide variant, insertion, deletion, tandem repeats, copy number variants, or gene rearrangements, and validated for specific sizes and lengths, as applicable.

(ii) The indicated specimen type(s) for which acceptable, as determined by FDA, validation data has been provided.

(2) The labeling required under § 809.10(b) of this chapter must include:

(i) The identification of, or the specifications for, the collection device or devices to be used for sample collection, as applicable.

(ii) A description of the reportable range, which is the region of the genome for which the assay is intended to provide results, as well as a description of the targeted regions of the genome that have enhanced coverage. This must include a description of any genomic regions that are excluded from the reportable region due to unacceptable risk of erroneous results, or for other reasons. A description of the clinically relevant genes excluded from the reportable range must also be included, if applicable.

(iii) A description of the design features and control elements, including the quality metrics and thresholds which are used for reporting the analytical range (the genomic DNA in the reportable range that passed the quality metrics in the run required for reporting to the user) that are incorporated into the testing procedure, that mitigate the risk of incorrect clinical results. The following metrics are considered applicable in the generation of high confidence data and the established thresholds for these metrics for reporting must be described and be determined to be acceptable by FDA: cluster density and percent of cluster pass quality filter, percent of bases meeting the minimum base quality score, average coverage of reads, percent of reads mapped on target, percent of reportable region with coverage meeting the minimum requirement, percent of unassigned read indices, percent of reads for non-human DNA, allele fraction, and strand bias. Any alternate metrics used must be described and an acceptable, as determined by FDA, rationale for applicability must be provided.

(iv) A representative sample of the device output report(s) provided to users, which must include any relevant limitations of the device, as determined applicable by FDA.

(3) Design verification and validation must include:

(i) A detailed description of the impact of any software, including software applications and hardware-based devices that incorporate software, on the device's function.

(ii) Acceptable data, as determined by FDA, demonstrating how the key quality metrics and quality metric thresholds in the list in paragraph (b)(2)(iii) of this section for reporting were established and optimized for accuracy using appropriate DNA standards with established reference genomic sequence. Data must include, as applicable, base quality score, allele fraction for heterozygosity and coverage, and other applicable metrics.

(iii) Data demonstrating acceptable, as determined by FDA, analytical device performance using patient specimens representing the full spectrum of expected variant types reported across the genome and in genomic regions that are difficult to sequence. The number of specimens tested must be sufficient to obtain estimates of device performance that are representative of the device performance that can be expected for the reportable region and clinically relevant subsets of the reportable region, as applicable. For each study, data must include a summary of the key quality metric data; the number and percentage of true positives (TP), false positives (FP), and false negatives (FN); number and percentage of no-calls; positive percent agreement (PPA); negative percent agreement (NPA); positive predictive value (PPV); technical positive percent value (TPPV); and non-reference concordance (NRC). These data must be provided per sample and stratified by variant type. The variant data must also be further stratified by size and zygosity (homozygous common allele, heterozygous, homozygous rare allele). Data demonstrating the accuracy assay based on guanine and cytosine (GC) content, pseudogenes, and proximity to short tandem repeats must also be presented. The data must be presented for the entire exome and also for clinically relevant subsets of the reportable region. For each study, the number of run failures and repeat/requeued specimens must be summarized.

(iv) Documentation of acceptance criteria that are applied to analytical and clinical validation studies, which must be justified based on the estimated risk of erroneous results on clinically significant genes and variants and must be clinically acceptable, as determined by FDA. The acceptance criteria must be pre-specified prior to clinical and analytical validation studies, and all validation testing results must be documented with respect to those acceptance criteria.

(v) Analytical validation must be demonstrated by conducting studies that provide:

(A) Data demonstrating acceptable, as determined by FDA, accuracy based on agreement with an acceptable, as determined by FDA, comparator method(s) that has been validated to have high accuracy and reproducibility. Accuracy of the test shall be evaluated with reference standards and clinical specimens for each indicated specimen type of a number determined acceptable by FDA, collected and processed in a manner consistent with the test's instructions for use.

(B) Data demonstrating acceptable, as determined by FDA, precision from a precision study using clinical samples to adequately evaluate intra-run, inter-run, and total variability across operator, instrument, lot, day, and site, as applicable. The samples must include the indicated range of DNA input. Precision, including repeatability and reproducibility, must be assessed by agreement between replicates, and also supported by sequencing quality metrics for targeted regions across the panel. Precision must be demonstrated per specimen and in aggregate. Precision data must be calculated and presented with and without no calls/invalid results.

(C) Data demonstrating acceptable, as determined by FDA, accuracy in the presence of clinically relevant levels of potential interfering substances that are present in the specimen type and intended use population, including, for example, endogenous substances, exogenous substances, and microbes, as applicable.

(D) Data demonstrating the absence of sample cross contamination due to index swapping (misassignment).

(E) Data demonstrating that the pre-analytical steps such as DNA extraction are robust such that sources of variability in these steps and procedures do not diminish the accuracy and precision of the device.

(F) Data demonstrating that acceptable, as determined by FDA, device performance is maintained across the range of claimed DNA input concentrations for the assay.

(vi) Design verification and validation for software within the whole exome sequencing constituent device must include the following:

(A) Detailed description of the software, including specifications and requirements for the format of data input and output, such that users can determine if the device conforms to user needs and intended uses.

(B) Device design must include a detailed strategy to ensure cybersecurity risks that could lead to loss of genetic data security, are adequately addressed and mitigated (including device interface specifications and how safe reporting of the genetic test is maintained when software is updated). Verification and validation must include security testing to demonstrate effectiveness of the associated controls.

(C) Device design must ensure that a record of critical events, including a record of all genetic test orders using the whole exome sequencing constituent device, device malfunctions, and associated acknowledgments, is stored and accessible for an adequate period to allow for auditing of communications between the whole exome sequencing constituent device and downstream clinical genetic tests, and to facilitate the sharing of pertinent information with the responsible parties for those devices.

(vii) A protocol reviewed and determined acceptable by FDA, that specifies the verification and validation activities that will be performed for anticipated bioinformatic software modifications to reevaluate performance claims or performance specifications. This protocol must include a process for assessing whether a modification to the bioinformatics software could significantly affect the safety or effectiveness of the device. The protocol must include assessment metrics, acceptance criteria, and analytical methods for the performance testing of changes, as applicable. The protocol must also include the process for communicating to developers of downstream clinical genetic tests the impact of the bioinformatics software change on the whole exome sequencing constituent system genetic data output so they may implement appropriate corresponding actions.