(57 days)
The MiSeqDx Platform is a sequencing instrument that measures fluorescence signals of labeled nucleotides through the use of instrument specific reagents and flow cells (MiSeqDx Universal Kit 1.0), imaging hardware, and data analysis software. The MiSeqDx Platform is intended for targeted sequencing of human genomic DNA from peripheral whole blood samples. The MiSeqDx Platform is not intended for whole genome or de novo sequencing.
The MiSeqDx Platform is a high throughput DNA sequence analyzer for clinical use.
The MiSeqDx Platform consists of the MiSeqDx instrument and data analysis software. It is for use with the MiSeqDx Universal Kit 1.0 [MiSeqDx reagent cartridge, MiSeqDx flow cell, SBS Solution (PR2 buffer)] for library preparation and sample indexing (K133136). The end-user inputs extracted genomic DNA to be sequenced and provides the Analyte Specific Reagents (ASRs) to develop a sequencing assay that targets their sequence of interest.
The Illumina MiSeqDx Platform is a high-throughput DNA sequencing instrument. The acceptance criteria and supporting studies are detailed below:
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria for the MiSeqDx Platform are primarily derived from the special controls stipulated in 21 CFR 862.2265, focusing on accuracy and reproducibility across various genomic features.
| Acceptance Criteria (from 21 CFR 862.2265 (B) items ii, iii, vii, viii) | Reported Device Performance (from Accuracy and Reproducibility Studies) |
|---|---|
| Accuracy: | |
| - Ability to detect single nucleotide variants (SNVs). | Study 1: All SNVs had 100% agreement with the reference sequence. PPA ranged from 89.5% to 95.8% (due to missed indels, not SNVs). NPA was 100%. |
| Study 2: PPA for SNVs and Indels was 94.1%, NPA was 100%. | |
| - Ability to detect insertions and deletions (indels). | Study 1: Variants missed were 1-base insertions or 1-base deletions in homopolymer regions (e.g., Amplicon 9 and 95). |
| Study 3 (CFTR Assay): 1-base insertion, 3-base deletion, and 2-base deletion were detected with 100% correct calls. | |
| Overall: Validated for detection of SNVs and up to 3-base deletions. Evaluation of 1-base insertions was limited to 3 different insertions on 3 separate chromosomes. The system has problems detecting 1-base insertions or deletions in homopolymer tracts. 2 out of 3 1-base insertions tested were called correctly (those in non-homopolymer regions). 3 out of 4 1-base deletions called correctly (those in non-homopolymer regions). | |
| - Performance across varying sequence context (e.g., GC-rich regions, homopolymer runs, different chromosomes). | Study 1 & 3 summary: |
- GC content > 19% and
§ 862.2265 High throughput genomic sequence analyzer for clinical use.
(a)
Identification. A high throughput genomic sequence analyzer for clinical use is an analytical instrument system intended to generate, measure and sort signals in order to analyze nucleic acid sequences in a clinical sample. The device may include a signal reader unit; reagent handling, dedicated instrument control, and other hardware components; raw data storage mechanisms; data acquisition software; and software to process detected signals.(b)
Classification. Class II (special controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9. The special controls for this device are:(1) The labeling for the instrument system must reference legally marketed pre-analytical and analytical reagents to be used with the instrument system and include or reference legally marketed analytical software that includes sequence alignment and variant calling functions, to be used with the instrument system.
(2) The labeling for the instrument system must include a description of the following information:
(i) The specimen type(s) validated as an appropriate source of nucleic acid for this instrument.
(ii) The type(s) of nucleic acids (
e.g., germline DNA, tumor DNA) validated with this instrument.(iii) The type(s) of sequence variations (
e.g. single nucleotide variants, insertions, deletions) validated with this instrument.(iv) The type(s) of sequencing (
e.g., targeted sequencing) validated with this instrument.(v) The appropriate read depth for the sensitivity claimed and validation information supporting those claims.
(vi) The nucleic acid extraction method(s) validated for use with the instrument system.
(vii) Limitations must specify the types of sequence variations that the instrument cannot detect with the claimed accuracy and precision (
e.g., insertions or deletions larger than a certain size, translocations).(viii) Performance characteristics of the instrument system must include:
(A) Reproducibility data generated using multiple instruments and multiple operators, and at multiple sites. Samples tested must include all claimed specimen types, nucleic acid types, sequence variation types, and types of sequencing. Variants queried shall be located in varying sequence context (
e.g., different chromosomes, GC-rich regions). Device results shall be compared to reference sequence data with high confidence.(B) Accuracy data for all claimed specimen types and nucleic acid types generated by testing a panel of well characterized samples to query all claimed sequence variation types, types of sequencing, and sequences located in varying sequence context (
e.g., different chromosomes, GC-rich regions). The well-characterized sample panel shall include samples from at least two sources that have highly confident sequence based on well-validated sequencing methods. At least one reference source shall have sequence generated independently of the manufacturer with respect to technology and analysis. Percent agreement and percent disagreement with the reference sequences must be described for all regions queried by the instrument.(C) If applicable, data describing endogenous or exogenous substances that may interfere with the instrument system.
(D) If applicable, data demonstrating the ability of the system to consistently generate an accurate result for a given sample across different indexing primer combinations.
(ix) The upper and lower limit of input nucleic acid that will achieve the claimed accuracy and reproducibility. Data supporting such claims must also be summarized.