(142 days)
The lon PGM™ Dx Instrument System is intended for targeted sequencing of human genomic DNA (gDNA) from peripheral whole-blood samples and DNA and RNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples. The lon PGM™ Dx Instrument System is not intended for whole genome or de novo sequencing.
The Ion PGM™ Dx System is used for detection of human variant sequences from DNA from whole blood samples or RNA and DNA from FFPE tissue samples. Detectable variants include substitutions, insertions, and deletions.
The Ion PGMTM Dx System consists of the following:
- Ion OneTouch™ Dx Instrument
- Ion OneTouch™ ES Dx Instrument
- Ion OneTouch™ Rack Kit
- Ion PGM™ Dx Chip Minifuge
- Ion PGM™ Dx Sequencer
- Ion PGMTM Wireless Scanner
- DynaMag™ Dx Kit—Tube & Plate
- Ion Torrent™ Server
- Torrent Suite™ Dx Software
The Ion PGM™ Dx System is used in conjunction with the following kits:
- Ion PGM™ Dx Library Kit
- Ion OneTouch™ Dx Template Kit
- Ion PGM™ Dx Sequencing Kit
- Ion 318™ Dx Chip Kit
The system should be used only by professionals trained in laboratory techniques and procedures and in the use of the system.
The provided text describes the acceptance criteria and the studies performed for the Ion PGM™ Dx System. Here's a structured breakdown of the requested information:
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are not explicitly stated in a single section as "acceptance criteria," but rather derived from the "Special conditions statement for performance" for both gDNA from whole blood and DNA/RNA from FFPE samples. The "Reported Device Performance" is taken from the "Non-Clinical Performance Data" section.
| Feature / Metric | Acceptance Criteria (from "Special Conditions") | Reported Device Performance (from "Non-Clinical Performance Data") |
|---|---|---|
| gDNA from Whole Blood (SVA Panel) | ||
| Sequencing output | > 0.7 gigabases | Not explicitly reported for gDNA from whole blood in the performance data, but indicated as "validated to deliver" in the special conditions. |
| Reads | > 4 million | Not explicitly reported for gDNA from whole blood in the performance data, but indicated as "validated to deliver" in the special conditions. |
| Read length | up to 200 base pairs | Not explicitly reported for gDNA from whole blood in the performance data, but indicated as "validated to deliver" in the special conditions. |
| Mean Raw Read Accuracy | 99.0% when compared to hg19 | Not explicitly reported for gDNA from whole blood in the performance data, but indicated as "validated to deliver" in the special conditions. |
| SNV Detection Reproducibility | 100% reproducibility for 440 unique SNV positions | Not explicitly reported in the Non-Clinical Performance Data for gDNA from whole blood. This is stated as a system capability in the "Special conditions statement for performance derived from gDNA from whole blood." |
| Indel Detection Reproducibility | 100% reproducibility for various insertion/deletion lengths (1-4 bp insertions, 1-14 bp deletions) | Not explicitly reported in the Non-Clinical Performance Data for gDNA from whole blood. This is stated as a system capability in the "Special conditions statement for performance derived from gDNA from whole blood." |
| HPT limitation | Variants in homoploymer tracts exceeding 8 bases called as no calls | Not applicable; this is a known limitation, not a performance metric to be achieved. |
| Min coverage for germline DNA | >30X | Not explicitly reported in performance data. (This is a recommended use parameter) |
| FFPE Samples (Representative Assay) | ||
| Sequencing output | > 0.7 gigabases | Not explicitly reported for a representative assay in the performance data, but indicated as "validated to deliver" in the special conditions. |
| Reads | > 3 million | Not explicitly reported for a representative assay in the performance data, but indicated as "validated to deliver" in the special conditions. |
| Read length | up to 141 base pairs | Not explicitly reported for a representative assay in the performance data, but indicated as "validated to deliver" in the special conditions. |
| PPA (excluding no calls) | Not explicitly stated as a minimum. | Variant: 98.5% (195/198)Bin: 97.2% (176/181)Sample: 96.9% (158/163) |
| NPA (excluding no calls) | Not explicitly stated as a minimum. | Variant: 100.0% (118,155/118,159)Bin: 99.8% (942/944)Sample: 98.4% (124/126) |
| OPA (excluding no calls) | Not explicitly stated as a minimum. | Variant: 100.0% (118,350/118,357)Bin: 99.4% (1,118/1,125)Sample: 97.6% (282/289) |
| Repeatability (DNA variants, excl. no calls) | ≥97.5% (95% CI lower limit) | ≥98.8% (95% CI lower limit of ≥97.5%) |
| Repeatability (RNA variants, excl. no calls) | Not explicitly stated as a minimum for individual RNA variant locations, but overall ≥87.5% for positive variant location. | 94.4% for each RNA clinical variant location. (For a specific ROS1 RNA variant for Sample C, it was 87.5% with 95% CI lower bound of 61.7%). |
| Call Rate (DNA pos. variants, excl. no calls) | Not explicitly stated as a minimum. | Mean: 96.60%, Median: 97.10% |
| Call Rate (RNA pos. variants, excl. no calls) | Not explicitly stated as a minimum. | Mean: 94.80%, Median: 95.50% |
| Call Rate (WT DNA, neg. calls, excl. no calls) | Not explicitly stated as a minimum. | Mean: 96.10%, Median: 95.00% |
| Call Rate (WT RNA, neg. calls, excl. no calls) | Not explicitly stated as a minimum. | Mean: 99.30%, Median: 99.30% |
| Tissue Input (% meeting conc.) | 98.3% (59/60) had DNA ≥0.83 ng/uL and RNA >1.43 ng/uL. (This is a study finding, implicitly demonstrating the ability to meet the given concentration requirements for the assay under specific tissue input conditions.) | 98.3% (59/60) met the concentration requirements (DNA ≥0.83 ng/uL, RNA >1.43 ng/uL). |
| DNA/RNA Input (Positive Call Rate) | 100% positive variant call rate within 5-15 ng input range for a representative assay. | 100% positive variant call rate within the input range tested (5-15 ng), supporting the 10 ng specified input. For clinical samples, one CD74-ROS1 fusion variant showed 100% positive calls at all input combinations, while the other showed rates as low as 50% at specific high input combinations (attributed to likely operator error). |
| DNA/RNA Input (Negative Call Rate) | Not explicitly stated as a minimum. | >95% for all except 4 sample/input combinations; cases with <95% were due to no calls. For clinical samples, DNA variants showed >95% negative call rates. The second CD74-ROS1 clinical sample showed 100% negative call rates for all test conditions where it was expected to be wild type. |
| Interfering Substances | Positive/Negative/Overall concordance with control was 100% (excluding no calls) for most interferents. For hemoglobin, positive concordance was 100%, negative 99.99%, overall 99.99%. (These are the observed results which met the study's goal of demonstrating non-interference). | For most interferents (Paraffin, Xylene, Ethanol, Protease, Wash buffer): 100% positive, negative, and overall concordance with control (excluding no calls). For Hemoglobin: 100% positive concordance, 99.99% negative concordance, 99.99% overall concordance (excluding no calls). |
| Cross Contamination Rate | Not explicitly stated as a specific rate, but the study aims to evaluate cross-contamination. | False-positive rate at DNA variant locations: 0% (0/100). False-positive rate at RNA variant locations: 1.25% (1/80), attributed to likely sample cross-contamination. |
| Minimal coverage needed for FFPE calling | ≥347X for SNV, MNV, deletion; ≥41X for fusion. | Not explicitly reported in performance data. (This is a recommended use parameter) |
2. Sample size used for the test set and the data provenance
- Accuracy Study (FFPE):
- Sample Size: 290 FFPE tumor samples.
- Data Provenance: The document implies these are clinical samples ("human specimens," "FFPE tumor samples") but does not specify country of origin. It is a retrospective study since variants were compared against "validated reference detection methods."
- Sample Reproducibility Study (FFPE):
- Sample Size: 2 WT (Wild Type) samples and 10 variant-positive samples. Each sample tested 8 times at each of 4 sites, for a total of 32 replicates per sample.
- Data Provenance: Not explicitly stated (e.g., country of origin, retrospective/prospective). These appear to be characterized samples used in a prospective, controlled study.
- Assay Reproducibility Study (FFPE):
- Sample Size: 18 DNA samples (6 plasmid/clinical DNA blends, 12 clinical DNA samples) and 9 RNA samples (1 WT, 8 with RNA variants). Each pre-extracted sample run in duplicate with 2 different reagent lots (3 sites) or 3 reagent lots (1 site), resulting in 72 test determinations per DNA sample and 144 per RNA sample, total of at least 1,296 sequencing reactions.
- Data Provenance: Mixtures of plasmid and clinical DNA/RNA. Not explicitly stated (e.g., country of origin, retrospective/prospective). This is a prospective, controlled study.
- Tissue Input Study (FFPE):
- Sample Size: 60 slide-mounted FFPE samples (30 resection with >20% tumor, 15 resection with <20% to ≥10% tumor (macrodissected), 15 CNB samples).
- Data Provenance: Clinical FFPE samples. Not explicitly stated (e.g., country of origin, retrospective/prospective). Presumed retrospective collection for the purpose of testing input requirements.
- DNA and RNA Input Study (FFPE):
- Sample Size: 8 cell-line samples (as FFPE sections) for blending. 540 individual DNA and RNA libraries tested. Additionally, 4 clinical samples (2 DNA variants, 2 CD74-ROS1 fusion variants).
- Data Provenance: Cell-line samples prepared as FFPE sections, and clinical FFPE samples. Not explicitly stated (e.g., country of origin, retrospective/prospective). This is a prospective, controlled study.
- Interfering Substances Study (FFPE):
- Sample Size: 8 FFPE samples (1 WT, 7 mutants) with 6 replicates each.
- Data Provenance: Not explicitly stated (e.g., country of origin, retrospective/prospective). This is a prospective, controlled study using selected samples.
- Cross Contamination Study (FFPE):
- Sample Size: 8 FFPE cell line samples. 100 DNA and 80 RNA data points analyzed.
- Data Provenance: FFPE cell line samples. Not explicitly stated (e.g., country of origin, retrospective/prospective). This is a prospective, controlled study.
3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts
The document does not specify the number or qualifications of experts used to establish ground truth for any of the studies.
4. Adjudication method for the test set
The document does not describe an adjudication method for the test set. For the accuracy study, variants were compared against "validated reference detection methods" (NGS assay, ROS1 FISH test), which implies pre-established ground truth, not an adjudication process during the study.
5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance
There is no indication that a multi-reader multi-case (MRMC) comparative effectiveness study was done, nor is there any mention of human readers or AI assistance. This device is a high-throughput genomic sequencer, not an imaging device typically involving human readers for interpretation in the way an MRMC study would apply.
6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done
The performance studies described (Accuracy, Reproducibility, etc.) are evaluating the Ion PGM™ Dx System as a whole, which includes the instrument and its associated software (Torrent Suite™ Dx Software). The "Performance Data" sections appear to describe the standalone performance of the entire system (instrument + software) without human intervention in the interpretation of the calls as the primary performance metric. The study results (PPA, NPA, call rates) reflect the algorithm's performance in detecting variants.
7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)
- Accuracy Study (FFPE): Ground truth was established using "validated reference detection methods," specifically:
- A validated NGS assay for SNV and deletion hotspot variants.
- A ROS1 FISH reference test for ROS1 fusions.
- Reproducibility Studies (Sample, Assay, DNA/RNA Input, Interfering Substances, Cross Contamination): Ground truth was based on:
- Characterized samples (WT samples, variant-positive samples, plasmid/clinical DNA blends, FFPE cell line samples). This implies a pre-existing understanding of the variants present in these samples, likely established through prior sequencing or genetic analysis, serving as the reference standard.
8. The sample size for the training set
The document does not provide a specific sample size for a "training set." The studies described are performance validation studies, not studies related to the development or training of an AI/ML algorithm. The Ion PGM™ Dx System measures hydrogen ions during sequencing; its algorithms for base calling and variant detection are typically deterministic or rule-based, or if they involve machine learning, the training details are not disclosed in this document.
9. How the ground truth for the training set was established
As no "training set" is described for an AI/ML context, this question is not directly applicable. If the underlying algorithms for variant calling involved machine learning, the method for establishing ground truth for their training is not provided in this document. The document describes validation against characterized samples and reference methods.
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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002
June 22, 2017
LIFE TECHNOLOGIES CORPORATION EMILY FINNEGAN, REGULATORY ANALYST 5781 VAN ALLEN WAY CARLSBAD CA 92008
Re: K170299
Trade/Device Name: Ion PGM Dx System Regulation Number: 21 CFR 862.2265 Regulation Name: High throughput genomic sequence analyzer for clinical use Regulatory Class: II (special controls); exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to 862.9. Product Code: PFF Dated: June 20, 2017 Received: June 21, 2017
Dear Ms. Finnegan:
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. 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 Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. 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
http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.
Sincerely yours,
Kellie B. Kelm -S
for Courtney H. Lias, Ph.D. Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K170299
Device Name Ion PGM™ Dx System
Indications for Use (Describe)
The Ion PGM™ Dx Instrument System is composed of a sequencing instrument that measures the hydrogen ions that are generated during the incorporation of nucleotides in the DNA sequencing reaction, and the ancillary instrumentation necessary for sample processing. This instrument system is used in conjunction with the instrument-specific Ion PGM™ Dx Library Kit, Ion OneTouch™ Dx Template Kit, Ion PGM™ Dx Sequencing Kit, and Ion 318™ Dx Chip Kit, and data analysis software. The lon PGM™ Dx Instrument System is intended for targeted sequencing of human genomic DNA (gDNA) from peripheral whole-blood samples and DNA and RNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples. The lon PGM™ Dx Instrument System is not intended for whole genome or de novo sequencing.
| Type of Use (Select one or both, as applicable) |
|---|
| ------------------------------------------------- |
X Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)
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510(k) Summary
Submitter Information - 21 CFR 807.92(a)(1):
| Submitter: | Life Technologies Corporation5781 Van Allen WayCarlsbad, CA 92008 |
|---|---|
| Manufacturer: | Life Technologies Holdings Pte LtdBlk 33, #07-06, Marsiling Industrial Estate, Road 3Singapore 739256 |
| Establishment Registration No: | 3003673482 |
| Contact: | Emily Finnegan, Regulatory Analyst |
| Phone: | 916-838-0714 |
| Fax: | 760-268-8393 |
| E-mail: | emily.finnegan@thermofisher.com |
| Alternate Contact: | Jody Schulz, Senior Manager, Regulatory Affairs |
| Phone: | 414-534-4809 |
| Fax: | 414-278-0688 |
| E-mail: | jody.schulz@thermofisher.com |
| Date Prepared: | June 20, 2017 |
| Name of Device and Classification – 21 CFR 807.92(a)(2): | |
| Product Name: | Ion PGM™ Dx System |
| Common Name: | High-throughput DNA sequencing |
Device Classification: Class II, exempt from the premarket notification requirement
subject to the limitations in 21 CFR 862.9
Product Code: PFF - High throughput DNA sequence analyzer
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Predicate Devices - 21 CFR 807.92(a)(3):
Predicates: Illumina MiSeqDx Platform, K123989 (DEN130011)
Device Description - 21 CFR 807.92(a)(4):
The Ion PGM™ Dx System is used for detection of human variant sequences from DNA from whole blood samples or RNA and DNA from FFPE tissue samples. Detectable variants include substitutions, insertions, and deletions.
The Ion PGMTM Dx System consists of the following:
- Ion OneTouch™ Dx Instrument
- Ion OneTouch™ ES Dx Instrument
- Ion OneTouch™ Rack Kit
- · Ion PGM™ Dx Chip Minifuge
- · Ion PGM™ Dx Sequencer
- · Ion PGMTM Wireless Scanner
- DynaMag™ Dx Kit—Tube & Plate
- · Ion Torrent™ Server
- Torrent Suite™ Dx Software
The Ion PGM™ Dx System is used in conjunction with the following kits:
- Ion PGM™ Dx Library Kit
- Ion OneTouch™ Dx Template Kit
- · Ion PGM™ Dx Sequencing Kit
- Ion 318™ Dx Chip Kit
The system should be used only by professionals trained in laboratory techniques and procedures and in the use of the system.
Intended Use/Indications for Use - 21 CFR 807.92(a)(5):
Ion PGM Dx System intended use and special conditions statement:
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Intended use:
The Ion PGM™ Dx Instrument System is composed of a sequencing instrument that measures the hydrogen ions that are generated during the incorporation of nucleotides in the DNA sequencing reaction, and the ancillary instrumentation necessary for sample processing. This instrument system is used in conjunction with the instrument-specific Ion PGM™ Dx Library Kit, Ion OneTouch™ Dx Template Kit, Ion PGM™ Dx Sequencing Kit, and Ion 318TM Dx Chip Kit, and data analysis software. The Ion PGM™ Dx Instrument System is intended for targeted sequencing of human genomic DNA (gDNA) from peripheral wholeblood samples and DNA and RNA extracted from formalin-fixed, paraffinembedded (FFPE) samples.
The Ion PGMTM Dx Instrument System is not intended for whole genome or de novo sequencing.
Indications for use:
Same as intended use.
Special conditions statement
For in vitro diagnostic use. For prescription use only.
Special conditions statement for performance derived from gDNA from whole blood:
-
- The Ion PGM™ Dx System has been validated to deliver the following using the
System Variant Assay (SVA) panel:
- The Ion PGM™ Dx System has been validated to deliver the following using the
-
· Sequencing output > 0.7 gigabases
-
Reads > 4 million
-
· Read length up to 200 base pairs
-
· Mean Raw Read Accuracy of 99.0% when compared to hg19
Note:
- · Mean Raw Read Accuracy is defined as the average raw accuracy across each individual base position in a read, where raw read accuracy is calculated as 100 * (1 - (sum(per base error)/sum(per base depth)))
- · The 632 primer pairs of the SVA panel are designed to amplify regions across 23
{6}------------------------------------------------
chromosomes in the two well-characterized cell lines. The regions were selected based on the presence of well characterized insertions/deletions (Indels) and singlenucleotide variant (SNV) positions. The amplicons produced range in size from 80 to 200 base pairs, with a GC content of 20-80%.
-
- The system has been evaluated for the detection of single-nucleotide variants (SNVs) and insertions and deletions of various lengths on 23 chromosomes. The system identified 440 unique SNV positions in the SVA panel with 100% reproducibility.
The following tables illustrate the lengths and locations of insertions and deletions in the SVA panel that were detected with 100% reproducibility.
- The system has been evaluated for the detection of single-nucleotide variants (SNVs) and insertions and deletions of various lengths on 23 chromosomes. The system identified 440 unique SNV positions in the SVA panel with 100% reproducibility.
| Insertion Length(base pairs) | Total Number ofDistinct chromosomallocations | Total Number of Uniquechromosomes |
|---|---|---|
| 1 | 14 | 10 |
| 2 | 4 | 4 |
| 3 | 5 | 4 |
| 4 | 5 | 4 |
| Deletion Length(base pairs) | Total Number ofDistinct chromosomallocations | Total Number of Uniquechromosomes |
|---|---|---|
| 1 | 15 | 11 |
| 2 | 10 | 6 |
| 3 | 3 | 3 |
| 4-14[1] | 7 | 7 |
[1] Deletions of ≥ 4 bp have been grouped for clarity
-
- The system may exhibit a limitation in detecting one-base insertions or deletions in homoploymer tracts (e.g., polyA). Variants in homopolymer runs exceeding 8 bases are called as no calls in the VCF file.
-
- The system is designed to deliver qualitative (i.e., genotype) results.
-
- As with any hybridization-based workflow, underlying polymorphisms or mutations in primer-binding regions can affect the regions being sequenced and, consequently, the calls made.
-
- The recommended minimal coverage per amplicon needed for accurate variant calling
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for germline DNA is >30X.
-
- Special instrument requirements for the Ion PGM™ Dx Library Kit, Ion OneTouch™ Dx Template Kit, Ion PGMTM Dx Sequencing Kit, and Ion 318™ Dx Chip Kit: For use with the Ion PGM™ Dx System.
Special Conditions statement for performance derived from a representative assay using RNA and gDNA from FFPE samples:
-
- The Ion PGM™ Dx System has been validated to deliver the following using a representative assay:
- Sequencing output > 0.7 gigabases .
- Reads > 3 million .
- Read length up to 141 base pairs .
-
- A representative assay, consisting of two sets of primer panels was used to detect DNA and RNA variants in key regions of cancer-related genes. The Ion PGM™ Dx System has been evaluated for the detection of SNVs, multi-nucleotide variants (MNVs), and deletions of various lengths in FFPE tissue samples using this representative assay. The types and numbers of variants detected by the assay are listed below.
| Type of | Number of variantsdetected with arepresentative assay | Number of samples tested for detection by sample type | ||
|---|---|---|---|---|
| Variant | Plasmid/FFPESample Blend | FFPE Cell Line orFFPE Cell LineBlend | FFPE ClinicalSample | |
| MNV | 9 | 9 | 0 | 2 |
| SNV | 326 | 329 | 8 | 113 |
| 3-bp deletion | 4 | 6 | 0 | 0 |
| 6-bp deletion | 4 | 8 | 0 | 0 |
| 9-bp deletion | 4 | 8 | 0 | 1 |
| 12-bp deletion | 7 | 7 | 0 | 0 |
| 15-bp deletion | 7 | 10 | 3 | 23 |
| 18-bp deletion | 7 | 8 | 0 | 11 |
-
- The following studies were used to evaluate the performance of the Ion PGMTM Dx System using a representative assay:
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- Accuracy ●
- Sample reproducibility ●
- Assay reproducibility ●
- Tissue input ●
- DNA and RNA input ●
- . Interfering substances
-
- The system is designed to deliver qualitative results.
-
- As with any hybridization-based workflow, underlying polymorphisms or mutations in primer-binding regions can affect the regions being sequenced and, consequently, the ability to make calls.
-
- The minimal coverage required to call an SNV, MNV or deletion variant is ≥347X. The minimal coverage required to call a fusion variant is ≥41X.
-
- Special instrument requirements for the Ion PGM™ Dx Library Kit, Ion OneTouch™ Dx Template Kit, Ion PGM™ Dx Sequencing Kit, and Ion 318TM Dx Chip Kit: For use with the Ion PGM™ Dx System.
Summary of technological characteristics of the device compared to the predicate devices-21 CFR 807.92(a)(6):
The Ion PGM™ Dx System ("Subject Device") and the legally marketed device Illumina MiSeqDx Platform are compared and contrasted as described in the table below:
| Item for Comparison | Proposed Device:Ion PGM DxSystem | Predicate Device:MiSeqDxPlatform | Comparison |
|---|---|---|---|
| Intended Use | The Ion PGMTM DxSystem is composedof a sequencinginstrument thatmeasures thehydrogen ions thatare generated duringthe incorporation ofnucleotides in the | The MiSeqDxPlatform is asequencinginstrument thatmeasuresfluorescencesignals of labelednucleotides throughthe use of | The intended uses aredifferent due to theexpansion of the Ion PGMDx System sample input toinclude DNA and RNAextracted from FFPEsamples, however the twodevices continue to use thesame basic technology and |
| Item for Comparison | Proposed Device:Ion PGM DxSystem | Predicate Device:MiSeqDxPlatform | Comparison |
| sequencing reactionand the ancillaryinstrumentationnecessary for sampleprocessing. Thisinstrument system isused in conjunctionwith the instrumentspecificIon PGMTM DxLibrary Kit, IonOneTouchTM DxTemplate Kit, IonPGMTM DxSequencing Kit, Ion318TM Dx Chip Kits,and data analysissoftware. The IonPGMTM DxSystem is intendedfor targetedsequencing ofhuman RNA andDNA derived fromFFPE tissue samplesand human gDNAfrom peripheralwhole bloodsamples. TheIon PGMTM DxSystem is notintended for wholegenome or de novosequencing. | instrument specificreagents and flowcells (MiSeqDxUniversal Kit 1.0),imaging hardware,and data analysissoftware. TheMiSeqDx Platformis intended fortargeted sequencingof human genomicDNA fromperipheral wholeblood samples. TheMiSeqDx Platformis not intended forwhole genome orde novosequencing. | are therefore under thesame regulation (21 CFRPart 862.2265). | |
| Environment of Use | Clinical Laboratories | ClinicalLaboratories | The environment of use isthe same. |
| Specimen Type | whole-blood orformalin fixedparaffin embedded(FFPE) samples | whole-bloodsamples | The specimen type isdifferent with the inclusionof FFPE samples |
| Input Sample | Genomic DNA andcDNA | Genomic DNA | The input sample isdifferent with the inclusionof cDNA as an option foruse on the Ion PGM Dxsystem. However, genomicDNA and cDNA arebiochemically equivalent asthey are both made up ofDeoxyribonucleic acid andare interpreted in the samemanner by the PGM Dxsequencer to generate |
| Item for Comparison | Proposed Device:Ion PGM DxSystem | Predicate Device:MiSeqDxPlatform | Comparison |
| Regulation/Classification | 21 CFR 862.2265Class II, exemptfrom the premarketnotificationrequirement subjectto the limitations in21 CFR 862.9 | 21 CFR 862.2265Class II, exemptfrom the premarketnotificationrequirement subjectto the limitations in21 CFR 862.9 | Theregulation/classification isthe same. For the purposeof this 510(k), Ion PGM Dxsystem is not exempt frompremarket notificationrequirements due to theexpansion in the intendeduse to add FFPE and RNA. |
| Technology | A sequencinginstrument thatmeasures thehydrogen ions thatare generated duringthe incorporation ofnucleotides in theDNA sequencingreaction and theancillaryinstrumentationnecessary for sampleprocessing. | A sequencinginstrument thatmeasuresfluorescencesignals of labelednucleotides throughthe use ofinstrument specificreagents and flowcells (MiSeqDxUniversal Kit 1.0),imaging hardware,and data analysissoftware. | The technology used forDNA sequencing areequivalent NGStechnologies. Both the IonPGM Dx system and theMiSeqDx platform detectthe incorporation ofnucleotides by DNApolymerases. However,they differ in that the IonPGM Dx system measuresthe incorporation ofhydrogen ions, while theMiSeqDx platformmeasures the fluorescencesignal. The differences inthe signal measured duringDNA sequencing do notraise questions to the safetyand effectiveness of the IonPGM Dx system. |
| Software DescriptionComparison | Combined Functionssoftware with AssayDevelopment modefor Research UseOnly | CombinedFunctions softwarewith ability to useResearch Use Onlymode | The software description isequivalent. |
| Equipment included inthe device system | • Ion OneTouch™Dx Instrument• Ion OneTouch™ES Dx Instrument• Ion PGM™ DxChip Minifuge• Ion PGM™ DxSequencer• Ion PGM™Torrent Server• Torrent Suite™ DxSoftware | • MiSeqDxInstrument• MOS -MiSeqDxOperatingSoftware• RTA - RealtimeAnalysisSoftware• MiSeqReporter | The equipment included inthe device system isdifferent as the technologycharacteristics differ. |
Table 1. Predicates Comparison - Thermo Fisher Scientific Ion PGM™ Dx System and Illumina MiSeqDx Platform
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Performance Data – 21 CFR 807.92(b):
This section provides a summary of the non-clinical and clinical performance studies with DNA and RNA from FFPE samples using a representative assay, which demonstrates instrument performance when using the Ion PGM™ Dx system.
Non-Clinical Performance Data– 21 CFR 807.92(b)(1):
-
a. Accuracy study
To evaluate the ability of a representative assay DNA and RNA panels to identify somatic variants in human specimens, 290 FFPE tumor samples were analyzed using a representative assay to demonstrate positive percent agreement (PPA) and negative percent agreement (NPA) concordance with validated reference detection methods. The following reference detection methods were used: -
· A validated NGS assay, to detect SNV and deletion hotspot variants
-
A ROS1 FISH reference test, to detect ROS1 fusions
Variants detected by a representative assay that were not covered by the reference methods were not included in the PPA/NPA concordance calculation. Variants detected by arepresentative assay test for which the reference method testing failed and did not yield a valid result were not included in the PPA/NPA calculation.
Accuracy data was analyzed by the following:
- Each variant location
- · Bins (or categories) of variants: RNA fusions , simple SNVs, complex SNVs, and deletions
- Each FFPE sample
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| PPAMeasure | Excluding no calls | Including no calls | ||
|---|---|---|---|---|
| Percentagreement | 95% CI | Percentagreement | 95% CI | |
| Variant | 98.5% (195/198) | 95.6%,99.7% | 98.5% (195/198) | (95.6%,99.7%) |
| Bin | 97.2% (176/181) | 93.7%,99.1% | 97.2% (176/181) | (93.7%,99.1%) |
| Sample | 96.9% (158/163) | 93.0%,99.0% | 96.9% (158/163) | (93.0%,99.0%) |
Table 2. PPA Results
Table 3. NPA results
| NPAMeasure | Excluding no calls | Including no calls | ||
|---|---|---|---|---|
| Percentagreement | 95% CI | Percentagreement | 95% CI | |
| Variant | 100.0%(118,155/118,159) | 99.99%,100.0% | 96.8%(118,155/122,012) | (96.7%, 96.9%) |
| Bin | 99.8% (942/944) | 99.2%, 100.0% | 70.0% (657/939) | (66.9%, 72.9%) |
| Sample | 98.4% (124/126) | 94.4%, 99.8% | 23.4% (29/124) | (16.3%, 31.8%) |
Table 4. OPA Results
| Excluding no calls | Including no calls | |||
|---|---|---|---|---|
| OPAMeasure | Percentagreement | 95% CI | Percentagreement | 95% CI |
| Variant | 100.0%(118,350/118,357) | 99.99%,100.0% | 96.8%(118,350/122,210) | (96.74%,96.94%) |
| Bin | 99.4% (1,118/1,125) | 98.72%,99.75% | 74.4% (833/1,120) | (71.71%,76.91%) |
| Sample | 97.6% (282/289) | 95.07%,99.02% | 65.2% (187/287) | (59.34%,70.66%) |
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b. Sample Reproducibility study
The reproducibility and repeatability of variant detection using a representative assay were assessed with 2 WT samples and 10 variant-positive samples at 4 testing sites. Each site had 4 Ion PGM™ Dx instrument systems and 4 operators.
Each sample was tested 8 times at each site, for a total of 32 replicates per sample. After repeat testing, the final number of invalid reactions was 15/768 (1.95%), possibly due to low sample quality or lack of sample, though the cause was not definitively determined.
The call rate, no call rate, positive call rate, negative call rate, and within-run repeatability were computed at each variant location of interest. Including no calls and excluding known positive variant locations, the negative call rate at each clinical variant location for all samples was 100%.
The results at positive variant locations are shown in Table 5. Including no calls, all positive call rates from positive variant locations were >84%.
Excluding no calls and combining data across all study samples, the estimate of repeatability was 100% for DNA variants and 87.5% for the RNA variant. The lower limit of the 95% two-sided confidence interval (CI) for repeatability exceeded 96% at all variant locations.
Including no calls from the data, the estimate of repeatability was 100% at 218 out of 605 variant locations, 94-99.9% at 186 out of 605 variant locations, and 71.6-93.9% at 184 out of 605 variant locations. Including no calls, the lower limit of the 95% two-sided confidence interval for repeatability exceeded 64.6% at all variant locations.
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| Sample | Variantidentification | Variantlocation | # of validsampleresults(N) | # ofpositivecalls (A) | # ofnegat-ivecalls(B) | # ofNoCalls(C) | Positive call rate+ 95% CI | Negative call rate+ 95% CI | Within-run repeatability + 95% CI | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Including nocalls (A/N) | Excludingno calls(A/(A+B)) | Including nocalls (B/N) | Excluding nocalls(B/(A+B)) | Including no calls | Excluding no calls | |||||||
| B | COSM6223 | EGRFExon19del | 32 | 32 | 0 | 0 | 100%(89.1%,100%) | 100%(89.1%,100%) | 0% (0%,10.9%) | 0% (0%,10.9%) | 100%(79.4%,100%) | 100%(79.4%,100%) |
| B | COSM763 | PIK3CAE545K | 32 | 32 | 0 | 0 | 100%(89.1%,100%) | 100%(89.1%,100%) | 0% (0%,10.9%) | 0% (0%,10.9%) | 100%(79.4%,100%) | 100%(79.4%,100%) |
| C | ROS1 | N/A | 32 | 30 | 2 | 0 | 93.8%(79.2%,99.2%) | 93.8%(79.2%,99.2%) | 6.3%(0.8%,20.8%) | 6.3%(0.8%,20.8%) | 87.5%(61.7%,98.4%) | 87.5%(61.7%,98.4%) |
| D | COSM6225 | EGFRExon19del | 32 | 32 | 0 | 0 | 100%(89.1%,100%) | 100%(89.1%,100%) | 0% (0%,10.9%) | 0% (0%,10.9%) | 100%(79.4%,100%) | 100%(79.4%,100%) |
| E | COSM476 | BRAF V600E | 32 | 32 | 0 | 0 | 100%(89.1%,100%) | 100%(89.1%,100%) | 0% (0%,10.9%) | 0% (0%,10.9%) | 100%(79.4%,100%) | 100%(79.4%,100%) |
| F | COSM521 | KRAS G12D | 32 | 30 | 0 | 2 | 93.8%(79.2%,99.2%) | 100%(88.4%,100%) | 0% (0%,10.9%) | 0% (0%,11.6%) | 87.5%(61.7%,98.4%) | 100%(76.8%,100%) |
| Positive call rate+ 95% CI | Negative call rate+ 95% CI | Within-run repeatability + 95% CI | ||||||||||
| Sample | Variantidentification | Variantlocation | # of validsampleresults(N) | # ofpositivecalls (A) | # ofnegativecalls(B) | # ofNoCalls(C) | Including nocalls (A/N) | Excludingno calls(A/(A+B)) | Including nocalls (B/N) | Excluding nocalls(B/(A+B)) | Including no calls | Excluding no calls |
| F | COSM29313 | PIK3CA M1043I | 32 | 30 | 0 | 2 | 93.8%(79.2%,99.2%) | 100%(88.4%,100%) | 0% (0%,10.9%) | 0% (0%,11.6%) | 87.5%(61.7%,98.4%) | 100%(76.8%,100%) |
| G | COSM6224 | EGFR L858R | 32 | 32 | 0 | 0 | 100%(89.1%,100%) | 100%(89.1%,100%) | 0% (0%,10.9%) | 0% (0%,10.9%) | 100%(79.4%,100%) | 100%(79.4%,100%) |
| J | COSM87298 | KRAS Q61K | 32 | 32 | 0 | 0 | 100%(89.1%,100%) | 100%(89.1%,100%) | 0% (0%,10.9%) | 0% (0%,10.9%) | 100%(79.4%,100%) | 100%(79.4%,100%) |
| J | COSM172423 | ERBB3V104M | 32 | 32 | 0 | 0 | 100%(89.1%,100%) | 100%(89.1%,100%) | 0% (0%,10.9%) | 0% (0%,10.9%) | 100%(79.4%,100%) | 100%(79.4%,100%) |
| K | COSM775 | PIK3 H1047R | 30 | 29 | 0 | 1 | 96.7%(82.8%,99.9%) | 100%(88.1%,100%) | 0% (0%,11.6%) | 0% (0%,11.9%) | 93.3%(68.1%,99.8%) | 100%(76.8%,100%) |
| M | COSM715 | FGR3 S249C | 32 | 32 | 0 | 0 | 100%(89.1%,100%) | 100%(89.1%,100%) | 0% (0%,10.9%) | 0% (0%,10.9%) | 100%(79.4%,100%) | 100%(79.4%,100%) |
Table 5. Call Rates at Positive Variant Locations
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c. Assay Reproducibility Study
The reproducibility and repeatability of a representative assay was evaluated for 30 representative variants from 18 DNA and 9 RNA samples.
The study was designed to evaluate within-run precision performance (repeatability) and variability across sites, operators, and instrument platforms (reproducibility). Six of the 18 DNA samples were mixtures of plasmid and clinical DNA. Seven of the 12 deletion variants were represented by these plasmid blends. All other variant types were represented by clinical sample DNA.
Due to the large number of variants detected by the test and the rarity of some of the variants, a representative variant approach was used. Variants were selected in the following categories:
| Variant Category | No. of Plasmid BlendsUsed | No. of Clinical SamplesUsed |
|---|---|---|
| 6-bp deletion | 6 | 0 |
| 9-bp deletion | 4 | 2 |
| 15-bp deletion | 2 | 4 |
| 18-bp deletion | 2 | 4 |
| Simple SNV | 0 | 8 |
| Complex SNVs andMNPs[1] | 0 | 6 |
| Fusion | 0 | 12 |
Table 6. Categories
[1] Including SNVs in di- or tri-nucleotide repeat regions and SNVs in high-GC (>60%) or low-
GC (<40%) content regions
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Two of the 18 DNA samples were WT at all locations, and the remaining 16 contained DNA from one or more DNA variants. One of the 9 RNA samples contained no fusion molecules, and the remaining 8 each contained RNA from an RNA variant. Each pre- extracted DNA or RNA sample was sequenced at 4 sites by 4 operators on 2 systems at each site.
At each site, operators were grouped into 2 pairs, with each pair assigned to2 instrument systems and responsible for testing 9 DNA samples and all 9 RNA samples. Samples were run in duplicate using 2 different reagent lots at 3 of the study sites and on all 3 reagent lots at one study site. The design resulted in a total of 72 test determinations per DNA sample. Because there were half as many RNA samples as DNA samples, each RNA sample was tested twice as many times (n=144). In total, at least 1,296 sequencing reactions were performed, and all variant locations were assessed for each sample.
The reproducibility results are summarized in the following table.
| Description | No. of VariantSamples | Call rate excluding nocalls | Call rate including nocalls | ||
|---|---|---|---|---|---|
| Mean | Median | Mean | Median | ||
| DNA positive variants (positivecalls) | 46 | 96.60% | 97.10% | 94.50% | 95.80% |
| RNA positive variants (positivecalls) | 6 | 94.80% | 95.50% | 94.80% | 95.50% |
| WT DNA variant locations(negative calls) | 872 | 96.10% | 95.00% | 96.10% | 95.00% |
| WT RNA variant locations(negative calls) | 170 | 99.30% | 99.30% | 99.30% | 99.30% |
Table 7. Reproducibility Results
Excluding no calls, the estimate of repeatability at each DNA variant location across all the samples was ≥98.8% (95% CI lower limit of ≥97.5%). The coefficient of variation (CV) across all DNA clinical variants ranged from 9.8% to 39%. The highest CVs (24.9-39.2%) were observed for the BRAF V600E variant. The higher percent CV for this sample was possibly due to poor sample
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quality, but the cause was not definitively determined. The CVs for the EGFR L858R variant ranged from 9.8% to 11.3%, and the CVs for the EGFR deletion variants ranged from 11.2% to 25.5%.
Excluding no calls, the estimate of repeatability at each RNA clinical variant location was 94.4%. The CV across all RNA locations ranged from 72% to 78%.
d. Tissue Input Study
Sixty slide-mounted FFPE samples were analyzed to determine if samples extracted using the Ion Torrent Dx Total Nucleic Acid Isolation Kit yield DNA and RNA at the concentrations required by a representative assay when tissue input requirements are met. The test requires DNA at a concentration of ≥0.83 ng/uL and RNA at a concentration of >1.43 ng/uL.
Thirty resection samples with >20% tumor content were prepared without macrodissection, 15 resection samples with <20% to ≥10% tumor cell content were macrodissected, and 15 samples were collected by core needle biopsy (CNB). For the resection samples, 2 × 5 µm sections were used per extraction. For CNBs, 9 × 5 um sections were used per extraction. DNA and RNA concentrations were determined using the Ion Torrent Dx DNA and RNA Quantification Kits, respectively. No sequencing was performed on the extracted samples.
Of the 60 samples tested, 98.3% (59/60) had a DNA concentration of ≥0.83 ng/uL and an RNA concentration of >1.43 ng/uL. One CNB sample failed the minimum DNA and RNA concentration specifications, with values of 0.52 ng/uL and 1.23 ng/uL respectively. The low concentrations were likely caused by the small tissue size and low tumor content (5%).
e. DNA and RNA Input Study
Eight cell-line samples were prepared as FFPE sections, and DNA and RNA
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were extracted and quantified from multiple sections from each cell line for blending and testing. Sample blends were prepared with known variants at various DNA and RNA input-level combinations within the range of 5-15 ng. The DNA and RNA blends had a target allele frequency of 15% for SNVs and deletions and target fusion reads of 300–600 for the ROS1 variant. A total of 540 individual DNA and RNA libraries were tested, including positive controls and NTC controls, with 6 replicate libraries each for DNA and RNA per test condition.
The study demonstrated a 100% positive variant call rate within the input range tested, supporting the specified input amount of 10 ng each for DNA and RNA for a representative assay.
The negative variant call rate was >95% for all except 4 sample and DNA/RNA input- level combinations. All cases with a negative variant call rate of <95% were due to no calls, 3 of which occurred with a DNA or RNA input amount of 5 ng and 1 of which occurred in a single sample with DNA and RNA inputs of 10 ng each. There were no false-positive calls.
Additionally, 4 clinical samples prepared as FFPE sections were tested: two samples containing DNA variants and two containing the CD74-ROS1 fusion variant.
The DNA variant samples were paired with wild-type RNA from the same sample at various input combinations within the range of 5–15 ng, and the RNA variant samples were paired with wild-type DNA at input combinations within the same range.
The study demonstrated positive and negative call rates of >95% for the DNA variants at all input combinations, and 100% for one of the CD74-ROS1 fusion variants at all input combinations. The second CD74-ROS1 clinical sample showed 100% negative call rates for all test conditions, and 100% positive call rates except for Test Condition 4 (8.5 ng RNA/15 ng DNA), where the call rate was 83%, and Test Condition 6 (15 ng RNA/15 ng DNA), where the call rate was 50%. The false negatives for these test conditions were possibly due to
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operator error during library preparation, since the remaining replicates in these test conditions had both high total mappable reads and fusion reads, but the cause was not definitively determined.
The results support the DNA and RNA 10-ng input requirement for a representative assay.
Interfering Substances Study f.
Five potentially interfering substances used to extract DNA and RNA from FFPE tissue samples were evaluated using a representative assay on the Ion PGM™ Dx System.
The guidelines for testing are defined in section 7.1 of CLSI EP07A2E, which describes testing substances at a relatively high concentration as an interference screen. One potentially interfering endogenous substance, hemoglobin, was tested at twice the concentration recommended in CLSI EP07A2E, Appendix D.
In addition to the substances tested in this study, data from the clinical studies demonstrated that 10-20% necrotic tissue in the region of interest in FFPE tissue samples does not appear to interfere with the assay. However, users should macrodissect highly necrotic areas or select alternate samples if possible.
| Potentialinterferingsubstance | Step | Amount of substance |
|---|---|---|
| Paraffin | At the deparaffinization step,extra paraffin was added to thexylene bath that contained 250mL of xylene. | 4X of normally expected levels |
| Xylene | Extra xylene was added into theethanol bath that contained 250mL of ethanol. | 6X of normally expectedresidual volume |
Table 8. Interfering substances and amounts
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| Potentialinterferingsubstance | Step | Amount of substance |
|---|---|---|
| Ethanol | Extra ethanol was added intothe Protease digestion stepbefore digestion. | >4X of normally expectedresidual volume |
| Hemoglobin | After deparaffinization,hemoglobin was added to theDigestion Buffer used to pre-wet the tissue section | 4 mg/mL |
| Protease | Extra Protease was added intothe reaction after the digestionstep and before columnpurification. | >10X of expected residualProtease after the heat-kill step |
| Wash buffer | Wash buffer used to isolateDNA and RNA fromdeparaffinized and digestedsamples was added into analiquot of Dilution Solution,which was subsequently used todilute the RNA and DNA to theappropriate concentration beforelibrary preparation. | 1% wash buffer (equivalent to~10% wash buffer carried overinto eluate) |
| Control | Tissue sections were processedusing the standard protocol,without the addition of anypotentially interferingsubstances. | N/A |
A total of 8 FFPE samples (1 WT and 7 mutants) with 6 replicates each were processed through the entire assay workflow. The mutant samples included variants from all variant categories that can be detected by the test. The samples were spiked with additional concentrations or amounts of the listed substances at the relevant processing step, as shown in the table. Replicates of a control sample with no spiked substances were also analyzed. The concordance between variant calls in samples with and without interfering substances was computed for each substance under investigation.
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With no calls excluded, for each potential interferent used in sample extraction, the positive and negative concordance with the control condition across all samples was 100%, and the overall concordance with the control condition across all samples was 100%
With no calls excluded, the results of testing with hemoglobin showed positive concordance with the control condition of 100% (only samples with a positive control condition were analyzed), negative concordance of 99.99%, and overall concordance of 99.99%.
g. Cross Contamination Study
A total of 8 FFPE cell line samples were evaluated to determine the percentage of false positive results caused by cross-contamination (contamination from one sample to another within the same sequencing run) and carryover contamination (contamination from a previous run on the same instrument system). Samples that were WT and mutant were tested in consecutive runs on the same instruments, and 5 DNA variant locations and 2 RNA variant locations that were expected to be WT for a sample were evaluated for contamination.
Out of 100 DNA and 80 RNA data points analyzed, no false positive results were reported in the DNA variants, and 1 false positive result was reported in a ROS1 fusion variant. The false positive was likely caused by sample crosscontamination from an adjacent well. Therefore, the false-positive rate at DNA variant locations was 0% (0/100) and the false-positive rate at RNA variant locations was 1.25% (1/80).
§ 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.