The MiSeqDx Universal Kit 1.0 is a set of reagents and consumables used in the processing of human genomic DNA samples derived from peripheral whole blood, and in the subsequent targeted re-sequencing of the resulting sample libraries. User-supplied analyte specific reagents are required for the preparation of libraries targeting specific genomic regions of interest. The MiSeqDx Universal Kit 1.0 is intended for use with the MiSeqDx instrument.

Device Description

The MiSeqDx Universal Kit 1.0 is intended for use with the MiSeqDx Platform. The MiSeqDx Universal Kit 1.0 consists of 5 boxes.

AI/ML Overview

This document describes the acceptance criteria and the study proving the MiSeqDx Universal Kit 1.0 meets those criteria, based on the provided text.

1. Table of Acceptance Criteria and Reported Device Performance

The provided text focuses on the classification and intended use of the MiSeqDx Universal Kit 1.0 as a component for high-throughput genomic sequencing. While specific numerical acceptance criteria for all performance characteristics are not explicitly detailed in the provided excerpt (many refer to an associated 510(k) K123989), the text does outline some critical operational specifications and general requirements.

Acceptance Criteria (from text)	Reported Device Performance (from text)
Operational Specifications:
Sequencing output > 1 Gb	The system is designed to deliver this.
Reads > 3 million	The system is designed to deliver this.
Read length (in paired end run) 2 x 150 bp	The system is designed to deliver this.
Bases higher than Q30 > 75% (Greater than 75% of bases have Phred scale quality score greater than 30, indicating base call accuracy > 99.9%)	The system is designed to deliver this.
Detection of SNVs and up to 3 base deletions	The system has been validated for this.
Qualitative (genotype) results	The system is designed to deliver this.
Recommended minimal coverage per amplicon for accurate variant calling (Q(max_gt \| poly_site) >= 100) is 75x	This is a recommended use condition.
Limitations/Known Performance:
Problems detecting 1 base insertions or deletions in homopolymer tracts (e.g., polyA)	This is a known limitation.
Evaluation of 1 base insertions limited to 3 different insertions on 3 separate chromosomes	This states the extent of validation.
Underlying polymorphisms or mutations in oligonucleotide-binding regions can affect calls	This is an inherent characteristic of hybridization-based workflows.
Stability:
Shelf life of 12 months when stored at appropriate conditions	Real-time stability testing for 6 months supports this; ongoing for 12 months.

2. Sample Size Used for the Test Set and Data Provenance

The provided text repeatedly refers to an associated 510(k) submission, K123989, for detailed performance studies. Therefore, the specific sample sizes used for the test set and the data provenance (e.g., country of origin, retrospective/prospective) are not detailed in this document. The studies mentioned in this document are for:

Precision/Reproducibility
Stability (related to blood samples)
Detection Limit
Analytical Specificity
Accuracy

All these reference K123989, meaning the details about the specific test sets, sample sizes, and provenance would be found in that separate submission.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

The document does not provide this information. The MiSeqDx Universal Kit 1.0 is a reagent kit that facilitates the creation of a library for genomic sequencing. The 'ground truth' for variant calling (SNVs, deletions, insertions) would typically be established based on orthogonal sequencing methods or well-characterized reference materials, rather than expert human interpretation of images or other subjective data. Details relating to the validation of variant calling would reside in K123989.

4. Adjudication Method for the Test Set

This information is not available in the provided text. Given the nature of a reagent kit for genomic sequencing, human adjudication methods like 2+1 or 3+1 are typically not applied to the primary sequencing data itself. The 'ground truth' for genomic variants is usually based on molecular methods.

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

No MRMC comparative effectiveness study was mentioned or implied. The MiSeqDx Universal Kit 1.0 is a reagent kit used in a genomic sequencing workflow. It does not involve human "readers" in the context of interpreting diagnostic images or output in a way that would necessitate an MRMC study with AI assistance. The device's role is to prepare genetic material for sequencing, not to interpret the sequencing results themselves.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

The device itself is a reagent kit. It is not an algorithm, and therefore the concept of "standalone" algorithm performance as typically applied to AI/software clinical decision support systems does not directly apply here. The performance of the sequencing system (which the kit is a part of) would be evaluated in terms of its ability to accurately detect genetic variants, which is an "algorithm only" type of assessment in terms of sequence analysis (without human interpretation of the raw data). However, the specific details of this would be in K123989.

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)

The document refers to studies establishing the "accuracy of high throughput DNA targeted sequencing of human genomic DNA" in K123989. For genomic sequencing, ground truth is typically established using:

Orthogonal Sequencing Methods: Such as Sanger sequencing, whole genome sequencing (WGS), or other highly reliable sequencing technologies on the same samples.
Reference Materials: Certified reference materials with known genetic variants.
Synthetic DNA Constructs: With precisely known mutations.

The specific type of ground truth used would be detailed in K123989.

8. The Sample Size for the Training Set

The provided text does not contain any information regarding a "training set." The MiSeqDx Universal Kit 1.0 is a reagent kit, not a machine learning algorithm that requires a training set. The performance studies referenced in K123989 would evaluate the analytical performance of the kit and the sequencing platform, not the training of an algorithm.

9. How the Ground Truth for the Training Set Was Established

As no training set is mentioned or applicable to a reagent kit in the context of machine learning, this information is not provided and is irrelevant for this specific device.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR MISEQDX UNIVERSAL KIT 1.0

DECISION SUMMARY

A. 510(k) Number:	K133136
B. Purpose for Submission:	De novo request for evaluation of automatic classIII designation of the MiSeqDx Universal Kit 1.0
C. Measurand:	Not applicable. Reagents for the creation of aLibrary specimen from a sample of human genomicDNA obtained from peripheral whole blood
D. Type of Test:	Not applicable: Reagents for preparation of alibrary specimen for use with high-throughputgenomic sequence analyzer
E. Applicant:	Illumina Inc.
F. Proprietary and Established Name:	MiSeqDx Universal Kit 1.0

G. Regulatory Information:

FDA identifies this type of device as:

1. New regulation number: 21 CFR 862.3800
1. Classification: Class I
1. Product code: PFT Reagents for molecular diagnostic test systems
1. Panel: Toxicology (91)

H. Intended Use:

1.Intended uses(s):
The MiSeqDx Universal Kit 1.0 is a set of reagents and consumables used in the processing of human genomic DNA samples derived from peripheral whole blood, and in the subsequent targeted re-sequencing of the resulting sample libraries. User-supplied analyte specific reagents are required for the preparation of libraries targeting specific genomic regions of interest. The MiSeqDx Universal Kit 1.0 is intended for use with the MiSeqDx instrument.

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2. Indication for uses(s):

Same as Intended use above.

1. Special conditions for use statement(s):
  For in vitro diagnostic use.

For prescription use only.

1. This product is limited to delivering:
- . Sequencing output >1 Gb
- . Reads >3 million
- Read length (in paired end run) 2 x 150 bp
- . Bases higher than Q30 >75% (Greater than 75% of bases have Phred scale quality score greater than 30, indicating base call accuracy greater than 99.9% )
1. The system has been validated for the detection of SNVs and up to 3 base deletions. Evaluation of 1 base insertions was been limited to 3 different insertions on 3 separate chromosomes.
1. The system has problems detecting 1 base insertions or deletions in homopolymer tracts (e.g. polyA).
1. This MiSeqDx system is designed to deliver qualitative (i.e. genotype) results.
1. As with any hybridization-based workflow, underlying polymorphisms or mutations in oligonucleotide-binding regions can affect the alleles being probed and, consequently, the calls made.
1. Recommended minimal coverage per amplicon needed for accurate variant calling (Q(max_gt | poly_site) >= 100) is 75x.

4. Special instrument requirements:

For use with the Illumina MiSeqDx platform (K123989).

I. Device Description:

The MiSeqDx Universal Kit 1.0 is intended for use with the MiSeqDx Platform.

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The MiSeqDx Universal Kit 1.0 consists of 5 boxes :

Component	Active Ingredients
Hybridization Buffer	Buffered aqueous solution containing salts andformamide
Extension-Ligation Mix	Buffered aqueous solution containingproprietary blend of DNA polymerases, DNAligase, and dNTPs
Index Primers A (A501) - H (A508)	PCR primers with index sequences andsequencing adapters
Index Primers 1 (A701) - 12 (A712)	PCR primers with index sequences andsequencing adapters
PCR Polymerase	Proprietary DNA polymerase
PCR Master Mix	Buffered aqueous solution containing salts anddNTPs

Box 1A Pre-Amp reagents :

Box 1B Post-Amp Reagents

Component	Active Ingredients
Library NormalizationDiluent	Buffered aqueous solution containing salts, 2-Mercaptoethanol, and formamide
Library DilutionBuffer	Buffered aqueous solution
PhiX Internal Control	Buffered aqueous solution containing PhiX genomic DNA

Box 2 Post-Amp Reagents

Component	Contents
MiSeqDx	Single-use cartridge that contains cluster generation and
Reagent	sequencing reagents for use with the MiSeqDx, including
Cartridge	formamide, 2-Mercaptoethanol, and < 2% DMSO

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Component	Active Ingredients
Stringent WashBuffer	Buffered aqueous solution containing salts, 2-Mercaptoethanol and formamide
Universal WashBuffer	Buffered aqueous solution containing salts

Box 3A Pre-Amp Reagents

Box 3B Post-Amp Reagents

Component	Active Ingredients
PCR Clean-UpBeads	Buffered aqueous solution containing solid phaseparamagnetic beads and polyethylene glycol
LibraryNormalizationWash	Buffered aqueous solution containing salts, 2-Mercaptoethanol and formamide
Library Beads	Buffered aqueous solution containing solid phaseparamagnetic beads
MiSeqDx Flow Cell	Glass substrate with covalently boundoligonucleotides

Box 4 Post-Amp Reagents

Component	Active Ingredients
MiSeqDx SBS Solution (PR2)	Buffered aqueous solution

Box 5 Pre-Amp Reagents

Component	Active Ingredients
Filter Plate	Polypropylene microtiter plate with a modifiedpolyethersulfone membrane

Box 5 Post-Amp Reagents

Component	Active Ingredients
Elution Buffer	Buffered aqueous solution
Library Storage Buffer	Buffered aqueous solution

J. Substantial Equivalence Information:

1. Predicate Device Name(s):

No predicate device exists.

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2. Predicate 510(k) numbers:

Not applicable.

2. Comparison with Predicate Device:

Not applicable.

K. Special Control/Guidance Document Referenced (if applicable):

Not applicable.

L. Test Principle:

Library Preparation using the MiSeqDx Universal Kit 1.0 includes the following steps:

1. Hybridization—The first step, Hybridization, hybridizes a pool of upstream and downstream analyte specific reagents (ASRs) specific to the regions of interest to the sample genomic DNA. At the end of this process, a three-step wash procedure with a filter capable of size selection removes unbound oligonucleotides from the genomic DNA.
1. Extension-Ligation-The second step, Extension-Ligation, connects the hybridized upstream and downstream oligonucleotides. A DNA polymerase extends from the upstream oligonucleotides through the targeted region, followed by ligation to the 5' end of the downstream oligonucleotide using a DNA ligase. The result is the formation of products that contain the oligonucleotides specific to the regions of interest flanked by sequences required for amplification.
1. PCR Amplification-The third step, PCR Amplification, amplifies the extension-ligation products using primers that add index sequences for sample multiplexing, as well as flow cell capture sequences required for cluster generation on the MiSeqDx. At the end of this process, a PCR clean-up procedure purifies the PCR products (referred to as a library).
1. Library Normalization-The final step, Library Normalization, normalizes the quantity of each library to ensure more equal library representation in the final pooled library.

M. Performance Characteristics:

1. Analytical Performance:
- a. Precision/Reproducibility:

See K123989 for the studies used to establish the reproducibility of high throughput DNA targeted sequencing of human genomic DNA using the reagents found in the MiSeqDx Universal Kit 1.0 and the test system known as MiSeqDx Platform.

b. Linearity/assay reportable range:
Not applicable.

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Traceability, Stability, Expected values (controls, calibrators, or methods): C.
Controls: A PhiX internal control (i.e. genomic DNA from the bacteriophage ΦΧ174) provided as a kit component is added to each pooled library prior to placement on the instrument. Successful sequencing of the PhiX genome indicates that the sequencing chemistry worked as expected. It is also recommended that a negative control, or no template control, (not provided in the kit) in used with every run in order to detect the presence of contamination in the environment or run.

Stability: Kit components have specific storage conditions (-15 to -25°C, 2 to 8°C, or 15 to 30°C). The shelf life of all components will be 12 months when stored at the appropriate conditions. Real time stability testing of the kit components has been performed to support 6 months of shelf life stability; stability testing is ongoing to support a 12 month shelf life claim.

Stability testing protocols and acceptance criteria for stability testing have been reviewed and found to be acceptable.

See K123989 for the studies used to verify the storage conditions and handling of blood samples for high throughput DNA targeted sequencing of human genomic DNA using the reagents found in the MiSeqDx Universal Kit 1.0 and the test system known as MiSeqDx Platform.

d. Detection Limit:
See K123989 for the studies used to establish the minimum DNA input for high throughput DNA targeted sequencing of human genomic DNA using the reagents found in the MiSeqDx Universal Kit 1.0 and the test system known as MiSeqDx Platform.
e. Analytical specificity:
See K123989 for the studies used to evaluate the impact of potential interferents on high throughput DNA targeted sequencing of human genomic DNA using the reagents found in the MiSeqDx Universal Kit 1.0 and the test system known as MiSeqDx Platform.
f. Assay cut-off:
Not applicable.
1. Comparison studies:
- a. Method comparison with a predicate device:

Not applicable.

b. Matrix comparison:

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Not applicable. For use only with human genomic DNA obtained from peripheral whole blood.

1. Clinical studies:
- a. Clinical Sensitivity:

Not applicable.

b. Clinical Specificity:
Not applicable.
Other clinical supportive data (when a. and b. are not applicable): C.
See K123989 for the studies used to establish the accuracy of high throughput DNA targeted sequencing of human genomic DNA using the reagents found in the MiSeqDx Universal Kit 1.0 and the test system known as MiSeqDx Platform.
1. Clinical cut-off:
  Not applicable.
1. Expected values/Reference range:
  Not applicable.
1. Other Supportive Instrument Performance Data Not Covered Above:
  None.

K. Proposed Labeling:

Labeling satisfies the requirements of 21 CFR 809.10, 21 CFR 801.109, including an appropriate prescription statement as required by 21 CFR 801.109(b).

L. Risks to Health and Mitigation Measures:

Identified Potential Risk	Required Mitigation Measure
Inaccurate test results due to inconsistentlymanufactured test system reagents	General controls, including current goodmanufacturing practices

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M. Benefit/Risk Analysis:

Summary
Summary ofthe Benefit(s)	• Well-characterized and reliable reagents are essential to the use of the test system. The test system is a tool for clinical laboratories that can be used in sequencing assays to provide high throughput genomic sequencing of genomic regions of interest at greater sequencing depth than current sequencing technology.• No other devices are available for generation of a genomic library of a region of genomic DNA to use with a high throughput DNA sequence analyzer. There is an unmet medical and public health need for a device that generates genomic libraries of a region of genomic DNA to use with a high throughput genomic sequence analyzer.
Summary ofthe Risk(s)	Patients are subject to blood specimen collection, which is a standard procedure inclinical care and carries minimal risk.
	Risk is related to inaccurate test results as follows:
	False positive: The risks to the individual of a false positive result obtained fromthis test system (library generation using the subject device followed by sequenceanalysis using a high throughput DNA sequence analyzer) could includeunnecessary testing or treatment related to an inaccurate test result. Often, the resultfrom this test system would be used with results from other diagnostic tests andclinical signs and symptoms to identify the genetic cause or contribution for apatient's disease or condition.
	False negative: The risks to the individual of a false negative result due to aninaccurate test result obtained from this test system (library generation using thesubject device followed by sequence analysis using a high throughput DNAsequence analyzer) could delay further evaluation and appropriate therapy whichwill vary depending on the disease or condition.
	Public Health Risk from Incorrect Test Results:The consequences to public health for both false positive and false negative resultsare similar.
	This type of reagent is a component of the test that will ultimately be designed by alaboratory or other manufacturer. This type of reagent contributes only a smallportion of the risk to the overall risk of the test. Therefore if the reagents aredesigned and validated for use with the instrument, they may be considered lowrisk by themselves. Requiring that reagents are subject to general controls(including the manufacture of the reagents under current Good ManufacturingPractices) is sufficient to provide reasonable assurance of the device's safety andeffectiveness.
Summary ofOtherFactors	Not applicable

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Conclusions

Do the probable benefits outweigh the probable risks?

Given robust analytical performance characteristics and risk mitigation (i.e., general controls), the probable benefits to both the individual and public health outweigh the probable risks of this device.

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N. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class I under regulation 21 CFR 862.3800. FDA believes that general controls provide reasonable assurance of the safety and effectiveness of the device type. This device, and similar devices, is classified under the following:

Product Code:	PFT
Device Type:	Reagents for molecular diagnostic test systems
Class:	I (general controls)
Regulation:	21 CFR 862.3800

(a)Identification. Reagents for molecular diagnostic test systems are reagents other than analyte specific reagents used as part of molecular diagnostic test systems, such as polymerases, nucleotides and nucleotide mixes, master mixes in which individual reagents are optimized to be used together, and labeled nucleic acid molecules.

(b)Classification. Class I (general 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.

Regulation Number and Section

§ 862.3800 Reagents for molecular diagnostic instrument test systems.

(a)
Identification. Reagents for molecular diagnostic test systems are reagents other than analyte specific reagents used as part of molecular diagnostic test systems, such as polymerases, nucleotides and nucleotide mixes, master mixes in which individual reagents are optimized to be used together, and labeled nucleic acid molecules.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedure in subpart E of part 807 of this chapter, subject to the limitations in § 862.9.