ILLUMINA MISEQDX PLATFORM

• K Number: DEN130011
• Device Name: ILLUMINA MISEQDX PLATFORM
• Manufacturer: ILLUMINA, INC.
• Date Cleared: 2013-11-19 ( 57 days)
• Product Code: PFF
• Regulation Number: 862.2265

Intended Use

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.

Device Description

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.

Predicate Devices

K133136

Reference Devices

K133136

AI/ML (Artificial Intelligence / Machine Learning)

No
The document describes a DNA sequencing platform and its performance studies, focusing on accuracy and reproducibility of genetic variant detection. There is no mention of AI or ML in the intended use, device description, or performance study summaries. The data analysis software is mentioned but not described in a way that suggests AI/ML capabilities.

Therapeutic

No
The device is a sequencing instrument used for targeted sequencing of human genomic DNA. It is a diagnostic tool that measures fluorescence signals and analyzes data, rather than directly treating a disease or condition.

Diagnostic

Yes.

The "Intended Use / Indications for Use" section states that the "MiSeqDx Platform is intended for targeted sequencing of human genomic DNA...". Targeted sequencing of human genomic DNA for clinical use, as indicated in the "Device Description," is performed to diagnose, monitor, or assess a patient's health condition, thereby classifying it as a diagnostic device.

SaMD (Software as a Medical Device)

No

The device description explicitly states that the MiSeqDx Platform consists of both the MiSeqDx instrument (hardware) and data analysis software. It also mentions imaging hardware and instrument-specific reagents and flow cells. Therefore, it is not a software-only medical device.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

• Intended Use: The intended use explicitly states it is for "targeted sequencing of human genomic DNA from peripheral whole blood samples." This is a diagnostic purpose, analyzing biological samples to provide information about a patient's health.
• Device Description: It is described as a "high throughput DNA sequence analyzer for clinical use." The term "clinical use" strongly indicates a diagnostic application.
• Performance Studies: The document details extensive performance studies including Accuracy, Reproducibility, Carryover, Interfering Substances, DNA Extraction, DNA Input, Thermal Cycler, Sample Indexing, and Specimen Storage. These types of studies are standard requirements for demonstrating the analytical and clinical validity of an IVD.
• Clinical Laboratories: The intended user is specified as "Clinical laboratories," which are the typical settings for performing IVD tests.
• Analyte Specific Reagents (ASRs): While the end-user provides the ASRs, the platform itself is designed to be used with these reagents for diagnostic purposes.

All these factors align with the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

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.

Product codes

PFF

Device Description

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.

Mentions image processing

After the flow cell images are captured by the MiSeqDx instrument following each sequencing cycle, primary analysis is performed without user intervention.

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Accuracy Study 1: This accuracy study used a representative assay designed to query a variety of genes covering 24,434 bases across 19 different chromosomes, and containing potentially clinically relevant exons. The 13 unique samples used in this study are from two parents and 11 children that have been frequently sequenced by multiple laboratories and sequencing methodologies. There are six samples from females and seven from males. Accuracy was determined for single nucleotide variants (SNVs) by comparing the study data to well-characterized composite reference information. The reference database sequence was derived from the combination of multiple sequencing methodologies, publicly available data, and hereditary information. No repeat testing was done for this study.

Accuracy Study 2: The sequencing results for the amplicon panel above were compared to a highly confident genotype established for NA12878 by the National Institutes of Standards and Technology (NIST) (v.2.152). Out of the 195 amplicons, 184 amplicons lied within highly confident reference calls in the NIST sequence and were compared. Non-variant base calls were compared to human genome reference sequence build 19.

Accuracy Study 3: An additional accuracy study was performed to assess the performance of small insertions and deletions within a representative assay, the Illumina MiSeqDx Cystic Fibrosis 139 Variant Assay, that included a subset of CFTR clinically significant genetic variations analyzed with the MiSeq Reporter (MSR) software v2.2.29.2 using the MiSeqDx Platform targeted DNA sequencing workflow. The queried insertions and deletions were detected where expected with high confidence. These samples were characterized by bidirectional Sanger sequencing as a reference method to establish the expected sequence.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical Performance Studies

Study 1: Accuracy Study:

• Study Type: Accuracy
• Sample Size: 13 unique samples (run to total 15 samples due to 2 replicates) for a representative assay covering 24,434 bases across 19 different chromosomes.
• Key Results: Accuracy was determined for single nucleotide variants (SNVs).
  • One 1-base insertion was not called in a homopolymer run of 14 A's on chromosome 2.
  • One 1-base deletion was not called in a homopolymer run of 14 A's on chromosome 9.
  • For amplicon 9 (Poly A (14)), for 15 total samples analyzed, there were 9 incorrect calls and 99.54% correct calls.
  • For amplicon 95 (Poly A (14)), for 15 total samples analyzed, there were 15 incorrect calls and 99.23% correct calls.
  • All non-variant bases had 100% agreement with the reference sequence.
  • All SNVs had 100% agreement with the reference sequence.
  • Variants that were missed were either 1 base insertions or 1 base deletions in the homopolymer regions.

Study 2: Accuracy Study:

• Study Type: Accuracy
• Sample Size: NA12878 for 184 amplicons.
• Key Results:
  • PPA: 94.1%
  • NPA: 100%
  • One missed variant: a one base pair deletion in amplicon 9 in the homopolymer run of 14 A's that was present in the NIST sequence.

Study 3: Accuracy Study:

• Study Type: Accuracy for small insertions and deletions.
• Sample Size: Not explicitly stated, but based on the table, it involved multiple amplicon types.
• Key Results: Insertions and deletions were detected where expected with high confidence. All amplicon types shown in the table had 100% correct calls and 0 incorrect calls.
  • Accuracy confirmed for GC content > 19% (100% correct calls for 135/135 amplicons).
  • Accuracy confirmed for GC content G (HET)3), each with 2 miscalls across all sites combined.
  • Some "No Calls" observed in specific samples (e.g., Y122X/R1158X (HET)2 had 135 no calls at Site 2, F508del/2184delA (HET) had 1 no call at Site 2, F508del (HET)- had 132 no calls at Site 2).
  • Footnotes indicate reasons for some lower performance or discrepancies: Variant N1303K not called in one sample, 0% call rate in some replicates due to possible sample addition error, samples likely switched by operator, and specific variants (I506V, I507V, F508C, M1V) not present or not called.

Carryover Study:

• Study Type: Carryover evaluation (intra-run and inter-run)
• Sample Size: Intra-run: one 48-sample library with two samples (high and low concentration) in checkerboard pattern, plus 4 NTCs. Inter-run: Two libraries, each with 47 replicates of a single genomic DNA sample and one NTC.
• Key Results: Demonstrated minimal to no carryover. Study met sponsor's acceptance criteria.

Interfering Substances Study:

• Study Type: Interference evaluation
• Sample Size: 8 whole blood samples. 48 replicates for each interfering substance test.
• Key Results: No interference observed with tested concentrations of bilirubin, cholesterol, hemoglobin, triglyceride, and EDTA. 15% wash buffer also showed 100% correct calls after addition to purified genomic DNA.

DNA Extraction Study:

• Study Type: Evaluation of extraction methods
• Sample Size: 14 unique blood samples per method, tested by 2 operators, 3 runs per operator/method. Total for each method: 168 (14x2x3x2 replicates).
• Key Results: Alcohol Precipitation (Qiagen Gentra PureGene), Silica Filter Column Isolation (Qiagen Blood Mini), and Magnetic Bead extraction (Biomerieux Easy Mag) all showed 100% call rate, 100% accuracy, and 100% first pass rate.

DNA Input Study:

• Study Type: DNA input range evaluation
• Sample Size: 14 representative DNA samples for serial dilution (9 input levels from 1 ng to 1250 ng, tested in duplicate). Further testing with 4 samples (80 replicates) and 14 samples (280 replicates) for specific input levels.
• Key Results: 1250 ng and 25 ng identified as upper and lower bounds for DNA input with >=95% sample first pass rate and no incorrect calls. Accuracy and sample first pass rate were 100% at all tested DNA input levels. Two no calls observed at 25 ng DNA input, resulting in 99.26% sample call rates.

Thermal Cycler Study:

• Study Type: Evaluation of thermal cyclers
• Sample Size: Three unique sample sets processed through three different thermal cyclers across 3 days, each set in triplicate per day.
• Key Results: Demonstrated that any commercially available thermal cycler is adequate for library preparation.

Sample Indexing Study:

• Study Type: Evaluation of sample index primers
• Sample Size: 96 sample indexes tested with 8 unique DNA samples. Each sample tested with 12 combinations.
• Key Results: Reproducibility and accuracy were 100% for all sample/index primer combinations.

Specimen Storage and Freeze-Thaw Studies:

• Study Type: Evaluation of storage conditions and freeze-thaw on gDNA samples.
• Sample Size: Six K2EDTA anticoagulated blood samples divided into 6 aliquots for storage conditions. 15 DNA samples for freeze-thaw.
• Key Results: No miscalls or no calls observed, demonstrating that tested blood and gDNA storage conditions did not affect assay results.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Accuracy Study 1:

• Positive Percent Agreement (PPA): Ranging from 89.5% to 95.8% (for variants).
• Negative Percent Agreement (NPA): 100% (for non-variant bases).

Accuracy Study 2:

• Positive Percent Agreement (PPA): 94.1%
• Negative Percent Agreement (NPA): 100%

Reproducibility Study 2 (Cystic Fibrosis 139 Variant Assay):

• Sample Pass Rate: 99.9%
• Positive Agreement (%): Ranging from 94.44% to 100%.
• Negative Agreement (%): Ranging from 94.40% to 100%.
• Overall Agreement (%): Ranging from 94.44% to 100%.

Predicate Device(s)

Not applicable.

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 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.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR MISEQDX PLATFORM

DECISION SUMMARY INSTRUMENT ONLY TEMPLATE Correction Date: February 24, 2017

This Decision Summary contains corrections to the November 19, 2013 Decision Summary.

A. 510(k) Number:	K123989
B. Purpose for Submission:	De novo request for evaluation of automatic class III designation for the Illumina MiSeqDx Platform
C. Type of Test or Tests Performed:	High-throughput DNA sequencing
D. Applicant:	Illumina Inc.
E. Device Name:	MiSeqDx Platform

F. Regulatory Information:

FDA identifies this type of device as:

• 1. New regulation number: 21 CFR 862.2265
• 2. Classification: Class II.
• 3. Product code: PFF High throughput DNA sequence analyzer
• 4. Panel: Toxicology (91)

G. Intended Use:

• 1.Intended uses(s):
  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.

• 2. Indication for uses(s):
     Same as intended use above.

1

3. Special conditions for use statement(s):

• 1. This product is limited to delivering:
  • Sequencing output >1 Gb o
  • Reads >3 million o
  • o Read length (in paired end run) 2 x 150 bp
  • Bases higher than 030 >75% (Greater than 75% of bases have Phred scale . quality score greater than 30, indicating base call accuracy greater than 99.9%)
• 2. Variants in homopolymer runs exceeding eight bases will be filtered out in the VCF files (R8 filter).
• The system has been validated for the detection of SNVs and up to 3 base deletions. 3. Evaluation of 1 base insertions was been limited to 3 different insertions on 3 separate chromosomes.
• The system has problems detecting 1 base insertions or deletions in homopolymer 4. tracts (e.g., polyA).
• 5. This MiSeqDx system is designed to deliver qualitative (i.e. genotype) results.
• 6. 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.
• 7. Recommended minimal coverage per amplicon needed for accurate variant calling (Q(max gt | poly site) >= 100) is 75x.

H. System Descriptions:

1. Device Description:

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.

2. Principles of Operation:

Testing begins with genomic DNA extracted from a peripheral whole blood sample. The genomic DNA is processed through library preparation. which specifically amplifies the intended genomic regions of each sample while also adding the indexes and flow cell capture sequences to the amplified products. The resulting sample libraries are then transferred into a MiSeqDx reagent cartridge which contains all of the reagents required for cluster generation and sequencing (Sequencing By Synthesis - SBS). The MiSeqDx cartridge, MiSeqDx flow cell, and MiSeqDx SBS Solution (PR2 buffer) are then inserted

2

into the MiSeqDx instrument which performs cluster generation, sequencing and data analysis.

The instrument uses cluster generation on the flow cell surface followed by sequencing using the Sequencing by Synthesis (SBS) process.

After the flow cell images are captured by the MiSeqDx instrument following each sequencing cycle, primary analysis is performed without user intervention. Primary analysis is performed by the RTA (Real Time Analysis) software, and consists of base calling of each cluster at each cycle. In addition to calling the bases, RTA assigns an analytical quality score (O-score) to each base call. Calculations of O-scores are based on the ratio of the signal intensity of the highest base in a given cluster during a given cycle to the signal intensity of the three other bases. The quality score Q is calculated as -10 log10 P, where P is the probability that base call is incorrect.

Secondary analysis is performed by the MiSeqDx Reporter software. It also occurs without user intervention and consists of de-multiplexing and FASTO file generation. De-multiplexing is the process of using the index sequences to assign clusters to the sample from which they originated.

After base calling and de-multiplexing, the software generates FASTQ files that contain sequence and quality information. Due to the massively parallel nature of the SBS biochemistry, hundreds of independent sequencing reads, each with their own quality score, are generated for each amplicon in each sample. The FASTO file which is a widely accepted text based format for storing both a nucleotide sequence and its corresponding quality score. FASTQ files serve as input files for various sequence alignment and subsequent variant calling algorithms.

The MiSeqDx has a sequence alignment and variant calling program available for use.

• 3. Modes of Operation:
     The MiSeqDx is a high throughput nucleic acid analyzer.

4. Specimen Identification:

Samples up to 96 unique specimens can be analyzed. Eight unique index primer sequences (forward), named i5 primers, and 12 unique index primer (reverse) sequences, named i7 primers, are provided. These 8 unique forward index primers and 12 unique reverse index primers, when combined in a pair wise manner, produce 96 unique index combinations allowing for up to 96 samples to be processed in parallel during the library preparation process. These are added during the library preparation process. The sample sheet, a file that the user provides the software, contains the link between each of the sample names and their associated index sequences.

3

After completion of the sequence run, MiSeq Reporter software de-multiplexes the samples using the index sequences and creates FASTQ files as the data analysis output. The user can also utilize the MiSeq Reporter Software for sequence alignment and variant calling.

5. Specimen Sampling and Handling:

The MiSeqDx specimen is a pooled library (or libraries) derived from genomic DNA extracted from peripheral whole blood that then undergoes the following steps to create the pooled library: the genomic DNA is quantified and then used to make a library, the library sample is processed to remove remaining library preparation reagents (e.g. unused primers), normalized, and then pooled for input on the analyzer. Library normalization is used to ensure that each library is equally represented in the pooled sample.

At a minimum, eight samples must be present. If six unique samples (excluding the positive and negative controls) are not available, it is acceptable to fill the run with sample replicates or any human genomic DNA sample.

• 6. Calibration:
     There is no end-user calibration of the system. During installation of the platform, a company representative (Field Applications Scientist) begins a series of tests to validate the performance of the instrument subsystems, which include optical alignment, fluidic delivery, and thermal calibration, among others. In the case of a test failure, the MiSeqDx company representative uses a set of instrument-specific tools to adjust and/or repair the instrument to meet operational specifications. Re-calibration occurs during the preventive maintenance visit.
• 7. Quality Control:
     A PhiX internal control (i.e. genomic DNA from the bacteriophage ΦΧ174) 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. A negative control, or no template control, (not provided by the sponsor) should be included in every run in order to detect the presence of contamination in the environment or run.
• 8. Software:
     FDA has reviewed applicant's Hazard Analysis and Software Development processes for this line of product types:

Yes X

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H. Substantial Equivalence Information:

• 1. Predicate Device Name(s) and 510(k) numbers:
     Not applicable.
• 2. Comparison with Predicate Device:
     Not applicable.

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

Not applicable.

J. Performance Characteristics:

• 1. Analytical Performance:
  • a. Accuracy:

Three accuracy studies were conducted.

Study 1: This accuracy study used a representative assay designed to query a variety of genes covering 24,434 bases across 19 different chromosomes, and containing potentially clinically relevant exons. The 13 unique samples used in this study are from two parents and 11 children that have been frequently sequenced by multiple laboratories and sequencing methodologies. There are six samples from females and seven from males.

Accuracy was determined for single nucleotide variants (SNVs) by comparing the study data to well-characterized composite reference information. The reference database sequence was derived from the combination of multiple sequencing methodologies, publicly available data, and hereditary information. The following table to evaluate accuracy of the system was compiled based on data from the first run in the study. No repeat testing was done for this study.

| Amp-
licon | Chr. | Ana-
lyzed
frag-
ment
size1 | Amplicon
Genomic
Content | # of
unique
samples | total # of
samples
analyzed2 | #
calls/
sample
that
could
be
made3 | # of
no
calls4 | # of
correct
calls/
sample5 | # in-
correct
calls6 | %
correct
calls7 |
|---------------|------|-----------------------------------------|------------------------------------------------------------------------------------|---------------------------|------------------------------------|-------------------------------------------------------|----------------------|--------------------------------------|----------------------------|------------------------|
| 1 | 1 | 132 | Poly C (5),
63% GC | 13 | 15 | 132 | 0 | 132 | 0 | 100.00 |
| 2 | 1 | 128 | Poly T (5) | 13 | 15 | 128 | 0 | 128 | 0 | 100.00 |
| 3 | 2 | 133 | - | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| Amp-
licon | Chr. | Ana-
lyzed
frag-
ment
size¹ | Amplicon
Genomic
Content | # of
unique
samples | total # of
samples
analyzed² | #
calls/
sample
that
could
be
made³ | # of
no
calls⁴ | # of
correct
calls/
sample⁵ | # in-
correct
calls⁶ | %
correct
calls⁷ |
| 4 | 2 | 119 | - | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |
| 5 | 2 | 127 | Poly T (5) | 13 | 15 | 127 | 0 | 127 | 0 | 100.00 |
| 6 | 2 | 135 | Poly A (6) | 13 | 15 | 135 | 0 | 135 | 0 | 100.00 |
| 7 | 2 | 122 | Poly T (5),
Poly C (5) | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 8 | 2 | 110 | Poly T (5) | 13 | 15 | 110 | 0 | 110 | 0 | 100.00 |
| 9⁸ | 2 | 131 | Poly A (14) | 13 | 15 | 130-
131 | 0 | 130-
131 | 9 | 99.54 |
| 10 | 2 | 117 | - | 13 | 15 | 117 | 0 | 117 | 0 | 100.00 |
| 11 | 2 | 121 | - | 13 | 15 | 121 | 0 | 121 | 0 | 100.00 |
| 12 | 2 | 114 | - | 13 | 15 | 114 | 0 | 114 | 0 | 100.00 |
| 13 | 2 | 129 | Poly A (5) | 13 | 15 | 129 | 0 | 129 | 0 | 100.00 |
| 14 | 3 | 131 | Poly A (5),
Poly T (5) | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 15 | 3 | 130 | - | 13 | 15 | 130 | 0 | 130 | 0 | 100.00 |
| 16 | 3 | 130 | - | 13 | 15 | 130 | 0 | 130 | 0 | 100.00 |
| 17 | 3 | 117 | - | 13 | 15 | 117 | 0 | 117 | 0 | 100.00 |
| 18 | 3 | 136 | Poly T (5) | 13 | 15 | 136 | 0 | 136 | 0 | 100.00 |
| 19 | 3 | 131 | Poly T (5),
SNV | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 20 | 3 | 123 | Poly A (5) | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 21 | 3 | 117 | Poly A (6),
Poly T (5),
Homologous
region on a
different
chromosome | 13 | 15 | 117 | 0 | 117 | 0 | 100.00 |
| 22 | 3 | 119 | Homologous
region on a
different
chromosome | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |
| 23 | 3 | 120 | - | 13 | 15 | 120 | 0 | 120 | 0 | 100.00 |
| 24 | 3 | 129 | Poly T (5) | 13 | 15 | 129 | 0 | 129 | 0 | 100.00 |
| 25 | 4 | 133 | Poly C (7),
66% GC | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 26 | 4 | 135 | Poly C (5),
60% GC | 13 | 15 | 135 | 0 | 135 | 0 | 100.00 |
| Amp-
licon | Chr. | Analyzed
fragment
size1 | Amplicon
Genomic
Content | # of
unique
samples | total # of
samples
analyzed2 | # calls/
sample that
could be
made3 | # of
no
calls4 | # of
correct
calls/
sample5 | # in-
correct
calls6 | %
correct
calls7 |
| 27 | 4 | 123 | SNV | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 28 | 4 | 134 | - | 13 | 15 | 134 | 0 | 134 | 0 | 100.00 |
| 29 | 4 | 132 | - | 13 | 15 | 132 | 0 | 132 | 0 | 100.00 |
| 30 | 4 | 121 | Poly A (5),
SNV | 13 | 15 | 121 | 0 | 121 | 0 | 100.00 |
| 31 | 4 | 125 | - | 13 | 15 | 125 | 0 | 125 | 0 | 100.00 |
| 32 | 4 | 134 | Poly T (5) | 13 | 15 | 134 | 0 | 134 | 0 | 100.00 |
| 33 | 4 | 118 | - | 13 | 15 | 118 | 0 | 118 | 0 | 100.00 |
| 34 | 4 | 122 | Poly A (5) | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 35 | 4 | 131 | - | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 36 | 4 | 133 | - | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 37 | 4 | 128 | Poly T (6) | 13 | 15 | 128 | 0 | 128 | 0 | 100.00 |
| 38 | 4 | 131 | - | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 39 | 4 | 129 | Poly A (5),
Poly T (5),
SNV | 13 | 15 | 129 | 0 | 129 | 0 | 100.00 |
| 40 | 4 | 133 | Poly T (5),
SNV | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 41 | 4 | 112 | SNV | 13 | 15 | 112 | 0 | 112 | 0 | 100.00 |
| 42 | 4 | 133 | - | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 43 | 4 | 135 | - | 13 | 15 | 135 | 0 | 135 | 0 | 100.00 |
| 44 | 4 | 122 | - | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 45 | 4 | 117 | - | 13 | 15 | 117 | 0 | 117 | 0 | 100.00 |
| 46 | 4 | 124 | - | 13 | 15 | 125 | 0 | 125 | 0 | 100.00 |
| 47 | 4 | 117 | Poly T (5) | 13 | 15 | 117 | 0 | 117 | 0 | 100.00 |
| 48 | 4 | 128 | Poly A (7) | 13 | 15 | 128 | 0 | 128 | 0 | 100.00 |
| 49 | 4 | 123 | Poly A (6) | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 50 | 4 | 133 | - | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 51 | 4 | 112 | - | 13 | 15 | 112 | 0 | 112 | 0 | 100.00 |
| 52 | 4 | 129 | - | 13 | 15 | 129 | 0 | 129 | 0 | 100.00 |
| 53 | 4 | 126 | - | 13 | 15 | 126 | 0 | 126 | 0 | 100.00 |
| 54 | 4 | 132 | - | 13 | 15 | 132 | 0 | 132 | 0 | 100.00 |
| 55 | 5 | 131 | - | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 56 | 5 | 119 | - | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |
| 57 | 5 | 120 | Poly A (5) | 13 | 15 | 120 | 0 | 120 | 0 | 100.00 |
| 58 | 5 | 119 | - | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |
| Amp-
licon | Chr. | Ana-
lyzed
frag-
ment
size1 | Amplicon
Genomic
Content | # of
unique
samples | total # of
samples
analyzed2 | #
calls/
sample
that
could
be
made3 | # of
no
calls4 | # of
correct
calls/
sample5 | # in-
correct
calls6 | %
correct
calls7 |
| 59 | 5 | 118 | - | 13 | 15 | 118 | 0 | 118 | 0 | 100.00 |
| 60 | 5 | 112 | - | 13 | 15 | 112 | 0 | 112 | 0 | 100.00 |
| 61 | 5 | 120 | - | 13 | 15 | 120 | 0 | 120 | 0 | 100.00 |
| 62 | 5 | 120 | Poly A (5) | 13 | 15 | 120 | 0 | 120 | 0 | 100.00 |
| 63 | 5 | 115 | CT(5) | 13 | 15 | 115 | 0 | 115 | 0 | 100.00 |
| 64 | 5 | 112 | SNV | 13 | 15 | 112 | 0 | 112 | 0 | 100.00 |
| 65 | 5 | 135 | Poly T (6) | 13 | 15 | 135 | 0 | 135 | 0 | 100.00 |
| 66 | 5 | 131 | 63% GC | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 67 | 5 | 121 | - | 13 | 15 | 121 | 0 | 121 | 0 | 100.00 |
| 68 | 5 | 132 | Poly A (6),
Poly T (8) | 13 | 15 | 132 | 0 | 132 | 0 | 100.00 |
| 69 | 7 | 133 | - | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 70 | 7 | 120 | 60% GC | 13 | 15 | 120 | 0 | 120 | 0 | 100.00 |
| 71 | 7 | 135 | - | 13 | 15 | 135 | 0 | 135 | 0 | 100.00 |
| 72 | 7 | 126 | Poly A (5),
59% GC | 13 | 15 | 126 | 0 | 126 | 0 | 100.00 |
| 73 | 7 | 134 | - | 13 | 15 | 134 | 0 | 134 | 0 | 100.00 |
| 74 | 7 | 122 | Poly C (5),
63% GC | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 75 | 7 | 127 | 59% GC;
SNV | 13 | 15 | 127 | 0 | 127 | 0 | 100.00 |
| 76 | 7 | 123 | - | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 77 | 7 | 125 | - | 13 | 15 | 125 | 0 | 125 | 0 | 100.00 |
| 78 | 7 | 133 | Poly A (5),
Poly T (5) | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 79 | 7 | 116 | - | 13 | 15 | 116 | 0 | 116 | 0 | 100.00 |
| 80 | 7 | 135 | - | 13 | 15 | 135 | 0 | 135 | 0 | 100.00 |
| 81 | 7 | 118 | - | 13 | 15 | 118 | 0 | 118 | 0 | 100.00 |
| 82 | 7 | 136 | 67% GC | 13 | 15 | 136 | 0 | 136 | 0 | 100.00 |
| 83 | 7 | 131 | 58% GC | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 84 | 7 | 119 | Poly G (6),
61% GC | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |
| 85 | 7 | 122 | Poly T (5) | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 86 | 7 | 123 | Poly A (6) | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 87 | 8 | 127 | 60% GC | 13 | 15 | 127 | 0 | 127 | 0 | 100.00 |
| 88 | 8 | 129 | 57% GC | 13 | 15 | 129 | 0 | 129 | 0 | 100.00 |
| Amp-
licon | Chr. | Ana-
lyzed
frag-
ment
size¹ | Amplicon
Genomic
Content | # of
unique
samples | total # of
samples
analyzed² | #
calls/
sample
that
could
be
made³ | # of
no
calls4 | # of
correct
calls/
sample5 | # in-
correct
calls6 | %
correct
calls7 |
| 89 | 9 | 130 | Poly T (5) | 13 | 15 | 130 | 0 | 130 | 0 | 100.00 |
| 90 | 9 | 116 | - | 13 | 15 | 116 | 0 | 116 | 0 | 100.00 |
| 91 | 9 | 119 | Homologous
region on a
different
chromosome | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |
| 92 | 9 | 121 | - | 13 | 15 | 121 | 0 | 121 | 0 | 100.00 |
| 93 | 9 | 117 | Homologous
region on a
different
chromosome | 13 | 15 | 117 | 0 | 117 | 0 | 100.00 |
| 94 | 9 | 114 | - | 13 | 15 | 114 | 0 | 114 | 0 | 100.00 |
| 9510 | 9 | 129 | Poly A (14) | 13 | 15 | 130 | 0 | 129 (of
130) | 15 | 99.23 |
| 96 | 9 | 114 | Homologous
region on a
different
chromosome;
SNV | 13 | 15 | 114 | 0 | 114 | 0 | 100.00 |
| 97 | 9 | 122 | - | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 98 | 9 | 127 | Poly A (5),
Poly C (5) | 13 | 15 | 127 | 0 | 127 | 0 | 100.00 |
| 99 | 9 | 133 | - | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 100 | 9 | 138 | 64% GC | 13 | 15 | 138 | 0 | 138 | 0 | 100.00 |
| 101 | 9 | 139 | - | 13 | 15 | 139 | 0 | 139 | 0 | 100.00 |
| 102 | 9 | 116 | - | 13 | 15 | 116 | 0 | 116 | 0 | 100.00 |
| 103 | 9 | 133 | Poly A (5),
57% GC | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 104 | 9 | 138 | 57% GC | 13 | 15 | 138 | 0 | 138 | 0 | 100.00 |
| 105 | 9 | 136 | Poly C (5),
67% GC | 13 | 15 | 136 | 0 | 136 | 0 | 100.00 |
| 106 | 9 | 118 | 70% GC | 13 | 15 | 118 | 0 | 118 | 0 | 100.00 |
| 107 | 10 | 128 | 62% GC | 13 | 15 | 128 | 0 | 128 | 0 | 100.00 |
| 108 | 10 | 120 | 60% GC | 13 | 15 | 120 | 0 | 120 | 0 | 100.00 |
| 109 | 10 | 139 | 58% GC;
SNV | 13 | 15 | 139 | 0 | 139 | 0 | 100.00 |
| 110 | 10 | 118 | 57% GC | 13 | 15 | 118 | 0 | 118 | 0 | 100.00 |

5

6

7

8

9

| Amp-
licon | Chr. | Ana-
lyzed
frag-
ment
size' | Amplicon
Genomic
Content | # of
unique
samples | total # of
samples
analyzed2 | #
calls/
sample
that
could
be
made3 | # of
no
calls
4 | # of
correct
calls/
sample5 | # in-
correct
calls6 | %
correct
calls' |
|---------------|------|-----------------------------------------|--------------------------------------------------------------------------------|---------------------------|------------------------------------|-------------------------------------------------------|--------------------------|--------------------------------------|----------------------------|------------------------|
| 111 | 10 | 123 | Poly T (5) | 13 | ો ર | 123 | 0 | 123 | 0 | 100.00 |
| 112 | 10 | 121 | | 13 | ા ર | 121 | 0 | 121 | 0 | 100.00 |
| 113 | 10 | 129 | 26% GC | 13 | ો ર | 129 | 0 | 129 | 0 | 100.00 |
| 114 | 10 | 122 | | 13 | ા ર | 122 | 0 | 122 | 0 | 100.00 |
| ા । ર | 10 | 124 | Poly T (5);
Homologous
region on a
different
chromosome | 13 | ો ર | 124 | 0 | 124 | 0 | 100.00 |
| 116 | 10 | ા ૩૨ | CA(4) | 13 | ો ર | ા ૩૨ | 0 | ા રેર | 0 | 100.00 |
| 117 | 10 | ા ૩૨ | Poly A (6);
Homologous
region on a
different
chromosome | 13 | ાં ર | ા રેર | 0 | । ૩૨ | 0 | 100.00 |
| 118 | 10 | 119 | Poly C (5);
SNV | 13 | ા ર | 119 | 0 | 119 | 0 | 100.00 |
| 119 | 10 | ા ટેર | - | 13 | ો ર | ા ટેર | 0 | ા ટેર | 0 | 100.00 |
| 120 | 10 | 131 | - | 13 | ા ર | 131 | 0 | 131 | 0 | 100.00 |
| 121 | 10 | 117 | - | 13 | ો ર | 117 | 0 | 117 | 0 | 100.00 |
| 122 | 10 | 116 | - | 13 | ા ર | 116 | 0 | 116 | 0 | 100.00 |
| 123 | 10 | 129 | 58% GC | 13 | ો ર | 129 | 0 | 129 | 0 | 100.00 |
| 124 | 11 | 117 | Poly T (10) | 13 | ા ર | 117 | 0 | 117 | 0 | 100.00 |
| । ટેર | 11 | 117 | Poly T (5) | 13 | ો ર | 117 | 0 | 117 | 0 | 100.00 |
| 126 | 11 | 113 | Poly A (5) | 13 | ા ર | 113 | 0 | 113 | 0 | 100.00 |
| 127 | 11 | 129 | - | 13 | ા ર | 129 | 0 | 129 | 0 | 100.00 |
| 128 | 11 | 121 | Poly T (5) | 13 | ો ર | 121 | 0 | 121 | 0 | 100.00 |
| 129 | 11 | 123 | - | 13 | ો ર | 123 | 0 | 123 | 0 | 100.00 |
| 130 | 11 | 127 | Poly A (6) | 13 | ા ર | 127 | 0 | 127 | 0 | 100.00 |
| 131 | 11 | 136 | Poly T (6) | 13 | ા ર | 136 | 0 | 136 | 0 | 100.00 |
| 132 | 11 | 132 | Poly T (5) | 13 | ો ર | 132 | 0 | 132 | 0 | 100.00 |
| 133 | 11 | ા । ર | | 13 | ો ર | ા । ર | 0 | ા । ર | 0 | 100.00 |
| 134 | 11 | 117 | Poly T (8);
19% GC | 13 | ા ર | 117 | 0 | 117 | 0 | 100.00 |
| ા રેર | 11 | 134 | Poly A (5);
Poly T (5) | 13 | ા ર | 134 | 0 | 134 | 0 | 100.00 |
| 136 | 11 | 131 | Poly A (5) | । ਤੇ | ા ર | 131 | 0 | 131 | 0 | 100.00 |
| Amp-
licon | Chr. | Ana-
lyzed
frag-
ment
size¹ | Amplicon
Genomic
Content | # of
unique
samples | total # of
samples
analyzed² | #
calls/
sample
that
could
be
made³ | # of
no
calls
4 | # of
correct
calls/
sample5 | # in-
correct
calls6 | %
correct
calls7 |
| 137 | 11 | 133 | 26% GC;
SNV | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 138 | 11 | 137 | Poly T (8);
SNV | 13 | 15 | 137 | 0 | 137 | 0 | 100.00 |
| 139 | 11 | 131 | Poly A (5) | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 140 | 12 | 131 | - | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 141 | 12 | 128 | - | 13 | 15 | 128 | 0 | 128 | 0 | 100.00 |
| 142 | 12 | 133 | Poly A (5) | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 143 | 12 | 136 | - | 13 | 15 | 136 | 0 | 136 | 0 | 100.00 |
| 144 | 12 | 124 | - | 13 | 15 | 124 | 0 | 124 | 0 | 100.00 |
| 145 | 12 | 122 | 59% GC | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 146 | 13 | 122 | - | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 147 | 13 | 116 | Poly C (5) | 13 | 15 | 116 | 0 | 116 | 0 | 100.00 |
| 148 | 13 | 133 | - | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 149 | 13 | 117 | SNV | 13 | 15 | 117 | 0 | 117 | 0 | 100.00 |
| 150 | 13 | 124 | Poly T (6) | 13 | 15 | 124 | 0 | 124 | 0 | 100.00 |
| 151 | 13 | 123 | Poly T (5);
26% GC | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 152 | 13 | 115 | Poly A (5) | 13 | 15 | 115 | 0 | 115 | 0 | 100.00 |
| 153 | 13 | 125 | - | 13 | 15 | 125 | 0 | 125 | 0 | 100.00 |
| 154 | 13 | 121 | - | 13 | 15 | 121 | 0 | 121 | 0 | 100.00 |
| 155 | 13 | 123 | - | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 156 | 13 | 114 | - | 13 | 15 | 114 | 0 | 114 | 0 | 100.00 |
| 157 | 13 | 119 | - | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |
| 158 | 14 | 122 | 58% GC | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 159 | 16 | 122 | - | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 160 | 16 | 121 | - | 13 | 15 | 121 | 0 | 121 | 0 | 100.00 |
| 161 | 16 | 123 | Poly C (5) | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 162 | 17 | 119 | - | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |
| 163 | 17 | 119 | 61% GC | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |
| 164 | 17 | 135 | - | 13 | 15 | 135 | 0 | 135 | 0 | 100.00 |
| 165 | 17 | 116 | Poly C (6);
60% GC;
SNV | 13 | 15 | 116 | 0 | 116 | 0 | 100.00 |
| 166 | 17 | 123 | - | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 167 | 17 | 116 | 62% GC | 13 | 15 | 116 | 0 | 116 | 0 | 100.00 |
| Amp-
licon | Chr. | Ana-
lyzed
frag-
ment
size¹ | Amplicon
Genomic
Content | # of
unique
samples | total # of
samples
analyzed² | #
calls/
sample
that
could
be
made³ | # of
no
calls
4 | # of
correct
calls/
sample5 | # in-
correct
calls6 | %
correct
calls7 |
| 168 | 17 | 118 | Poly C (5);
65% GC | 13 | 15 | 118 | 0 | 118 | 0 | 100.00 |
| 169 | 17 | 129 | - | 13 | 15 | 129 | 0 | 129 | 0 | 100.00 |
| 170 | 17 | 131 | Poly G (6);
67% GC;
SNV | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 171 | 17 | 127 | 61% GC | 13 | 15 | 127 | 0 | 127 | 0 | 100.00 |
| 172 | 17 | 118 | Poly C (5) | 13 | 15 | 118 | 0 | 118 | 0 | 100.00 |
| 173 | 17 | 138 | 61% GC | 13 | 15 | 138 | 0 | 138 | 0 | 100.00 |
| 174 | 17 | 131 | 58% GC | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 175 | 18 | 112 | - | 13 | 15 | 112 | 0 | 112 | 0 | 100.00 |
| 176 | 18 | 124 | - | 13 | 15 | 124 | 0 | 124 | 0 | 100.00 |
| 177 | 18 | 134 | Poly A (6) | 13 | 15 | 134 | 0 | 134 | 0 | 100.00 |
| 178 | 18 | 129 | - | 13 | 15 | 129 | 0 | 129 | 0 | 100.00 |
| 179 | 18 | 133 | - | 13 | 15 | 133 | 0 | 133 | 0 | 100.00 |
| 180 | 18 | 118 | - | 13 | 15 | 118 | 0 | 118 | 0 | 100.00 |
| 181 | 18 | 114 | 60% GC | 13 | 15 | 114 | 0 | 114 | 0 | 100.00 |
| 182 | 18 | 118 | - | 13 | 15 | 118 | 0 | 118 | 0 | 100.00 |
| 183 | 19 | 122 | Poly G (6);
66% GC | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 184 | 19 | 139 | 64% GC | 13 | 15 | 139 | 0 | 139 | 0 | 100.00 |
| 185 | 19 | 131 | 67% GC | 13 | 15 | 131 | 0 | 131 | 0 | 100.00 |
| 186 | 19 | 141 | 59% GC;
Homologous
region on a
different
chromosome | 13 | 15 | 141 | 0 | 141 | 0 | 100.00 |
| 187 | 19 | 121 | Poly C (5);
72% GC;
Homologous
region on a
different
chromosome | 13 | 15 | 121 | 0 | 121 | 0 | 100.00 |
| 188 | 19 | 138 | 58% GC | 13 | 15 | 138 | 0 | 138 | 0 | 100.00 |
| 189 | 19 | 123 | 64% GC | 13 | 15 | 123 | 0 | 123 | 0 | 100.00 |
| 190 | 19 | 138 | - | 13 | 15 | 138 | 0 | 138 | 0 | 100.00 |
| 191 | 20 | 117 | Poly T (5) | 13 | 15 | 117 | 0 | 117 | 0 | 100.00 |
| Amp-
licon | Chr. | Ana-
lyzed
frag-
ment
size1 | Amplicon
Genomic
Content | # of
unique
samples | total # of
samples
analyzed2 | #
calls/
sample
that
could
be
made3 | # of
no
calls4 | # of
correct
calls/
sample5 | # in-
correct
calls6 | %
correct
calls7 |
| 192 | 22 | 136 | Poly A (7) | 13 | 15 | 136 | 0 | 136 | 0 | 100.00 |
| 193 | 22 | 122 | Poly A (5);
Poly C (5) | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 194 | 22 | 122 | 62% GC;
SNV | 13 | 15 | 122 | 0 | 122 | 0 | 100.00 |
| 195 | 22 | 119 | 66% GC | 13 | 15 | 119 | 0 | 119 | 0 | 100.00 |

10

11

12

1. Analyzed fragment means the size of the sequenced genomic region in bases, not including target-specific primers.

2 Total # of samples analyzed is 15 because two of the 13 unique samples were run in two independent replicates.

3 # calls/sample that could be made is the number of bases that had adequate quality to be called by the system

4 # of no calls is the number of bases in an amplicon that results in a no call in the run

5 # correct calls per sample is number of bases in the amplicon that were called that had results that matched the human genome reference sequence build 19+ and the well characterized composite reference.

6 # incorrect calls were the total number of incorrect calls for the SNV or indel in that amplicon; addition details on incorrect calls are presented in footnotes below.

7 % correct calls equals the correct call rate for all of the bases in the amplicon, where the correct call for the SNV or indel is based on the well characterized composite reference information and the correct call for the bases in the remainder of the amplicon sequence is based on comparison to human genome reference sequence build 19. This column may have more than one expected result for a given amplicon if some samples contain an indel while some do not, e.g., amplicon 9.

8 Amplicon 9 has a homopolymer run of 14 A's according to the human genome reference sequence build 19. However, the well characterized composite reference information for 7 out of 13 samples have 13 A 's in this homopolymer run. In these 7 samples, this one base pair deletion represents a false negative in the MiSeqDx sequencing accuracy study.

¹ Human Feb. 2009 (GRCh37/hg19) assembly available from NCBI

13

9 Amplicon 46 has a one base insertion which is reported in 9 samples in the well characterized reference database and is correctly detected in all analyzed samples.

10 Amplicon 95 has a homopolymer run of 14 A's according to human genome reference sequence build 19. However, the well characterized composite reference sequences for 13 out of 13 samples have 15 A 's in this homopolymer run. In these 13 samples, this one base pair insertion is a false negative in the MiSeqDx sequencing accuracy study.

The following table contains data from study 1 presented with positive and negative percent agreement (PPA and NPA, respectively), where the variant results are compared to the well characterized composite reference information for PPA calculations. Since the composite reference information only provides results for the single nucleotide variants and insertions/deletions. non-variant base results are compared to human genome reference sequence build 19 for NPA calculations. All non-variant bases had 100% agreement with the reference sequence. All SNVs had 100% agreement with the reference sequence. Variants that were missed were either 1 base insertions or 1 base deletions in the homopolymer regions.

| Sample | #
amplicons | %
Amplicon1
Coverage1 | Variants
expected
per
sample2 | Variants
Correctly
Called | Variants
Missed3 | Non-variant
bases called
correctly | PPA4 | NPA5 |
|---------|----------------|-----------------------------|----------------------------------------|---------------------------------|---------------------|------------------------------------------|------|------|
| NA12877 | 195 | 100 | 19 | 17 | 2 | 24418 | 89.5 | 100 |
| NA12878 | 195 | 100 | 19 | 17 | 2 | 24417 | 89.5 | 100 |
| NA12879 | 195 | 100 | 20 | 19 | 1 | 24416 | 95 | 100 |
| NA12880 | 195 | 100 | 20 | 18 | 2 | 24417 | 90 | 100 |
| NA12881 | 195 | 100 | 22 | 20 | 2 | 24415 | 90.9 | 100 |
| NA12882 | 195 | 100 | 16 | 15 | 1 | 24419 | 93.8 | 100 |
| NA12883 | 195 | 100 | 24 | 23 | 1 | 24412 | 95.8 | 100 |
| NA12884 | 195 | 100 | 21 | 20 | 1 | 24415 | 95.2 | 100 |
| NA12885 | 195 | 100 | 19 | 17 | 2 | 24417 | 89.5 | 100 |
| NA12886 | 195 | 100 | 22 | 20 | 2 | 24415 | 90.9 | 100 |
| NA12887 | 195 | 100 | 19 | 18 | 1 | 24416 | 94.7 | 100 |
| NA12888 | 195 | 100 | 24 | 23 | 1 | 24412 | 95.8 | 100 |
| NA12893 | 195 | 100 | 20 | 18 | 2 | 24417 | 90 | 100 |

1 % Amplicon coverage is number of bases in the amplicons sequenced with confidence

2 Variants expected per sample includes both SNVs and indels

3 For the variants missed, please see the first table for study 1 and the footnotes 8-10.

4 Positive percent agreement (PPA) = 100xTP/(TP+FN) where the true positives (TP) are the number of positive variant calls at genomic coordinates where variants are

14

present according to the reference sequence and mutant allele called is concordant with reference sequence (column named "Variants called correctly") and the false negatives (FN) are the number of negative variant calls at genomic coordinates where variants are present according to the reference sequence (column named "Variants missed).

5 Negative percent agreement (NPA) = 100xTN/(FP+TN) where the false positives (FP) are the number of positive variant calls at genomic coordinates where variants are absent according to the reference sequence, or if mutant allele called is discordant with reference sequence (not in the table; no false positive variants calls were made in this study) and true negatives (TN) are the number of negative variant calls at genomic coordinates where variants are absent according to the reference standard (column named "non-variant bases called correctly").

Study 2: The sequencing results for the amplicon panel above were compared to a highly confident genotype established for NA12878 by the National Institutes of Standards and Technology (NIST) (v.2.152). Out of the 195 amplicons, 184 amplicons lied within highly confident reference calls in the NIST sequence and were compared. Non-variant base calls were compared to human genome reference sequence build 19.

| Sample | #
Amplicons | %
Amplicon1
Coverage | Variants
expected | Variants
Correctly
Called | Variants
Missed | Non-variant
bases called
correctly | PPA2
(%) | NPA3
(%) |
|---------|----------------|----------------------------|----------------------|---------------------------------|--------------------|------------------------------------------|-------------|-------------|
| NA12878 | 184 | 100 | 17 | 16 | 14 | 23066 | 94.1 | 100 |

1 % Amplicon coverage is number of bases in the amplicons sequenced with confidence

2 Positive percent agreement (PPA) = 100xTP/(TP+FN)

3 Negative percent agreement (NPA) = 100xTN/(FP+TN)

4 The missed variant is the one base pair deletion in amplicon 9 in the homopolymer run of 14 A's not called by the MiSeqDx that is present in the NIST sequence. Note that the NIST sequence does not include the one base pair insertion in the other homopolymer of A's that was present in the other reference database used above in study 1.

2 Zook, JM et al. Integrating sequencing datasets to form highly confident SNP and indel genotype calls for a whole human genome. arXiv:1307.4661 [q-bio.GN]

15

Study 3: An additional accuracy study was performed to assess the performance of small insertions and deletions within a representative assay, the Illumina MiSeqDx Cystic Fibrosis 139 Variant Assay, that included a subset of CFTR clinically significant genetic variations analyzed with the MiSeq Reporter (MSR) software v2.2.29.2 using the MiSeqDx Platform targeted DNA sequencing workflow. The queried insertions and deletions were detected where expected with high confidence. These samples were characterized by bidirectional Sanger sequencing as a reference method to establish the expected sequence.

Amplicon	Analyzed fragment size1	Amplicon Genomic Content	# calls/ sample that could be made	# of bases called/ sample	# of no calls	# of correct calls/ sample	# of incorrect calls	% correct calls
1	129	1 base insertion	130	130	0	130	0	100.00
2	154	3 base deletion	151	151	0	151	0	100.00
3	167	2 base deletion	165	165	0	165	0	100.00
4	134	1 base deletion	133	133	0	133	0	100.00
5	132	1 base deletion	131	131	0	131	0	100.00
6	129	1 base deletion	128	128	0	128	0	100.00

The data provided by these three accuracy studies supports the claim that the MiSeqDx Instrument can accurately sequence:

• GC content > 19% (all bases in 135 out of 135 sequenced amplicons with 19% GC 0 content called correctly)
• o GC content Study2: A reproducibility study performed with a representative assay, the Illumina MiSeqDx Cystic Fibrosis 139 Variant Assay, included a subset of CFTR clinically significant genetic variations analyzed with the MiSeq Reporter (MSR) software v2.2.29.2 using the MiSeqDx Platform targeted DNA sequencing workflow. The blinded study used 3 trial sites and 2 operators at each site. Two well-characterized panels of 46 samples each were tested by each of the operators at each site for a total

26

of 810 calls per site. The panels contained a mix of genomic DNA from cell lines with known variants in the CFTR gene, as well as leukocyte-depleted blood spiked with cell lines with known variants in the CFTR gene. The blood samples were provided to allow incorporation of the extraction steps used to prepare gDNA that serves as the primary input for the assay workflow. The sample pass rate, defined as the number of samples passing QC metrics on the first attempt, was 99.9%. All test results are based on initial testing. No repeat testing was done for the reproducibility study.

Sample Genotype	Total calls per site	Positive Agreeing Calls (Variants) Site 1	Positive Agreeing Calls (Variants) Site 2	Positive Agreeing Calls (Variants) Site 3	Negative Agreeing Calls (Wild type) Site 1	Negative Agreeing Calls (Wild type) Site 2	Negative Agreeing Calls (Wild type) Site 3	# Miscalls	# No Calls	Positive Agreement (%)	Negative Agreement (%)	Overall Agreement (%)
S549N (HET)	810	6	6	6	804	804	804	0	0	100	100	100
1812-1 G->A (HET)	810	6	6	6	804	804	804	0	0	100	100	100
Q493X/F508del (HET)	810	12	12	12	798	798	798	0	0	100	100	100
F508del/2184delA (HET)	810	12	12	12	797	798	798	0	1	100	100	100
Y122X/R1158X (HET)2	810	12	11	12	798	664	798	0	135	97.22	94.40	94.44
F508del/2183AA>G	810	12	12	12	798	798	798	0	0	100	100	100
R75X (HET)	810	6	6	6	804	804	804	0	0	100	100	100
I507del/F508del (HET)	810	12	12	12	798	798	798	0	0	100	100	100
F508del/W1282X (HET)3	810	12	11	12	798	797	798	2	0	97.22	99.96	99.9
F508del/3272-26A>G (HET)3	810	12	11	12	798	797	798	2	0	97.22	99.96	99.9
F508del/3849+10kbC>T (HET)	810	12	12	12	798	798	798	0	0	100	100	100
621+1G>T/3120+1G>A (HET)	810	12	12	12	798	798	798	0	0	100	100	100
E60X/F508del (HET)	810	12	12	12	798	798	798	0	0	100	100	100
M1101K (HET)	810	6	6	6	804	804	804	0	0	100	100	100
M1101K (HOM)	810	6	6	6	804	804	804	0	0	100	100	100
F508del (HOM)4	828	6	6	6	822	822	822	0	0	100	100	100
F508del/3659delC (HET)	810	12	12	12	798	798	798	0	0	100	100	100
	Total
calls		Positive Agreeing
Calls (Variants)		Negative Agreeing
Calls (Wild type)		#		Positive	Negative	Overall
Sample Genotype	per
site	Site
l	Site
2	Site
3	Site
l	Site
2	Site
3	Miscall
S	# No
Calls	t (%)	Agreemen Agreement Agreement
(%)	(%)
R117H/F508del (HET) ³	816	18	18	18	798	798	798	0	0	100	100	100
621+1G>T/711+1G>T
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
G85E/621+1G>T (HET)	810	12	12	12	798	798	798	0	0	100	100	100
A455E/F508del (HET)	810	12	12	12	798	798	798	0	0	100	100	100
F508del/R560T (HET)	810	12	12	12	798	798	798	0	0	100	100	100
F508del/Y1092X (C>A)
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
N1303K (HET)	810	6	6	6	804	804	804	0	0	100	100	100
G542X (HOM)	810	6	6	6	804	804	804	0	0	100	100	100
G542X (HET)	810	6	6	6	804	804	804	0	0	100	100	100
G551D/R553X (HET)	810	12	12	12	798	798	798	0	0	100	100	100
3849+10kbC>T (HOM)	810	6	6	6	804	804	804	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
F508del (HET)	810	6	6	6	804	804	804	0	0	100	100	100
1717-1G>A (HET)	810	6	6	6	804	804	804	0	0	100	100	100
R1162X (HET)	810	6	6	6	804	804	804	0	0	100	100	100
R347P/G551D (HET)	810	12	12	12	798	798	798	0	0	100	100	100
R334W (HET)	810	6	6	6	804	804	804	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	1 00
G85E (HET)	810	6	6	6	804	804	804	0	0	100	100	100
I336K (HET)	810	6	6	6	804	804	804	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
F508de1/3849+10kbC>T
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
621+1G>T/3120+1G>A
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
F508del/3659de1C
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
R117H/F508del (HET)S	816	18	18	18	798	798	798	0	0	100	100	100
G85E/621+1G>T (HET)	810	12	12	12	798	798	798	0	0	100	100	100
A455E/F508del (HET)	810	12	12	12	798	798	798	0	0	100	100	100
	Total
calls	Positive Agreeing
Calls (Variants)			Negative Agreeing
Calls (Wild type)			#		Positive	Negative	Overall
Sample Genotype	per	Site
1	Site
2	Site
3	Site
1	Site
2	Site
3	Miscall	# No
Calls	Agreement
(%)	Agreement
(%)	Agreement
(%)
	site							s
N1303K (HET)	810	6	6	6	804	804	804	0	0	100	100	100
G551D/R553X (HET)	810	12	12	12	798	798	798	0	0	100	100	100
2789+5G>A (HOM)	810	6	6	6	804	804	804	0	0	100	100	100
F508del/1898+1G>A
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
F508del/2143delT
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
3876delA (HET)	810	6	6	6	804	804	804	0	0	100	100	100
3905insT (HET)	810	6	6	6	804	804	804	0	0	100	100	100
394delTT (HET)	810	6	6	6	804	804	804	0	0	100	100	100
F508del (HET)	810	6	6	6	804	804	804	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
F508del (HET)	810	6	6	6	804	804	804	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
L206W (HET)	810	6	6	6	804	804	804	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
G330X (HET)	810	6	6	6	804	804	804	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
R347H (HET)	810	6	6	6	804	804	804	0	0	100	100	100
1078delT (HET)	810	6	6	6	804	804	804	0	0	100	100	100
G178R/F508del (HET)	810	12	12	12	798	798	798	0	0	100	100	100
S549R (c.1647T>G)
(HET)	810	6	6	6	804	804	804	0	0	100	100	100
S549N (HET)	810	6	6	6	804	804	804	0	0	100	100	100
W846X (HET)	810	6	6	6	804	804	804	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
E92X/F508del (HET)	810	12	12	12	798	798	798	0	0	100	100	100
621+1G>T/1154insTC
(HET)	810	12	12	12	798	798	797	0	1	100	99.96	99.96
G542X (HET)	810	6	6	6	804	804	804	0	0	100	100	100
Sample Genotype	Total
calls
per
site	Site
1	Positive Agreeing
Calls (Variants)
Site
2	Site
3	Site
l	Negative Agreeing
Calls (Wild type)
Site
2	Site
3	#
Miscall
ഗ	# No
Calls	Positive
t (%)	Negative
(%)	Overall
Agreemen Agreement Agreement
(%)
F508del (HET)	810	6	6	6	804	804	804	0	0	100	100	100
F508del (HET)-	810	6	5	6	804	670	804	0	ા ૩૨	94.44	94.44	94.44
F508del (HET)	810	6	6	6	804	804	804	0	0	100	100	100
621+1G>T/A455E
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
1812-1 G->A (HET)	810	6	6	6	804	804	804	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
F508del/R553X (HET)	810	12	12	12	798	798	798	0	0	100	100	100
F508del/G551D (HET)	810	12	12	12	798	798	798	0	0	100	100	100
R347P/F508del (HET)	810	12	12	12	798	798	798	0	0	100	1 00	100
R117H/F508del (HET)	816	18	18	18	798	798	798	0	0	100	100	100
I507del (HET)	810	6	6	6	804	804	804	0	0	100	100	100
2789+5G>A (HOM)	810	6	6	6	804	804	804	0	0	100	100	100
F508del/1898+1G>A
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
WT	810	0	0	0	810	810	810	0	0	N/A	100	100
F508del/2143delT
(HET)	810	12	12	12	798	798	798	0	0	100	100	100
3905insT (HET)	810	6	6	6	804	804	804	0	0	100	100	100
394delTT (HET)	810	6	6	6	804	804	804	0	0	100	100	100
F508del (HET)	810	6	6	6	804	804	804	0	0	100	100	100

27

28

29

¹ Variant N1303K was not called in this sample

2 One replicate of samples 5 and 75 had a 0% call rate. Further investigation indicates that samples may not have been added to the sample plate prior to library preparation

3 Evidence indicates that samples 9 and 10 were likely switched by the operator prior to library preparation.

4 I506V, I507V, F508C not present in sample

5 Sample also has the (TG)10 (T)9/(TG) 12(T)5 variant

6 Variant M1V was not called in these samples

c. Linearity:

30

Not applicable.

d. Carryover:

A study was performed to evaluate the potential for inter-run and intra-run sample carryover. Intra-run sample carryover tested the system in the most challenging scenario for sample carryover within a single sequencing run. One 48-sample library composed of two samples with unique variants was setup in a checkerboard matrix pattern at alternating high (500 ng) and low (100 ng) concentrations, along with 4 NTC's (no template control samples).

Inter-run sample carryover tested the system for sample carryover between successive sequencing runs. Two libraries were prepared; each library was composed of 47 replicates of a single genomic DNA sample and one NTC. Each library used a different sample from the other.

For both inter- and intra-run, sample carryover was determined by measuring the error rate at the position of variant calls for all samples used in the study. Acceptance criteria for this study were reviewed and deemed acceptable. This study met the sponsor's acceptance criteria and demonstrated that there is minimal to no carryover on the MiSeqDx.

e. Interfering Substances:

To assess the impact of interfering substances on the MiSeqDx Platform, a representative assay (Assay 2) was evaluated in the presence and absence of potential interferents. Eight whole blood samples representing eight unique genotypes were utilized in the study. Four endogenous interfering substances (bilirubin, cholesterol, hemoglobin, and triglycerides) were tested by spiking them into the blood specimens prior to DNA extraction. To assess interference resulting from blood collection (short draw), potassium EDTA was spiked into blood samples at two concentrations. The concentration limits for each substance is shown in the following table. Additionally, to assess interference resulting from sample preparation, 15% wash buffer was added to 8 purified genomic DNA with 100% correct calls.

31

| Test
Substance | Total Number
of Replicates | Concentration
Tested in Blood
(Upper Limit) | Concentration
Tested in Blood
(Lower Limit) | Call
Rate |
|-------------------|-------------------------------|---------------------------------------------------|---------------------------------------------------|--------------|
| Bilirubin | 48 | 684 µmol/L | 137 µmol/L | 100% |
| Cholesterol | 48 | 13 mmol/L | 2.6 mmol/L | 100% |
| Hemoglobin | 48 | 2 g/L | 0.4 g/L | 100% |
| Triglyceride | 48 | 37 mmol/L | 7.4 mmol/L | 100% |
| EDTA | 48 | 7.0 mg/mL | 2.8 mg/mL | 100% |

2. Other Supportive Instrument Performance Data Not Covered Above:

DNA extraction study: Three different extraction methods, magnetic bead extraction, alcohol precipitation and silica filter column isolation were evaluated using K2EDTA anticoagulated whole blood. Fourteen unique blood samples were used in the study representing a range of genotypes from one representative gene. The three DNA extraction methods were tested independently by 2 different operators who each performed 3 runs per extraction method. Each extraction was performed by each operator on different days. The DNA concentration and A260/A280 ratio of the extracted gDNA samples was determined using spectrophotometry. The total sample size for each extraction method in this study was 168 (14 samples x 2 operators/extraction method x 3 runs/operator x 2 replicates/extracted gDNA sample).

| Extraction Method | Number of
samples tested | Call Rate | Accuracy | Sample First
Pass Rate* |
|----------------------------------------------------------|-----------------------------|-----------|----------|----------------------------|
| Alcohol Precipitation
(Qiagen Gentra PureGene) | 168 | 100% | 100% | 100% |
| Silica Filter Column
Isolation (Qiagen Blood
Mini) | 168 | 100% | 100% | 100% |
| Magnetic Bead extraction
(Biomerieux Easy Mag) | 168 | 100% | 100% | 100% |

32

DNA input study: The DNA input range for the MiSeqDx Platform was evaluated by performing a serial dilution study using 14 representative DNA samples containing 16 unique single gene variants. Each sample was tested in duplicate at 9 DNA input levels ranging from 1250 ng to 1 ng (1250 ng, 500 ng, 250 ng, 100 ng, 50 ng, 10 ng, 5 ng, and 1 ng). For determination of accuracy, sample genotypes were compared to bidirectional Sanger sequencing data and the deletions were compared to PCR assay. 1250 ng and 25 ng were identified as the upper and lower bound for DNA input respectively as they had ≥95% sample first pass rate with no incorrect calls (100% accuracy and call rate).

DNA inputs of 1250 ng, 250 ng, and 100 ng were further tested with 4 representative DNA samples and 20 replicates per DNA input level for each sample (n=420=80 samples), while the lower bound of 25 ng was tested with 14 samples, 20 replicates for each sample (n=1420=280 samples). The accuracy and sample first pass rate was 100% at all DNA input levels. There were 2 no calls overall observed at the 25 ng DNA input level, with sample call rates of 99.26%.

Thermal cycler study: Three different commercially available thermal cyclers were evaluated using the representative assay, the Illumina MiSeaDx Cystic Fibrosis 139 Variant Assay. Thermal cycles are used in the library preparation. Three unique sample sets were processed through all three thermal cyclers across 3 days. This enabled performance assessment across different thermal cyclers on different days. Each sample set was processed in triplicate each day (i.e. one replicate per thermal cycler). Acceptance criteria for this study were reviewed and deemed acceptable. This study met the sponsor's acceptance criteria and demonstrated that any commercially available thermal cycler would be adequate for library preparation for use with the MiSeqDx.

Sample indexing study: Sample index primers are used in the kit to assign a unique barcode to each sample DNA, allowing the ability to pool multiple samples together into a single sequencing run.

A total of 96 samples indexes were tested with Assay 2 using 8 unique DNA samples to verify the ability of the assay to consistently make a genotyping call for a given sample across different indexing primer combinations. Each sample was tested with 12 different indexing primer combinations. Sample results were compared against bidirectional Sanger sequencing data for all positions/variants. Reproducibility and accuracy were 100% for all sample/index primer combinations.

Specimen Storage: To verify the storage conditions and handling of blood samples for use with the MiSeqDx test system, six K2EDTA anti-coagulated blood samples were divided to six aliquots, one aliquot of each blood sample were stored under 6 different conditions: 2°C to 8°C for 1 day; -15°C to -25°C for 1 day; 2°C to 8°C for 30 days; -15°C to -25°C for 30 days: room temperature (20-25°C) for 7 days: and controlled room temperature (30°C) for 7 days. Genomic DNA was isolated from each aliquot using a commonly used commercial DNA extraction kit. All extractions were performed by a single operator. The extracted gDNA samples were stored at -15°C to -25°C until the libraries were prepared and sequenced.

33

The impact of repeated freeze-thaws on gDNA samples were tested by subjecting 15 DNA samples to 6 freeze thaw cycles.

Library preparations for both the samples from both the specimen storage and gDNA freeze-thaw studies were performed at the same time point. The samples from a single library preparation were pooled into one run of 48 samples and a second run of 32 samples prior to sequencing. Impact on call rate, reproducibility, and sample first pass rate were determined for each sample as compared to a respective control sample. No miscalls or no calls were observed for any of the specimens and demonstrated that the blood and gDNA storage conditions tested did not affect assay results.

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), and the special controls for this type of device.

L. Other Supportive Instrument Characteristics Data Not Covered In The "Performance Characteristics" Section above:

None.

Identified Potential Risk	Required Mitigation Measure
Inaccurate test results due to unavailability
of necessary components of the instrument
system	The labeling for the instrument system must
reference 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.
Inaccurate results due to unknown
performance of the instrument system	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. | |
| Identified Potential Risk | Required Mitigation Measure | |
| | 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 |
| Identified Potential Risk | Required Mitigation Measure | |
| | 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. | |

M. Identified Potential Risks and Required Mitigation Measures:

34

35

N. Benefit/Risk Analysis:

Summary

36

| Summary of
the Benefit(s) | This is a tool for clinical laboratories that can provide accurate and reproducible
high throughput genomic sequencing of genomic regions of interest at greater
sequencing depth than current sequencing technology. No other instruments are available for high throughput genomic sequence analysis.
There is an unmet medical and public health need for a well-validated IVD labelled
high throughput genomic sequence analyzer. |
|--------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Summary of
the Risk(s) | Patients are subject to blood specimen collection, which is a standard procedure in
clinical 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 could include
unnecessary testing or treatment related to an inaccurate test result. Often, the result
from this test would be used with results from other diagnostic tests and clinical
signs and symptoms to identify the genetic cause or contribution for a patient's
disease or condition.
False negative: The risks to the individual of a false negative result due to an
inaccurate test result could delay further evaluation and appropriate therapy which
will 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 results
are similar. |
| Summary of
Other
Factors | Not applicable. |
| Conclusions | Do the probable benefits outweigh the probable risks? |

Given robust analytical performance characteristics and risk mitigation (i.e. extensive performance data provided in the labeling), the probable benefits to both the individual and public health outweigh the probable risks of this device.

0. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 862.2265. FDA believes that special controls, along with the applicable general controls, provide reasonable assurance of the safety and

37

effectiveness of the device type. This device, and similar devices, is classified under the following:

Product Code:	PFF
Device Type:	High throughput genomic sequence analyzer for clinical use
Class:	II (special controls)
Regulation:	21 CFR 862.2265

(a)Identification. A high throughput genetic 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). A high throughput genetic sequence analyzer for clinical use must comply with the following special controls:

• 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.
  • The type(s) of nucleic acids (e.g., germline DNA, tumor DNA) ii) 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.
  • The appropriate read depth for the sensitivity claimed and v) validation information supporting those claims.
  • The nucleic acid extraction method(s) validated for use with vi) the instrument system.

38

• 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 wellcharacterized 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.