Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The document describes a standard targeted sequencing assay and data analysis software. There is no mention of AI or ML in the device description, intended use, or performance studies. The data analysis appears to be based on comparing sequencing results to reference methods (Sanger and PCR) and calculating agreement metrics, which is typical for traditional genetic testing analysis.

Therapeutic

No

Explanation: This device is an in vitro diagnostic (IVD) system used for sequencing the CFTR gene to aid in the diagnosis of cystic fibrosis. It detects genetic mutations and does not directly treat or alleviate a disease, injury, or disability.

Diagnostic

Yes

Explanation: The "Intended Use / Indications for Use" section explicitly states, "The test is intended to be used as an aid in the diagnosis of individuals with suspected cystic fibrosis (CF)."

SaMD (Software as a Medical Device)

No

The device description explicitly states that the system consists of "library preparation and sample indexing reagents, sequencing reagents and consumables, MiSeqDx instrument and data analysis software." This includes hardware components (reagents, consumables, instrument) in addition to the software.

IVD (In Vitro Diagnostic)

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

Here's why:

Explicit Statement: The "Intended Use / Indications for Use" section explicitly states: "The Illumina MiSeqDx(TM) Cystic Fibrosis Clinical Sequencing Assay is a targeted sequencing in vitro diagnostic system..."
Intended Use: The intended use is to aid in the diagnosis of individuals with suspected cystic fibrosis, which is a diagnostic purpose.
Sample Type: It uses genomic DNA isolated from human peripheral blood, which is a biological specimen.
Testing Method: It performs targeted sequencing of the CFTR gene, which is a laboratory test performed outside of the body (in vitro).
Regulatory Context: The presence of a "Predicate Device(s)" section with a K number (K083845) indicates that this device has gone through a regulatory submission process, which is typical for IVDs.

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

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Illumina MiSeqDx™ Cystic Fibrosis Clinical Sequencing Assay is a targeted sequencing in vitro diagnostic system that re-sequences the protein coding regions and intron/exon boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in genomic DNA isolated from human peripheral whole blood specimens collected in K2EDTA. The test detects single nucleotide variants, and small InDels within the region sequenced, and additionally reports on two deep intronic mutations and two large deletions. The test is intended to be used on the Illumina MiSeqDx Instrument.

The test is intended to be used as an aid in the diagnosis of individuals with suspected cystic fibrosis (CF). The test is most appropriate when the patient has an atypical or non-classic presentation of CF or when other mutation panels have failed to identify both causative mutations. The results of the test are intended to be interpreted by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available information including clinical symptoms, other diagnostic tests, and family history. This test is not indicated for use for stand-alone diagnostic purposes, fetal diagnostic testing, for preimplantation testing, carrier screening, newborn screening, or population screening.

Product codes

PFS

Device Description

The Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay consists of library preparation and sample indexing reagents, sequencing reagents and consumables, MiSeqDx instrument and data analysis software. Testing begins with genomic DNA from a peripheral whole blood sample. The genomic DNA is processed through the library preparation steps, which specifically amplifies the intended genomic regions of each sample while also adding the indexes for sample identification. Flow cell capture sequences are also added 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). The MiSeqDx Cartridge, MiSeqDx Flow Cell, and MiSeqDx SBS Solution (PR2) are then inserted into the MiSeqDx instrument, which performs cluster generation, sequencing and data analysis.

Mentions image processing

Not Found

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

The results of the test are intended to be interpreted by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available information including clinical symptoms, other diagnostic tests, and family history.

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 of the Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay was assessed by evaluating 500 samples representing a wide variety of CFTR variants from four separate sources. The primary source of accuracy data was a clinical accuracy study conducted using a panel of 366 samples. The majority (n = 355) of samples consisted of archived, anonymized clinical gDNA specimens isolated from human blood, the remaining 11 samples were obtained from commercially available cell line specimens.
Data from this study was supplemented with accuracy data from 68 cell line samples evaluated in the reproducibility study, 14 clinical samples from the extraction method evaluation analytical study, and 52 synthetic plasmid samples. The synthetic plasmids were designed to include the genomic context of rare variants, and contained anywhere from 1 to 10 variants within the same construct. They were linearized, diluted to genomic DNA equivalent copy numbers, and blended with human genomic DNA samples of wild type genotype at equivalent copy numbers to mimic a heterozygous sample.
For the MiSeqDx Cystic Fibrosis Clinical Sequencing Assay, a total of 5,206 positions were compared to the reference methods of Sanger bi-directional sequencing and PCR testing. The genotyping results for SNV and small InDel sites, including the PolyTG/PolyT region, were compared to Sanger bi-directional sequence analysis.
Two validated PCR based assays were used as the reference method for the two large deletions in the panel. Each duplex PCR assay made use of 2 primer sets to discriminate between wild type, heterozygous; and homozygous genotypes. One of the primer sets was designed to flank the deletion breakpoints, whereas the other amplified a region internal to the deletion. The two products were detected by size separation on an agarose gel. The PCR assays were validated using a panel of 28 samples in all (22 samples for each deletion) consisting of cell line and blood derived genomic DNA samples, and synthetic plasmids which encompassed the WT, HET and HOM genotypes for each large deletion. The PCR assays were confirmed to have 100% specificity and reproducibility for all samples tested, by evaluation of PCR products on an agarose gel. The accuracy of the PCR assays was confirmed using Sanger Sequencing and found to be 100% for all samples.

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

Accuracy Study:

Sample Size: 500 samples (366 primary clinical samples, 68 cell line samples, 14 clinical samples from extraction study, 52 synthetic plasmid samples). Total of 5,206 positions compared.
Study Type: Clinical accuracy study comparing MiSeqDx results to Sanger bi-directional sequencing (for SNV and small InDel sites including PolyTG/PolyT) and validated PCR based assays (for large deletions).
Key Results:
- Genotype-level Positive Agreement (PA): 99.66% (including PolyTG/PolyT variants), 100% (excluding PolyTG/PolyT variants).
- Negative Agreement (NA) for all wild types: >99.99%.
- Overall Agreement (OA) for all WT and variants: >99.99%.
- PolyTG/PolyT variant PA: 98.44%.
- All results based on initial testing; no repeat testing.

Reproducibility Study:

Sample Size: 62 samples (genomic DNA from lymphoblastoid cell lines and leukocyte cell lines, and extracted from normal whole blood). Tested across 3 trial sites with 2 operators at each site, performing 3 runs per sample for a total of 18 replicates each. Total of 1,116 replicates for PolyTG/PolyT variants.
Study Type: Multi-site reproducibility study.
Key Results:
- Overall sample pass rate (defined as number of samples passing QC metrics on the first attempt): 99.7%.
- Genotype-level PA for all variants including PolyTG/PolyT: 99.22%.
- Genotype-level PA excluding PolyTG/PolyT variants: 99.60%.
- NA for all WT: 99.70%.
- OA for all reported positions: 99.70%.
- PolyTG/PolyT variants PA: 97.83%.

DNA Extraction Study:

Sample Size: 14 blood samples (2 wild type, 12 with 9 different variants) tested using 3 extraction methods. Each method tested by 2 operators, 3 runs/method, 2 replicates/sample. Total of 168 runs per extraction method.
Study Type: Evaluation of DNA extraction methods.
Key Results:
- Call Rate: >99.99% for all methods.
- Accuracy: >99.99% for all methods.
- Sample First Pass Rate: 100% for all methods.

DNA Input Study:

Sample Size: 14 representative DNA samples with 16 unique CF variants. Tested in duplicate at 9 DNA input levels (1250 ng to 1 ng). Further tested with 4 representative DNA samples (20 replicates each, n=80) and 14 samples (20 replicates each, n=280) at specific input levels.
Study Type: Evaluation of DNA input range.
Key Results:
- 1250 ng and 25 ng identified as upper and lower bound for DNA input, respectively, with ≥95% sample first pass rate and no incorrect calls (100% accuracy and call rate).
- Accuracy and sample first pass rate was 100% at all DNA input levels tested.

Interfering Substances Study:

Sample Size: Sixteen whole blood specimens.
Study Type: Evaluation of performance in presence and absence of potential interferents.
Key Results:
- Achieved 100% call rate for all samples.
- 100% reproducibility in genotype calls between samples in presence and absence of interfering substances.
- No interference observed for endogenous substances (bilirubin, cholesterol, hemoglobin, triglycerides) and sample collection/preparation-related interferents (EDTA, wash buffer).
- Cross contamination study showed no change in variant calling with contamination levels

Regulation Number and Section

§ 866.5900 Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

(a)
Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It is intended as an aid in confirmatory diagnostic testing of individuals with suspected cystic fibrosis (CF), carrier identification, and newborn screening. This device is not intended for stand-alone diagnostic purposes, prenatal diagnostic, pre-implantation, or population screening.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: CFTR Gene Mutation Detection System.” See § 866.1(e) for the availability of this guidance document.

Summary

0

: --

K132750

510(k) Summary 1.3

The following 510(k) summary was prepared in accordance with 21 CFR 807.92.

NOV 1 9 2013

1

510(k) Summary

GENERAL INFORMATION

Illumina Inc. Submitted by: 5200 Illumina Way San Diego, CA 92122 858-202-4500 (phone) 858-202-4600 (fax)

Company Contact: Bryan Schneider Associate Director, Regulatory Affairs 858-255-5228 (phone) bschneider@illumina.com . :
:

: ,

Date Prepared: November 18, 2013

DEVICE IDENTIFICATION

Assay: ..... ... ... ..

Trade or Proprietary Name:

| | Illumina MiSeqDx™ Cystic Fibrosis Clinical
Sequencing Assay |
|----------------------|-------------------------------------------------------------------------------------------------------------------------------|
| Common Name: | Sequencing by synthesis cystic fibrosis test |
| Classification Name: | CFTR (cystic fibrosis transmembrane conductance
regulatory) gene mutation detection (21 CFR 866.5900,
Product Code PFS) |
| Predicate Device: | x-TAG Cystic Fibrosis 60 Kit v2 (K083845) |

2

DEVICE DESCRIPTION

The Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay consists of library preparation and sample indexing reagents, sequencing reagents and consumables, MiSeqDx instrument and data analysis software. Testing begins with genomic DNA from a peripheral whole blood sample. The genomic DNA is processed through the library preparation steps, which specifically amplifies the intended genomic regions of each sample while also adding the indexes for sample identification. Flow cell capture sequences are also added 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). The MiSeqDx Cartridge, MiSeqDx Flow Cell, and MiSeqDx SBS Solution (PR2) are then inserted into the MiSeqDx instrument, which performs cluster generation, sequencing and data analysis.

INTENDED USE

Illumina MiSeqDx™ Cystic Fibrosis Clinical Sequencing Assay

The Illumina MiSeqDx(TM) Cystic Fibrosis Clinical Sequencing Assay is a targeted sequencing in vitro diagnostic system that re-sequences the protein coding regions and intron/exon boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene in genomic DNA isolated from human peripheral whole blood specimens collected in K2EDTA. The test detects single nucleotide variants, and small InDels within the region sequenced, and additionally reports on two deep intronic mutations and two large deletions. The test is intended to be used on the Illumina MiSeqDx Instrument.

The test is intended to be used as an aid in the diagnosis of individuals with suspected cystic fibrosis (CF). The test is most appropriate when the patient has an atypical or non-classic presentation of CF or when other mutation panels have failed to identify both causative mutations. The results of the test are intended to be interpreted by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available information including clinical symptoms, other diagnostic tests, and family history. This test is not indicated for use for stand-alone diagnostic purposes, fetal diagnostic testing, for preimplantation testing, carrier screening, newborn screening, or population screening. i : . . . . . . . . .

3

SUBSTANTIAL EQUIVALENCE

・・・・

Characteristic	Illumina	Luminex (K083845)
Assay Name	Illumina MiSeqDx Cystic Fibrosis
Clinical Sequencing Assay	Luminex xTAG®Cystic
Fibrosis 60 Kit v2
Intended
Use	The Illumina MiSeqDx Cystic
Fibrosis Clinical Sequencing
Assay is a targeted sequencing in
vitro diagnostic system that re-
sequences the protein coding
regions and intron/exon
boundaries of the Cystic Fibrosis
Transmembrane Conductance
Regulator (CFTR) gene in
genomic DNA isolated from
human peripheral whole blood
specimens collected in K2EDTA.
The test detects single nucleotide
variants, and small indels within
the region sequenced, and
additionally reports on two deep
intronic mutations and two large
deletions. The test is intended to
be used on the Illumina MiSeqDx
Instrument.

The test is intended to be used as
an aid in the diagnosis of
individuals with suspected cystic
fibrosis (CF).This assay is most
appropriate when the patient has | The xTAG® Cystic Fibrosis 60
kit v2 is a device used to
simultaneously detect and
identify a panel of mutations
and variants in the cystic
fibrosis transmembrane
conductance regulator
(CFTR) gene in human blood
specimens. The panel
includes mutations and
variants currently
recommended by the
American College of Medical
Genetics and American
College of Obstetricians and
Gynecologists
(ACMG/ACOG) plus some
of the world's most common
and North American
prevalent mutations. The
xTAG Cystic Fibrosis 60 kit
v2 is a qualitative
genotyping test which
provides information
intended to be used for
carrier testing in adults of |
| Confidential 4 | | |
| Characteristic | Illumina | Luminex (K083845) |
| | an atypical or non-classic
presentation of CF or when other
mutation panels have failed to
identify both causative mutations.
The results of the test are
intended to be interpreted by a
board-certified clinical molecular
geneticist or equivalent and
should be used in conjunction
with other available information
including clinical symptoms,
other diagnostic tests, and family
history. This test is not indicated
for use for fetal diagnostic testing,
for pre-implantation testing,
carrier screening, newborn
screening, or population
screening.
The test is intended to be used on
the Illumina MiSeqDx™
Instrument. | reproductive age, as an aid
in newborn screening, and in
confirmatory diagnostic
testing in newborns and
children.
The kit is not indicated for
use in fetal diagnostic or pre-
implantation testing. The kit
is also not indicated for
stand-alone diagnostic
purposes. |
| Assay type | Sequencing by synthesis test | Qualitative nucleic acid
multiplex test |
| Variants
Detected | Mutations and variants from the
protein coding regions and
intron/exon boundaries of the
CFTR gene, including two deep
intronic mutations and two large
deletions. | 60 CFTR mutations and 4
variants (benign
polymorphisms) |
| Characteristic | Illumina | Luminex (K083845) |
| Technology | PCR-based amplification of
regions of interest that are then
hybridized to a flow cell to allow
sequencing by synthesis | Multiplex PCR followed by
multiplex allele specific
primer extension for
genotyping, hybridized to
multiplex fluorescent
microparticles, detected by
flow cytometry. |
| Sample Type | Nucleic acid from K2EDTA
anticoagulated blood | Nucleic acid from whole
blood anticoagulated with
either EDTA or citrate. |
| Sample
Preparation | DNA extraction using validated
laboratory method | Same |
| Contra-
indications | This test is not indicated for use
for fetal diagnostic testing, for
pre-implantation testing, carrier
screening, newborn screening, or
population screening. | Not indicated for fetal
diagnostic testing, for pre-
implantation testing, or for
stand-alone diagnostic
purposes. |
| Assay
Controls | Positive and negative controls
required, not supplied | Negative controls required,
not supplied. Positive
controls recommended, not
supplied. |
| Instrument
System | MiSeqDx | Luminex 100 or 200 IS |

4

MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

.

、

・・・・・・・・・・・・・・・・・・・

Confidential 5

: :

5

Image /page/5/Picture/1 description: The image contains the word "illumina" in a bold, sans-serif font. The letters are closely spaced, and the word appears to be a logo or brand name. The overall impression is clean and modern.

PERFORMANCE.CHARACTERISTICS

Accuracy

Accuracy of the Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay was assessed by evaluating 500 samples representing a wide variety of CFTR variants from four separate sources. The primary source of accuracy data was a clinical accuracy study conducted using a panel of 366 samples. The majority (n = 355) of

6

samples consisted of archived, anonymized clinical gDNA specimens isolated from human blood, the remaining 11 samples were obtained from commercially available cell line specimens.

Data from this study was supplemented with accuracy data from 68 cell line samples evaluated in the reproducibility study, 14 clinical samples from the extraction method evaluation analytical study, and 52 synthetic plasmid samples. The synthetic plasmids were designed to include the genomic context of rare variants, and contained anywhere from 1 to 10 variants within the same construct. They were linearized, diluted to genomic DNA equivalent copy numbers, and blended with human genomic DNA samples of wild type genotype at equivalent copy numbers to mimic a heterozygous sample.

For the MiSeqDx Cystic Fibrosis Clinical Sequencing Assay, a total of 5,206 positions were compared to the reference methods of Sanger bi-directional sequencing and PCR testing. The genotyping results for SNV and small InDel sites, including the PolyTG/PolyT region, were compared to Sanger bi-directional sequence analysis.

Two validated PCR based assays were used as the reference method for the two large deletions in the panel. Each duplex PCR assay made use of 2 primer sets to discriminate between wild type, heterozygous; and homozygous genotypes. One of the primer sets was designed to flank the deletion breakpoints, whereas the other amplified a region internal to the deletion. The two products were detected by size separation on an agarose gel. The PCR assays were validated using a panel of 28 samples in all (22 samples for each deletion) consisting of cell line and blood derived genomic DNA samples, and synthetic plasmids which encompassed the WT, HET and HOM genotypes for each large deletion. The PCR assays were confirmed to have 100% specificity and reproducibility for all samples tested, by evaluation of PCR products on an agarose gel. The accuracy of the PCR assays was confirmed using Sanger Sequencing and found to be 100% for all samples.

Accuracy was determined for each genotype through three statistical measures. Positive Agreement (PA) was calculated for each variant genotype by dividing the number of samples with agreeing variant calls by the total number of samples with that variant as identified by the reference methods. Negative Agreement (NA) was calculated across all wild type (WT) positions by dividing the number of concordant WT positions by the total number of WT positions as

7

MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

illumına

defined by the reference methods. Overall Agreement (OA) was calculated across all reported positions by dividing the number of concordant WT and variant positions by the total number of reported positions as determined by the reference methods.

The MiSeqDx Cystic Fibrosis Clinical Sequencing Assay had a genotype-level PA of 99.66% including the PolyTG/PolyT variants and 100%, excluding PolyTG/PolyT variants (Table 1). The NA for all wild types was >99.99% and the OA for all WT and variants was >99.99%. The PolyTG/PolyT variant PA was 98.44%. All results are based on initial testing. No repeat testing was done for this study.

Accuracy of the PolyTG/PolyT variants is demonstrated in Table 2.

(),,一个人

" " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " " "

・・・・・・・・・・・・・・・・・・・

and the country of the capital of the county of

、『『『』『

... .. . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

Company of the was and

		A
	All - Alle - A deller a more - comment - comments



.

                                                                                      |                                                   |   |

| 1 | | | | | | | | | | | | | | | | | | | | | | . | | | | | | | | | | | | | | | | 1 | | | | | | | | | | | | A September 2017 A 1 A 2 A 2 A 2 A 2 2 A 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 | | | | | | | | | | 0011136 | | | | | | 1 | | | | | | the us dife which and | | | | | | | | A M A S A T W S M A M & P W B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B | | 1210 1 | | | | | | | | | | | | |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |
| |

| Genotype
(Common
Name) | cDNA name
coordinate | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | | | No
Calls | Miscalls | Positive
Agreement |
|---------------------------------------------------------|--------------------------------|-----------------|----------------------------------|----------------------------------|---------------------------|-------------------------|----------------------|-------------|----------|-----------------------|
| | | | | | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | | | |
| 117120141 | N/A | SNV | Exon1 | 25 | 3 | 0 | 0 | 0 | 100 | |
| 117120145 | N/A | SNV | Exon1 | 3 | 2 | 0 | 0 | 0 | 100 | |
| M1V | c.1A>G | SNV | Exon1 | 0 | 0 | 1 | 0 | 0 | 100 | |
| CFTR dele2, 3 | c.54-
5940_273+10250del21kb | Del | Intron1 | 4 | 1 | 0 | 0 | 0 | 100 | |
| R31C | c.91C>T | SNV | Exon2 | 3 | 1 | 0 | 0 | 0 | 100 | |
| Q39X | c.115C>T | SNV | Exon2 | 0 | 0 | 1 | 0 | 0 | 100 | |
| E60X | c.178G>T | SNV | Exon3 | 6 | 1 | 0 | 0 | 0 | 100 | |
| P67L | c.200C>T | SNV | Exon3 | 1 | 0 | 1 | 0 | 0 | 100 | |
| R74W | c.220C>T | SNV | Exon3 | 0 | 2 | 0 | 0 | 0 | 100 | |
| R74Q | c.221G>A | SNV | Exon3 | 2 | 0 | 0 | 0 | 0 | 100 | |
| R75X | c.223C>T | SNV | Exon3 | 3 | 1 | 0 | 0 | 0 | 100 | |
| R75Q | c.224G>A | SNV | Exon3 | 20 | 1 | 0 | 0 | 0 | 100 | |
| G85E | c.254G>A | SNV | Exon3 | 6 | 2 | 0 | 0 | 0 | 100 | |
| Genotype
(Common
Name
cDNA name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | | | | | | Positive
Agreement |
| | | | | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | No
Calls | Miscalls | | |
| 394delTT | c.262_263delTT | DIV | Exon3 | 3 | 1 | 0 | 0 | 0 | 100 | |
| 405+1G>A | c.273+1G>A | SNV | Intron3 | 0 | 0 | 1 | 0 | 0 | 100 | |
| 406-1G>A | c.274-1G>A | SNV | Exon4 | 4 | 0 | 0 | 0 | 0 | 100 | |
| E92K | c.274G>A | SNV | Exon4 | 0 | 0 | 1 | 0 | 0 | 100 | |
| E92X | c.274G>T | SNV | Exon4 | 0 | 1 | 1 | 0 | 0 | 100 | |
| Q98X | c.292C>T | SNV | Exon4 | 0 | 0 | 2 | 0 | 0 | 100 | |
| 444delA | c.312delA | DIV | Exon4 | 0 | 2 | 0 | 0 | 0 | 100 | |
| 457TAT>G | c.325_327delTATinsG | DIV | Exon4 | 0 | 0 | 1 | 0 | 0 | 100 | |
| D110H | c.328G>C | SNV | Exon4 | 1 | 0 | 1 | 0 | 0 | 100 | |
| R117C | c.349C>T | SNV | Exon4 | 4 | 0 | 0 | 0 | 0 | 100 | |
| R117H | c.350G>A | SNV | Exon4 | 17 | 2 | 0 | 0 | 0 | 100 | |
| Y122X | c.366T>A | SNV | Exon4 | 0 | 1 | 0 | 0 | 0 | 100 | |
| F143LfsX10 | c.425delT | DIV | Exon4 | 0 | 1 | 0 | 0 | 0 | 100 | |
| 574delA | c.442delA | DIV | Exon4 | 0 | 0 | 2 | 0 | 0 | 100 | |
| Q151K | c.451C>A | SNV | Exon4 | 1 | 0 | 0 | 0 | 0 | 100 | |

9

10

| | કુ
ﻟﻤﺴﺎ
n
S
0 |
|----|---------------------------|
| ﻣﻨ | 0
œ
œ
œ |
| 1 | S
1 |
| | |

| Genotype
(Common
Name
cDNA name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | | | No
Calls | Miscalls | Positive
Agreement | | |
|----------------------------------------------------------|---------------------------------------|-----------------|----------------------------------|----------------------------------|---------------------------|-------------------------|-------------------------|----------------------|-----------------------|-----------------------|-----------------------|
| 621+1G>T | c.489+1G>T | SNV | Intron4 | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | 0 | 0 | 100 | | |
| 621+3A>G | c.489+3A>G | SNV | Intron4 | 1 | 0 | 0 | 0 | 0 | 100 | | |
| 663delT | c.531delT | DIV | Exon5 | 1 | 0 | 1 | 0 | 0 | 100 | | |
| G178R | c.532G>A | SNV | Exon5 | 1 | 1 | 0 | 0 | 0 | 100 | | |
| 711+1G>T | c.579+1G>T | SNV | Intron5 | 3 | 1 | 0 | 0 | 0 | 100 | | |
| 711+3A>G | c.579+3A>G | SNV | Intron5 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| 711+5 G->A | c.579+5G>A | SNV | Intron5 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| 712-1 G->T | c.580-1G>T | SNV | Exon6 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| H199Y | c.595C>T | SNV | Exon6 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| P205S | c.613C>T | SNV | Exon6 | 1 | 0 | 1 | 0 | 0 | 100 | | |
| L206W | c.617T>G | SNV | Exon6 | 8 | 1 | 0 | 0 | 0 | 100 | | |
| A209S | c.625G>T | SNV | Exon6 | 0 | 1 | 0 | 0 | 0 | 100 | | |
| Q220X | c.658C>T | SNV | Exon6 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| L227R | c.680T>G | SNV | Exon6 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| 852del22 | c.720_741delAGGGAGAA
TCATCATCACTAC | DIV | Exon6 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| Genotype
(Common
Name
cDNA name,
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | No
Calls" | Miscalls | Positive
Agreement | |
| E279D | c.837A>T | SNV | Exon7 | 1 | 0 | 0 | 0 | 0 | 100 | | |
| R297Q | c.890G>A | SNV | Exon8 | 2 | 0 | 0 | 0 | 0 | 100 | | |
| 1078delT | c.948delT | DIV | Exon8 | 1 | 1 | 0 | 0 | 0 | 100 | | |
| L320V | c.958T>G | SNV | Exon8 | 1 | 0 | 0 | 0 | 0 | 100 | | |
| G330X | c.988G>T | SNV | Exon8 | 1 | 1 | 0 | 0 | 0 | 100 | | |
| R334W | c.1000C>T | SNV | Exon8 | 6 | 1 | 0 | 0 | 0 | 100 | | |
| I336K | c.1007T>A | SNV | Exon8 | 0 | 1 | 0 | 0 | 0 | 100 | | |
| T338I | c.1013C>T | SNV | Exon8 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| 1154insTC | c.1022_1023insTC | DIV | Exon8 | 0 | 1 | 0 | 0 | 0 | 100 | | |
| S341P | c.1021T>C | SNV | Exon8 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| R347H | c.1040G>A | SNV | Exon8 | 6 | 1 | 1 | 0 | 0 | 100 | | |
| R347P | c.1040G>C | SNV | Exon8 | 3 | 2 | 0 | 0 | 0 | 100 | | |
| R352Q | c.1055G>A | SNV | Exon8 | 5 | 0 | 0 | 0 | 0 | 100 | | |
| Q359K/T360K | c.[1075C>A;1079C>A] | SNV | Exon8 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| 1213delT | c.1081delT | DIV | Exon8 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| Genotype
(Common
Name | cDNA name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | No
Calls | Miscalls | Positive
Agreement |
| 1248+1G>A | c.1116+1G>A | SNV | Intron8 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| 1259insA | c.1127_1128insA | DIV | Exon9 | 0 | 0 | 2 | 0 | 0 | 100 | | |
| W401X
(c.1202G>A) | c.1202G>A | SNV | Exon9 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| W401X
(c.1203G>A) | c.1203G>A | SNV | Exon9 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| 1341+1G->A | c.1209+1G>A | SNV | Intron9 | 0 | 0 | 2 | 0 | 0 | 100 | | |
| PolyTGPolyT | N/A | PolyTGPolyT | Intron9 | 369 | 79 | 52 | 3 | 4 | 98.60 | | |
| 1461ins4 | c.1329_1330insAGAT | DIV | Exon10 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| A455E | c.1364C>A | SNV | Exon10 | 4 | 2 | 0 | 0 | 0 | 100 | | |
| 1525-1G->A | c.1393-1G>A | SNV | Exon11 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| S466X (C->A) | c.1397C>A | SNV | Exon11 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| S466X (C->G) | c.1397C>G | SNV | Exon11 | 1 | 0 | 1 | 0 | 0 | 100 | | |
| L467P | c.1400T>C | SNV | Exon11 | 0 | 0 | 1 | 0 | 0 | 100 | | |
| V470M | c.1408G>A | SNV | Exon11 | 311 | 71 | 0 | 0 | 0 | 100 | | |

.

ﺗ

i

11

T

1

T

12

13

.

| Variant
Type | CFTR
gene
region
(hg19) | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | No
Calls | Miscalls | Positive
Agreement |
|-----------------|----------------------------------|---------------------|-------------------------|----------------------|-------------|----------|-----------------------|
| DIV | Exon11 | 1 | 0 | 1 | 0 | 0 | 100 |
| SNV | Exon11 | 0 | 1 | 0 | 0 | 0 | 100 |
| SNV | Exon11 | 1 | 0 | 0 | 0 | 0 | 100 |
| SNV | Exon11 | 0 | 0 | 2 | 0 | 0 | 100 |
| SNV | Exon11 | 0 | 0 | 1 | 0 | 0 | 100 |
| SNV | Exon11 | 4 | 2 | 0 | 0 | 0 | 100 |
| SNV | Exon11 | 7 | 0 | 0 | 0 | 0 | 100 |
| DIV | Exon11 | 4 | 2 | 0 | 0 | 0 | 100 |
| DIV | Exon11 | 84 | 29 | 0 | 0 | 0 | 100 |
| SNV | Exon11 | 0 | 1 | 0 | 0 | 0 | 100 |
| SNV | Exon11 | 1 | 1 | 0 | 0 | 0 | 100 |
| DIV | Exon11 | 1 | 0 | 0 | 0 | 0 | 100 |
| SNV | Exon11 | 2 | 0 | 0 | 0 | 0 | 100 |
| SNV | Exon11 | 0 | 0 | 1 | 0 | 0 | 100 |
| SNV | Exon11 | 3 | 2 | 0 | 0 | 0 | 100 |

'r

14

| | greemer
Positive | 100 | 100 | 00 I | 00 T | 00 T | 100 | 00 I | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
|---------------------------------------------------------|-----------------------------------------------------------------------------------|-----------------|----------------------------------|---------------------------|-------------------------|----------------------|-------------|-----------------|-----------------------|---------|----------|-----------|-----------|----------|---------|
| | Aiscall | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | ાદ) તાલુકાઓ પૈકીના એક એવા ગુજરાત રાજ્યના | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | ynthetiI | .1585-8G> | .1585-1G>/ | .1624G> | :.1645A> | .1646G> | :1647T>G | :1652G>/ | .1654C> | .1657C>" | :.1666A>( | c.1673T>A | .1679G> |
| | DNA name
្រុកប្រចេះ សារបោះបាន សុវណ្ណបក្សត្រ សុំសុប្រជុំសូលប្រជុំស្
oordinat | E528E | 717-8G-> | 717-1G>/ | G542X | S549R c.1645A>C | 5549N | S549R c.1647T>G | G551D | Q552X | R553X | 556V | resss | A559T | R560K |
| | | | | | | | | | | | | | | | |
| | | | | Positive calls (Variants) | | | | | | | | | | | |
| Genotype
(Common
Name
cDNA name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | No
Calls | Miscalls | Positive
Agreement | | | | | | |
| R560T | c.1679G>C | SNV | Exon12 | 6 | 1 | 0 | 0 | 0 | 100 | | | | | | |
| 811+1.6kb A->G | c.1679+1.6kbA>G | SNV | Intron12 | 0 | 0 | 1 | 0 | 0 | 100 | | | | | | |
| 1812-1 G->A | c.1680-1G>A | SNV | Exon13 | 1 | 2 | 0 | 0 | 0 | 100 | | | | | | |
| A561T | c.1681G>A | SNV | Exon13 | 1 | 0 | 0 | 0 | 0 | 100 | | | | | | |
| V562I | c.1684G>A | SNV | Exon13 | 1 | 0 | 0 | 0 | 0 | 100 | | | | | | |
| Y569D | c.1705T>G | SNV | Exon13 | 0 | 0 | 1 | 0 | 0 | 100 | | | | | | |
| P574H | c.1721C>A | SNV | Exon13 | 0 | 1 | 0 | 0 | 0 | 100 | | | | | | |
| G576A | c.1727G>C | SNV | Exon13 | 4 | 1 | 0 | 0 | 0 | 100 | | | | | | |
| D579G | c.1736A>G | SNV | Exon13 | 0 | 1 | 0 | 0 | 0 | 100 | | | | | | |
| E585X | c.1753G>T | SNV | Exon13 | 0 | 1 | 0 | 0 | 0 | 100 | | | | | | |
| 1898+1G>A | c.1766+1G>A | SNV | Intron13 | 2 | 1 | 0 | 0 | 0 | 100 | | | | | | |
| 1898+3A>G | c.1766+3A>G | SNV | Intron13 | 0 | 1 | 0 | 0 | 0 | 100 | | | | | | |
| H609R | c.1826A>G | SNV | Exon14 | 0 | 1 | 0 | 0 | 0 | 100 | | | | | | |
| D614G | c.1841A>G | SNV | Exon14 | 0 | 0 | 2 | 0 | 0 | 100 | | | | | | |
| R668C | c.2002C>T | SNV | Exon14 | 5 | 2 | 0 | 0 | 0 | 100 | | | | | | |

15

16

| Genotype
(Common Name:
cDNA name coordinate) | cDNA name | Variant Type | CFTR gene region (hg19) | Positive calls (Variants)
Clinical Samples | Positive calls (Variants)
Cell Line Samples | Positive calls (Variants)
Synthetic Samples | No Calls | Miscalls | Positive Agreement |
|---------------------------------------------------------|--------------------------------|-----------------|----------------------------------|-----------------------------------------------|------------------------------------------------|------------------------------------------------|-----------------------|-----------------------|-----------------------|
| R668H | c.2003G>A | SNV | Exon14 | 1 | 0 | 0 | 0 | 0 | 100 |
| 2143delT | c.2012delT | DIV | Exon14 | 2 | 1 | 0 | 0 | 0 | 100 |
| K684TfsX4 | c.2046_2047delAA | DIV | Exon14 | 0 | 0 | 1 | 0 | 0 | 100 |
| 2183AA>G | c.2051_2052delAAinsG | DIV | Exon14 | 3 | 1 | 0 | 0 | 0 | 100 |
| 2184delA | c.2052delA | DIV | Exon14 | 1 | 1 | 0 | 0 | 0 | 100 |
| 2184insA | c.2052_2053insA | DIV | Exon14 | 3 | 0 | 1 | 0 | 0 | 100 |
| S686Y | c.2057C>A | SNV | Exon14 | 0 | 1 | 0 | 0 | 0 | 100 |
| R709X | c.2125C>T | SNV | Exon14 | 1 | 0 | 2 | 0 | 0 | 100 |
| K710X | c.2128A>T | SNV | Exon14 | 3 | 0 | 0 | 0 | 0 | 100 |
| E725K | c.2173G>A | SNV | Exon14 | 2 | 0 | 0 | 0 | 0 | 100 |
| 2307insA | c.2175_2176insA | DIV | Exon14 | 3 | 0 | 2 | 0 | 0 | 100 |
| L732X | c.2195T>G | SNV | Exon14 | 0 | 0 | 2 | 0 | 0 | 100 |
| 2347delG | c.2215delG | DIV | Exon14 | 0 | 0 | 2 | 0 | 0 | 100 |
| P750L | c.2249C>T | SNV | Exon14 | 1 | 0 | 0 | 0 | 0 | 100 |
| V754M | c.2260G>A | SNV | Exon14 | 2 | 1 | 0 | 0 | 0 | 100 |
| Genotype
(Common
Name
cDNA name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | | | | | Positive
Agreement |
| | | | | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | No
Calls | Miscalls | |
| R764X | c.2290C>T | SNV | Exon14 | 1 | 0 | 2 | 0 | 0 | 100 |
| 2585delT | c.2453delT | DIV | Exon14 | 0 | 0 | 2 | 0 | 0 | 100 |
| E822X | c.2464G>T | SNV | Exon14 | 0 | 0 | 2 | 0 | 0 | 100 |
| 2622+1G>A | c.2490+1G>T | SNV | Intron14 | 0 | 0 | 2 | 0 | 0 | 100 |
| E831X | c.2491G>T | SNV | Exon15 | 0 | 0 | 1 | 0 | 0 | 100 |
| D836Y | c.2506G>T | SNV | Exon15 | 0 | 1 | 0 | 0 | 0 | 100 |
| W846X | c.2537G>A | SNV | Exon15 | 0 | 1 | 0 | 0 | 0 | 100 |
| R851X | c.2551C>T | SNV | Exon15 | 0 | 0 | 1 | 0 | 0 | 100 |
| T854T | c.2562T>G | SNV | Exon15 | 212 | 44 | 0 | 0 | 0 | 100 |
| 2711delT | c.2583delT | DIV | Exon15 | 0 | 0 | 1 | 0 | 0 | 100 |
| V868V | c.2604A>G | SNV | Exon15 | 2 | 0 | 0 | 0 | 0 | 100 |
| 2657+2_2657
+3insA | c.2657+2_2657+3insA | DIV | Intron16 | 0 | 0 | 1 | 0 | 0 | 100 |
| 2789+5G>A | c.2657+5G>A | SNV | Intron16 | 9 | 1 | 0 | 0 | 0 | 100 |
| Q890X | c.2668C>T | SNV | Exon17 | 1 | 0 | 0 | 0 | 0 | 100 |
| A923A | c.2769C>T | SNV | Exon17 | 1 | 0 | 0 | 0 | 0 | 100 |
| Genotype
(Common
Name
cDNA name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | | | No
Miscalls | Positive
Agreement | |
| | | | | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | | | |
| L927P | c.2780T>C | SNV | Exon17 | 0 | 0 | 1 | 0 | 100 | |
| S945L | c.2834C>T | SNV | Exon17 | 0 | 0 | 1 | 0 | 100 | |
| M952T | c.2855T>C | SNV | Exon17 | 1 | 0 | 0 | 0 | 100 | |
| 3007delG | c.2875delG | DIV | Exon17 | 0 | 0 | 1 | 0 | 100 | |
| T966T | c.2898G>A | SNV | Exon17 | 5 | 0 | 0 | 0 | 100 | |
| G970R | c.2908G>C | SNV | Exon17 | 0 | 0 | 1 | 0 | 100 | |
| S977F | c.2930C>T | SNV | Exon18 | 0 | 0 | 1 | 0 | 100 | |
| 3120G>A | c.2988G>A | SNV | Exon18 | 1 | 0 | 0 | 0 | 100 | |
| 3120+1G>A | c.2988+1G>A | SNV | Intron18 | 7 | 1 | 0 | 0 | 100 | |
| 3121-1G->A | c.2989-1G>A | SNV | CF
Syn_Ex19 | 0 | 0 | 1 | 0 | 100 | |
| L997F | c.2991G>C | SNV | Exon19 | 2 | 1 | 0 | 0 | 100 | |
| I1027T | c.3080T>C | SNV | Exon19 | 1 | 2 | 0 | 0 | 100 | |
| 3272-26A>G | c.3140-26A>G | SNV | Intron19 | 0 | 1 | 0 | 0 | 100 | |
| F1052V | c.3154T>G | SNV | Exon20 | 0 | 1 | 0 | 0 | 100 | |
| L1065P | c.3194T>C | SNV | Exon20 | 0 | 0 | 1 | 0 | 100 | |
| Genotype
(Common
Name
cDNA name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | | | Positive
Agreement | | |
| | | | | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | No
"Calls" | Miscalls | |
| R1066C | c.3196C>T | SNV | Exon20 | 6 | 0 | 0 | 0 | 0 | 100 |
| R1066H | c.3197G>A | SNV | Exon20 | 1 | 0 | 1 | 0 | 0 | 100 |
| G1069R | c.3205G>A | SNV | Exon20 | 0 | 1 | 0 | 0 | 0 | 100 |
| R1070W | c.3208C>T | SNV | Exon20 | 0 | 2 | 0 | 0 | 0 | 100 |
| R1070Q | c.3209G>A | SNV | Exon20 | 0 | 1 | 0 | 0 | 0 | 100 |
| L1077P | c.3230T>C | SNV | Exon20 | 0 | 0 | 1 | 0 | 0^ | 100 |
| W1089X | c.3266G>A | SNV | Exon20 | 4 | 0 | 0 | 0 | 0 | 100 |
| Y1092X (C>A) | c.3276C>A | SNV | Exon20 | 3 | 1 | 0 | 0 | 0 | 100 |
| Y1092X (C>G) | c.3276C>G | SNV | Exon20 | 0 | 0 | 1 | 0 | 0 | 100 |
| T1095T | c.3285A>T | SNV | Exon20 | 7 | 0 | 0 | 0 | 0 | 100 |
| M1101K | c.3302T>A | SNV | Exon20 | 2 | 2 | 0 | 0 | 0 | 100 |
| E1104X | c.3310G>T | SNV | Exon20 | 0 | 0 | 1 | 0 | 0 | 100 |
| c.3368-2A>T | c.3368-2A>T | SNV | Intron20 | 0 | 1 | 0 | 0 | 0 | 100 |
| D1152H | c.3454G>C | SNV | Exon21 | 10 | 1 | 0 | 0 | 0 | 100 |
| V1153E | c.3458T>A | SNV | Exon21 | 1 | 0 | 0 | 0 | 0 | 100 |
| Genotype
(Common
Name
cDNA name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | | | | | Positive
Agreement |
| | | | | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | No
Calls | Miscalls | |
| R1158X | c.3472C>T | SNV | Exon22 | 7 | 1 | 0 | 0 | 0 | 100 |
| R1162X | c.3484C>T | SNV | Exon22 | 5 | 1 | 0 | 0 | 0 | 100 |
| R1162L | c.3485G>T | SNV | Exon22 | 0 | 2 | 0 | 0 | 0 | 100 |
| 3659delC | c.3528delC | DIV | Exon22 | 4 | 1 | 0 | 0 | 0 | 100 |
| S1196X | c.3587C>G | SNV | Exon22 | 1 | 0 | 0 | 0 | 0 | 100 |
| W1204X
(c.3611G>A) | c.3611G>A | SNV | Exon22 | 0 | 0 | 1 | 0 | 0 | 100 |
| W1204X
(c.3612G>A) | c.3612G>A | SNV | Exon22 | 0 | 0 | 1 | 0 | 0 | 100 |
| 3791delC | c.3659delC | DIV | Exon22 | 2 | 0 | 0 | 0 | 0 | 100 |
| I1234V | c.3700A>G | SNV | Exon22 | 1 | 0 | 1 | 0 | 0 | 100 |
| S1235R | c.3705T>G | SNV | Exon22 | 9 | 1 | 0 | 0 | 0 | 100 |
| 3849+10kbC>T | c.3717+12191C>T | SNV | Intron22 | 11 | 2 | 0 | 0 | 0 | 100 |
| G1244E | c.3731G>A | SNV | Exon23 | 0 | 0 | 1 | 0 | 0 | 100 |
| 3876delA | c.3744delA | DIV | Exon23 | 6 | 1 | 0 | 0 | 0 | 100 |
| S1251N | c.3752G>A | SNV | Exon23 | 1 | 0 | 1 | 0 | 0 | 100 |
| Genotype
(Common
Name
cDNA name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | | | | | |
| | | | | Clinical
Samples | Cell
Line
Samples | Synthetic
Samples | No
Calls | Miscalls | Positive
Agreement |
| 3905insT | c.3773_3774insT | DIV | Exon23 | 3 | 1 | 0 | 0 | 0 | 100 |
| D1270N | c.3808G>A | SNV | Exon23 | 0 | 2 | 0 | 0 | 0 | 100 |
| W1282X | c.3846G>A | SNV | Exon23 | 9 | 1 | 0 | 0 | 0 | 100 |
| P1290P | c.3870A>G | SNV | Exon23 | 10 | 3 | 0 | 0 | 0 | 100 |
| 4005+1G->A | c.3873+1G>A | SNV | Intron23 | 0 | 0 | 1 | 0 | 0 | 100 |
| 4016insT | c.3884_3885insT | DIV | Exon24 | 0 | 0 | 1 | 0 | 0 | 100 |
| T1299T | c.3897A>G | SNV | Exon24 | 3 | 0 | 0 | 0 | 0 | 100 |
| N1303K | c.3909C>G | SNV | Exon24 | 9 | 1 | 0 | 0 | 0 | 100 |
| Q1313X | c.3937C>T | SNV | Exon24 | 0 | 0 | 1 | 0 | 0 | 100 |
| G1349D | c.4046G>A | SNV | Exon25 | 0 | 1 | 0 | 0 | 0 | 100 |
| 4209TGTT>A
A | c.4077_4080delTGTTinsAA | DIV | Exon25 | 0 | 0 | 1 | 0 | 0 | 100 |
| CFTRdele22,2
3 | c.3964-78_4242+577del | Del | Intron24 | 1 | 0 | 1 | 0 | 0 | 100 |
| 4382delA | c.4251delA | DIV | Exon27 | 0 | 0 | 1 | 0 | 0 | 100 |
| Y1424Y | c.4272C>T | SNV | Exon27 | 6 | 2 | 0 | 0 | 0 | 100 |
| Genotype
(Common
Name
coordinate) | cDNA name | Variant
Type | CFTR
gene
region
(hg19) | Positive calls (Variants) | | No
Synthetic
Samples | Miscalls | Positive
Agreement | |
| Q1463Q | cDNA name
c.4389G>A | SNV | Exon27 | Clinical
Samples
150 | Cell
Line
Samples
32 | 0 | 0 | 100 | |
| | Total All Variants (PA)† | | | 2072 | | 3 | 4 | 99.66 | |
| | Total All WT (NA) | | | | 2600928 | 1 | 2** | >99.99 | |
| | Total All WT and Variants (OA) | | | | 2603000 | 4 | 6 | >99.99 | |

17

illumina*

:

..

::

.…"

18

・・

19

•

20 Confidential

. + T

1

20

21

22

amples were not retested

nowever indicated that the variant was in fact homozygous and incorrectly reported. MiSeqDx reported the variant as The Sanger report listed the P205S variant as heterozygous for the clinical sample. A review of the Sanger trac omozygous.

One of the discordant results was from the reproducibility study. The PolyTG/PolyT result for the sampl ncordant across all 18 replicates, but discordant with Sanger bi-directional sequencing.

The original synthetic heterozygous specimen was determined to be improperly prepared. When it was subsequen ested after it was re-prepared, using the same plasmid, it would be detected

ole, Sanger synthetic sample heterozygous for exon 8 was reported as heterozygous for the variant CFTR dele22, 23. Fur nvestigation revealed that this result was likely from low level contamination. Additionally, for a second samply ners could not fully detect the variant Q1463Q due to indels both upstream and downstream of the variant PA excluding PolyTG/PolyT calls was 100%

23

PolyTGPolyT Genotype	# Clinical Samples	# Cell Line Samples	# Synthetic Samples	# Miscalls	# No Calls^	% Accuracy
(TG)9(T)7/(TG)11(T)7	2	0	0	0	1	50.00
(TG)9(T)9/(TG)10(T)7	1	0	0	0	0	100
(TG)9(T)9/(TG)11(T)7	5	1	0	0	0	100
(TG)9(T)9/(TG)11(T)9	1	0	0	0	0	100
(TG)10(T)7/(TG)10(T)7	25	8	0	0	0	100
(TG)10(T)7/(TG)10(T)9	39	16	0	0	0	100
GPolyT Genotype	# Clinical Samples	# Cell Line Samples	# Synthetic Samples	# Miscalls	# No Calls^	% Accuracy
10(T)9/(TG)12(T)7	13	0	0	0	1	92.31
11(T)5/(TG)11(T)7	6	0	0	1	0	83.33
11(T)7/(TG)11(T)7	52	8	0	0	0	100
11(T)7/(TG)11(T)9*	2	1	0	3	0	0
11(T)7/(TG)12(T)5	2	0	0	0	0	100
11(T)7/(TG)12(T)7	37	3	0	0	0	100
11(T)9/(TG)12(T)7	3	0	0	0	0	100
12(T)7/(TG)12(T)7	2	2	0	0	0	100
Total		448		4	3	98.44

.

-.

, "

. ..

: .

: : : :

・・

e 2: PolyTG/PolyT variant accuracy for the MiSeqDx Cystic Fibrosis Clinical Sequencing A

←

onfidentia

ব্য

90.91

I

0

I

OL

G)10(T)7/(TG)12(T)

100

0

9

ש

G)10(T)9/(TG)10(T)

100

0

ഹ

G)10(T)9/(TG)11(T

100

0

07

92

G)10(T)9/(TG)11(T)

100

0

ಳ

G)10(T)9/(TG)11(T)

100

0

ਟ

ಕ

G)10(T)9/(TG)12(T

100

0

โ

"G)10(T)7/(TG)12(T)

100

0

ਟ

S)10(T)7/(TG)11(T

100

0

II

と

G)10(T)7/(TG)11(T

24

:

amples were not reteste

*One of the discordant results was from the reproducibility study. The PolyT result for the sample
was concordant across all 18 replicates, but discordant with Sanger bi-dire

25

Reproducibili

o oreators at each it for a total one one on the parel contand a mix of genomic DNA
iphoblastoid cell lines with the CFTT gene a well as ome leukory on energer on and order he reproductibility of the MiSeqDx Cystic Fibrosis Clinical Sequencing Assay was determined through a blir
sing 3 trial sites and 2 operators at each site. Two well characte

nple pass rate, defined as the number of samples passing QC metrics on the first attempt, was 99.7%. All result ed on initial testir he genotype-level PA for all variants including the PolyTwas 99.22% and excluding the PolyTG/PolyT v
ras 99.60%. The NA for all WT was 99.70% and the OA for all reported pos A was 97.83%

26

MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

illumina:

| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | | Agreeing Calls | | | | Total #
(All Sites) | | %
Agreement |
|-----------------------|---------------------------------------|------------------|---------------------------|-------------------------------|--------------------------|--------------------------|--------------------------|---------------------------------------|------------------------------------|------------------------------------------|----------------|
| | | | Per Site | All
Sites | Site 1 | Site 2 | Site 3 | No
Calls | Miscalls | | |
| 1 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 1 | c.1646G>A | S549N | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 1 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 2 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 2 | c.1581A>G | E527E | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 2 | c.1680-1G>A | 1812-1 G>A | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 2 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 2 | c.312delA | 444delA | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 2 | c.3870A>G | P1290P | 6 | 18 | 6 | 5 | 6 | 0 | 1 | 94.44 | |
| Sample | HGVS Name (or Location if no HGVS) | Mutation Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No Calls" | Total #
(All Sites)
Miscalls | %
Agreement | |
| | | | | | Site 1 | Site 2 | Site 3 | | | | |
| 2 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 3 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 3 | c.1477C>T | Q493X | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 3 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 3 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 3 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 4 | c.1408G>A | V470M | 6 | 18 | 5 | 6 | 6 | 1 | 0 | 94.44 | |
| 4 | c.1521_1523delCTT | F508del | 6 | 18 | 5 | 6 | 6 | 1 | 0 | 94.44 | |
| 4 | c.2052delA | 2184delA | 6 | 18 | 5 | 6 | 6 | 1 | 0 | 94.44 | |
| 5 | c.1408G>A | V470M | 6 | 18 | 6 | 5 | 6 | 1" | 0 | 94.44 | |
| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results
Per Site | Total Results
All
Sites | Agreeing Calls | | | Total #
(All Sites)
No
Calls | Total #
(All Sites)
Miscalls | Total #
(All Sites)
%
Agreement | |
| | | | | | Site 1 | Site 2 | Site 3 | | | | |
| 5 | c.224G>A | R75Q | 6 | 18 | 6 | 5 | 6 | 11 | 0 | 94.44 | |
| 5 | c.2562T>G | T854T | 6 | 18 | 6 | 5 | 6 | 11 | 0 | 94.44 | |
| 5 | c.3472C>T | R1158X | 6 | 18 | 6 | 5 | 6 | 11 | 0 | 94.44 | |
| 5 | c.366T>A | Y122X | 6 | 18 | 6 | 5 | 6 | 11 | 0 | 94.44 | |
| 5 | c.625G>T | A209S | 6 | 18 | 6 | 5 | 6 | 11 | 0 | 94.44 | |
| 6 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 6 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 6 | c.2051_2052delAAinsG | 2183AA>G | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 7 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 7 | c.223C>T | R75X | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| % | greemer | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 94.44 |
| (All Sites
Total # | Miscalls | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | i I |
| | Callsª
No | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | Site 3 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
| greeing Call | Site 2 | 9

      | 9                         | 9                             | 9                        | 9                        | 9                        | 9                                     | 9                                  | 9                                        | g              |

| | Site 1 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
| otal Results | Sites
All | 18 | 81 | 18 | 81 | 81 | 81 | 81 | 81 | 18 | 81 |
| | Per Site | !
9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 | 9 |
| Iutation | Name | 1854T | V470M | 507del | F508de | T854T | Q1463Q | V470M | F508de | T854T | V1282X |
| GVS Name | cation if no HGV: | 2562T>G | c.1408G> | 519_1521delAT | 1521_1523delCT | .2562T>G | .4389G>/ | c.1408G> | 1521_1523delC | 2562T>G | 3846G> |
| Sample | | 1 | 8 | 8 | 8 | 8 | 8 | 6 | б | 6 | б |
| | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | Agreeing Calls | | | Total #
(All Sites) | | %
Agreement | | |
| ample | | | Per Site | All
Sites | Site 1 | Site 2 | Site 3 | No
Calls | Miscalls | | |
| 9 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 10 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 10 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 10 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 10 | c.3140-26A>G | 3272-26A>G | 6 | 18 | 6 | 5 | 6 | 0 | 1' | 94.44 | |
| 10 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 11, 39 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 11, 39 | c.1521_1523delCTT | F508del | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 11, 39 | c.2002C>T | R668C | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 11, 39 | c.2562T>G | T854T | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | Agreeing Calls | | | Total #
(All Sites) | | | %
Agreement | |
| | | | Per Site | All
Sites | Site 1 | Site 2 | Site 3 | No
Calls | Miscalls | | |
| 11, 39 | c.3717+12191C>T | 3849+10kbC>T | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 11, 39 | c.4389G>A | Q1463Q | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 12, 40 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 12, 40 | c.2562T>G | T854T | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 12, 40 | c.2988+1G>A | 3120+1G>A | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 12, 40 | c.4389G>A | Q1463Q | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 12, 40 | c.489+1G>T | 621+1G>T | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 13 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 13 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 13 | c.178G>T | E60X | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | Agreeing Calls | | | Total #
(All Sites) | | %
Agreement | | |
| | | | Per Site | All
Sites | Site 1 | Site 2 | Site 3 | No
Calls" | Miscalls | | |
| 13 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 14 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 14 | c.1584G>A | E528E | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 14 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 14 | c.3302T>A | M1101K | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 15 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 15 | c.1584G>A | E528E | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 15 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 15 | c.3302T>A | M1101K | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 16 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| Sample | HGVS Name (or Location if no HGVS) | Mutation Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No Calls | Total #
(All Sites)
Miscalls | %
Agreement | |
| | | | | | Site 1 | Site 2 | Site 3 | | | | |
| 16 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 16 | c.3080T>C | I1027T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 17, 41 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 17, 41 | c.1521_1523delCTT | F508del | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 17, 41 | c.3528delC | 3659delC | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 18, 42 | 117120145 | 117120145 | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 18, 42 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 18, 42 | c.1521_1523delCTT | F508del | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 18, 42 | c.350G>A | R117H | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 19 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | | Agreeing Calls | | | Total #
(All Sites) | | | %
Agreement |
| | | | Per Site | All
Sites | Site 1 | Site 2 | Site 3 | No
Calls | Miscalls | | |
| 19 | c.489+1G>T | 621+1G>T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 19 | c.579+1G>T | 711+1G>T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 20, 43 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 20, 43 | c.254G>A | G85E | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 20, 43 | c.489+1G>T | 621+1G>T | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 21, 44 | c.1364C>A | A455E | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 21, 44 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 21, 44 | c.1521_1523delCTT | F508del | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 22 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 22 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| Sample | HGVS Name (or Location if no HGVS) | Mutation Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls
Site 1 | Agreeing Calls
Site 2 | Agreeing Calls
Site 3 | Total #
No Calls | Total #
Miscalls | Total #
(All Sites)
% Agreement | |
| 22 | c.1679G>C | R560T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 22 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 22 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 23 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 23 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 23 | c.3276C>A | Y1092X (C>A) | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 24, 45 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 24, 45 | c.3909C>G | N1303K | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 24, 45 | c.4046G>A | G1349D | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 25 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| | | | | | | | | | Total # | | |
| Sample | IGVS Name | lutation | otal Result | | | greeing Call | | | (All Sites | 90 | |
| | ation if no HGV | Name | Per Site | Sites
All | Site 1 | Site 2 | Site 3 | Callsª
No | Miscalls | greeme | |
| ਤੀ | .1624G> | G542X | 9 | 18 | 9 | 9 | 9 | 0 | 0 | 001 | |
| 97 | 1712014 | 17120141 | 9 | 18 | 9 | 9 | 9 | 0 | 0 | 100 | |
| 97 | c.1408G>A | /470M | 9 | 18 | 9 | 9 | 9 | 0 | 0 | 100 | |
| 97 | c.1624G> | G542X | 9 | 81 | 9 | 9 | 9 | 0 | 0 | 100 | |
| 27, 46 | .1408G>r | /470M | टा | 98 | ਟਾ | टा | 12 | 0 | 0 | 100 | |
| 27, 46 | c.1652G> | G551D | ਟ I | 9€ | ਟਾ | 12 | 12 | 0 | 0 | 100 | |
| 27, 46 | c.1657C>1 | R553X | ਟ I | 98 | ਟ I | ਟਾ | 12 | 0 | 0 | 100 | |
| 27, 46 | 2562T>G | T854T | ਟ I | 96 | ਟ। | ਟ L | 12 | 0 | 0 | 100 | |
| 27, 46 | :4389G>A | Q1463Q | ਟ I | 9€ | ਟਾ | ਟਾ | 12 | 0 | 0 | 100 | |
| 87 | .1408G> | /470M | 9 | 81 | 9 | 9 | 9 | 0 | 0 | 100 | |
| Sample | HGVS Name (or Location if no HGVS) | Mutation Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No Calls | Total #
(All Sites)
Miscalls | %
Agreement | |
| 28 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 28 | c.3717+12191C>T | 3849+10kbC>T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 28 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 29 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 29 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 29 | c.91C>T | R31C | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 30 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 30 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 30 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 30 | c.3485G>T | R1162L | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| Sample | HGVS Name (or Location if no HGVS) | Mutation Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No Calls | Total #
(All Sites)
Miscalls | % Agreement | |
| | | | | | Site 1 | Site 2 | Site 3 | | | | |
| 30 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 31 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 31 | c.1585-1G>A | 1717-1G>A | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 31 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 31 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 32 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 32 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 32 | c.3484C>T | R1162X | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 32 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 33 | c.1040G>C | R347P | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |

27 Confidential

...
... : : . : •

27

.

28

:

29

. .

30

:

31

. Li

،

32

Confidential

33

Confidential

34

35

,;'

36

37

38 Confidential

:『

38

39

| | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | Agreeing Calls | | | Total #
(All Sites) | | %
Agreement | | |
|--------|---------------------------------------|------------------|---------------------------|-------------------------------|--------------------------|--------------------------|--------------------------|---------------------------------------|------------------------------------|----------------|----------------|
| ample | | | Per Site | Site 1 | Site 2 | Site 3 | No
Calls | Miscalls | | | |
| 33 | c.1408G>A | V470M | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| 33 | c.1652G>A | G551D | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| 33 | c.2562T>G | T854T | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| 33 | c.4272C>T | Y1424Y | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| 33 | c.4389G>A | Q1463Q | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| 34 | c.1000C>T | R334W | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| 34 | c.3368-2A>T | c.3368-2A>T | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| 35 | c.1523T>G | F508C | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| 36 | c.254G>A | G85E | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| 36 | c.3454G>C | D1152H | 6 | 6 | 6 | 6 | 0 | 0 | 100 | | |
| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No Calls | Total #
(All Sites)
Miscalls | %
Agreement | |
| | | | | | Site 1 | Site 2 | Site 3 | | | | |
| 37 | c.1007T>A | I336K | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 37 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 37 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 37 | c.3705T>G | S1235R | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 38 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 38 | c.1727G>C | G576A | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 38 | c.2002C>T | R668C | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 38 | c.2057C>A | S686Y | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 38 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 38 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results
Per Site | Total Results
All
Sites | Agreeing Calls
Site 1 | Agreeing Calls
Site 2 | Agreeing Calls
Site 3 | Total #
(All Sites)
No
Calls | Total #
(All Sites)
Miscalls | %
Agreement | |
| 47, 85 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 47, 85 | c.2562T>G | T854T | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 47, 85 | c.2657+5G>A | 2789+5G>A | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 47, 85 | c.4389G>A | Q1463Q | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 48, 86 | 5940_273+10250del21kb
c.54- | CFTRdele2,3 | 12 | 36 | 12 | 11 | 12 | 1 | 0 | 97.22 | |
| 48, 86 | c.1408G>A | V470M | 12 | 36 | 12 | 11 | 12 | 1 | 0 | 97.22 | |
| 48, 86 | c.1521_1523delCTT | F508del | 12 | 36 | 12 | 11 | 12 | 1 | 0 | 97.22 | |
| 49, 87 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 49, 87 | c.1521_1523delCTT | F508del | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| Sample | HGVS Name (or Location if no HGVS) | Mutation Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites) | | | %
Agreement |
| | | | | | Site 1 | Site 2 | Site 3 | No Calls | Miscalls | | |
| 49, 87 | c.1766+1G>A | 1898+1G>A | 12 | 36 | 12 | 12 | 12 | 0 | 0 | | 100 |
| 50, 88 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | | 100 |
| 50, 88 | c.220C>T | R74W | 12 | 36 | 12 | 12 | 12 | 0 | 0 | | 100 |
| 50, 88 | c.2562T>G | T854T | 12 | 36 | 12 | 12 | 12 | 0 | 0 | | 100 |
| 50, 88 | c.3808G>A | D1270N | 12 | 36 | 12 | 12 | 12 | 0 | 0 | | 100 |
| 51, 89 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | | 100 |
| 51, 89 | c.1521_1523delCTT | F508del | 12 | 36 | 12 | 12 | 12 | 0 | 0 | | 100 |
| 51, 89 | c.2012delT | 2143delT | 12 | 36 | 12 | 12 | 12 | 0 | 0 | | 100 |
| 52 | c.3744delA | 3876delA | 6 | 18 | 6 | 6 | 6 | 0 | 0 | | 100 |
| 53, 90 | c.3773_3774insT | 3905insT | 12 | 36 | 12 | 12 | 12 | 0 | 0 | | 100 |
| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | | Agreeing Calls | | | Total #
(All Sites) | | %
Agreement | |
| | | | Per Site | All
Sites | Site 1 | Site 2 | Site 3 | No
Calls" | Miscalls | | |
| 54, 91 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 54, 91 | c.262_263delTT | 394delTT | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 55,92 | c.1408G>A | V470M | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 55,92 | c.1519A>G | I507V | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 55,92 | c.1521_1523delCTT | F508del | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 55,92 | c.2562T>G | T854T | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 55,92 | c.3080T>C | I1027T | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 55,92 | c.4389G>A | Q1463Q | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 | |
| 56 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 56 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |

Confidential

40

.

..

41

:

42

T

Confidential

43

44

| HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | Agreeing Calls | Total #
(All Sites) | %
Agreement | | | | | |
|---------------------------------------|---------------------------------------|------------------|---------------------------|----------------------------|----------------|--------|-------------|------------------------------------|------------------------------------|----------------|
| | | Per Site | All
Sites | Site 1 | Site 2 | Site 3 | No
Calls | Miscalls | | |
| c.3154T>G | F1052V | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| 117120141 | 117120141 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| c.3209G>A | R1070Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| c.2991G>C | L997F | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 | |
| | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No Calls | Total #
(All Sites)
Miscalls | %
Agreement |
| Sample | | | | | Site 1 | Site 2 | Site 3 | | | |
| 59 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 59 | c.3205G>A | G1069R | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 60 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 60 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 60 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 60 | c.617T>G | L206W | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 61 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 61 | c.2260G>A | V754M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 61 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 62 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |

45

46

Sample	HGVS Name (or Location if no HGVS)	Mutation Name	Total Results Per Site	Total Results All Sites	Agreeing Calls			Total # (All Sites) No Calls	Total # (All Sites) Miscalls	% Agreement
					Site 1	Site 2	Site 3
62	c.2562T>G	T854T	6	18	6	6	6	0	0	100
62	c.988G>T	G330X	6	18	6	6	6	0	0	100
64	c.1040G>A	R347H	6	18	6	6	6	0	0	100
64	c.1408G>A	V470M	6	18	6	6	6	0	0	100
64	c.2562T>G	T854T	6	18	6	6	6	0	0	100
64	c.4389G>A	Q1463Q	6	18	6	6	6	0	0	100
65	c.948delT	1078delT	6	18	6	6	6	0	0	100
66	c.1408G>A	V470M	6	18	6	6	6	0	0	100
66	c.1521_1523delCTT	F508del	6	18	6	6	6	0	0	100
66	c.532G>A	G178R	6	18	6	6	6	0	0	100

47

Sample	HGVS Name (or Location if no HGVS)	Mutation Name	Total Results Per Site	Total Results All Sites	Agreeing Calls Site 1	Agreeing Calls Site 2	Agreeing Calls Site 3	% Agreement
67	c.1408G>A	V470M	6	18	6	6	6	100
67	c.1647T>G	S549R (c.1647T>G)	6	18	6	6	6	100
68	c.1408G>A	V470M	6	18	6	6	6	100
68	c.1646G>A	S549N	6	18	6	6	6	100
68	c.2562T>G	T854T	6	18	6	6	6	100
68	c.4389G>A	Q1463Q	6	18	6	6	6	100
69	c.2506G>T	D836Y	6	18	6	6	6	100
69	c.2537G>A	W846X	6	18	6	6	6	100
70	c.1408G>A	V470M	6	18	6	6	6	100

48

| Sample | HGVS Name (or Location if no HGVS) | Mutation Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No Calls | Total #
(All Sites)
Miscalls | %
Agreement |
|--------|------------------------------------|---------------|---------------------------|----------------------------|----------------|-------------|-------------|------------------------------------|------------------------------------|----------------|
| 70 | c.2562T>G | T854T | 6 | 18 | Site 1
6 | Site 2
6 | Site 3
6 | 0 | 0 | 100 |
| 70 | c.3485G>T | R1162L | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 70 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 71 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 71 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 71 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 71 | c.274G>T | E92X | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 71 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 72 | c.1022_1023insTC | 1154insTC | 6 | 18 | 6 | 6 | 5 | 1 | 0 | 94.44 |
| 72 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 5 | 1 | 0 | 94.44 |

Confidential

.

49

| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites) | | %
Agreement |
|--------|---------------------------------------|------------------|---------------------------|----------------------------|----------------|--------|--------|------------------------|----------|----------------|
| | | | | | Site 1 | Site 2 | Site 3 | No
Calls | Miscalls | |
| 72 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 5 | 1 | 0 | 94.44 |
| 72 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 5 | 1 | 0 | 94.44 |
| 72 | c.489+1G>T | 621+1G>T | 6 | 18 | 6 | 6 | 5 | 1 | 0 | 94.44 |
| 73 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 73 | c.1624G>T | G542X | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 73 | c.1826A>G | H609R | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 74 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 5 | 0 | 1 | 94.44 |
| 74 | c.1429C>T | P477S | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 74 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 75 | c.1408G>A | V470M | 6 | 18 | 6 | 5 | 6 | 11 | 0 | 94.44 |

Confidential

50

| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No
Calls | Total #
(All Sites)
Miscalls | %
Agreement |
|--------|---------------------------------------|------------------|---------------------------|----------------------------|----------------|--------|--------|---------------------------------------|------------------------------------|----------------|
| | | | | | Site 1 | Site 2 | Site 3 | | | |
| 75 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 5 | 6 | 1a | 0 | 94.44 |
| 75 | c.1721C>A | P574H | 6 | 18 | 6 | 5 | 6 | 1a | 0 | 94.44 |
| 76 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 76 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 76 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 76 | c.425delT | F143LfsX10 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 76 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 77 | c.1364C>A | A455E | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 77 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 77 | c.489+1G>T | 621+1G>T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |

Confidential

51

| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | Agreeing Calls | Total #
(All Sites) | %
Agreement | | | | |
|--------|---------------------------------------|------------------|---------------|----------------|------------------------|----------------|--------------|-------------|----------|-----|
| | | | Per Site | Site 1 | Site 2 | Site 3 | All
Sites | No
Calls | Miscalls | |
| 78 | c.1408G>A | V470M | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |
| 78 | c.1581A>G | E527E | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |
| 78 | c.1680-1G>A | 1812-1 G>A | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |
| 78 | c.2562T>G | T854T | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |
| 78 | c.312delA | 444delA | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |
| 78 | c.3870A>G | P1290P | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |
| 78 | c.4389G>A | Q1463Q | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |
| 79 | c.1408G>A | V470M | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |
| 79 | c.220C>T | R74W | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |
| 79 | c.2562T>G | T854T | 6 | 6 | 6 | 6 | 18 | 0 | 0 | 100 |

Confidential

52

| Sample | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results | Agreeing Calls | | | Total #
(All Sites) | | | %
Agreement |
|--------|---------------------------------------|------------------|---------------|----------------|--------|--------|------------------------|--------------|----------|----------------|
| | | | Per Site | All
Sites | Site 1 | Site 2 | Site 3 | No
Calls" | Miscalls | |
| 79 | c.3808G>A | D1270N | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 80 | 117120141 | 117120141 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 80 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 80 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 80 | c.1657C>T | R553X | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 80 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 81 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 81 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 81 | c.1652G>A | G551D | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 81 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |

53

| Sample | HGVS Name (or Location if no HGVS) | Mutation Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No Calls" | Total #
(All Sites)
Miscalls | Total #
(All Sites)
% Agreement |
|--------|------------------------------------|---------------|---------------------------|----------------------------|----------------|--------|--------|-------------------------------------|------------------------------------|---------------------------------------|
| | | | | | Site 1 | Site 2 | Site 3 | | | |
| 81 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 82 | c.1040G>C | R347P | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 82 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 82 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 82 | c.4272C>T | Y1424Y | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 83 | 117120145 | 117120145 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 83 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 83 | c.1521_1523delCTT | F508del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 83 | c.350G>A | R117H | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 84 | c.1408G>A | V470M | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |

54

| | HGVS Name (or
Location if no HGVS) | Mutation
Name | Total Results
Per Site | Total Results
All Sites | Agreeing Calls | | | Total #
(All Sites)
No
Calls | Total #
(All Sites)
Miscalls | Total #
(All Sites)
%
Agreement |
|--------|-------------------------------------------------------------------------|------------------|---------------------------|----------------------------|----------------|---------|---------|---------------------------------------|------------------------------------|------------------------------------------|
| Sample | | | | | Site 1 | Site 2 | Site 3 | | | |
| 84 | c.1519_1521delATC | I507del | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 84 | c.2562T>G | T854T | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| 84 | c.4389G>A | Q1463Q | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| | Total All Variants (PA)* (including the
PolyTGPolyT data in Table 4) | | 2580 | 7740 | 2562 | 2553 | 2565 | 37 | 23 | 99.22 |
| | Total All WT (NA) | | 2871132 | 8613396 | 2865930 | 2855526 | 2865932 | 26006 | 2 | 99.70 |
| | Total All WT and variants (OA) | | 2873712 | 8621136 | 2868492 | 2858079 | 2868497 | 26043 | 25 | 99.70 |

Samples were not retest

¶ One replicate each of samples 5 and 75 had a 0% call rate. Further investigation indicated that the samples had likely no een added to the sample plate prior to library preparation

Upon review, samples 9 and 10 were likely switched by the operator prior to library preparati

55

.

Excluding PolyTG/PolyT variants, the PA was 99.60%

...

:

.

—

56

SSAV
4
1
Seatlencing


t

Clinical Sequencing
194
TOTOSIS :
11 9 8 8 8 0 8 0 8 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

99.89
ﺑ
1
UC
l

| ﭘ
1
1 |
| M1400
. |
| |
| CHE ITELUCULA |
| . |
| ﻟﻠﻘﺎﻧﻮﻥ ﺍﻟﻤﺴﺎﺣﺔ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘ |
| |
| |
| |
| |
| Kontod1101h111711111111
" In VIVUHUUUIII
11
to the |
| Parcel
1
. In course for a |
| ﻟ
TODA |
| ﻟﻤﺴﺎﺣﺔ ﺍﻟﻤﺴﺎﺣﺔ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮ
1 |
| |
| ﻟ
S
t
ﺎ ﺍﻟﻤﺴﺎﺣﺔ ﺍﻟﻤﺴﺎﺣﺔ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤ |
| 4.
4
: |
| Table |
| |
| |
| |

.

·

.

. ·

:

···

.

| Panel | Sample | Genotype | Total
Results | Agreeing Calls | | | Total #
(All Sites) | | %
Agreement | |
|-------|--------|-----------------------|------------------|------------------------|-----------|-----------|------------------------|-------------|----------------|-------|
| | | | Per
Site | Sites | Site
1 | Site
2 | Site
3 | No
Calls | Miscalls | |
| A | 1 | (TG)12(T)7/(TG)12(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 2 | (TG)10(T)9/(TG)10(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 3 | (TG)10(T)7/(TG)10(T)9 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 4 | (TG)10(T)9/(TG)11(T)7 | 6 | 18 | 5 | 6 | 6 | 1 | 0 | 94.44 |
| A | 5 | (TG)10(T)7/(TG)11(T)7 | 6 | 18 | 6 | 5 | 6 | 1 | 0 | 94.44 |
| A | 6 | (TG)10(T)9/(TG)10(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 7 | (TG)10(T)9/(TG)11(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 8 | (TG)10(T)7/(TG)10(T)9 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 9 | (TG)10(T)9/(TG)10(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 10 | (TG)10(T)9/(TG)10(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| | | Total
Results | Agreeing Calls | Total #
(All Sites) | | | | | | |
| A | 11, 39 | (TG)10(T)9/(TG)10(T)7 | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 |
| A | 12, 40 | (TG)10(T)9/(TG)11(T)7 | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 |
| A | 13 | (TG)10(T)9/(TG)11(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 14 | (TG)10(T)7/(TG)11(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 15 | (TG)10(T)7/(TG)11(T)7 | 6 | 18 | 6 | 5 | 6 | 1 | 0 | 94.44 |
| A | 16 | (TG)10(T)9/(TG)10(T)9 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 17, 41 | (TG)10(T)9/(TG)11(T)7 | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 |
| A | 18, 42 | (TG)10(T)9/(TG)12(T)5 | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 |
| A | 19 | (TG)10(T)9/(TG)11(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |
| A | 20, 43 | (TG)10(T)9/(TG)11(T)7 | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 |
| A | 21, 44 | (TG)10(T)9/(TG)10(T)9 | 12 | 36 | 12 | 12 | 12 | 0 | 0 | 100 |
| A | 22 | (TG)10(T)9/(TG)10(T)7 | 6 | 18 | 6 | 6 | 6 | 0 | 0 | 100 |

57

ﺮ ﺗ

··

58

| | | Total
Results | Agreeing Calls | Total #
(All Sites) | | |
|---|--------|------------------------|----------------|------------------------|---|-------|
| A | 23 | (TG)10(T)9/(TG)11(T)7 | 6 | 6 | 0 | 100 |
| A | 24, 45 | (TG)10(T)9/(TG)11(T)7 | 12 | 12 | 0 | 100 |
| A | 25 | (TG)10(T)9/(TG)10(T)9 | 6 | 6 | 0 | 100 |
| A | 26 | (TG)10(T)9/(TG)11(T)7 | 6 | 6 | 0 | 100 |
| A | 27, 46 | (TG)10(T)7/(TG)11(T)7 | 11 | 12 | 1 | 97.22 |
| A | 28 | (TG)10(T)7/(TG)10(T)7 | 6 | 6 | 0 | 100 |
| A | 29 | (TG)10(T)7/(TG)12(T)7 | 4 | 4 | 0 | 77.77 |
| A | 30 | :(TG)10(T)9/(TG)10(T)7 | 6 | 6 | 0 | 100 |
| A | 31 | (TG)10(T)7/(TG)11(T)7 | 6 | 6 | 0 | 100 |
| A | 32 | (TG)10(T)7/(TG)10(T)7 | 6 | 6 | 0 | 100 |
| A | 33 | (TG)10(T)7/(TG)11(T)7 | 5 | 6 | 0 | 94.44 |
| A | 34 | (TG)11(T)7/(TG)12(T)7 | 6 | 6 | 0 | 100 |

59

. .

| | Total
Results | Agreeing Calls | Total #
(All Sites) |
|---------------------------------|------------------|----------------|------------------------|
| 35
(TG)11(T)7/(TG)11(T)7 | 18 | 6 | 100 |
| 36
(TG)11(T)7/(TG)11(T)7 | 18 | 6 | 100 |
| 37
(TG)11(T)7/(TG)12(T)7 | 18 | 6 | 100 |
| 38
(TG)10(T)7/(TG)11(T)7 | 18 | 6 | 100 |
| 47, 85
(TG)10(T)7/(TG)10(T)7 | 36 | 12 | 100 |
| 48, 86
(TG)10(T)9/(TG)11(T)7 | 36 | 11 | 94.44 |
| 49, 87
(TG)10(T)9/(TG)11(T)7 | 36 | 12 | 100 |
| 50, 88
(TG)10(T)9/(TG)11(T)7 | 36 | 12 | 100 |
| 51, 89
(TG)10(T)9/(TG)10(T)9 | 36 | 12 | 100 |
| 52
(TG)11(T)7/(TG)11(T)7 | 18 | 6 | 100 |
| 53, 90
(TG)11(T)7/(TG)11(T)7 | 36 | 12 | 100 |
| 91, 54
(TG)10(T)9/(TG)11(T)7 | 36 | 12 | 100 |

t

60

| | Total
Results | Agreeing Calls | Total #
(All Sites) | | | | | |
|-----------------------|------------------|-----------------------|------------------------|----------------|----|------------------------|-------|-------|
| B | 92, 55 | (TG)10(T)9/(TG)10(T)7 | 12 | 12 | 12 | 0 | 0 | 100 |
| B | 56 | (TG)10(T)7/(TG)10(T)9 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 57 | (TG)12(T)7/(TG)12(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 58 | (TG)10(T)9/(TG)10(T)9 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 59 | (TG)11(T)7/(TG)12(T)7 | 6 | 5 | 6 | 1 | 0 | 94.44 |
| B | 60 | (TG)9(T)9/(TG)11(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 61 | (TG)10(T)9/(TG)11(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 62 | (TG)10(T)7/(TG)11(T)7 | 6 | 5 | 6 | 1 | 0 | 94.44 |
| B | 63 | (TG)11(T)7/(TG)11(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 64 | (TG)10(T)7/(TG)11(T)7 | 6 | 5 | 6 | 1 | 0 | 94.44 |
| B | 65 | (TG)11(T)7/(TG)11(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 66 | (TG)10(T)9/(TG)11(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| | | Total
Results | | Agreeing Calls | | Total #
(All Sites) | | |
| B | 67 | (TG)11(T)7/(TG)11(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 68 | (TG)10(T)7/(TG)11(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 69 | (TG)11(T)7/(TG)11(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 70 | (TG)10(T)7/(TG)10(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 71 | (TG)10(T)9/(TG)11(T)7 | 6 | 6 | 6 | 0 | 0 | 100 |
| B | 72 | (TG)10(T)7/(TG)10(T)9 | 6 | 5 | 6 | 5 | 2 | 88.88 |
| B | 73 | (TG)10(T)9/(TG)11(T)7 | 6 | 6 | 6 | 6 | 0 | 100 |
| B | 74 | (TG)10(T)9/(TG)11(T)7 | 6 | 6 | 6 | 6 | 0 | 100 |
| B | 75 | (TG)10(T)7/(TG)10(T)9 | 6 | 6 | 5 | 6 | 1 | 94.44 |
| B | 76 | (TG)10(T)7/(TG)10(T)9 | 6 | 6 | 6 | 6 | 0 | 100 |
| B | 77 | (TG)10(T)9/(TG)10(T)9 | 6 | 6 | 6 | 6 | 0 | 100 |
| B | 78 | (TG)10(T)7/(TG)10(T)9 | 6 | 5 | 6 | 6 | 1 | 94.44 |
| | Total
Results | Agreeing Calls | Total #
(All Sites) | | | | | |
| (TG)10(T)7/(TG)11(T)7 | 18 | 6 | 0 | | | | 100 | |
| (TG)11(T)7/(TG)11(T)9 | 18 | 0 | 18 | | | | 0 | |
| (TG)10(T)7/(TG)10(T)9 | 18 | 6 | 0 | | | | 100 | |
| (TG)10(T)9/(TG)11(T)7 | 18 | 6 | 0 | | | | 100 | |
| (TG)10(T)9/(TG)12(T)5 | 18 | 6 | 0 | | | | 100 | |
| (TG)10(T)7/(TG)10(T)7 | 18 | 6 | 0 | | | | 100 | |
| olyT Variants (PA) | 1656 | 537 | 17 | | | | 97.83 | |

... |

: .

61

62

·

.

, ·

..

.. -

.

·

··· . ...

・

ﺘ

·

.

63

DNA Extraction

Three commonly used, commercially available extraction methods representing magnetic bead extraction, alcohol precipitation and silica filter column isolation methods, were evaluated using K2EDTA anti-coagulated whole blood. A total of 14 blood samples were used during the study; two were wild type, while the remaining samples carried unique genotypes representing 9 different variants, including both common and rare variants. For the polyTG/polyT variation, samples with (T)5-9 and (TG)10-12 were included. 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 | 168 | >99.99% | >99.99% | 100% |
| Silica Filter
Column Isolation | 168 | >99.99% | >99.99% | 100% |
| Magnetic Bead
Extraction | 168 | >99.99% | >99.99% | 100% |

*Percent of samples having call rate of >99% in first run.

DNA input

The DNA input range of the Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay was evaluated by performing a serial dilution study using 14 representative DNA samples containing 16 unique CF 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, 25 ng, 10 ng, 5 ng, and 1 ng). For determination of accuracy, sample genotypes were compared to bidirectional Sanger sequencing

64

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 at least 20 replicates per DNA input level for each sample (n= 4 x 20=80 samples), while the lower bound of 25 ng was tested with 14 samples, 20 replicates for each sample (n=14 x 20=280 samples). The accuracy and sample first pass rate was 100% at all DNA input levels.

Interfering Substances

To assess the impact of interfering substances on the Illumina MiSeqDx Cystic Fibrosis System, the performance of the assay was evaluated in the presence and absence of potential interferents. Sixteen whole blood specimens having unique genotypes were included in the study. Four endogenous interfering substances (bilirubin, cholesterol, hemoglobin, and triglycerides) were tested by spiking them into blood specimens prior to DNA extraction. The concentration limits for each substance is shown in the table below. Additionally, to assess interference resulting from blood collection (short draw), EDTA was spiked into blood samples, and to assess interference resulting from sample preparation, the final wash buffer from a silica filter column isolation method was added to purified genomic DNA.

The MiSeqDx Cystic Fibrosis Clinical Sequencing Assay achieved 100% call rate for all samples tested, and 100% reproducibility in genotype calls between samples in the presence and absence of interfering substances. No interference was observed for any of the potential interferents.

To assess the impact of multiplexing index primer interference, a cross contamination study using two samples, each with unique homozygous genotypes at 4 different genomic positions, and two respective index primers was performed. NO change in variant calling was observed with contamination levels