(328 days)
Not Found
No
The document describes a genetic sequencing assay and associated instrument and software for detecting specific gene variants. It details the laboratory procedures, performance studies, and metrics like accuracy and reproducibility, which are standard for molecular diagnostic devices. There is no mention of AI, ML, or any techniques typically associated with these technologies for data analysis or interpretation. The analysis software is described as performing data analysis, but this is a general term and doesn't imply AI/ML. The interpretation is explicitly stated to be done by a board-certified clinical molecular geneticist.
No.
This device is an in vitro diagnostic system used to detect genetic mutations for carrier screening and diagnosis of cystic fibrosis, not to treat the condition.
Yes
Explanation: The "Intended Use / Indications for Use" section explicitly states that the system is a "qualitative in vitro diagnostic system" and is intended for "confirmatory diagnostic testing of newborns and children, and as an initial test to aid in the diagnosis of individuals with suspected cystic fibrosis."
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 indicates the device includes hardware components (reagents, consumables, instrument) in addition to software.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use/Indications for Use: The description explicitly states it is a "qualitative in vitro diagnostic system" and is used to detect mutations and variants in genomic DNA isolated from human peripheral whole blood specimens. It also specifies its intended use for carrier screening, confirmatory diagnostic testing, and aiding in diagnosis.
- Device Description: The description details the components and process for analyzing biological samples (genomic DNA from blood) to provide diagnostic information.
- Anatomical Site: It specifies the use of "human peripheral whole blood specimens," which are biological samples.
- Performance Studies: The document includes detailed performance studies (Accuracy, Reproducibility, DNA Extraction, DNA Input, Interfering Substances, Sample Indexing) which are typical for validating an IVD device.
- Key Metrics: The performance studies report metrics like Positive Agreement, Negative Agreement, Overall Agreement, Call Rate, Sample First Pass Rate, and Reproducibility, which are standard for evaluating the performance of diagnostic tests.
- Predicate Device(s): The mention of predicate devices (K083845; Luminex xTAG® Cystic Fibrosis 60 Kit v2) further indicates that this device is being compared to other legally marketed IVD devices.
All of these points align with the definition and characteristics of an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
The Illumina MiSeqDx Cystic Fibrosis 139-Variant Assay is a qualitative in vitro diagnostic system used to simultaneously detect 139 clinically relevant cystic fibrosis disease-causing mutations and variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in genomic DNA isolated from human peripheral whole blood specimens. The variants include those recommended in 2004 by the American College of Medical Genetics (ACMG) and in 2011 by the American College of Obstetricians and Gynecologists (ACOG). The test is intended for carrier screening in adults of reproductive age, in confirmatory diagnostic testing of newborns and children, and as an initial test to aid in the diagnosis of individuals with suspected cystic fibrosis. The results of this test are intended to be interpreted by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available laboratory and clinical information. This test is not indicated for use for newborn screening, fetal diagnostic testing, pre-implantation testing, or for stand-alone diagnostic purposes. The test is intended to be used on the Illumina MiSeqDxTM Instrument.
Product codes (comma separated list FDA assigned to the subject device)
PFR
Device Description
The Illumina MiSeqDx Cystic Fibrosis 139-Variant 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
Adults of reproductive age, newborns, and children.
Intended User / Care Setting
Board-certified clinical molecular geneticist or equivalent. Not Found for Care Setting.
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
The accuracy of the Illumina MiSeqDx Cystic Fibrosis 139-Variant 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 the rare variants, and contained anywhere from 1 to 9 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.
The genotyping results for 137 SNA/small InDel sites, including the PolyTG/Poly T region were compared to Sanger bi-directional sequence analysis. A PCR based assay was 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 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 sample.
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Accuracy Study:
- Study Type: Accuracy study
- Sample Size: 500 samples
- Data Sources: Clinical gDNA specimens (355), commercial cell line specimens (11), cell line samples from reproducibility study (68), clinical samples from extraction method evaluation (14), synthetic plasmid samples (52).
- Key Results:
- Genotype-level PA (Positive Agreement) for all variants: 100%.
- NA (Negative Agreement) for all wild types: >99.99%.
- OA (Overall Agreement) for all reported positions: >99.99%.
- Metrics: Positive Agreement (PA), Negative Agreement (NA), Overall Agreement (OA).
Reproducibility Study:
- Study Type: Reproducibility study
- Sample Size: Two panels of 46 samples each, tested by 3 trial sites and 2 operators at each site, for a total of 810 calls per site.
- Data Sources: Lymphoblastoid cell lines with known mutations in CFTR gene, leukocyte-depleted blood spiked with lymphoblastoid cell lines with known mutations in CFTR gene.
- Key Results:
- Sample pass rate (samples passing QC metrics on first attempt): 99.9%.
- Genotype-level Positive Agreement for all variants: 99.77%.
- Negative Agreement for all WT positions: 99.88%.
- Overall Agreement for all reported positions: 99.88%.
DNA Extraction Study:
- Study Type: Evaluation of DNA extraction methods
- Sample Size: 14 unique blood samples (wild type and three mutant genotypes: F508del, I506V, D110H). Each method tested by 2 operators, 3 runs/operator, 2 replicates/extracted gDNA sample. Total 168 per method.
- Key Results: All three evaluated methods (Alcohol Precipitation, Silica Filter Column Isolation, Magnetic Bead Extraction) showed 100% call rate, 100% accuracy, and 100% sample first pass rate.
DNA Input Study:
- Study Type: Serial dilution study to evaluate DNA input range
- Sample Size: 14 representative DNA samples containing 16 unique CF variants. Tested in duplicate at 9 DNA input levels (1250 ng to 1 ng). Further testing with 4 samples, 20 replicates for 1250 ng, 250 ng, 100 ng (n=80), and 14 samples, 20 replicates for 25 ng (n=280).
- Key Results: 1250 ng and 25 ng identified as upper and lower bounds 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 tested DNA input levels.
Interfering Substances Study:
- Study Type: Assessment of impact of interfering substances
- Sample Size: Eight whole blood specimens, including 3 CF positive samples with unique genotypes.
- Test Substances: Bilirubin (684 µmol/L, 137 µmol/L), Cholesterol (13 mmol/L, 2.6 mmol/L), Hemoglobin (2 g/L, 0.4 g/L), Triglyceride (37 mmol/L, 7.4 mmol/L), EDTA (7.0 mg/mL, 2.8 mg/mL). All tested with 16 replicates. Also included final wash buffer from silica filter column.
- Key Results: 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 observed.
Sample Indexing Study:
- Study Type: Verification of sample indexing capability
- Sample Size: 96 sample indexes tested using 8 unique DNA samples. Each sample tested with 12 different indexing primer combinations.
- Key Results: Reproducibility and accuracy were 100% for all sample/index primer combinations.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Positive Agreement (PA), Negative Agreement (NA), Overall Agreement (OA)
Accuracy, Call Rate, Sample First Pass Rate, Reproducibility
Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.
Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).
Not Found
§ 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.
0
MiSeqDx Cystic Fibrosis System Premarket Notification
510(k) Summary
GENERAL INFORMATION
-
Submitted by: Illumina Inc. 5200 Illumina Way San Diego, CA 92122 858-202-4500 (phone)
NOV 1 9 2013 -
Leanne M. Kiviharju Company Contact: Sr. Director, Regulatory Affairs 858-246-8811 (phone) lkiviharju@illumina.com
Date Prepared: November 18, 2013
DEVICE IDENTIFICATION
Assay:
| Trade or Proprietary Name: Illumina MiSeqDx™ Cystic Fibrosis 139-Variant Assay | |
|---|---|
| Assay Common Name: | Next generation sequencing cystic fibrosis test |
| Classification Name: | CFTR (cystic fibrosis transmembrane conductance |
| regulator) gene mutation detection (21 CFR 866.5900, | |
| Product Code PFR) | |
| Predicate Device: | Luminex xTAG® Cystic Fibrosis 60 Kit v2 (K083845) |
1
DEVICE DESCRIPTION
The Illumina MiSeqDx Cystic Fibrosis 139-Variant 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 139-Variant Assay
The Illumina MiSeqDx Cystic Fibrosis 139-Variant Assay is a qualitative in vitro diagnostic system used to simultaneously detect 139 clinically relevant cystic fibrosis disease-causing mutations and variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in genomic DNA isolated from human peripheral whole blood specimens. The variants include those recommended in 2004 by the American College of Medical Genetics (ACMG) and in 2011 by the American College of Obstetricians and Gynecologists (ACOG). The test is intended for carrier screening in adults of reproductive age, in confirmatory diagnostic testing of newborns and children, and as an initial test to aid in the diagnosis of individuals with suspected cystic fibrosis. The results of this test are intended to be interpreted by a board-certified clinical molecular geneticist or equivalent and should be used in conjunction with other available laboratory and clinical information. This test is not indicated for use for newborn screening, fetal diagnostic testing, pre-implantation testing, or for stand-alone diagnostic purposes.
The test is intended to be used on the Illumina MiSeqDxTM Instrument.
2
Image /page/2/Picture/0 description: The image shows the word "illumina" in a simple, sans-serif font. The letters are black and appear to be bolded. There is a superscript symbol after the last letter.
SUBSTANTIAL EQUIVALENCE
MiSeqDx Cystic Fibrosis 139-Variant Assay
| Characteristic | Illumina | Luminex (K083845) |
|---|---|---|
| Assay Name | Illumina MiSeqDx Cystic Fibrosis 139- | |
| Variant Assay | Luminex xTAG®Cystic | |
| Fibrosis 60 Kit v2 | ||
| Intended Use | The Illumina MiSeqDx Cystic Fibrosis 139- | |
| Variant Assay is a qualitative in vitro | ||
| diagnostic system used to simultaneously | ||
| detect 139 clinically relevant cystic fibrosis | ||
| disease-causing mutations and variants of | ||
| the cystic fibrosis transmembrane | ||
| conductance regulator (CFTR) gene in | ||
| genomic DNA isolated from human | ||
| peripheral whole blood specimens. The | ||
| variants include those recommended in 2004 | ||
| by the American College of Medical Genetics | ||
| (ACMG) and in 2011 by the American | ||
| College of Obstetricians and Gynecologists | ||
| (ACOG). The test is intended for carrier | ||
| screening in adults of reproductive age, in | ||
| confirmatory diagnostic testing of newborns | ||
| and children, and as an initial test to aid in | ||
| the diagnosis of individuals with suspected | ||
| cystic fibrosis. The results of this test are | ||
| intended to be interpreted by a board- | ||
| certified clinical molecular geneticist or | ||
| equivalent and should be used in | ||
| conjunction with other available laboratory | ||
| and clinical information. This test is not | ||
| indicated for use for newborn screening, | ||
| fetal diagnostic testing, pre-implantation | ||
| testing, or for stand-alone diagnostic | ||
| purposes. |
The test is intended to be used on the
Illumina MiSeqDx™ instrument. | 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
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 | Next generation sequencing test | Qualitative nucleic acid
multiplex test |
| Variants | 139 clinically relevant variants | 60 CFTR mutations and 4 |
| Characteristic | Illumina | Luminex (K083845) |
| Detected | | variants (benign
polymorphisms) |
| Technology | Sequencing by Synthesis (SBS) | 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 EDTA anticoagulated
blood | Nucleic acid from whole blood
anticoagulated with either
EDTA or citrate. |
| Sample
Preparation | DNA extraction using validated laboratory
method | Same |
| Contra-
indications | Not indicated for newborn screening, fetal
diagnostic testing, pre-implantation testing,
or for stand-alone diagnostic purposes. | The kit is not indicated for use
in fetal diagnostic or pre-
implantation testing. This kit is
also not indicated 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 instrument | Luminex 100 or 200 IS |
3
PERFORMANCE CHARACTERISTICS
Accuracy
Accuracy of the Illumina MiSeqDx Cystic Fibrosis 139-Variant 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
4
were designed to include the genomic context of the rare variants, and contained anywhere from 1 to 9 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.
The genotyping results for 137 SNA/small InDel sites, including the PolyTG/Poly T region were compared to Sanger bi-directional sequence analysis. A PCR based assay was 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 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 sample
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 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 MiSegDx Cystic Fibrosis 139-Variant Assay had a genotype-level PA of 100%. The NA for all wild types was >99.99% and the OA for all reported positions was >99.99%. All test results were based on initial testing.
5
able 1: Overall Accuracy for the MiSeqDx Cystic Fibrosis 139-Variant Assa
| Mutation
(Common Name) | Total calls
per
mutation | Positive calls (Variants) | | | Negative calls
(Wild Type) | # Miscalls | # No
Calls | Positive
Agreement
(%) | Negative
Agreement
(%) | Overall
Agreement
(%) |
|---------------------------|--------------------------------|---------------------------|---------------------------------------------------|----------------------|-------------------------------|------------|---------------------|------------------------------|------------------------------|-----------------------------|
| | | Clinical
Samples | Cell Line
Samples | Synthetic
Samples | | | | | | |
| CFTR dele2, 3 | 500 | 4 | 1 | 0 | 495 | 0 | 0 | 100 | 100 | 100 |
| E60X | 500 | 6 | 1 | 0 | 493 | 0 | 0 | 100 | 100 | 100 |
| P67L | 500 | 1 | 0 | 1 | 498 | 0 | 0 | 100 | 100 | 100 |
| R75X | 500 | 3 | 1 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| G85E | 500 | 6 | 2 | 0 | 492 | 0 | 0 | 100 | 100 | 100 |
| 394delTT | 500 | 3 | 1 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| 406-1G>A | 500 | 4 | 0 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| E92X | 500 | 0 | 1 | 1 | 498 | 0 | 0 | 100 | 100 | 100 |
| D110H | 500 | 1 | 0 | 1 | 498 | 0 | 0 | 100 | 100 | 100 |
| R117C | 500 | 4 | 0 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| R117H | 500 | 17 | 2 | 0 | 481 | 0 | 0 | 100 | 100 | 100 |
| Y122X | 500 | 0 | 1 | 0 | 499 | 0 | 0 | 100 | 100 | 100 |
| 621+1G>T | 500 | 7 | 5 | 0 | 488 | 0 | 0 | 100 | 100 | 100 |
| 663delT | 500 | 1 | 1 | 0 | 498 | 0 | 0 | 100 | 100 | 100 |
| G178R | 500 | 1 | 1 | 0 | 498 | 0 | 0 | 100 | 100 | 100 |
| 711+1G>T | 500 | 3 | 1 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| P205S | 500 | 1 | 0 | 1 | 498 | 0* | 0 | 100 | 100 | 100 |
| L206W | 500 | 8 | 1 | 0 | 491 | 0 | 0 | 100 | 100 | 100 |
| 1078delT | 500 | 1 | 1 | 0 | 498 | 0 | 0 | 100 | 100 | 100 |
| G330X | 500 | 1 | 1 | 0 | 498 | 0 | 0 | 100 | 100 | 100 |
| R334W | 500 | 6 | 1 | 0 | 493 | 0 | 0 | 100 | 100 | 100 |
| | otal call | Positiv | calls (Variants | | Negative calls (Wild Type) | Miscal | ମାସେ ଚନ୍ଦ୍ର ମଧ୍ୟ # | Positive | Negative | Overal |
| Mutation ommon Nam | per nutation | ລາວປັນຈ ເຖິງແມງດ | Cell Line
Sample: | Synthetic
Samples | | | | greement | Agreemen
196 | Agreemer
1961 |
| 1336K | 500 | | T | 0 | ਪਰੇਰੇ | 0 | 0 | 100 | 100 | 100 |
| 154insT | 500 | 0 | I | 0 | ਪਰੇਰੇ | 0 | 0 | 100 | 100 | 100 |
| R347H | ર૦૮ | 9 | โ | t | 492 | 0 | 0 | 100 | 100 | 100 |
| R347F | 500 | t | ਟ | 0 | 495 | 0 | 0 | IDT | 100 | 100 |
| R352C | 500 | S | 0 | 0 | 495
ﺴﻨ | 0 | 0 | 100 | 100 | 100 |
| A455E | 500 | ਸ | て | 0 | ਪਰੇਵ | 0 | 0 | 100 | 100 | 100 |
| 466X (C->G | 500 | โ | 0 | T | 498
r | 0 | i T T
0 | 100 | 100 | 100 |
| 548del | ર૦૮ | โ | 0 | T | 498 | 0 | 0 | 100 | 100 | 00T |
| Q493) | ર૦૮ | t | ਟ | 0 | 494 | | 0 | 100 | 100 | 100 |
| 507de | ક્વવ | 7 | ਟ | 0 | 4 ਰੋਪ | 0 : 0 | 0 | 100 | 100 | 100 |
| 508de | 500 | 78 | රිට | 0 | 387 | 0 | 0 | 100 | 100 | 100 |
| 677delT | 500 | L | 0 | 0 | ਧਰੇਰੇ | 0 | 0 | 100 | 100 | 100 |
| V520F | 500 | て | 0 | 0 | 498 | 0 | 0 | 100 | 100 | 100 |
| 717-1G> | 500 | b | I | 0 | 495 | 0 | 0 | 100 | 100 | 100 |
| G542) | 200 | ਟ ਦ | € | 0 | 485 | 0 | 0 | 100 | 100 | 100 |
| 549N | 500 | ਟ | ਟ | T | ਕਰੇ ਟ | 0 | 0 | 100 | 100 | 100 |
| S549R 1647T>G | 500 | ಕ | T | 0 | ૫૦૦ | 0 | 0 | 100 | 100 | 100 |
| GSS10 | నంద | 8 | દ | 0 | 489 | 0 | 0 | 100 | 100 | 100 |
| R553X | ડવ્યુ | 8 | ਟ | 0 | 490 | 0 | 0 | 100 | 100 | 100 |
| 4559. | 500 | 7 | 0 | T | વવાયુ | 0 | 0 | 100 | 100 | 100 |
| R5601 | 500 | 9 | T | 0 | ਖਰੇਤ | 0 | 0 | 100 | 100 | 100 |
| 812-1 G-> | ર૦ત | 0 | ਟ | 0 | 498 | 0 | 0 | 100 | 100 | 100 |
| Mutation
(Common Name) | Total calls
per
mutation | Positive calls (Variants) | | Synthetic
Samples | Negative calls
(Wild Type) | # Miscalls | # No
Calls | Positive
Agreement
(%) | Negative
Agreement
(%) | Overall
Agreement
(%) |
| | | Clinical
Samples | Cell Line
Samples | | | | | | | |
| 1898+1G>A | 500 | 2 | 1 | 0 | 497 | 0 | 0 | 100 | 100 | 100 |
| 2143delT | 500 | 2 | 1 | 0 | 497 | 0 | 0 | 100 | 100 | 100 |
| 2183AA>G | 500 | 3 | 1 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| 2184insA | 500 | 3 | 0 | 1 | 496 | 0 | 0 | 100 | 100 | 100 |
| 2184delA | 500 | 1 | 1 | 0 | 498 | 0 | 0 | 100 | 100 | 100 |
| R709X | 500 | 1 | 0 | 2 | 497 | 0 | 0 | 100 | 100 | 100 |
| K710X | 500 | 3 | 0 | 0 | 497 | 0 | 0 | 100 | 100 | 100 |
| 2307insA | 500 | 3 | 0 | 2 | 495 | 0 | 0 | 100 | 100 | 100 |
| R764X | 500 | 1 | 0 | 2 | 497 | 0 | 0 | 100 | 100 | 100 |
| W846X | 500 | 0 | 1 | 0 | 499 | 0 | 0 | 100 | 100 | 100 |
| 2789+5G>A | 500 | 9 | 1 | 0 | 490 | 0 | 0 | 100 | 100 | 100 |
| Q890X | 500 | 1 | 0 | 0 | 499 | 0 | 0 | 100 | 100 | 100 |
| 3120G>A | 500 | 1 | 0 | 0 | 499 | 0 | 0 | 100 | 100 | 100 |
| 3120+1G>A | 500 | 7 | 1 | 0 | 492 | 0 | 0 | 100 | 100 | 100 |
| 3272-26A>G | 500 | 0 | 1 | 0 | 499 | 0 | 0 | 100 | 100 | 100 |
| R1066C | 500 | 6 | 0 | 0 | 494 | 0 | 0 | 100 | 100 | 100 |
| R1066H | 500 | 1 | 0 | 1 | 498 | 0 | 0 | 100 | 100 | 100 |
| W1089X | 500 | 4 | 0 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| Y1092X (C>A) | 500 | 3 | 1 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| M1101K | 500 | 2 | 2 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| R1158X | 500 | 7 | 1 | 0 | 492 | 0 | 0 | 100 | 100 | 100 |
| R1162X | 500 | 5 | 1 | 0 | 494 | 0 | 0 | 100 | 100 | 100 |
| 3659delC | 500 | 4 | 1 | 0 | 495 | 0 | 0 | 100 | 100 | 100 |
| Mutation
(Common Name) | Total calls
per
mutation | Positive calls (Variants) | | | Negative calls
(Wild Type) | # Miscalls | # No
Calls | Positive
Agreement
(%) | Negative
Agreement
(%) | Overall
Agreement
(%) |
| | | Clinical
Samples | Cell Line
Samples | Synthetic
Samples | | | | | | |
| S1196X | 500 | 1 | 0 | 0 | 499 | 0 | 0 | 100 | 100 | 100 |
| 3791delC | 500 | 2 | 0 | 0 | 498 | 0 | 0 | 100 | 100 | 100 |
| 3849+10kbC>T | 500 | 11 | 2 | 0 | 487 | 0 | 0 | 100 | 100 | 100 |
| 3876delA | 500 | 6 | 1 | 0 | 493 | 0 | 0 | 100 | 100 | 100 |
| S1251N | 500 | 1 | 0 | 1 | 498 | 0 | 0 | 100 | 100 | 100 |
| 3905insT | 500 | 3 | 1 | 0 | 496 | 0 | 0 | 100 | 100 | 100 |
| W1282X | 500 | 9 | 1 | 0 | 490 | 0 | 0 | 100 | 100 | 100 |
| N1303K | 500 | 9 | 1 | 0 | 490 | 0 | 0 | 100 | 100 | 100 |
| CFTRdele22,23 | 500 | 1 | 0 | 1 | 498 | 1" | 0 | 100 | 99.8 | 99.8 |
| M1V | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| Q39X | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 405+1 G->A | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| E92K | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| Q98X | 500 | 0 | 0 | 2 | 498 | 0 | 0 | 100 | 100 | 100 |
| 457TAT->G | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 574delA | 500 | 0 | 0 | 2 | 498 | 0 | 0 | 100 | 100 | 100 |
| 711+3A>G | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 711+5 G->A | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 712-1 G->T | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| H199Y | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| Q220X | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 852del22 | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| Mutation
(Common Name) | Total calls
per
mutation | Clinical
Samples | Positive calls (Variants)
Cell Line
Samples | Synthetic
Samples | Negative calls
(Wild Type) | # Miscalls | # No
Calls | Positive
Agreement
(%) | Negative
Agreement
(%) | Overall
Agreement
(%) |
| T3381 | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| S341P | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 1213delT | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 1248+1G>A | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 1259insA | 500 | 0 | 0 | 2 | 498 | 0 | 0 | 100 | 100 | 100 |
| W401X
(c.1202G>A) | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| W401X
(c.1203G>A) | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 1341+1G->A | 500 | 0 | 0 | 2 | 498 | 0 | 0 | 100 | 100 | 100 |
| 1461ins4 | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 1525-1G->A | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| S466X (C->A) | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| L467P | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| S489X | 500 | 0 | 0 | 2 | 498 | 0 | 0 | 100 | 100 | 100 |
| S492F | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| Q525X | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 1717-8G->A | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| S549R
(c.1645A>C) | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| Q552X | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| R560K | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 1811+1.6kb A->G | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| E585X | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
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iSeqDx Cystic Fibrosis System
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MiSeqDx Cystic Fibrosis System
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MiSeqDx Cystic Fibrosis System
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MiSeqDx Cystic Fibrosis System
| Overal | greemen
ಳಿಕ | 100 | 100 | 100 | 100 | 00T | 001 00T | | 100 | 00T | 100 | 00T | 100 | 100 | 100 | 00T | 00T | 100 | 100 | 100 | 100 | 100 |
|-----------------------------|--------------------------|---------|-------|---------|---------|-------|-----------|-------|-------|-----------|-------|--------|---------|-------|----------|------------|--------|------------|--------|--------|--------------------------------------------------------------------------------------------------|--------------|
| Negative | Agreemer
(%) | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | от | 100 | 100 | 00T | 100 | 100 | 100 | 100 | 100 | 100 |
| Positive | greemer
(%) | 100 | 100 | 100 | 100 | 100 | 00 T | 100 | 100 | 100 | 100 | 100 | 100 | 00T | 100 | 00T | 00 I | 100 | 100 | 100 | 100 | 100 |
| siles ON # | | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Miscal | | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0
. ﻟ . | VO | 0 | 0 | 0 | 0 | 0 |
| Negative calls (Wild Type) | | ਥ ਰੇਰੇ | 498 | 498 | 498 | 198 | 498 | 499 | ਰੇਰੇ | ਕਰੇਰੇ | ਖਰੇਰੇ | ਰ ਰੇਰੇ | aa | 499 | ਥ ਰੇਰੇ | 499 | 499 | ਖਰੇਰੇ | 499 | ਧ ਰੇਖੇ | ਧਰੇਤੇ | 499 |
| | Synthetic
Samples | โ | ਟ | ਟ | ट | ट | で | સ્ન | T | T | पु | :
T | I | I | โ | I | T | I | T | I | T | · , i '
T |
| calls (Variants) | Cell Line
mples
୧୮ | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | | 0) 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Positive | Samples
Clinical | 0 | 0 | 0 | 0 | 0 | 0 | (0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total calls | per mutatio | 500 | 500 | ടറവ | 200 | റ്ററ | నంది. సంర | ક્તભ | నం | నంది. వైద | ર૦૮ | રૂભવ | నంర | నం | રેજિ | 500 | 200 | ર૦૮ | ર૦૮ | 500 | ર૦૮ | SOC |
| Mutation | ommon Nam | 898+3A> | L732) | 347delG | 2585del | E822) | 622+1G> | E831X | R851) | 2711del | L927F | 59451 | 007delG | G9708 | 121-1G-> | 1065b | L1077P | 1092X (C>G | E1104) | | היסטורים אימריקע א לפני נייני נייני מיני מ לפני נייני נייני ניי אמטרוני אינטער א אמטע מעניינע א | |
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| 2 |
|---|
| g |
| - |
| S |
| 1 |
| - |
| œ |
| m |
| n |
| Chamble Comment |
| ages of the program and contrôleant |
| . . |
| I |
iSeqDx Cystic Fibrosis Syste
| Mutation
(Common Name) | Total calls
per
mutation | Positive calls (Variants) | | | Negative calls
(Wild Type) | # Miscalls | # No
Calls | Positive
Agreement
(%) | Negative
Agreement
(%) | Overall
Agreement
(%) |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------|---------------------------|----------------------|----------------------|-------------------------------|------------|---------------|------------------------------|------------------------------|-----------------------------|
| | | Clinical
Samples | Cell Line
Samples | Synthetic
Samples | | | | | | |
| 4005+1G->A | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 4016insT | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| Q1313X | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 4209TGTT>AA | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| 4382delA | 500 | 0 | 0 | 1 | 499 | 0 | 0 | 100 | 100 | 100 |
| PolyTG/PolyT | 19 | 17 | 2 | 0 | 0 | 0 | 0 | 100 | N/A | 100 |
| I506V* | 1 | 0 | 0 | 0 | 1 | 0 | 0 | N/A | 100 | 100 |
| I507V* | 1 | 0 | 0 | 0 | 1 | 0 | 0 | N/A | 100 | 100 |
| F508C* | 1 | 0 | 0 | 0 | 1 | 0 | 0 | N/A | 100 | 100 |
| Total | 67522 | | 557 | | 66965 | 1 | 0 | 100.00 | >99.99 | >99.99 |
| * The Sanger report listed the P205S variant as heterozygous for the clinical sample. A review of the Sanger trace data however indicated that the variant was in fact homozygous and incorrectly | | | | | | | | | | |
The Sanger report listed le P2055 var
as heterozygous for the
sam
reported. MiSeqDx reported the variant as homozygous
Jaus Jouel contamination
he original syntheic hetercozygous specient was determined when it was subsequently tested after it was re prepared, using the same plasmid, it would be detece A youtheric sample heterozygous for exon 8 was reported on the variant CFTR dele2, 23. Further investigation revealed that this result was likely from lov level contanimal
When R117H is positive, the PolyTG/PolyT variant is additionally reported
In the case of one homozygous F508del variant, three additional wild type bases (1506V, I507V, F508C) were additionally reported
able 2: Accuracy of the MiSeqDx Cystic Fibrosis 139-Variant Assay for 1506V, 1507V and F5086
| Variant
(Common
Name) | Total
Calls per
Variant | Positive Calls (Variants) | | Negative
Calls (Wild
Type) | =
Miscalls | = No
Calls | Positive
Agreement
(%) | Negative
Agreement
(%) | Overall
Agreement
(%) |
|-----------------------------|-------------------------------|---------------------------|----------------------------------------|----------------------------------|---------------|---------------|------------------------------|------------------------------|-----------------------------|
| 1506V | 500 | Clinical
Samples
7 | Cell Line
Synthetic
Samples
0 | 493 | 0 | 0 | 100 | 100 | 100 |
Confidentia
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MiSeqDx Cystic Fibrosis System
| Variant
(Common
Name) | Total
Calls per
Variant | Positive Calls (Variants) | | | Negative
Calls (Wild
Type) | Miscalls
= No
Calls | Positive
Agreement
(%) | Negative
Agreement
(%) | Overall
Agreement
(%) |
|-----------------------------|-------------------------------|---------------------------|---------------------------|---------------------------|----------------------------------|---------------------------|------------------------------|------------------------------|-----------------------------|
| 1507V | 500 | Clinical
Samples
0 | Cell Line
Samples
1 | Synthetic
Samples
0 | 499 | 0 | 100 | 100 | 100 |
| F508C | 500 | Clinical
Samples
1 | Cell Line
Samples
1 | Synthetic
Samples
0 | 498 | 0 | 100 | 100 | 100 |
Table 3. Accuracy of the MiSeqDx Cystic Fibrosis 139-Variant Assay for PolyTG/PolyT Variants
| 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 | 50.0 |
| (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 |
| (TG)10(T)7/(TG)11(T)5 | 2 | 0 | 0 | 0 | 0 | 100 |
| (TG)10(T)7/(TG)11(T)7 | 72 | 11 | 0 | 0 | 0 | 100 |
| (TG)10(T)7/(TG)12(T)5 | 1 | 0 | 0 | 0 | 0 | 100 |
| (TG)10(T)7/(TG)12(T)7 | 10 | 1 | 0 | 0 | 1 | 90.9 |
34
13
iSeqDx Cystic Fibrosis System
Premarket Notificati
| 100 | 100 | 00 I | 100 | 100 | 92.3 | 83.3 | 00 I | 0.0 | 100 | 100 | 100 | 100 | 98.44 | a suala firm the complex |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 0 | 0 | 0 | 0 | լ. | 0 | በ | 0 | 0 | 0 | 0 | 0 | ప్రాంత | |
| 0 | 0 | 0 | 0 | 0 | 0 | L | 0 | C | 0 | 0 | 0 | 0 | ోగా | 0 |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | -r'l- | |
| . | ||||||||||||||
| 9 | 0 | 07 | 0 | ਨ। | ﮨﮯ | 0 | 8 | I | 0 | € | 0 | で | ||
| 1 | ഗ്ര | 9 ﻳ | ಕ್ಕೆ | tt, | g I | 9 | ਟ ਉ | Z | ਟ | 1 g | € | ಗಳ | 148 | |
| (TC)10(T)9/(TC)10(T)9 | G)10(T)9/(TG)11(T)5 | TG)10(T)9/(TG)11(T)7 | TG)10(T)9/(TG)11(T)9 | (G)10(T)9/(TG)12(T) | TG)10(T)9/(TG)12(T)7 | G)11(T)5/(TG)11(T)7 | (TG)11(T)7/(TG)11(T)7 | TG)11(T)7/(TG)11(T)9 | TG)11(T)7/(TG)12(T)5 | G)11(T)7/(TC)12(T)7 | TG)11(T)9/(TG)12(T) | (TC)12(T)7/(TC)12( | Total** | ( |
One of the discordant results was from the repoducibility study. The PolyTC/PolyT result for the sample was concertains
across all 18 replicates, but discordant with Sanger b
Samples were not retested
" The bala ample count for the Roy Crise est a synhuic sample (n=2) were built by bending linerized plannis with cell him
smules which were also part of the spresented from
14
Image /page/14/Picture/0 description: The image shows the word "illumina" in a simple, sans-serif font. The letters are black against a white background, creating a clear contrast. A small superscript symbol appears to the right of the final "a", adding a subtle detail to the logo.
Reproducibility - 139-Variant Assay
The reproducibility of the MiSeqDx Cystic Fibrosis System was determined through a blinded study using 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 of 810 calls per site. The panels contained a mix of genomic DNA from lymphoblastoid cell lines with known mutations in the CFTR gene as well as some leukocyte-depleted blood spiked with lymphoblastoid cell lines with known mutations 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%.
The genotype-level Positive Agreement for all variants was 99.77%. The Negative Agreement for all WT positions was 99.88% and the Overall Agreement for all reported positions was 99.88%. All test results are based on initial testing. No repeat testing was done for the reproducibility study.
15
Table 4: Reproducibility Panel Variants
.
| Panel | Sample # | Sample Genotype | Variants | Total calls per site | Positive Agreeing calls (Variants) | Negative Agreeing calls (Wild type) | # Miscalls | # No Calls | Positive Agreement (%) | Negative Agreement (%) | Overall Agreement (%) | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Site 1 | Site 2 | Site 3 | Site 1 | Site 2 | Site 3 | |||||||||||
| A | 1 | S549N (HET) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | ||
| A | 2 | 1812-1 G>A (HET) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | ||
| A | 3 | Q493X/F50 8del (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||
| A | 4* | F508del/21 84delA (HET) | 810 | 12 | 12 | 12 | 797 | 798 | 798 | 0 | 1* | 100 | 100 | 100 | ||
| A | 5^ | Y122X/R115 8X (HET) | 810 | 12 | 10 | 12 | 798 | 665 | 798 | 0 | 135 | 94.44 | 94.44 | 94.44 | ||
| A | 6 | F508del/21 83AA>G (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||
| A | 7 | R75X (HET) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | ||
| A | 8 | 1507del/F50 8del (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||
| A | 9** | F508del/W1 282X (HET) | 810 | 12 | 11 | 12 | 798 | 797 | 798 | 2* | 0 | 97.22 | 99.96 | 99.92 | ||
| A | 10* | F508del/32 72-26A>G (HET) | 810 | 12 | 11 | 12 | 798 | 797 | 798 | 2* | 0 | 97.22 | 99.96 | 99.92 | ||
| A | 11 | F508del/38 49+10kbC>T (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||
| A | 12 | 621+1G>T/3 120+1G>A (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||
| A | 13 | E60X/F508del (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||
| A | 14 | M1101K (HET) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | ||
| A | 15 | M1101K (HOM) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | ||
| A | 16 | F508del | ||||||||||||||
| (HOM) | I506V, | |||||||||||||||
| I507V, | ||||||||||||||||
| F508C | ||||||||||||||||
| not | ||||||||||||||||
| present | 828 | 6 | 6 | 6 | 822 | 822 | 822 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 17 | F508del/36 | ||||||||||||||
| 59delC | ||||||||||||||||
| (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 18 | R117H/F508 | ||||||||||||||
| del (HET) | (TG)10(T) | |||||||||||||||
| 9/(TG)12 | ||||||||||||||||
| (T)5 | 816 | 18 | 18 | 18 | 798 | 798 | 798 | 0 | 0 | 100 | 100.0 | |||||
| 0 | 100 | |||||||||||||||
| A | 19 | 621+1G>T/7 | ||||||||||||||
| 11+1G>T | ||||||||||||||||
| (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 20 | G85E/621+1 | ||||||||||||||
| G>T (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 21 | A455E/F508 | ||||||||||||||
| del (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 22 | F508del/R5 | ||||||||||||||
| 60T (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 23 | F508del/Y1 | ||||||||||||||
| 092X (C>A) | ||||||||||||||||
| (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 24 | N1303K | ||||||||||||||
| (HET) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 25 | G542X | ||||||||||||||
| (HOM) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 26 | G542X | ||||||||||||||
| (HET) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 27 | G551D/R55 | ||||||||||||||
| 3X (HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | ||||
| A | 28 | 3849+10kbC |
T (HOM) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| A | 29 | WT | | 810 | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 | |
| A | 30 | F508del
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| A | 31 | 1717-1G>A
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| A | 32 | R1162X
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| A | 33 | R347P/G55
1D (HET) | | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | |
| A | 34 | R334W
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| A | 35 | WT | | 810 | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 | |
| A | 36 | G85E (HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| A | 37 | I336K (HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| | | | | | | | | | | | | | | | | |
| A | 39 | F508del/38
49+10kbC>T
(HET) | | 810 | | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| A | 40 | 621+1G>T/3
120+1G>A
(HET) | | 810 | | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| A | 41 | F508del/36
59delC
(HET) | | 810 | | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| A | 42 | R117H/F508
del (HET) | (TG)10(T)
9/(TG)12
(T)5 | 816 | 18 | 18 | 18 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | |
| A | 43 | G85E/621+1
G>T (HET) | | 810 | | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| A | 44 | A455E/F508
del (HET) | | 810 | | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| A | 45 | N1303K
(HET) | | 810 | | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| A | 46 | G551D/R55
3X (HET) | | 810 | | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| B | 47 | 2789+5G>A
(HOM) | | 810 | | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| B | 48 | CFTR dele2,
3/F508del
(HET) | | 810 | | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| B | 49 | F508del/18
98+1G>A
(HET) | | 810 | | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| B | 50 | WT | | 810 | | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 |
| B | 51 | F508del/21
43delT
(HET) | | 810 | | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| B | 52 | 3876delA
(HET) | | 810 | | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| B | 53 | 3905insT
(HET) | | 810 | | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| B | 54 | 394delTT
(HET) | | 810 | | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| B | 55 | F508del
(HET) | | 810 | | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| B | 56 | WT | | 810 | | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 |
| B | 57 | WT | | 810 | | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 |
| B | 58 | F508del
(HET) | | 810 | | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| B | 59 | WT | | 810 | | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 |
| B | 60 | L206W
(HET) | | 810 | | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| B | 61 | WT | | 810 | | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 |
| B | 62 | G330X
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 63 | WT | | 810 | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 | |
| B | 64 | R347H
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 65 | 1078delT
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 66 | G178R/F508
del (HET) | | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | |
| B | 67 | S549R
(c.1647T>G)
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 68 | S549N (HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 69 | W846X
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 70 | WT | | 810 | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 | |
| B | 71 | E92X/F508del (HET) | | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | |
| B | 72" | 621+1G>T/1
154insTC
(HET) | | 810 | 12 | 12 | 12 | 798 | 798 | 797 | 0 | 1" | 100 | 99.96 | 99.96 | |
| B | 73 | G542X
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 74 | F508del
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 75^ | F508del
(HET) | | 810 | 6 | 5 | 6 | 804 | 670 | 804 | 0 | 135 | 94.44 | 94.44 | 94.44 | |
| B | 76 | F508del
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 77 | 621+1G>T/
A455E (HET) | | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | |
| B | 78 | 1812-1 G->A (HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 79 | WT | | 810 | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 | |
| B | 80 | F508del/R5
53X (HET) | | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | |
| B | 81 | F508del/G5
51D (HET) | | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | |
| B | 82 | R347P/F508
del (HET) | | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | |
| B | 83 | R117H/F508
del (HET) | (TG)10(T)
9/(TG)12
(T)S | 816 | 18 | 18 | 18 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 | |
| B | 84 | I507del
(HET) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
| B | 85 | 2789+5G>A
(HOM) | | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 | |
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| B | 86" | CFTR dele2,
3/F508del
(HET) | 810 | 12 | 12 | 12 | 798 | 797 | 798 | 0 | 1" | 100 | 99.96 | 99.96 |
|---|-----|-----------------------------------|-------|----|------|----|-----|--------|-----|---|-----|-------|-------|-------|
| B | 87 | F508del/18
98+1G>A
(HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| B | 88 | WT | 810 | 0 | 0 | 0 | 810 | 810 | 810 | 0 | 0 | N/A | 100 | 100 |
| B | 89 | F508del/21
43delT
(HET) | 810 | 12 | 12 | 12 | 798 | 798 | 798 | 0 | 0 | 100 | 100 | 100 |
| B | 90 | 3905insT
(HET) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| B | 91 | 394delTT
(HET) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| B | 92 | F508del
(HET) | 810 | 6 | 6 | 6 | 804 | 804 | 804 | 0 | 0 | 100 | 100 | 100 |
| | | Total | 74556 | | 2209 | | | 221182 | | 4 | 273 | 99.77 | 99.88 | 99.88 |
- The wild type location corresponding to the N1303K variant for one replicate resulted in a No Call due to insufficient coverage.
^ 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.
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- Evidence indicates that samples 9 and 10 were likely switched by the operator prior to library preparation.
The wild type location corresponding to the M1V variant for one replicate of each of two samples resulted in a No Call due to insufficient coverage.
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| Variation (Common
Name) | Variant Type | CFTR Gene
Region |
|----------------------------|---------------------------------|---------------------|
| polyTG/PolyT | Compound DIV* | Intron 9 |
| 2183AA>G | Compound DIV* | Exon 14 |
| CFTR dele2, 3 | DEL | Intron1-Intron3 |
| 1154insTC | DIV* | Exon 8 |
| 1507del | DIV* | Exon 11 |
| F508del | DIV* | Exon 11 |
| 2143delT | DIV* | Exon 14 |
| 3659delC | DIV* | Exon 22 |
| 3876delA | DIV* | Exon 23 |
| 394delTT | DIV in homopolymeric
region* | Exon 3 |
| 1078delT | DIV in homopolymeric
region* | Exon 8 |
| 2184delA | DIV in homopolymeric
region* | . Exon 14 |
| 3905insT | DIV in homopolymeric
region* | Exon 23 |
| E60X | SNV | Exon 3 |
| R75X | SNV | Exon 3 |
| G85E | SNV | Exon 3 |
| E92X | SNV | Exon 4 |
| R117H | SNV | Exon 4 |
| Y122X | SNV | Exon 4 |
| Table 4: Reproducibility Panel Variants | ||
|---|---|---|
| Variation (Common | ||
| Name) | Variant Type | CFTR Gene |
| Region | ||
| 621+1G>T | SNV | Intron 4 |
| G178R | SNV | Exon 5 |
| 711+1G>T | SNV | Intron 5 |
| L206W | SNV | Exon 6 |
| G330X | SNV | Exon 8 |
| R334W | SNV | Exon 8 |
| I336K | SNV | Exon 8 |
| R347P | SNV | Exon 8 |
| R347H | SNV | Exon 8 |
| A455E | SNV | Exon 10 |
| Q493X | SNV | Exon 11 |
| 1717-1G>A | SNV | Intron 11 |
| G542X | SNV | Exon 12 |
| S549N | SNV | Exon 12 |
| S549R (c.1647T>G) | SNV | Exon 12 |
| G551D | SNV | Exon 12 |
| R553X | SNV | Exon 12 |
| R560T | SNV | Exon 12 |
| 1812-1 G->A | SNV | Intron 12 |
| 1898+1G>A | SNV | Intron 13 |
| W846X | SNV | Exon 15 |
| 2789+5G>A | SNV | Intron 16 |
| Variation (Common | ||
| Name) | Variant Type | CFTR Gene |
| Region | ||
| 3120+1G>A | SNV | Intron 18 |
| 3272-26A>G | SNV | Intron 19 |
| Y1092X (C>A) | SNV | Exon 20 |
| M1101K | SNV | Exon 20 |
| R1158X | SNV | Exon 22 |
| R1162X | SNV | Exon 22 |
| 3849+10kbC>T | SNV | Intron 22 |
| W1282X | SNV | Exon 23 |
| N1303K | SNV | Exon 24 |
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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 unique blood samples were used in the study representing wild type and three mutant genotypes (3 samples with F508del, 1 sample with I506V, and 1 sample with D110H). 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 | 100% | 100% | 100% |
| Silica Filter Column
Isolation | 168 | 100% | 100% | 100% |
| Magnetic Bead Extraction | 168 | 100% | 100% | 100% |
- Percent of samples having call rate of >99% in first run
DNA input
The DNA input range of the Illumina MiSeqDx Cystic Fibrosis 139-Variant 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 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.
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Interfering Substances
To assess the impact of interfering substances on the MiSeqDx Cystic Fibrosis 139-Variant Assay, the performance of the assay was evaluated in the presence and absence of potential interferents. Eight whole blood specimens were tested in the study including 3 CF positive samples with unique genotypes. 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 following table. 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 139-Variant 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.
To access 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