illumhina-

1.3 510(k) Summary

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

NOV 921

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Mlummia- MiSeq~x Cystic Fibrosis Clinical Sequencing Assay

510(k) Summary

GENERAL INFORMATION

Submitted by: Illumina Inc.5200 Illumnina WaySan Diego, CA 92122858-202-4500 (phone)858-202-4600 (fax)

Company Contact: Bryan SchneiderAssociate Director, Regulatory Affairs858-255-5228 (phone)bschneider@illumina.com

Date Prepared: November 18, 2013

DEVICE IDENTIFICATION

Assay: *

Trade or Proprietary Name:

Illumina MiSeqDxTM Cystic Fibrosis ClinicalSequencing Assay

Common Name: Sequencing by synthesis cystic fibrosis test

Classification Name: CFTR (cystic fibrosis transmemnbrane conductanceregulatory) gene mutation detection (21 CFR 866.5900,Product Code PFS)

Predicate Device: i -TAG Cystic Fibrosis 60 Kit v2 (k083845)

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ilium: iha7 MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

DEVICE DESCRIPTION

The Illumiria MiSeqDx Cystic Fibrosis Clinical Sequencing Assay consists of

library preparation and sample indexing reagents, sequencing reagents andconsumnables, MiSeqDx instrument and data analysis software. Testing beginswith genomic DNA from a peripheral whole blood sample. The genomic DNA isprocessed through the library preparation steps, which specifically amplifies theintended genomic regions of each sample while also adding the indexes forsample identification. Flow cell capture sequences are also added to theamplified products. The resulting sample libraries are then transferred into aMiSeqDx reagent cartridge which contains all of the reagents required for clustergeneration and sequencing (Sequencing By Synthesis). The MiSeq~x Cartridge,MiSeqDx Flow Cell, and MiSeqDx SBS Solution (PR2) are then inserted into theMiSeqDx instrument, which performs cluster generation, sequencing and dataanalysis.

INTENDED USE

Illumnina MiSeqDxTM Cystic Fibrosis Clinical Sequencing Assay

The Illumina MiSeqDx(TM) Cystic Fibrosis Clinical Sequencing Assay is atargeted sequencing in vitro diagnostic system that re.-sequences the proteincoding regions and intron/exon boundaries of the.Cystic FibrosisTransmembrane Conductance Regulator (CFTR) gene in genomic DNA isolatedfrom human peripheral whole blood specimens collected in K2EDTA. The testdetects 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 MiSeql x Instrument.

The test is .-i nt 'en ded t o be usedas an aid in the diagnosis of individuals withsuspected cystic fibrosis (CF). The test is most appropriate when the patient hasan atypical or non-classic presentation of CF or when other mutation panels havefailed to identify both causative mutations. The results of the test are intended tobe interpreted by a board-certified clinical molecular geneticist or equivalent and

should be used in conjunction with other available information including clinicalsymptoms, other diagnostic tests, and family history. This test is not indicated foruse for stand-alone diagnostic purposes, fetal diagnostic testing, for pre-implantation testing, carrier screening, newborn screening, or population

screening.-

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Mlummna' MiSeqox Cystic Fibrosis Clinical Sequencing Assay

SUBSTANTIAL EQUIVALENCE

Characteristic Illumina Luminex (K083845)

Assay Name Illumina MiSeqDx Cystic Fibrosis Luminex xTAG® CysticClinical Sequencing Assay Fibrosis 60 Kit v2

Intended The Illumina MiSeqDx Cystic The xTAG® Cystic Fibrosis 60Use - Fibrosis Clinical Sequencing -kit v2 is a device used to

Assay is a targeted sequencing in simultaneously detect andvitro diagnostic system that re- identify a panel of mutationssequences the protein coding and variants in the cysticregions and intron/exon fibrosis transmembraneboundaries of the Cystic Fibrosis conductance regulatorTransmembrane Conductance (CFTR) gene in human bloodRegulator (CFTR) gene in specimens. The panelgenomic DNA isolated from includes mutations andhuman peripheral whole blood variants currentlyspepimens collected in K2EDTA. recommended by theThe test detects single nucleotidle American College of Medicalvariants, and small indels within Genetics and Americanthe region sequenced, and College of Obstetricians andadditionally reports on two deep Gynecologistsintronic mutations and two large (ACMG/ACOG) plus somedeletions. The test is intended to of the world's most commonbe used on the Illumina MiSeqDx and North AmericanInstrument, prevalent mutations. The

xTAG Cystic Fibrosis 60 kit

The iest is intende .d . to be u sed as v2 is a qualitative

an aid in the diagnosis of genotyping test wkhich

individuals with suspected cystic provides information

fibrosis (CF).This .assay is most intended to be used for

_____________ when the patient has carrier testing in adults of

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Mlummia MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

Characteristic Illuinina Luminex (K083845)

an atypical or non-classic reproductive age, as an aidpresentation of CF or when other in newborn screening, and inmutation panels have failed to confirmatory diagnosticidentify both causative mutations. testing in newborns andThe results of the test are children.intended to be interpreted by a The kit is not indicated forboard-certified clinical molecular' use in fetal diagnostic or pre-geneticist or equivalent and implantation testing. The kitshould be used in conjunction is also not indicated forwith other available information stand-alone diagnosticincluding clinical symptoms, purposes.other diagnostic tests, and familyhistory. This test is not indicatedfor use for fetal diagnostic testing,for pre-implantation testing,carrier screening, newbornscreening, or population

screening.The test is intended to be used onthe Illumia MiSeqDxTMInstrument.

Assay type Sequencing by synthesis test Qualitative nucleic acidmultiplex test

Variants Mutations and variants from the 60 CFTR mutations and 4Detected protein coding regions ahd' variants (benign

intron/exon boundaries df the polymorphisms)CFTR gene, including two deepintronic mutations and two largedeletions.

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MIummia; MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

Characteristic Illumina Luminex (K083845)

Technology PCR-based amplification of Multiplex PCR followed byregions of interest that are then multiplex allele specifichybridized to a flow cell to allow primer extension forsequencing by synthesis genotyping, hybridized to

multiplex fluorescentmicroparticles, detected byflow cytometry.

Sample Type Nucleic acid from K2EDTA Nucleic acid from wholeanticoagulated blood blood anticoagulated with

either EDTA or citrate.

Sample DNA extraction using validate d Same

Preparation laboratory method

Contra- This test is not indicated for use Not indicated for fetalindications for fetal diagnostic testing, for diagnostic testing, for pre-

pre-implantation testing, carrier implantation testing, or forscreening, newborn. screening, or stand-alone diagnosticpopulation screening. purposes.

Assay Positive and negative controls Negative controls required,Controls required, not supplied not supplied. Positive

controls recommended, not____ ___ ___ ____ ___ ___ ____ ___ ___ supplied.

Instrument Mi~eqDx Luminex 100 or 200 IS

System _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

PERFORMANCE CHARACTERISTICS

Accuracy

Accuracy of the Illumina MiSeqDx Cystic Fibrosis Clinical Sequencing Assay wasassessed by evaluating 500 samples representing a wide variety of CFTR variantsfrom four separate sources"Theprimary source of accuracy data was a clinicalaccuracy study conducted using a panel of 366 samples. The majority (n = 355) of

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Mlummna MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

samples consisted of archived, anonymized clinical gDNA specimens isolatedfrom human blood, the remaining 11 samples were obtained from commerciallyavailable cell line specimens.Data from this study was supplemented with accuracy data from 68 cell linesamples evaluated in the reproducibility study, 14 clinical samples from theextraction method evaluation analytical study, and 52 synthetic plasmid samples.The synthetic plasmids were designed to include the genomic context of rarevariants, and contained anywhere from I to 10 variants within the sameconstruct. They were linearized, diluted to genomic DNA equivalent copynumbers, and blended with human genomic DNA samples of wild typegenotype at equivalent copy numbers to mimic a heterozygous sample.

For the MiSeqDx Cystic Fibrosis Clinical Sequencing Assay, a total of 5,206positions were compared to the reference methods of Sanger bi-directionalsequencing and PCR testing. The genotyping results for SNV and small InDelsites, including the PolyTG/P6IyT region, were compared to Sanger bi-directionalsequence analysis.

Two validated PCR based assays were used as the reference method for the twolarge deletions in the panel. Each duplex PCR assay made use of 2 primer sets todiscriminate between wild type, heterozygous; and homozygous genotypes. One

of the primer sets was designed to flank the deletion breakpoints, whereas theother amplified a region internal to the deletion. The two products were detectedby size separation on an agarose gel. The PCR assays were validated using apanel of 28 samples in all (22 samples for each deletion) consisting of cell line andblood derived gen6mic DNA samples, and syhthetic plasmids whichencompassed the WT, HET and HOM genotypes for each large deletion. ThePCR assays were confirmed to have 100% specificity and reproducibility for allsamples tested, by evaluation of PCR products on an agarose gel. The accuracy ofthe 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 dividingthe number of samples with agreeing variant calls by the total number of

samples with that variant as identified by the reference methods. NegativeAgreement (NA) was calculated across all wild type (WI) positions by dividing

the number of concoidaiit'WT positions by the total number of WT positions as

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Mlummna MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

defined by the reference methods. Overall Agreement (GA) was calculatedacross all reported positions by dividing the number of concordant WT andvariant positions by the total number of reported positions as determined by thereference methods.

The MiSeqDx Cystic Fibrosis Clinical Sequencing Assay had a genotype-level PAof 99.66% including the PolyTGIPolyT variants and 100%, excludingPolyTGIPolyT variants (Table 1). The NA for all wild types was >99.99% and theOA for all WT and variants was >99.99%. The PolyiG/PolyT variant PA was98.44%. All results are based on initial testing. No repeat testing was done forthis study.

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

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Mlummna MiSeq~x Cystic Fibrosis Clinical Sequencing Assay

DNA Extraction

Three commonly used, commercially available extraction methods representingmagnetic bead extraction, alcohol precipitation and silica filter column isolationmethods, were evaluated using K2EDTA anti-coagulated whole blood. A total of14 blood samples were used during the study; two were wild type, while theremaining 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 extractionmethods were tested independently by 2 different operators who each performed3 runs per extraction method. Each extraction was performed by each operatoron different days. The DNA concentration and A260/A280 ratio of the extractedgDNA samples was determined using spectrophotometry. The total sample sizefor each extraction method in this study was 168 (14 samples x 2operators/extraction method x 3 runs/operator x 2 replicates/extracted gDNAsample).

rExtraction Number of Call Accuracy Sample First

--M~h.-. samples tested Rate Pass Rate*

:'l ~ 168 >99.99% >99.99% 100%jPrecipitation

I.Silit a Filte r 168 >99.99% >99.99% 100%'Column Isola6tion-

LMiagneticiBead_ -> 168 >99.99% >99.99% 100%

E x tr 6 hoti n-".-

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

DNA inputThe DNA input range of the Illumina MiSeqDx Cystic Fibrosis Clinical

Sequencing Assay was evaluated by performing a serial dilution study using 14representative DNA samples containing 16 unique CF variants. Each sample wastested in duplicate at 9 DNA input levels ranging from 1250 ng to 1 ng (1250 ng,500 ng, 250 hg, 100 ng, 50 ng, 25 ng, 10 ng, 5 ng, and 1 ng). For determination ofaccuracy, sample genotypes were compared to bidirectional Sanger sequencing

Confidential 64

ilIlIUrnin 6a, MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

data and the deletions were compared to PCR assay. 1250 ng and 25 ng wereidentified as the upper and lower bound for DNA input respectively as they had95% 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 4representative DNA samples and at least 20 replicates per DNA input level foreach sample (n= 4 x 20=80 samples), while the lower bound of 25 ng was testedwith 14 samples, 20 replicates for each sample (n=14 x 20=280 samples). Theaccuracy 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 CysticFibrosis System, the performance of the assay was evaluated in the presence andabsence of potential interferents. Sixteen whole blood specimens having uniquegenotypes were included in the study. Four endogenous interfering substances(bilirubin, cholesterol, hemoglobin, and triglycerides) were tested by spikingthem into blood specimens prior to DNA extraction. the concentration limits foreach substanceisshown in the table below. Additionally, to assess interferenceresulting from blood collection (short draw), EDTA was spiked into bloodsamples, and to assess interference resulting from sample preparation, the finalwash buffer from a silica filter column. isolation method was added to purifiedgenomic DNA.

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

To assess the impact of rnultiplexing index primer interference, a crosscontamination study using two samples, each with unique homozygousgenotypes at 4 different genomic positions, and two respective index primerswas performed. NO change in variant calling was observed with contaminationlevels <40%. The sample genotype became heterozygous when contaminationlevels were >40%.'-

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illur6ina:, MiSeqDx Cy stic Fibrosis Clinical Sequencing Assay

Test Substance Concentrationtested in blood Concentration tested in(upper limit) blood (lower limit)

Bilirubin 684 pmol/L 137 pmol/L

Cholesterol 13 mmol/L 2.6mmol/L

Hemoglobin 2gIL 0.4 g/L

EDTA 7.0 mg/mL 2.8mg/mL

Triglycerides 37 mmol/L 7.4 mmol/L

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illurn-ina, MiSeqDx Cystic Fibrosis Clinical Sequencing Assay

Confidential 67

I0

DEPARTMIENT OF H EAL-1-1 &H UMAN SER%'ICKS Public Hlth Sn

IDoujunin Contfrol ('enter - W066-G6(9

Silver Spring. MD 20993.0002

November I19. 201I3

ILLUMINA, INC.2RYAN SCHNEIDERASSOCIATE DIRECTOR. REGUJLATORY AFI:AIRS5200 ILLUMINA \VAYSAN DIEGO. CA 92122

Re: k132750TradelDevice Name: IllUmIina, MiSeqDx Cystic Fihrosis Clinical Sequencing AssayRegulation Number: 21 CER 866.5900Regulation Name: Cystic fibrosis transmemrbranc conductance regulator (CFTR) gene

Mutation detection systemnRegulatory Class: 11Product Code: PFSDated: ALuust 30. 2013Received: September 3.,2013

Dear Mr. Schneider:

We have reviewed your Section 5 10(k) premarket notification of intent to market the devicereferenced above and have determined the device is substantially equivalent (for the indicationsfor use stated in the enclosure) to legally marketed predicate devices marketed in interstatecommerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or todevices that have been reclassified in accordance with the provisions of the Federal Food. Drug.and Cosmetic Act (Act) that do not require approval of a premnarket approval application (P'MA).You may. therefore, market the device, subject to the general controls provisions of the Act. Thegeneral controls provisions of the Act include requirements for annual registration, listing of'devices, good manufacturing practice, labeling, and prohibitions against misbranding andadulteration. Please note: CDRI- does not evaluate information related to contract liabilitywarranties. We remind you, however, that device labeling must be truthful and not misleading.

Ifvyotir device is classified (see above) into either class 11 (Special Controls) or class III (PMA).it may ,be subject to additionl conitrols. Existing major regulations affecting your device can befound in the Code of Federal Regulations. Title 2 1. Parts 8'00 to 898. [In addition. FDA maypublish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equiivalence determination does not meanthat FDA has niade a determination that your device complies with other rep' remnts of the Actor any Federal statutes and regulations administered by other Federal agencies. You mustcomply with all the Act's requirements. including, but not limited to: registration and listing (21CFR Part 807); labeling (21 CER Parts 801 and 809); medical device reporting (reporting ofmedical device-related adverse events) (21 CFR 803); good mianufacturing practice requirementsas set Forth in the quality systems (QS) regulation (2 I CFR P)art 820); and if-applicable, the

Page 2-Mr. Schneider

electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 80! and809), please contact the Division of Small Manufacturers, International and ConsumerAssistance at its toll-free number (800) 638 204! or (301) 796-7 100 or at its Internet addresslitto://www.fda.gov/MedicaDevices/RsotrccsforYou/ndustv/defatlt.htin. Also, please notethe regulation entitled, "Misbranding by reference to premarkec notification" (2ICFR Part807.97). For questions regarding the reporting of adverse events under the MDR regulation (21CFR Pant 803), please go tohtp://www.fda.gov/MedicalDeviccs/Sat'etv/Rep~ortaProblei-n/defauilt.him for the CDRH's Officeof Surveillance and Biometrics/Division of Posunarket Surveillance.

You may obtain other general information on your responsibilities under the Act from theDivision of Small Manufacturers, International and Consumer Assistance at its toll-free number(800) 638-2041 or (30!) 796-7 100 or at its Internet addresshttp://wwwfda.gov/MedicalDev iccs/RcsotircesforYou/I ndutstrv/dctboult.htim.

Sincerely yours,

Reena Philip -Sfor

Maria M. Chan, Ph.D.DirectorDivision of Immunology and Hematology DevicesOffice of In Vitro Diagnostics and Radiological

HealthCenter for Devices and Radiological Health

Enclosure

510(k) Number (if known)

k132750

Device Name

Illumina MiSeqDx(TM) Cystic Fibrosis Clinical Sequencing Assay

Indications for Use (Describe)

The Illumina MiSeqDx(TM) Cystic Fibrosis Clinical Sequencing Assay is a targeted sequencing in vitro diagnostic system thatre-sequences the protein coding regions and iniman/exon boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator(CFTR) gene in genomic DNA isolated frorn human peripheral whale blood specimens collected in K2EDTA. The test detects singlenucleotide variants, and small InDels within the region sequenced, and additionally reports on two deep intronic mutations and twolarge 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 mostappropriate when the patient has an atypical or non-classic presentation of CF or when other mutation panels have failed to identifyboth causative mutations. Ihe results of the test are intended to be interpreted by a board-certified clinical molecular geneticist orequivalent 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 pre-imuplantationtesting, carrier screening, newborn screening, or population screening.

Type of Use (Select one or both, as applicable)

I& Prescription Use (Part 21 CFR B01 Subpart 0) 0Over-The-Counter Use (21 CFR 801 Subpart C)

PLEASE DO NOT WRITE BELOW THIS UINE - CONTINUE ON A SEPARATE PAGE IF NEEDED.

.FOR FDA USE ONLYConcurrence of Center for Devices and Radiological Health (CDRH) (Signature)

K132750 - November 19, 2013

DonTRA--t,"' 51 o

FORM FDA 3881 (8113) Page 1 of 2 fcsftM4OE