Data Sheet: Molecular Diagnostics

MiSeqDx™ Cystic Fibrosis ClinicalSequencing Assay
The first FDA-cleared in vitro diagnostic next-generation sequencing assay toprovide a complete view of the CFTR gene for increased confidence in cystic
fibrosis variant detection.

Highlights• Complete View of the CFTR Gene
Capture all variants in the protein coding regions
and intron/exon boundaries of the CFTR gene• Accurate Results
Deep coverage (> 3,000×) ensures detection
accuracy with a Positive Agreement (PA) of99.66%*• Removed Demographic Bias
Sequencing the CFTR gene removes the bias
inherent in existing genotyping panels

Figure 1: MiSeqDx Cystic Fibrosis Clinical Sequencing Assay. The
MiSeqDx Cystic Fibrosis Clinical Sequencing Assay is the first FDAcleared NGS test to provide a comprehensive view of the CFTR gene.

*Looking at the CFTR Sequence

PA is the number of samples with agreeing variant calls divided
by the total number of samples with that variant as identified by
the reference method.

Introduction
Cystic fibrosis (CF) affects approximately 70,000 children
and adults worldwide1. The disease appears whenan individual inherits two mutated copies of the cystic
fibrosis transmembrane receptor (CFTR) gene. Early
diagnosis and treatment of CF can improve both survivaland quality of life2. Unfortunately, current moleculargenotyping tests target a limited number of mutations,omitting variants that may have some clinical relevance.
As a result, families may endure long periods ofadditional testing and patients may experience a delay inreceiving needed treatment.
To overcome this challenge, Illumina offers the MiSeqDx
Cystic Fibrosis Clinical Sequencing Assay (Figure 1), thefirst FDA-cleared in vitro diagnostic (IVD) next-generation
sequencing (NGS) test for obtaining a comprehensive
view of the CFTR gene.

Using Illumina NGS technology and sequencing bysynthesis (SBS) chemistry, the MiSeqDx Cystic Fibrosis
Clinical Sequencing Assay sequences all protein coding
regions and intron/exon boundaries of the CFTR gene.
In addition, the assay detects two large deletions, two
deep intronic mutations, and indels in homopolymericregions such as the 2184delA deletion (Table 1). Theassay also automatically detects polyTG/polyT variants.
Looking at the full CFTR sequence minimizes thedemographic bias inherent in existing CF genotyping
panel tests. As a result, the MiSeqDx Cystic Fibrosis
Clinical Sequencing Assay can uncover rare mutations
not accounted for in standard tests.

How it Works
The MiSeqDx Cystic Fibrosis Clinical Sequencing Assaytakes advantage of Illumina NGS technology and SBS
chemistry to produce a reliable CFTR sequence. The
streamlined sample preparation workflow replaces themultiple rounds of PCR required by Sanger sequencing

Data Sheet: Molecular Diagnostics
with a single PCR step. For higher throughput, theassay enables multiplexing capabilities for simultaneoussequencing of 8 samples (Figure 2). Additionally, Illumina
NGS is highly accurate and reproducible with ≥ 99.7%
overall agreement for all reported positions and asample first pass rate of 99.7%.

Complete Kit Convenience
The MiSeqDx Cystic Fibrosis Clinical Sequencing Assay
includes everything needed for library preparation,
sample multiplexing, and sequencing in a single kit.
All reagents are packaged in a convenient ready-touse format, minimizing hands-on time and increasing
uniformity in all tests.

Efficient Sample Preparation Increases
Throughput
Library preparation begins with 250 ng genomic DNA(gDNA) isolated from a blood sample. The DNA is mixed
with a pool of oligonucleotide probes. Each probe
includes sequences designed to capture the designatedvariant and an adapter sequence used in a subsequent
amplification reaction. The probes hybridize to theDNA, one upstream and one downstream of specific
CFTR variants (Figure 3). A proprietary extensionligation reaction extends across the region of interest,followed by ligation, to unite the two probes. This
reaction creates a template strand and gives the assay
excellent specificity.
To increase the number of samples analyzed in a single
sequencing run, individual libraries are “tagged” witha unique identifier, or index. These unique samplespecific indices are added to each extensionligation
template in a PCR amplification step. The final reaction
product contains the CFTR variants with the necessary
sequencing adapters and indices for sequencing on
the MiSeqDx instrument. An automated three-readsequencing strategy identifies each tagged sample forindividual downstream analysis. Using this approach,sample identification is highly accurate, maintaining thehigh integrity of the assay. Eight samples can be pooled
in a single sequencing run.

Table 1: CFTR Regions Sequenced in the MiSeqDx
Cystic Fibrosis Clinical Sequencing Assay.
All protein coding regions
Intron/exon boundaries
Large deletions (exon 2,3 and exon 22,23)
Deep intronic mutations (1811+1.6Kb A>G, 3849+10Kb C>T)
Indels in homopolymeric regions
PolyTG/PolyT

PREPARE LIBRARIES
Generate sample libraries using theMiSeqDx Cystic Fibrosis Clinical Sequencing Assay
Attach sample indices to libraries
and amplify with one PCR step
Normalize and pool libraries
Hands-on time: 2 hr 10 min

SEQUENCE
Load pooled library on toMiSeqDx reagent cartridge
MiSeqDx instrument performs automatedcluster generation and sequencing
Hands-on time: 5 min

ANALYZE
View report generated by theMiSeq Reporter software
Hands-on time: 5–30 min
Figure 2: Integrated Clinical Sequencing Assay Workflow. TheMiSeqDx Cystic Fibrosis Clinical Sequencing Assay offers a fully
integrated end-to-end solution for sequencing the protein coding
regions and intron/exon boundaries of the CFTR gene.

Data Sheet: Molecular Diagnostics

Easy Results Interpretation
Probe 1

Region of interest

Probe 2

Results from the MiSeqDx Cystic Fibrosis ClinicalSequencing Assay are presented in an easy-to-readfashion that a board-certified molecular geneticist
or equivalent can easily interpret. The assay report
includes assay name, sample ID, dbSNP ID, HGVS
nomenclature, and the call rates for each base(Figure 4). Call rates for each sample must be ≥ 99% tobe considered valid.

Variant–specifc probes hybridize to flanking
regions of interest in unfragmented gDNA
Probe 1

Probe 2

Extension/Ligation between variant–specifc
probes across regions of interest

Data Quality

PCR adds indices and sequencing primers
P7

Index 1

P5

Index 2

Uniquely tagged library readyfor cluster generation and sequencing
P7

Index 1

Index 2

P5

Figure 3: MiSeqDx Cystic Fibrosis Clinical Sequencing Assay
Chemistry. The MiSeqDx Cystic Fibrosis Clinical Sequencing Assay
offers a highly specific method for sequencing the CFTR gene.

Widely Adopted NGS Platform
Illumina SBS technology is a widely adopted NGS
platform in the sequencing community. Throughmassively parallel sequencing using a proprietary
reversible terminator-based method, it enables detectionof single bases as they are incorporated into growing
DNA strands. A fluorescently labeled terminator
is imaged as each dNTP (deoxyribonucleotide
triphosphates: dATP, dCTP, dGTP, or dTTP) is added
and then cleaved to allow incorporation of the nextbase. Since all four reversible terminator-bound dNTPs
are present during each sequencing cycle, naturalcompetition minimizes incorporation bias. The result
is base-by-base sequencing that enables highly
accurate data.

The MiSeqDx Cystic Fibrosis Clinical Sequencing Assay
supports 8 samples per run, while providing excellent
specificity and uniformity. To assess performance,accuracy, and reproducibility, studies were conductedusing Sanger sequencing and PCR as reference
methods. Total accuracy and reproducibility were
≥ 99.7%.

Summary
The MiSeqDx Cystic Fibrosis Clinical Sequencing Assayis the first FDA-cleared NGS diagnostic test for CF.
Sequencing the CFTR gene provides a comprehensive
genetic view not available using standard moleculargenotyping panels. The additional data eliminates
demographic bias and enables accurate detectionof two large deletions, two deep intronic mutations,and indels in homopolymeric regions. This single
assay can lead to faster identification of clinically
relevant mutations.

Data Sheet: Molecular Diagnostics

Figure 4: Easy Visualization Using the MiSeq Reporter Software.

Learn MoreIntended Use

To learn more about the MiSeqDx Cystic Fibrosis ClinicalSequencing Assay, visit www.illumina.com/cysticfibrosis.

References
1. Cystic Fibrosis Foundation (www.cff.org/AboutCF/Faqs/)
2. U.S. National Library of Medicine. PubMed Health (www.ncbi.nlm.
nih.gov/pubmedhealth/PMH0001167/)
Ordering Information.
Product

Catalog No.

MiSeqDx Cystic Fibrosis Clinical Sequencing
Assay (6 runs, up to 48 samples)

DX-102-1001

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 ingenomic DNA isolated from human peripheral whole blood specimens
collected in K2EDTA. The test detects single nucleotide variants andsmall indels within the region sequenced, and additionally reports
on two deep intronic mutations and two large deletions. The test isintended 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 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 aboard-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 notindicated for use for stand-alone diagnostic purposes, fetal diagnostic
testing, pre-implantation testing, carrier screening, newborn screening,or population screening.

Illumina • 1.800.809.4566 toll-free (U.S.) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For In Vitro Diagnostic Use. Contact an Illumina Representative for Regional Availability.
© 2015 Illumina, Inc. All rights reserved.
Illumina, MiSeqDx, and the pumpkin orange color are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/orother countries. Pub. No. 670-2013-007 Current as of 26 August 2015
Document # 1000000006361 v00