Clinical investigational studies for validation of a next-generation sequencing in vitro diagnostic device for cystic fibrosis testing.
OBJECTIVE
Clinical investigational studies were conducted to demonstrate the accuracy and reproducibility of the Illumina MiSeqDx CF System, a next-generation sequencing (NGS) in vitro diagnostic device for cystic fibrosis testing.


METHODS
Two NGS assays - a Clinical Sequencing Assay (Sequencing Assay) and a 139-Variant Assay (Variant Assay) - were evaluated in both an Accuracy Study and a Reproducibility Study, with comparison to bi-directional Sanger sequencing and PCR as reference methods. For each study, positive agreement (PA), negative agreement (NA), and overall agreement (OA) were evaluated.


RESULTS
In the Accuracy Study, the Sequencing Assay achieved PA of 99.7% including the polyTG/polyT region and PA of 100% excluding the region. The Variant Assay achieved PA of 100%. NA and OA were >99.99% for both Assays. In the Reproducibility Study, the Sequencing Assay achieved PA of 99.2%; NA and OA were both 99.7%. The Variant Assay achieved PA of 99.8%; NA and OA were both 99.9%. Sample pass rates were 99.7% in both studies for both assays.


CONCLUSIONS
This is the first systematic evaluation of a NGS platform for broad clinical use as an in vitro diagnostic, including accuracy validation with multiple reference methods and reproducibility validation at multiple clinical sites. These NGS-based Assays had accurate and reproducible results which were comparable to or better than other methods currently in clinical use for clinical genetic testing of cystic fibrosis.
Illumina, Inc., San Diego, CA 92122, USA.
Daniel S
DS
Grosu
Lynda
L
Hague
Manjula
M
Chelliserry
Kristina M
KM
Kruglyak
Ross
R
Lenta
Brandy
B
Klotzle
Jonathan
J
San
Wendy M
WM
Goldstein
Sharmili
S
Moturi
Patricia
P
Devers
Julie
J
Woolworth
Eric
E
Peters
Barbara
B
Elashoff
Jay
J
Stoerker
Daynna J
DJ
Wolff
Kenneth J
KJ
Friedman
W Edward
WE
Highsmith
Erick
E
Lin
Frank S
FS
Ong
D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards*
clinical validation; cystic fibrosis; in vitro diagnostics; next-generation sequencing; sequencing-by-synthesis