Clinical investigational studies for validation of a next-generation sequencing in vitro diagnostic device for cystic fibrosis testing

Expert Rev Mol Diagn. 2014 Jun;14(5):605-22. doi: 10.1586/14737159.2014.916618.

Abstract

Purpose: 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.

Conclusion: 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.

Keywords: clinical validation; cystic fibrosis; in vitro diagnostics; next-generation sequencing; sequencing-by-synthesis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Cystic Fibrosis / diagnosis*
  • Cystic Fibrosis / genetics
  • High-Throughput Nucleotide Sequencing / methods
  • High-Throughput Nucleotide Sequencing / standards*
  • Humans
  • Molecular Diagnostic Techniques / methods
  • Molecular Diagnostic Techniques / standards*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Analysis, DNA / methods
  • Sequence Analysis, DNA / standards*