Multi-Institutional FASTQ File Exchange as a Means of Proficiency Testing for Next-Generation Sequencing Bioinformatics and Variant Interpretation

Kurtis D. Davies, Midhat S. Farooqi, Mike Gruidl, Charles E. Hill, Julie Woolworth-Hirschhorn, Heather Jones, Kenneth L. Jones, Anthony Magliocco, Midori Mitui, Philip H. O'Neill, Rebecca O'Rourke, Nirali M. Patel, Dahui Qin, Erica Ramos, Michael R. Rossi, Thomas M. Schneider, Geoffrey H. Smith, Linsheng Zhang, Jason Y. Park, Dara L. Aisner

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


Next-generation sequencing is becoming increasingly common in clinical laboratories worldwide and is revolutionizing clinical molecular testing. However, the large amounts of raw data produced by next-generation sequencing assays and the need for complex bioinformatics analyses present unique challenges. Proficiency testing in clinical laboratories has traditionally been designed to evaluate assays in their entirety; however, it can be alternatively applied to separate assay components. We developed and implemented a multi-institutional proficiency testing approach to directly assess custom bioinformatics and variant interpretation processes. Six clinical laboratories, all of which use the same commercial library preparation kit for next-generation sequencing analysis of tumor specimens, each submitted raw data (FASTQ files) from four samples. These 24 file sets were then deidentified and redistributed to five of the institutions for analysis and interpretation according to their clinically validated approach. Among the laboratories, there was a high rate of concordance in the calling of single-nucleotide variants, in particular those we considered clinically significant (100% concordance). However, there was significant discordance in the calling of clinically significant insertions/deletions, with only two of seven being called by all participating laboratories. Missed calls were addressed by each laboratory to improve their bioinformatics processes. Thus, through our alternative proficiency testing approach, we identified the bioinformatic detection of insertions/deletions as an area of particular concern for clinical laboratories performing next-generation sequencing testing.

Original languageEnglish (US)
Pages (from-to)572-579
Number of pages8
JournalJournal of Molecular Diagnostics
Issue number4
StatePublished - Jul 1 2016

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine


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