title: Multi-Institutional FASTQ File Exchange as a Means of Proficiency Testing for Next-Generation Sequencing Bioinformatics and Variant Interpretation.
abstract: 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.
authors.affiliation: Department of Pathology, University of Colorado-Anschutz Medical Campus, Denver, Colorado.
authors.forename: Kurtis D
authors.initials: KD
authors.lastname: Davies
authors.affiliation: Department of Pathology, Children's Health & UT Southwestern Medical Center, Dallas, Texas.
authors.forename: Midhat S
authors.initials: MS
authors.lastname: Farooqi
authors.affiliation: Pathology Department, H. Lee Moffitt Cancer Center, Tampa, Florida.
authors.forename: Mike
authors.initials: M
authors.lastname: Gruidl
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Hill
authors.affiliation: Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina.
authors.forename: Julie
authors.initials: J
authors.lastname: Woolworth-Hirschhorn
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Heather
authors.initials: H
authors.lastname: Jones
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado-Anschutz Medical Campus, Denver, Colorado.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Jones
authors.affiliation: Pathology Department, H. Lee Moffitt Cancer Center, Tampa, Florida.
authors.forename: Anthony
authors.initials: A
authors.lastname: Magliocco
authors.affiliation: Department of Pathology, Children's Health & UT Southwestern Medical Center, Dallas, Texas.
authors.forename: Midori
authors.initials: M
authors.lastname: Mitui
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado-Anschutz Medical Campus, Denver, Colorado.
authors.forename: Philip H
authors.initials: PH
authors.lastname: O'Neill
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado-Anschutz Medical Campus, Denver, Colorado.
authors.forename: Rebecca
authors.initials: R
authors.lastname: O'Rourke
authors.affiliation: Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina.
authors.forename: Nirali M
authors.initials: NM
authors.lastname: Patel
authors.affiliation: Pathology Department, H. Lee Moffitt Cancer Center, Tampa, Florida.
authors.forename: Dahui
authors.initials: D
authors.lastname: Qin
authors.affiliation: Department of Pathology, Children's Health & UT Southwestern Medical Center, Dallas, Texas.
authors.forename: Erica
authors.initials: E
authors.lastname: Ramos
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Rossi
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Schneider
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Geoffrey H
authors.initials: GH
authors.lastname: Smith
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Linsheng
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Pathology, Children's Health & UT Southwestern Medical Center, Dallas, Texas.
authors.forename: Jason Y
authors.initials: JY
authors.lastname: Park
authors.affiliation: Department of Pathology, University of Colorado-Anschutz Medical Campus, Denver, Colorado. Electronic address: dara.aisner@ucdenver.edu.
authors.forename: Dara L
authors.initials: DL
authors.lastname: Aisner
mesh_terms: D019295:Computational Biology / Q000379:methods* / Q000592:standards*; D005820:Genetic Testing / Q000379:methods* / Q000592:standards*; D019538:Health Care Surveys; D059014:High-Throughput Nucleotide Sequencing / Q000592:standards*; D006801:Humans; D007753:Laboratories / Q000592:standards; D059021:Laboratory Proficiency Testing