title: Orthogonal NGS for High Throughput Clinical Diagnostics.
abstract: Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Niru
authors.initials: N
authors.lastname: Chennagiri
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: White
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Frieden
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Edgardo
authors.initials: E
authors.lastname: Lopez
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Lieber
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Nikiforov
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Tristen
authors.initials: T
authors.lastname: Ross
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Batorsky
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Sherry
authors.initials: S
authors.lastname: Hansen
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Va
authors.initials: V
authors.lastname: Lip
authors.affiliation: Harvard Medical School, Boston MA, USA.
authors.forename: Lovelace J
authors.initials: LJ
authors.lastname: Luquette
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Evan
authors.initials: E
authors.lastname: Mauceli
authors.affiliation: Claritas Genomics, Cambridge MA, USA.|Harvard Medical School, Boston MA, USA.|Boston Children's Hospital, Boston MA, USA.
authors.forename: David
authors.initials: D
authors.lastname: Margulies
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Patrice M
authors.initials: PM
authors.lastname: Milos
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Nichole
authors.initials: N
authors.lastname: Napolitano
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Marcia M
authors.initials: MM
authors.lastname: Nizzari
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Yu
authors.affiliation: Claritas Genomics, Cambridge MA, USA.
authors.forename: John F
authors.initials: JF
authors.lastname: Thompson
mesh_terms: D059472:Exome; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*