Benchmarking challenging small variants with linked and long reads

Genome in a Bottle benchmarks are widely used to help validate clinical sequencing pipelines and develop variant calling and sequencing methods. Here we use accurate linked and long reads to expand benchmarks in 7 samples to include difficult-to-map regions and segmental duplications that are challenging for short reads. These benchmarks add more than 300,000 SNVs and 50,000 insertions or deletions (indels) and include 16% more exonic variants, many in challenging, clinically relevant genes not covered previously, such as PMS2. For HG002, we include 92% of the autosomal GRCh38 assembly while excluding regions problematic for benchmarking small variants, such as copy number variants, that should not have been in the previous version, which included 85% of GRCh38. It identifies eight times more false negatives in a short read variant call set relative to our previous benchmark. We demonstrate that this benchmark reliably identifies false positives and false negatives across technologies, enabling ongoing methods development.

Cell Genomics Volume 2, Issue 5, 11 May 2022, 100128


Other Contributors

Justin Wagner,Nathan D. Olson,Lindsay Harris,Ziad Khan,Jesse Farek,Medhat Mahmoud,Ana Stankovic,Vladimir Kovacevic,Byunggil Yoo,Neil Miller,Jeffrey A. Rosenfeld,Bohan Ni,Samantha Zarate,Melanie Kirsche,Sergey Aganezov,Michael C. Schatz,Giuseppe Narzisi,Marta Byrska-Bishop,Wayne Clarke,Uday S. Evani,Charles Markello,Kishwar Shafin,Xin Zhou,Arend Sidow,Vikas Bansal,Peter Ebert,Tobias Marschall,Peter Lansdorp,Vincent Hanlon,Carl-Adam Mattsson,Alvaro Martinez Barrio,Ian T. Fiddes,Chunlin Xiao,Arkarachai Fungtammasan,Chen-Shan Chin,Aaron M. Wenger,William J. Rowell,Fritz J. Sedlazeck,Andrew Carroll,Marc Salit,Justin M. Zook

Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Dr, MS8312, Gaithersburg, MD 20899, USA
Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Seven Bridges, Omladinskih brigada 90g, 11070 Belgrade, Republic of Serbia
Children’s Mercy Kansas City, Kansas City, MO, USA
Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA
Department of Computer Science, Johns Hopkins University, Baltimore, MD, USA
New York Genome Center, 101 Avenue of the Americas, New York, NY, USA
University of California at Santa Cruz Genomics Institute, 1156 High Street, Santa Cruz, CA, USA
Department of Computer Science, Stanford University, Stanford, CA 94305, USA
Department of Pathology, Stanford University, Stanford, CA 94305, USA
Department of Genetics, Stanford University, Stanford, CA 94305, USA
Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA
Institute of Medical Biometry and Bioinformatics, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany
Terry Fox Laboratory, BC Cancer Research Institute and Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
10X Genomics, Pleasanton, CA 94588, USA
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, 8600 Rockville Pike, Bethesda, MD 20894, USA
DNAnexus, Inc., Mountain View, CA 94040, USA
Pacific Biosciences, Menlo Park, CA 94025, USA
Google Inc., 1600 Amphitheatre Pkwy., Mountain View, CA 94040, USA
Joint Initiative for Metrology in Biology, SLAC National Laboratory, Stanford, CA, USA