Comprehensive benchmarking of somatic single-nucleotide variant and indel detection at ultra-low allele fractions using short- and long-read data

Comprehensive benchmarking of somatic single-nucleotide variant and indel detection at ultra-low allele fractions using short- and long-read data - Find this author on Google Scholar - Find this author on PubMed - Search for this author on this site - ORCID record for Yoo-Jin Jiny Ha - For correspondence: yoojinha{at}hanyang.ac.kr kardlie{at}broadinstitute.org rfulton22{at}wustl.edu sgermer{at}nygenome.org agibbs{at}bcm.edu gmarth{at}genetics.utah.edu James.Bennett{at}seattlechildrens.org peter_park{at}hms.harvard.edu - Find this author on Google Scholar - Find this author on PubMed - Search for this author on this site - ORCID record for Kristin Ardlie - For correspondence: yoojinha{at}hanyang.ac.kr kardlie{at}broadinstitute.org rfulton22{at}wustl.edu sgermer{at}nygenome.org agibbs{at}bcm.edu gmarth{at}genetics.utah.edu James.Bennett{at}seattlechildrens.org peter_park{at}hms.harvard.edu - Find this author on Google Scholar - Find this author on PubMed - Search for this author on this site - ORCID record for Robert S. Fulton - For correspondence: yoojinha{at}hanyang.ac.kr kardlie{at}broadinstitute.org rfulton22{at}wustl.edu sgermer{at}nygenome.org agibbs{at}bcm.edu gmarth{at}genetics.utah.edu James.Bennett{at}seattlechildrens.org peter_park{at}hms.harvard.edu - Find this author on Google Scholar - Find this author on PubMed - Search for this author on this site - ORCID record for Soren Germer - For correspondence: yoojinha{at}hanyang.ac.kr kardlie{at}broadinstitute.org rfulton22{at}wustl.edu sgermer{at}nygenome.org agibbs{at}bcm.edu gmarth{at}genetics.utah.edu James.Bennett{at}seattlechildrens.org peter_park{at}hms.harvard.edu - Find this author on Google Scholar - Find this author on PubMed - Search for this author on this site - ORCID record for Richard Gibbs - For correspondence: yoojinha{at}hanyang.ac.kr kardlie{at}broadinstitute.org rfulton22{at}wustl.edu sgermer{at}nygenome.org agibbs{at}bcm.edu gmarth{at}genetics.utah.edu James.Bennett{at}seattlechildrens.org peter_park{at}hms.harvard.edu - Find this author on Google Scholar - Find this author on PubMed - Search for this author on this site - ORCID record for Gabor T. Marth - For correspondence: yoojinha{at}hanyang.ac.kr kardlie{at}broadinstitute.org rfulton22{at}wustl.edu sgermer{at}nygenome.org agibbs{at}bcm.edu gmarth{at}genetics.utah.edu James.Bennett{at}seattlechildrens.org peter_park{at}hms.harvard.edu - Find this author on Google Scholar - Find this author on PubMed - Search for this author on this site - ORCID record for James T. Bennett - For correspondence: yoojinha{at}hanyang.ac.kr kardlie{at}broadinstitute.org rfulton22{at}wustl.edu sgermer{at}nygenome.org agibbs{at}bcm.edu gmarth{at}genetics.utah.edu James.Bennett{at}seattlechildrens.org peter_park{at}hms.harvard.edu - Find this author on Google Scholar - Find this author on PubMed - Search for this author on this site - ORCID record for Peter J. Park - For correspondence: yoojinha{at}hanyang.ac.kr kardlie{at}broadinstitute.org rfulton22{at}wustl.edu sgermer{at}nygenome.org agibbs{at}bcm.edu gmarth{at}genetics.utah.edu James.Bennett{at}seattlechildrens.org peter_park{at}hms.harvard.edu Abstract Mosaic mutations in normal tissues occur at low variant allele fractions (VAFs), complicating detection. To benchmark strategies, the SMaHT Network created a cell-line mixture (1:49) and produced ultra-deep whole-genome sequencing using short and long reads (five centers, 180–500× each). We assembled a reference of 44,008 mosaic SNVs and 2,059 Indels, cross-validation between platforms to expose limits of short-read analysis. We also partitioned the genome by mappability to examine the impact of genomic context, added a negative reference set, and accounted for culture-derived mutations. When seven institutions applied eleven algorithms to mixture data, call sets were largely discordant across tools and replicates, partly reflecting stochastic presence of low-VAF mutations in biological replicants. For >2% VAF SNVs, sensitivity and precision approached ∼80% at ≥300×, with little gain from additional sequencing. This work provides a comprehensive framework for reliable detection of low-VAF mutations in non-cancer tissues and a valuable resource for the community. Competing Interest Statement Peter Park a member of the Scientific Advisory Board for Bioskryb Genomics. James Bennett is a consultant for Mosaica Medicines. Andrew Stergachis is a co-inventor on a patent relating to the Fiber-seq and DAF-seq methods. 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