Benchmarking of duplex sequencing approaches to reveal somatic mutation landscapes

Abstract Detecting somatic mutations in normal tissues is challenging due to sequencing errors and the low allele fractions of post-zygotic variants. Duplex sequencing greatly reduces errors and can detect mutations at any allele fraction, but systematic, cross-platform comparisons are lacking. We present a comprehensive benchmarking of six duplex sequencing technologies used by the SMaHT Network: CODEC, CompDuplex-seq, HiDEF-seq, NanoSeq, ppmSeq, and VISTA-seq. We evaluated their performance using cord blood DNA, a tumor-normal cell line mixture, and homogenates from six human tissues. Each method shows distinct profiles in genomic footprint, sensitivity, and cost. Despite differences in library construction and sequencing platforms, estimates of mutation rates and mutational signatures are highly concordant. Integration with ultra-deep whole-genome sequencing shows that duplex approaches sensitively capture mutations and signatures beyond embryonic or clonally expanded variants. These results provide a foundation for selecting duplex methods and interpreting their data, enabling scalable single-molecule analyses of somatic mutation landscapes. Competing Interest Statement V.A.A. and R.L. are co-inventors on patent applications filed by the Broad Institute on CODEC and Methyl-CODEC. V.A.A. is a co-inventor on patent applications filed by the Broad Institute on Duplex-Repair and MEDUSA, and on the MAESTRO MRD test which has been licensed to Exact Sciences. V.A.A. is a co-founder and advisor to Amplifyer Bio. G.D.E. has filed a patent application for HiDEF-seq and owns equity in DNA sequencing companies (Illumina, Oxford Nanopore Technologies, and Pacific Biosciences). C.Z. is a co-founder and equity holder of Pioneer Genomics and reports that Baylor College of Medicine filed a patent application related to the CompDuplex-seq or CompDup method. R.A.G and Baylor College of Medicine have equity interest in Codified Genomics, Ltd.