Whole genome error-corrected sequencing for sensitive circulating tumor DNA cancer monitoring

Circulating cell-free DNA (ccfDNA) sequencing for low-burden cancer monitoring is limited by sparsity of circulating tumor DNA (ctDNA), the abundance of genomic material within a plasma sample, and pre-analytical error rates due to library preparation, and sequencing errors. Sequencing costs have historically favored the development of deep targeted sequencing approaches for overcoming sparsity in ctDNA detection, but these techniques are limited by the abundance of ccfDNA in samples, which imposes a ceiling on the maximal depth of coverage in targeted panels. Whole genome sequencing (WGS) is an orthogonal approach to ctDNA detection that can overcome the low abundance of ccfDNA by supplanting sequencing depth with breadth, integrating signal across the entire tumor mutation landscape. However, the higher cost of WGS limits the practical depth of coverage and hinders broad adoption. Lower sequencing costs may thus allow for enhanced ctDNA cancer monitoring via WGS. We therefore applied emerging lower-cost WGS (Ultima Genomics, 1USD/Gb) to plasma samples at ~120x coverage. Copy number and single nucleotide variation profiles were comparable between matched Ultima and Illumina datasets, and the deeper WGS coverage enabled ctDNA detection at the parts per million range. We further harnessed these lower sequencing costs to implement duplex error-corrected sequencing at the scale of the entire genome, demonstrating a ~1,500x decrease in errors in the plasma of patient-derived xenograft mouse models, and error rates of ~10-7 in patient plasma samples. We leveraged this highly de-noised plasma WGS to undertake cancer monitoring in the more challenging context of resectable melanoma without matched tumor sequencing. In this context, duplex-corrected WGS allowed us to harness known mutational signature patterns for disease monitoring without matched tumors, paving the way for de novo cancer monitoring. ### Competing Interest Statement A.P.C. and D.A.L. have filed a provisional patent regarding certain aspects of this manuscript. D.A.L. and A.J.W. have also filed two additional patent applications regarding work presented in this manuscript. A.P.C. is listed as an inventor on submitted patents pertaining to cell-free DNA (US patent applications 63/237,367, 63/056,249, 63/015,095, 16/500,929) and receives consulting fees from Eurofins Viracor. D.A.L. received research support from Illumina, Inc., Ultima Genomics, Celgene, 10X genomics and Abbvie. D.A.L. is a scientific co-founder of C2i Genomics and an equity holder. Additional consulting was provided by D.A.L. for Illumina, Pharmacyclics, Mission Bio, Pangea, Alethiomics, and AstraZeneca. I.R., A.J. and D.L. are employees and shareholders of Ultima Genomics. J.D.W. is a consultant for Apricity, CellCarta, Ascentage Pharma, AstraZeneca, Astellas, Bicara Therapeutics. Boehringer Ingelheim, Bristol Myers Squibb, Daiichi Sankyo, Dragonfly, Georgiamune, Imvaq, Larkspur, Psioxus, Recepta, Tizona, Sellas; reports grant and research support from Bristol Myers Squibb and Sephora and has equity in Apricity, Arsenal IO, Ascentage, Beigene, Imvaq, Linneaus, Georgiamune, Maverick, Tizona Pharmaceuticals and Trieza. A.S. receives research funding from AstraZeneca, has served on Advisory Boards for AstraZeneca, Blueprint Medicines, and Jazz Pharmaceuticals, and has been a consultant for Genentech. M.A.P. has received consulting fees from BMS, Merck, Novartis, Eisai, Pfizer, Chugai and has received institutional support from RGenix, Infinity, BMS, Merck and Novartis. M.K.C. has received consulting fees from BMS, Merck, InCyte, Moderna, ImmunoCore, and AstraZeneca and receives institutional support from BMS. S.T. is funded by Cancer Research UK (grant reference number A29911); the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC10988), the UK Medical Research Council (FC10988), and the Wellcome Trust (FC10988); the National Institute for Health Research (NIHR) Biomedical Research Centre at the Royal Marsden Hospital and Institute of Cancer Research (grant reference number A109), the Royal Marsden Cancer Charity, The Rosetrees Trust (grant reference number A2204), Ventana Medical Systems Inc (grant reference numbers 10467 and 10530), the National Institute of Health (U01 CA247439) and Melanoma Research Alliance (Award Ref no 686061). S.T. has received speaking fees from Roche, Astra Zeneca, Novartis and Ipsen. S.T. has the following patents filed: Indel mutations as a therapeutic target and predictive biomarker PCTGB2018/051892 and PCTGB2018/051893. G.M.B. has sponsored research agreements through her institution with: Olink Proteomics, Teiko Bio, InterVenn Biosciences, Palleon Pharmaceuticals; served on advisory boards for: Iovance, Merck, Nektar Therapeutics, Novartis, and Ankyra Therapeutics; consulted for: Merck, InterVenn Biosciences, and Ankyra Therapeutics; and holds equity in Ankyra Therapeutics.