{"doi":"10.1101/2025.08.11.669689","title":"Paired plus-minus sequencing is an ultra-high throughput and accurate method for dual strand sequencing of DNA molecules","abstract":"Abstract Distinguishing real biological variation in the form of single-nucleotide variants (SNVs) from errors is a major challenge for genome sequencing technologies. This is particularly true in settings where SNVs are at low frequency such as cancer detection through liquid biopsy, or human somatic mosaicism. State-of-the-art molecular denoising approaches for DNA sequencing rely on duplex sequencing, where both strands of a single DNA molecule are sequenced to discern true variants from errors arising from single stranded DNA damage. However, such duplex approaches typically require massive over-sequencing to overcome low capture rates of duplex molecules. To address these challenges, we introduce paired plus-minus sequencing (ppmSeq) technology, in which both DNA strands are partitioned and clonally amplified on sequencing beads through emulsion PCR. In this reaction, both strands of a double-stranded DNA molecule contribute to a single sequencing read, allowing for a duplex yield that scales linearly with sequencing coverage across a wide range of inputs (1.8-98 ng). We benchmarked ppmSeq against current duplex sequencing technologies, demonstrating superior duplex recovery with ppmSeq, with a rate of 44%±5.5% (compared to ∼5-11% for leading duplex technologies). Using both genomic as well as cell-free DNA, we established error rates for ppmSeq, which had residual SNV detection error rates as low as 7.98×10 -8 for gDNA (using an end-repair protocol with dideoxy nucleotides) and 3.5×10 -7 ±7.5×10 -8 for cell-free DNA. To test the capabilities of ppmSeq for error-corrected whole-genome sequencing (WGS) for clinical application, we assessed circulating tumor DNA (ctDNA) detection for disease monitoring in cancer patients. We demonstrated that ppmSeq enables powerful tumor-informed ctDNA detection at concentrations of 10 -4 across most cancers, parts per million sensitivity in cancers with high mutation burden, and further increased sensitivity with higher sequencing depth. We then leveraged genome-wide trinucleotide mutation patterns characteristic of urothelial (APOBEC3-related and platinum exposure-related signatures) and lung (tobacco-exposure-related signatures) cancers to perform tumor-naive ctDNA detection, showing that ppmSeq can identify a disease-specific signal in plasma cell-free DNA without a matched tumor, and that this signal correlates with imaging-based disease metrics. Altogether, ppmSeq provides an error-corrected, cost-efficient and scalable approach for high-fidelity WGS that can be harnessed for challenging clinical applications and emerging frontiers in human somatic genetics where high accuracy is required for mutation identification.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":555714,"datarank":0.21029146533721052,"base_score":1.3862943611198906,"endowment":1.3862943611198906,"self_citation_contribution":0.20794415416798362,"citation_network_contribution":0.0023473111692269063,"self_endowment_contribution":0.20794415416798362,"citer_contribution":0.0023473111692269063,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":2,"citers_with_citation_signal":1,"citers_with_endowment":1,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9546,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":985715,"name":"Itai Rusinek","orcid":"0000-0001-5027-5764","position":1,"is_corresponding":false},{"id":818436,"name":"Aaron Sossin","orcid":"0000-0003-2546-3389","position":2,"is_corresponding":false},{"id":615859,"name":"Adam J. 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