Biology subjects

Shoag, J. E.

Publications and source records attributed to Shoag, J. E..

3 recordsLinked to original sources

bioRxiv2025

Paired plus-minus sequencing is an ultra-high throughput and accurate method for dual strand sequencing of DNA molecules

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 7.98x10-8 for gDNA (using an end-repair protocol with dideoxy nucleotides) and 3.5x10-7{+/-}7.5x10-8 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.

genomics↗

bioRxiv2023

Single-strand mismatch and damage patterns revealed by single-molecule DNA sequencing

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other genetic diseases1-4. Almost all of these mosaic mutations begin as nucleotide mismatches or damage in only one of the two strands of the DNA prior to becoming double-strand mutations if unrepaired or misrepaired5. However, current DNA sequencing technologies cannot resolve these initial single-strand events. Here, we developed a single-molecule, long-read sequencing method that achieves single-molecule fidelity for single-base substitutions when present in either one or both strands of the DNA. It also detects single-strand cytosine deamination events, a common type of DNA damage. We profiled 110 samples from diverse tissues, including from individuals with cancer-predisposition syndromes, and define the first single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumors deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples deficient in only polymerase proofreading. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. Since the double-strand DNA mutations interrogated by prior studies are only the endpoint of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable new studies of how mutations arise in a variety of contexts, especially in cancer and aging.

genomics↗

bioRxiv2019

Integrative genomic, transcriptomic, and epigenomic analyses of benign prostatic hyperplasia reveal new options for therapy

Benign prostatic hyperplasia (BPH), a nonmalignant enlargement of the prostate, is one of the most common diseases affecting aging men, but the underlying molecular features of BPH remain poorly understood, and therapeutic options are limited. Here we employed a comprehensive molecular investigation of BPH, including genomic, transcriptomic and epigenetic profiling of 18 BPH cases. At the molecular level, we found no evidence of neoplastic features in BPH: no evidence of driver genomic alterations, including low coding mutation rates, mutational signatures consistent with aging tissues, minimal copy number alterations, and no genomic rearrangements. Similarly at the epigenetic level, we found global hypermethylation was the dominant process (unlike most neoplastic processes). By integrating transcriptional and methylation signatures, we identified two BPH subgroups with distinct clinical features and associated signaling pathways, which were validated in two independent cohorts. Finally, our analyses nominated mTOR inhibitors as a potential subtype-specific therapeutic option. Supporting this, a cohort of men exposed to mTOR inhibitors showed a significant decrease in prostate size. Our results demonstrate that BPH consists of distinct molecular subgroups, with potential for subtype-specific precision therapy via mTOR inhibition.

genomics↗