Biology subjects

Glover, T. W.

Publications and source records attributed to Glover, T. W..

3 recordsLinked to original sources

bioRxiv2026

High-fidelity rare structural variant detection with HiFiRE3 reduced representation via restriction enzyme ends

High-fidelity detection of rare structural variants (SVs) remains challenging because library preparation and sequencing techniques generate artifactual junctions that obscure true single-molecule events. Here, we present HiFiRe3, an error-minimized sequencing framework that combines artifact-aware library design with error suppression and correction strategies to enable rare SV detection and frequency assessment across long and short-read sequencing platforms. We first systematically characterized major classes of SV artifacts, including chimeric PCR products, intermolecular ligation, sequencing platform-specific artifacts, and mapping errors. HiFiRe3 supports error correction of these artifact junctions by combining reduced representation restriction fragments with pre-ligation size selection to enable computational filtering via independent forced restriction enzyme end (FREE) and <1N size logics. In nanopore libraries, these approaches enabled targeted detection of single-molecule SVs at replication stress hotspots in cultured human cells exposed to genotoxicants and in long genes in untreated mouse brains, while markedly reducing singleton translocation artifacts. HiFiRe3 extends to PacBio sequencing for joint SV and SNV error correction and to short-read platforms for cost-efficient high-fidelity nonhomologous SV analysis. Together, HiFiRe3 is a flexible framework for accurately detecting rare genomic structural variation with broad applicability to targeted and genome-wide studies by selective application of its error correction approaches.

genomics↗

bioRxiv2024

POLQ mediates replication-stress induced structural variant formation throughout common fragile sites during mitosis

Genomic structural variants (SVs) greatly impact human health and disease, but much is unknown about their generative mechanisms, especially for the large class of nonrecurrent alterations. Common fragile sites (CFSs) are unstable loci that provide a model for SV formation, especially large deletions, under replication stress. We studied SV junction formation as it occurred in cells by applying error-minimized capture sequencing to CFS DNA harvested during replication stress. SV junctions formed throughout CFS genes at a 5-fold higher rate after cells passed from G2 into M-phase. Neither SV formation nor CFS expression depended on mitotic DNA synthesis (MiDAS), an error-prone form of conservative replication active at CFSs. Instead, analysis of tens of thousands of de novo SV junctions combined with DNA repair pathway inhibition revealed a primary role for DNA polymerase theta (POLQ)-mediated end-joining (TMEJ) in M-phase SV formation. We propose an important role for TMEJ in nonrecurrent SV formation genome wide.

genetics↗

bioRxiv2022

svCapture: Efficient and specific detection of very low frequency structural variant junctions by error-minimized capture sequencing

Error-corrected sequencing of genomic targets enriched by probe-based capture has become a standard approach for detecting single-nucleotide variants (SNVs) and small insertion/deletions (indels) present at very low variant allele frequencies. Less attention has been given to strategies for comparable detection of rare structural variant (SV) junctions, where different error mechanisms must be addressed. Working from cell samples with known SV properties, we demonstrate that Duplex Sequencing (DuplexSeq), which demands confirmation of variants on both strands of a source DNA molecule, eliminates false SV junctions arising from chimeric PCR. DuplexSeq could not address frequent intermolecular ligation artifacts that arise during Y-adapter addition prior to strand denaturation without requiring multiple source molecules. In contrast, tagmentation libraries coupled with data filtering based on strand family size greatly reduced both artifact classes and enabled efficient and specific detection of even single-molecule SV junctions. The throughput of SV capture sequencing (svCapture) and the high base-level accuracy of DuplexSeq provided detailed views of the microhomology profile and limited occurrence of de novo SNVs near the junctions of hundreds of sub-clonal and newly created SVs, suggesting end joining as a predominant formation mechanism. The open source svCapture pipeline enables rare SV detection as a routine addition to SNVs/indels in properly prepared capture sequencing libraries.

genomics↗