Enhanced SNP genotype recovery from low-template DNA using primary template-directed amplification and hybrid capture-based MPS

Enhanced SNP genotype recovery from low-template DNA using primary template-directed amplification and hybrid capture-based NGS | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced SNP genotype recovery from low-template DNA using primary template-directed amplification and hybrid capture-based NGS Su Min Joo, Ye‑Lim Kwon, Kyoung‑Jin Shin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7287428/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Forensic DNA analysis often faces challenges with low-template DNA (LT DNA) due to stochastic effects that compromise genotyping reliability. Although whole genome amplification (WGA) has previously been investigated as a strategy to amplify trace amounts of DNA, its forensic application remains limited by amplification bias and artifacts. Primary template-directed amplification (PTA), a recently developed low-bias WGA method, offers improved coverage uniformity and genome reproducibility. However, its applicability in forensic contexts has not yet been explored. In this study, we developed a workflow integrating PTA with hybrid capture-based next generation sequencing (NGS) targeting 1,225 identity-informative single nucleotide polymorphisms (SNPs). Serially diluted genomic DNA (gDNA) samples (1 ng to 6.25 pg) were amplified using PTA, followed by library preparation and hybrid capture-based sequencing. To evaluate the applicability of PTA for SNP genotyping of LT DNA, coverage uniformity, library complexity, SNP genotype recovery, and false-positive rates were investigated. The improvements in coverage uniformity and library complexity achieved by PTA led to a marked enhancement in SNP genotype recovery, reaching 96.0% at 100 pg and 39.7% (more than 400 autosomal SNPs) at a single-cell-equivalent input (6.25 pg). Furthermore, PTA maintained false-positive rates consistently below 1% regardless of input amounts, indicating high reliability of the developed PTA-based workflow. These findings demonstrate the utility of PTA in enhancing SNP genotype recovery for LT DNA analysis, highlighting its potential for forensic applications. Low-template DNA Whole genome amplification Primary template-directed amplification Single nucleotide polymorphisms Hybridization capture Next generation sequencing Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryTableS1.xlsx Supplementary Table S1 SupplementaryTableS2.xlsx Supplementary Table S2 SupplementaryTableS3.xlsx Supplementary Table S3 SupplementaryTableS4.xlsx Supplementary Table S4 SupplementaryTableS5.xlsx Supplementary Table S5 SupplementaryFigures.docx Supplementary Figures Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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