A novel target enrichment strategy in next-generation sequencing through 7-deaza-dGTP-resistant enzymatic digestion
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(2020) 13:445 Peng et al. BMC Res Notes https://doi.org/10.1186/s13104-020-05292-y
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RESEARCH NOTE
A novel target enrichment strategy in next‑generation sequencing through 7‑deaza‑dGTP‑resistant enzymatic digestion Peng Peng1,3, Yanjuan Xu1, Adrian M. Di Bisceglie1,2 and Xiaofeng Fan1,2*
Abstract Objective: Owing to the overwhelming dominance of human and commensal microbe sequences, low efficiency is a major concern in clinical viral sequencing using next-generation sequencing. DNA composed of 7-deaza-2′deoxyguanosine 5′-triphosphate (c7dGTP), an analog of deoxyguanosine triphosphate (dGTP), is resistant to selective restriction enzymes. This characteristic has been utilized to develop a novel strategy for target enrichment in nextgeneration sequencing. Results: The new enrichment strategy is named target enrichment via enzymatic digestion in next-generation sequencing (TEEDseq). It combined 7-deaza-2′-deoxyguanosine 5′-triphosphate (c7dGTP)-involved primer extension, splinter-assisted intracellular cyclization, c7dGTP)-resistant enzymatic digestion, and two-phase rolling cycle amplification. We first estimated c7dGTP for its efficiency in PCR amplification and its resistance to three restriction enzymes, AluI, HaeIII, and HpyCH4V. We then evaluated TEEDseq using a serum sample spiked with a 1311-bp hepatitis B virus (HBV) fragment. TEEDseq achieved an HBV on-target rate of 3.31 ± 0.39%, which was equivalent to 454× the enrichment of direct Illumina sequencing. Therefore, the current study has provided a concept proof for TEEDseq as an alternative option for clinical viral sequencing that requires an enrichment in next-generation sequencing. Keywords: Next-generation sequencing, Target enrichment, 7-deaza-2′-deoxyguanosine 5′-triphosphate, Hepatitis B virus Introduction In current clinical viral genome sequencing, next-generation sequencing (NGS) is a frequent choice that provides an unbiased high resolution of mutation profile in a genome-wide manner [1]. Because of an overwhelming dominance of human genetic content in clinical specimens, a major limitation of this approach is its low *Correspondence: [email protected] 1 Division of Gastroenterology & Hepatology, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO 63104, USA Full list of author information is available at the end of the article
efficiency, which is rarely higher than 1% of viral sequencing reads in NGS output [1]. Among numerous virusenriched methods, capture sequencing, employing a hybridization step after NGS library construction, comes out as the most efficient strategy to enrich viral sequences [1]. However, this strategy is associated with a dramatic cost increase as it requires the synthesis of expensive biotin labeled virus-specific probes (baits) and streptavidin beads [2]. The inclusion of such a hybridization step after initial library preparation also makes the entire NGS pipeline a lengthy procedure. Most human viruses, such as hepatitis B virus (HBV), hepatitis C
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