1. Academic Validation
  2. A novel target enrichment strategy in next-generation sequencing through 7-deaza-dGTP-resistant enzymatic digestion

A novel target enrichment strategy in next-generation sequencing through 7-deaza-dGTP-resistant enzymatic digestion

  • BMC Res Notes. 2020 Sep 18;13(1):445. doi: 10.1186/s13104-020-05292-y.
Peng Peng 1 2 Yanjuan Xu 1 Adrian M Di Bisceglie 1 3 Xiaofeng Fan 4 5
Affiliations

Affiliations

  • 1 Division of Gastroenterology & Hepatology, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO, 63104, USA.
  • 2 Wuhan Pulmonary Hospital, Wuhan, 430030, Hubei, China.
  • 3 Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, 63104, USA.
  • 4 Division of Gastroenterology & Hepatology, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, MO, 63104, USA. xiaofeng.fan@health.slu.edu.
  • 5 Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, 63104, USA. xiaofeng.fan@health.slu.edu.
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 next-generation 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

7-deaza-2′-deoxyguanosine 5′-triphosphate; Hepatitis B virus; Next-generation sequencing; Target enrichment.

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