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

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.