In:
Scientific Reports, Springer Science and Business Media LLC, Vol. 13, No. 1 ( 2023-08-25)
Abstract:
Although methods for sequencing library preparation from double-stranded DNA are well established, those from single-stranded DNA (ssDNA) have not been well studied. Further, the existing methods have limitations in efficiency and yield. Therefore, we developed a highly efficient procedure for sequencing library preparation from ssDNA. In this method, the first adaptor tagging of ssDNA is performed using terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation, which we reported recently. After complementary strand synthesis using the adaptor-tagged ssDNA, second adaptor tagging via Vaccinia virus topoisomerase I (VTopoI or TOPO)-based adaptor ligation is performed. With additional steps for degradation, repression, and removal of the adaptor dimer, the proposed TACS-TOPO scheme realizes adaptor dimer-free sequencing library preparation from ssDNA samples of 24 pg. The TACS-TOPO scheme was successfully applied to cell-free DNA analysis with amplification-free library preparation from 50 µL of human serum. A modified TACS-TOPO scheme was also applied to DNA extracted from ancient human bones, bringing two to eight times more library yields than those using a conventional library preparation protocol. The procedures for preparing VTopoI and its complex with a double-stranded oligonucleotide adaptor are also described. Overall, the proposed TACS-TOPO scheme can facilitate practical and sensitive sequencing analysis of ssDNA.
Type of Medium:
Online Resource
ISSN:
2045-2322
DOI:
10.1038/s41598-023-40890-3
Language:
English
Publisher:
Springer Science and Business Media LLC
Publication Date:
2023
detail.hit.zdb_id:
2615211-3
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