A highly efficient scheme for library preparation from single-stranded DNA

• Published in: Scientific reports Vol. 13; no. 1; p. 13913
• Main Authors: Miura, Fumihito, Kanzawa-Kiriyama, Hideaki, Hisano, Osamu, Miura, Miki, Shibata, Yukiko, Adachi, Noboru, Kakuda, Tsuneo, Shinoda, Ken-ichi, Ito, Takashi
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 25.08.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/2217; 631/1647/48; 631/1647/514/2254; 631/45/147; 631/45/607/1161; 631/45/607/1166; 631/45/607/1171; 692/53/2421; Antibiotics; Cloning; Deoxyribonucleic acid; DNA; DNA nucleotidylexotransferase; DNA sequencing; DNA topoisomerase; Efficiency; Enzymes; Humanities and Social Sciences; Kinases; Methods; multidisciplinary; Museums; Oligonucleotides; Protein expression; Science; Science (multidisciplinary); Sequence analysis; Single-stranded DNA
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-40890-3

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.