A highly efficient scheme for library preparation from single-stranded DNA.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
25 08 2023
25 08 2023
Historique:
received:
20
02
2023
accepted:
17
08
2023
medline:
28
8
2023
pubmed:
26
8
2023
entrez:
25
8
2023
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 37626096
doi: 10.1038/s41598-023-40890-3
pii: 10.1038/s41598-023-40890-3
pmc: PMC10457334
doi:
Substances chimiques
DNA, Single-Stranded
0
Oligonucleotides
0
Cell-Free Nucleic Acids
0
DNA Nucleotidylexotransferase
EC 2.7.7.31
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
13913Informations de copyright
© 2023. Springer Nature Limited.
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