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

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Established methods for double-stranded DNA (dsDNA) library preparation exist.
• Single-stranded DNA (ssDNA) library preparation methods are less studied and often inefficient.
• Existing ssDNA methods suffer from low efficiency and yield.

Purpose of the Study:

• To develop a highly efficient and reliable method for ssDNA sequencing library preparation.
• To overcome the limitations of existing ssDNA library preparation protocols.
• To enable sensitive sequencing analysis of low-input ssDNA samples.

Main Methods:

• Developed the TACS-TOPO scheme for ssDNA library preparation.
• Utilizes terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation for initial adaptor tagging.
• Employs Vaccinia virus topoisomerase I (VTopoI or TOPO)-based ligation for second adaptor tagging after complementary strand synthesis.
• Incorporates steps for adaptor dimer removal to ensure library purity.

Main Results:

• The TACS-TOPO scheme enables adaptor dimer-free library preparation from as little as 24 pg of ssDNA.
• Successfully applied to amplification-free library preparation from cell-free DNA in human serum (50 µL).
• A modified TACS-TOPO protocol increased library yields by two to eight times for ancient human bone DNA compared to conventional methods.

Conclusions:

• The TACS-TOPO scheme provides a highly efficient and practical solution for ssDNA sequencing library preparation.
• Demonstrates improved performance for low-input and degraded DNA samples.
• Facilitates sensitive and robust sequencing analysis of diverse ssDNA sources.