A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Cell-free DNA (cfDNA) in blood plasma holds critical health information.
• Single-stranded DNA (ssDNA) library prep methods are effective for cfDNA analysis.
• Existing ssDNA methods are expensive and time-consuming.

Purpose of the Study:

• To develop an efficient and cost-effective ssDNA library preparation method for cfDNA.
• To enable rapid and accurate genomic analysis of fragmented DNA.
• To overcome limitations of current ssDNA library preparation techniques.

Main Methods:

• Developed SRSLY (Single Reaction Single-stranded LibrarY), a ligation-based method.
• Utilized a one-step phosphorylation/ligation reaction without end-polishing.
• Tested with synthetic DNA oligos and cfDNA, comparing to existing methods.

Main Results:

• SRSLY produces complex libraries in under 2.5 hours from 1 ng of DNA.
• The method preserves the native ends and lengths of DNA molecules.
• SRSLY-generated cfDNA data enables analysis of DNA fragmentation patterns, nucleosome positioning, and transcription factor binding.

Conclusions:

• SRSLY is a versatile tool for preparing sequencing libraries from short, fragmented DNA like cfDNA.
• The method retains the native characteristics of DNA fragments.
• SRSLY offers an efficient and accessible approach for cfDNA analysis.