End-repair causes methylation underestimation in cell-free DNA sequencing libraries.

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Cell-free DNA (cfDNA) methylation sequencing is crucial for determining tissue of origin and chromatin structure.
• Library preparation workflows for cfDNA analysis often involve end-repair steps.

Purpose of the Study:

• To investigate the impact of end-repair during library preparation on cfDNA methylation inference.
• To compare the accuracy of tissue of origin determination using single-stranded versus double-stranded cfDNA libraries.

Main Methods:

• Matched single-stranded and double-stranded DNA libraries were prepared from identical cfDNA extracts.
• Inferred CpG methylation levels were analyzed across both library types.
• The accuracy of tissue of origin prediction was assessed.

Main Results:

• End-repair in double-stranded cfDNA libraries significantly reduced globally inferred CpG methylation.
• This reduction in methylation inference led to decreased accuracy in determining the tissue of origin.
• Trimming read termini in double-stranded libraries partially corrected the bias but reduced coverage and lost fragmentomic data.
• Single-stranded cfDNA libraries, which omit end-repair, preserved methylation accuracy and fragmentomic information.

Conclusions:

• End-repair is a critical step that introduces bias in cfDNA methylation sequencing, impacting tissue of origin accuracy.
• Single-stranded library preparation is a superior method for cfDNA methylation analysis, preserving crucial epigenetic and fragmentomic information.