Low Input Genome-Wide DNA Methylation Analysis with Minimal Library Amplification

Area of Science:

• Genomics
• Epigenetics
• Molecular Biology

Background:

• Whole genome bisulfite sequencing (WGBS) is crucial for mapping DNA methylation at base resolution.
• Existing WGBS methods often require substantial cell numbers, limiting their application.
• Post-bisulfite adaptor tagging (PBAT) offers a way to reduce cell input requirements.

Purpose of the Study:

• To describe a refined PBAT protocol for preparing WGBS libraries from a small number of cells (200).
• To introduce a bioinformatic analysis pipeline for PBAT-generated WGBS data.
• To demonstrate the efficiency of the PBAT method for low-input WGBS.

Main Methods:

• Development and optimization of a post-bisulfite adaptor tagging (PBAT) library preparation protocol.
• Application of the PBAT protocol to prepare WGBS libraries from 200 cells.
• Outline of a downstream bioinformatic analysis workflow for PBAT-WGBS data.
• Sequencing of prepared libraries using the Illumina NovaSeq 6000 platform.

Main Results:

• A robust PBAT protocol for WGBS library construction from 200 cells was established.
• The protocol yields sufficient sequencing reads (approx. 800 million) for comprehensive human and mouse genome coverage.
• The generated data allows for approximately 15x genome coverage on Illumina NovaSeq 6000.

Conclusions:

• The described PBAT protocol significantly lowers the cell input requirement for WGBS.
• This advancement expands the utility of WGBS for epigenomic studies using limited cell samples.
• The protocol and associated bioinformatic analysis provide a powerful tool for low-input genome-wide DNA methylation profiling.