Systematic evaluation of parameters in RNA bisulfite sequencing data generation and analysis

Area of Science:

• Epigenetics
• Molecular Biology
• Bioinformatics

Background:

• 5-methylcytosine (m⁵C) is a crucial RNA modification regulating RNA metabolism.
• Accurate detection and quantification of RNA m⁵C remain technically challenging despite recent advances.

Purpose of the Study:

• To compare library construction procedures for RNA bisulfite sequencing.
• To develop an analytical pipeline for assessing m⁵C calling parameters.
• To identify and mitigate artifacts in RNA m⁵C detection.

Main Methods:

• Comparison of four RNA library construction protocols for bisulfite sequencing.
• Implementation of a bioinformatics pipeline for m⁵C analysis.
• Utilizing Unique Molecular Identifiers (UMIs) to assess PCR bias.
• Evaluation of bisulfite conversion efficiency and sequencing quality impacts.

Main Results:

• RNA fragmentation post-bisulfite conversion significantly increased yield.
• High-temperature treatment enhanced bisulfite conversion efficiency, particularly for mitochondrial transcripts.
• PCR amplification showed bias towards unmethylated RNA templates.
• Low sequencing quality of bisulfite-converted bases contributed to methylation artifacts.
• Mitochondrial mRNAs were resistant to bisulfite conversion, yielding no high-confidence p-m5C sites.

Conclusions:

• Identified major sources of artifacts in RNA bisulfite sequencing data.
• Developed an improved experimental procedure and analytical methodology for RNA m⁵C analysis.
• Highlighted challenges in detecting m⁵C modifications in mitochondrial transcripts.