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

Abstract The presence of 5-methylcytosine (m5C) in RNA molecules has been known for decades and its importance in regulating RNA metabolism has gradually become appreciated. Despite recent advances made in the functional and mechanistic understanding of RNA m5C modifications, the detection and quant...

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Published inNAR genomics and bioinformatics Vol. 4; no. 2; p. lqac045
Main Authors Johnson, Zachary, Xu, Xiguang, Pacholec, Christina, Xie, Hehuang
Format Journal Article
LanguageEnglish
Published England Oxford University Press 01.06.2022
Subjects
Bioinformatics
Biology
Bisulfite
Editor's Choice
Genomics
Growth factors
Heat resistance
High temperature
Metabolism
Methods
Methylation
Mitochondria
Proteins
Ribonucleic acid
RNA
Stem cells
Transcriptomes
Transfer RNA
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Summary:Abstract The presence of 5-methylcytosine (m5C) in RNA molecules has been known for decades and its importance in regulating RNA metabolism has gradually become appreciated. Despite recent advances made in the functional and mechanistic understanding of RNA m5C modifications, the detection and quantification of methylated RNA remains a challenge. In this study, we compared four library construction procedures for RNA bisulfite sequencing and implemented an analytical pipeline to assess the key parameters in the process of m5C calling. We found that RNA fragmentation after bisulfite conversion increased the yield significantly, and an additional high temperature treatment improved bisulfite conversion efficiency especially for sequence reads mapped to the mitochondrial transcriptome. Using Unique Molecular Identifiers (UMIs), we observed that PCR favors the amplification of unmethylated templates. The low sequencing quality of bisulfite-converted bases is a major contributor to the methylation artifacts. In addition, we found that mitochondrial transcripts are frequently resistant to bisulfite conversion and no p-m5C sites with high confidence could be identified on mitochondrial mRNAs. Taken together, this study reveals the various sources of artifacts in RNA bisulfite sequencing data and provides an improved experimental procedure together with analytical methodology.
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The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
ISSN:2631-9268
2631-9268
DOI:10.1093/nargab/lqac045