DNA methylation profiling using long-read single molecule real-time bisulfite sequencing (SMRT-BS)

Yao Yang, Stuart A. Scott

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

13 Scopus citations

Abstract

For the past two decades, bisulfite sequencing has been a widely used method for quantitative CpG methylation detection of genomic DNA. Coupled with PCR amplicon cloning, bisulfite Sanger sequencing allows for allele-specific CpG methylation assessment; however, its time-consuming protocol and inability to multiplex has recently been overcome by next-generation bisulfite sequencing techniques. Although high-throughput sequencing platforms have enabled greater accuracy in CpG methylation quantitation as a result of increased bisulfite sequencing depth, most common sequencing platforms generate reads that are similar in length to the typical bisulfite PCR size range (~300–500 bp). Using the Pacific Biosciences (PacBio) sequencing platform, we developed single molecule real-time bisulfite sequencing (SMRT-BS), which is an accurate targeted CpG methylation analysis method capable of a high degree of multiplexing and long read lengths. SMRT-BS is reproducible and was found to be concordant with other lower throughput quantitative CpG methylation methods. Moreover, the ability to sequence up to ~1.5–2.0 kb amplicons, when coupled with an optimized bisulfite-conversion protocol, allows for more thorough assessment of CpG islands and increases the capacity for studying the relationship between single nucleotide variants and allele-specific CpG methylation.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages125-134
Number of pages10
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1654
ISSN (Print)1064-3745

Keywords

  • Bisulfite sequencing
  • CpG islands
  • DNA methylation
  • Long-read sequencing
  • Multiplex DNA methylation analysis
  • PacBio sequencing
  • SMRT sequencing

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