Chapter title |
DNA Methylation Profiling Using Long-Read Single Molecule Real-Time Bisulfite Sequencing (SMRT-BS)
|
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Chapter number | 8 |
Book title |
Functional Genomics
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7231-9_8 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7230-2, 978-1-4939-7231-9
|
Authors |
Yao Yang, Stuart A. Scott, Yang, Yao, Scott, Stuart A. |
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. |
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