A Rapid and Robust Protocol for Reduced Representation Bisulfite Sequencing in Multiple Myeloma

Methods Mol Biol. 2018;1792:179-191. doi: 10.1007/978-1-4939-7865-6_13.

Abstract

Reduced representation bisulfite sequencing (RRBS) is one of the most comprehensive yet economic ways of mapping whole genome DNA-methylation. Here, we have substantially modified the RRBS protocol by combining end-repair and A-tailing steps, and by introducing a bead-based method for rapid and easy size selection of the library molecules. The method has been optimized for myeloma clinical samples, where the input DNA concentration can be as low as 100 ng. The method developed can be accomplished in 3 days, including the initial overnight MspI enzyme digestion. Although the protocol has been optimized in myeloma samples, it is broadly applicable to any clinical sample, which is restricted by very low input DNA concentrations.

Keywords: Bisulfite conversion; DNA methylation; Library preparation; Multiple myeloma; Next generation sequencing; RRBS; Size selection.

MeSH terms

  • CpG Islands
  • DNA Barcoding, Taxonomic
  • DNA Methylation
  • Gene Library
  • Genomics / methods
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Multiple Myeloma / genetics*
  • Quality Control
  • Sequence Analysis, DNA* / methods