一种用于多发性骨髓瘤中简化代表性亚硫酸氢盐测序的快速且稳健的方案。

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

作者信息

Choudhury Samrat Roy, Walker Brian A

机构信息

Department of Internal Medicine, Myeloma Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

出版信息

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

DOI:10.1007/978-1-4939-7865-6_13

PMID:29797260

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.

摘要

简化代表性亚硫酸氢盐测序（RRBS）是全基因组DNA甲基化图谱绘制中最全面且经济的方法之一。在此，我们通过合并末端修复和加A步骤，并引入一种基于磁珠的方法来快速简便地筛选文库分子大小，对RRBS方案进行了重大改进。该方法已针对骨髓瘤临床样本进行了优化，其中输入DNA浓度可低至100 ng。所开发的方法可在3天内完成，包括最初的过夜MspI酶切。尽管该方案已在骨髓瘤样本中进行了优化，但它广泛适用于任何受极低输入DNA浓度限制的临床样本。