Application of microdroplet PCR for large-scale targeted bisulfite sequencing
- H. Kiyomi Komori1,
- Sarah A. LaMere1,
- Ali Torkamani1,
- G. Traver Hart1,
- Steve Kotsopoulos2,
- Jason Warner2,
- Michael L. Samuels2,
- Jeff Olson2,
- Steven R. Head3,
- Phillip Ordoukhanian3,
- Pauline L. Lee1,
- Darren R. Link2 and
- Daniel R. Salomon1,4
- 1Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA;
- 2RainDance Technologies, Lexington, Massachusetts 02421, USA;
- 3Next Generation Sequencing Core, The Scripps Research Institute, La Jolla California 92037, USA
Abstract
Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, few current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream of and downstream from their transcription start sites. This method yielded 96% coverage of the targeted CpGs and demonstrated high correlation between CpG island (CGI) DNA methylation and transcriptional regulation. The method was scaled to interrogate the methylation status of 77,674 CpGs in the promoter regions of 2100 genes in primary CD4 T cells. The 2100 gene library yielded 97% coverage of all targeted CpGs and 99% of the target amplicons.
Footnotes
-
↵4 Corresponding author.
E-mail dsalomon{at}scripps.edu.
-
[Supplemental material is available for this article.]
-
Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.116863.110.
- Received October 19, 2010.
- Accepted July 11, 2011.
- Copyright © 2011 by Cold Spring Harbor Laboratory Press
