Whole methylome analysis by ultra-deep sequencing using two-base encoding.
Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.
Bormann Chung
Christina A
CA
Life Technologies, Foster City, California, United States of America. Christina.Chung@lifetech.com
Boyd
Victoria L
VL
McKernan
Kevin J
KJ
Fu
Yutao
Y
Monighetti
Cinna
C
Peckham
Heather E
HE
Barker
Melissa
M
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015894:Genome, Human / Q000235:genetics*; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D013447:Sulfites / Q000737:chemistry
D013447:Sulfites; D004247:DNA