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Human Molecular Genetics Advance Access published online on February 17, 2009
Human Molecular Genetics, doi:10.1093/hmg/ddp054
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Massive parallel bisulfite sequencing of CG-rich DNA fragments reveals that methylation of many X-chromosomal CpG islands in female blood DNA is incomplete

Michael Zeschnigk1,*, Marcel Martin2, Gisela Betzl4, Andreas Kalbe4, Caroline Sirsch4, Karin Buiting1, Stephanie Gross1, Epameinondas Fritzilas3, Bruno Frey4, Sven Rahmann2 and Bernhard Horsthemke1

1 Institut für Humangenetik, Universitätsklinikum Essen, Hufelandstr. 55, D- 45122 Essen, Germany 2 Bioinformatics for High-Throughput Technologies, Computer Science 11, TU Dortmund, D-44221 Dortmund, Germany 3 International NRW Graduate School in Bioinformatics and Genome Research, Bielefeld University, D-33594 Bielefeld, Germany 4 Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany

* Corresponding author: Michael Zeschnigk, Institut für Humangenetik, Universitätsklinikum Essen, Hufelandstr. 55, D- 45122 Essen, Germany, Tel.: ++49 201 723 4558, Fax.: ++49 201 723 5900, Email. Michael.Zeschnigk{at}uni-due.de

Received October 27, 2008; Revised January 21, 2009; Accepted January 27, 2009

Methylation of CpG islands (CGIs) plays an important role in gene silencing. For genome-wide methylation analysis of CGIs in female white blood cells and in sperm, we used four restriction enzymes and a size selection step to prepare DNA libraries enriched with CGIs. The DNA libraries were treated with sodium bisulfite and subjected to a modified 454/Roche Genome Sequencer protocol. We obtained 163,034 and 129,620 reads from blood and sperm, respectively, with an average read length of 133 bp. Bioinformatic analysis revealed that 12,358 (7.6%) blood library reads and 10,216 (7.9%) sperm library reads map to 6,167 and 5,796 different CGIs, respectively. In blood and sperm DNA we identified 824 (13.7%) and 482 (8.5%) fully methylated autosomal CGIs, respectively. Differential methylation, which is characterized by the presence of methylated and unmethylated reads of the same CGI, was observed in 53 and 52 autosomal CGIs in blood and sperm DNA, respectively. Remarkably, methylation of X-chromosomal CGIs in female blood cells was most often incomplete (25-75%). Such incomplete methylation was mainly found on the X-chromosome, suggesting that it is linked to X-chromosome inactivation.


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