Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution.

Abstract

BACKGROUND:

New high-throughput sequencing technologies promise a very sensitive and high-resolution analysis of DNA methylation patterns in quantitative terms. However, a detailed and comprehensive comparison with existing validated DNA methylation analysis methods is not yet available. Therefore, a systematic cross-validation of 454 sequencing and conventional pyrosequencing, both of which offer exact quantification of methylation levels with a single CpG dinucleotide resolution, was performed.

RESULTS:

To this end the methylation patterns of 12 loci (GSTπ1, p16INK4a, RASSF1A, SOCS1, MAL, hsa-mir-1-1, hsa-mir-9-3, hsa-mir-34a, hsa-mir-596, hsa-mir-663, MINT31, and LINE-1) were analyzed in ten primary hepatocellular carcinoma specimens. After applying stringent quality control criteria, 35749 sequences entered further analysis. The methylation level of individual CpG dinucleotides obtained by 454 sequencing was systematically compared with the corresponding values obtained by conventional pyrosequencing. Statistical analyses revealed an excellent concordance of methylation levels for all individual CpG dinucleotides under study (r2 = 0.927).

CONCLUSIONS:

Our results confirm that 454 sequencing of bisulfite treated genomic DNA provides reliable high quality quantitative methylation data and identify MAL, hsa-mir-9-3, hsa-mir-596, and hsa-mir-663 as new targets of aberrant DNA methylation in human hepatocellular carcinoma. In addition, the single molecule resolution of 454 sequencing provides unprecedented information about the details of DNA methylation pattern heterogeneity in clinical samples.

• Authors: Anna Potapova, Cord Albat, Britta Hasemeier, Katrin Haeussler, Stella Lamprecht, Sebastian Suerbaum, Hans Kreipe, Ulrich Lehmann
• Journal: BMC biotechnology (BMC Biotechnol) Vol. 11 Pg. 6 (Jan 14 2011) ISSN: 1472-6750 [Electronic] England
• PMID: 21235780 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA

Topics

• Carcinoma, Hepatocellular (genetics, metabolism)
• DNA (chemistry)
• DNA Methylation
• Humans
• Liver Neoplasms (genetics, metabolism)
• Regression Analysis
• Reproducibility of Results
• Sequence Analysis, DNA (methods)