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

Anna Potapova1, Cord Albat1, Britta Hasemeier1, Katrin Haeussler2, Stella Lamprecht3, Sebastian Suerbaum3, Hans Kreipe1 and Ulrich Lehmann1*

Author Affiliations

1 Institute of Pathology, Medizinische Hochschule Hannover, D-30625 Hannover, Germany

2 Institute of Biometrics, Medizinische Hochschule Hannover, D-30625 Hannover, Germany

3 Institute of Medical Microbiology and Hospital Epidemiology, Medizinische Hochschule Hannover, D-30625 Hannover, Germany

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BMC Biotechnology 2011, 11:6  doi:10.1186/1472-6750-11-6

Published: 14 January 2011



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.


To this end the methylation patterns of 12 loci (GSTĪ€1, 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).


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 hepatocelluar carcinoma. In addition, the single molecule resolution of 454 sequencing provides unprecedented information about the details of DNA methylation pattern heterogeneity in clinical samples.