title: 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.affiliation: Institute of Pathology, Medizinische Hochschule Hannover, D-30625 Hannover, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Potapova
authors.forename: Cord
authors.initials: C
authors.lastname: Albat
authors.forename: Britta
authors.initials: B
authors.lastname: Hasemeier
authors.forename: Katrin
authors.initials: K
authors.lastname: Haeussler
authors.forename: Stella
authors.initials: S
authors.lastname: Lamprecht
authors.forename: Sebastian
authors.initials: S
authors.lastname: Suerbaum
authors.forename: Hans
authors.initials: H
authors.lastname: Kreipe
authors.forename: Ulrich
authors.initials: U
authors.lastname: Lehmann
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism; D012044:Regression Analysis; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004247:DNA