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Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution
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,...
Autores principales: | , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032674/ https://www.ncbi.nlm.nih.gov/pubmed/21235780 http://dx.doi.org/10.1186/1472-6750-11-6 |
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author | Potapova, Anna Albat, Cord Hasemeier, Britta Haeussler, Katrin Lamprecht, Stella Suerbaum, Sebastian Kreipe, Hans Lehmann, Ulrich |
author_facet | Potapova, Anna Albat, Cord Hasemeier, Britta Haeussler, Katrin Lamprecht, Stella Suerbaum, Sebastian Kreipe, Hans Lehmann, Ulrich |
author_sort | Potapova, Anna |
collection | PubMed |
description | 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, p16(INK4a), 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 (r(2 )= 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 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. |
format | Text |
id | pubmed-3032674 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-30326742011-02-03 Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution Potapova, Anna Albat, Cord Hasemeier, Britta Haeussler, Katrin Lamprecht, Stella Suerbaum, Sebastian Kreipe, Hans Lehmann, Ulrich BMC Biotechnol Methodology Article 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, p16(INK4a), 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 (r(2 )= 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 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. BioMed Central 2011-01-14 /pmc/articles/PMC3032674/ /pubmed/21235780 http://dx.doi.org/10.1186/1472-6750-11-6 Text en Copyright ©2011 Potapova et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Article Potapova, Anna Albat, Cord Hasemeier, Britta Haeussler, Katrin Lamprecht, Stella Suerbaum, Sebastian Kreipe, Hans Lehmann, Ulrich Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution |
title | Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution |
title_full | Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution |
title_fullStr | Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution |
title_full_unstemmed | Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution |
title_short | Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution |
title_sort | systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of dna methylation patterns with single cpg resolution |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032674/ https://www.ncbi.nlm.nih.gov/pubmed/21235780 http://dx.doi.org/10.1186/1472-6750-11-6 |
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