BISMA - Fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences

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Abstract

Background

Bisulfite sequencing is a popular method to analyze DNA methylation patterns at high resolution. A region of interest is targeted by PCR and about 20-50 subcloned DNA molecules are usually analyzed, to determine the methylation status at single CpG sites and molecule resolution.

Results

The BISMA (Bisulfite Sequencing DNA Methylation Analysis) software for analysis of primary bisulfite sequencing data implements sequencing data extraction and enhanced data processing, quality controls, analysis and presentation of the methylation state. It uses an improved strategy for detection of clonal molecules and accurate CpG site detection and it supports for the first time analysis of repetitive sequences.

Conclusions

BISMA works highly automated but still provides the user full control over all steps of the analysis. The BISMA software is freely available as an online tool for academic purposes for the analysis of bisulfite sequencing data from both unique and repetitive sequences http://biochem.jacobs-university.de/BDPC/BISMA/.

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Most cited references 16

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A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

M Frommer, L. E. McDonald, D Millar … (1992)

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

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A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements.

Allen Yang, Marcos R H Estécio, Ketan Doshi … (2004)

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15 000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.

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Biochemistry and biology of mammalian DNA methyltransferases.

A Jeltsch, H Gowher, Kay-Geert Hermann (2004)

DNA methylation is a stable but not irreversible epigenetic signal that silences gene expression. It has a variety of important functions in mammals, including control of gene expression, cellular differentiation and development, preservation of chromosomal integrity, parental imprinting and X-chromosome inactivation. In addition, it has been implicated in brain function and the development of the immune system. Somatic alterations in genomic methylation patterns contribute to the etiology of human cancers and ageing. It is tightly interwoven with the modification of histone tails and other epigenetic signals. Here we review our current understanding of the molecular enzymology of the mammalian DNA methyltransferases Dnmt1, Dnmt3a, Dnmt3b and Dnmt2 and the roles of the enzymes in the above-mentioned biological processes.

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Author and article information

Journal

Journal ID (nlm-ta): BMC Bioinformatics

Title: BMC Bioinformatics

Publisher: BioMed Central

ISSN (Electronic): 1471-2105

Publication date Collection: 2010

Publication date (Electronic): 6 May 2010

Volume: 11

Page: 230

Affiliations

[1 ]School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28725 Bremen, Germany

[2 ]Max Planck Institute for Molecular Genetics, Ihnestrasse 63-73, D-14195 Berlin-Dahlem, Germany

Article

Publisher ID: 1471-2105-11-230

DOI: 10.1186/1471-2105-11-230

PMC ID: 2877691

PubMed ID: 20459626

SO-VID: bd761165-ff0f-4c59-9fdf-e52502ee33e7

License:

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.

BISMA - Fast and accurate bisulfite sequencing data analysis of individual clones from unique and repetitive sequences

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Abstract

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Most cited references 16

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements.

Biochemistry and biology of mammalian DNA methyltransferases.

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