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Kismeth: analyzer of plant methylation states through bisulfite sequencing.

Gruntman E, Qi Y, Slotkin RK, Roeder T, Martienssen RA, Sachidanandam R - BMC Bioinformatics (2008)

Bottom Line: Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation.For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations.Kismeth can also be used to study methylation states in different tissues and disease cells compared to a reference sequence.

View Article: PubMed Central - HTML - PubMed

Affiliation: gruntman@cshl.edu

ABSTRACT

Background: There is great interest in probing the temporal and spatial patterns of cytosine methylation states in genomes of a variety of organisms. It is hoped that this will shed light on the biological roles of DNA methylation in the epigenetic control of gene expression. Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation. For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations. However, in contrast to the situation in animals, there aren't many tools that analyze bisulfite data in plants, which can exhibit methylation of cytosines in a variety of sequence contexts (CG, CHG, and CHH).

Results: Kismeth http://katahdin.mssm.edu/kismeth is a web-based tool for bisulfite sequencing analysis. Kismeth was designed to be used with plants, since it considers potential cytosine methylation in any sequence context (CG, CHG, and CHH). It provides a tool for the design of bisulfite primers as well as several tools for the analysis of the bisulfite sequencing results. Kismeth is not limited to data from plants, as it can be used with data from any species.

Conclusion: Kismeth simplifies bisulfite sequencing analysis. It is the only publicly available tool for the design of bisulfite primers for plants, and one of the few tools for the analysis of methylation patterns in plants. It facilitates analysis at both global and local scales, demonstrated in the examples cited in the text, allowing dissection of the genetic pathways involved in DNA methylation. Kismeth can also be used to study methylation states in different tissues and disease cells compared to a reference sequence.

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Detailed view of results for each sequence. The detailed view of matches highlights various types of cytosines (CG, CHG and CHH) and their fate under bisulfite treatment for each sequenced read. The first line, labeled in, of each set is the reference sequence with the cytosines colored to represent the type of cytosine (as in Figure 3), CG (red), CHG (blue) and CHH(green). The second line, labeled out, shows the result of bisulfite sequencing with colored T's, red for untransformed C's (which are protected by methylation) and tan for transformed C's (unmethylated). The mismatches number above each sequence gives the number of mismatches the sequence exhibited (besides the C to T conversions) and the total length of the particular sequence. This provides an estimate for sequence quality.
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Figure 4: Detailed view of results for each sequence. The detailed view of matches highlights various types of cytosines (CG, CHG and CHH) and their fate under bisulfite treatment for each sequenced read. The first line, labeled in, of each set is the reference sequence with the cytosines colored to represent the type of cytosine (as in Figure 3), CG (red), CHG (blue) and CHH(green). The second line, labeled out, shows the result of bisulfite sequencing with colored T's, red for untransformed C's (which are protected by methylation) and tan for transformed C's (unmethylated). The mismatches number above each sequence gives the number of mismatches the sequence exhibited (besides the C to T conversions) and the total length of the particular sequence. This provides an estimate for sequence quality.

Mentions: In addition, two kinds of detailed reports, on a sequence-by-sequence basis, are accessible through the Matches and dot plot links on the synopsis page (shown in figure 2). The detailed matches view highlights the various kinds of cytosines in the sequence and the result of the bisulfite treatment (figure 4) allowing the user to study individual alignments to estimate the quality of the sequencing effort that can lead to mismatches (besides the C to T conversions). The dot plot shows only the cytosines as circles, colored according to the type of cytosine (red for CG, blue for CHG and green for CHH), with filled circles representing methylated cytosines and empty circles representing un-methylated cytosines (figure 5). The program parameters, described in the algorithm section, can be changed on the front page of Kismeth. Kismeth also generates postscript files for various figures, which can be downloaded for use in publications.


Kismeth: analyzer of plant methylation states through bisulfite sequencing.

Gruntman E, Qi Y, Slotkin RK, Roeder T, Martienssen RA, Sachidanandam R - BMC Bioinformatics (2008)

Detailed view of results for each sequence. The detailed view of matches highlights various types of cytosines (CG, CHG and CHH) and their fate under bisulfite treatment for each sequenced read. The first line, labeled in, of each set is the reference sequence with the cytosines colored to represent the type of cytosine (as in Figure 3), CG (red), CHG (blue) and CHH(green). The second line, labeled out, shows the result of bisulfite sequencing with colored T's, red for untransformed C's (which are protected by methylation) and tan for transformed C's (unmethylated). The mismatches number above each sequence gives the number of mismatches the sequence exhibited (besides the C to T conversions) and the total length of the particular sequence. This provides an estimate for sequence quality.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553349&req=5

Figure 4: Detailed view of results for each sequence. The detailed view of matches highlights various types of cytosines (CG, CHG and CHH) and their fate under bisulfite treatment for each sequenced read. The first line, labeled in, of each set is the reference sequence with the cytosines colored to represent the type of cytosine (as in Figure 3), CG (red), CHG (blue) and CHH(green). The second line, labeled out, shows the result of bisulfite sequencing with colored T's, red for untransformed C's (which are protected by methylation) and tan for transformed C's (unmethylated). The mismatches number above each sequence gives the number of mismatches the sequence exhibited (besides the C to T conversions) and the total length of the particular sequence. This provides an estimate for sequence quality.
Mentions: In addition, two kinds of detailed reports, on a sequence-by-sequence basis, are accessible through the Matches and dot plot links on the synopsis page (shown in figure 2). The detailed matches view highlights the various kinds of cytosines in the sequence and the result of the bisulfite treatment (figure 4) allowing the user to study individual alignments to estimate the quality of the sequencing effort that can lead to mismatches (besides the C to T conversions). The dot plot shows only the cytosines as circles, colored according to the type of cytosine (red for CG, blue for CHG and green for CHH), with filled circles representing methylated cytosines and empty circles representing un-methylated cytosines (figure 5). The program parameters, described in the algorithm section, can be changed on the front page of Kismeth. Kismeth also generates postscript files for various figures, which can be downloaded for use in publications.

Bottom Line: Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation.For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations.Kismeth can also be used to study methylation states in different tissues and disease cells compared to a reference sequence.

View Article: PubMed Central - HTML - PubMed

Affiliation: gruntman@cshl.edu

ABSTRACT

Background: There is great interest in probing the temporal and spatial patterns of cytosine methylation states in genomes of a variety of organisms. It is hoped that this will shed light on the biological roles of DNA methylation in the epigenetic control of gene expression. Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation. For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations. However, in contrast to the situation in animals, there aren't many tools that analyze bisulfite data in plants, which can exhibit methylation of cytosines in a variety of sequence contexts (CG, CHG, and CHH).

Results: Kismeth http://katahdin.mssm.edu/kismeth is a web-based tool for bisulfite sequencing analysis. Kismeth was designed to be used with plants, since it considers potential cytosine methylation in any sequence context (CG, CHG, and CHH). It provides a tool for the design of bisulfite primers as well as several tools for the analysis of the bisulfite sequencing results. Kismeth is not limited to data from plants, as it can be used with data from any species.

Conclusion: Kismeth simplifies bisulfite sequencing analysis. It is the only publicly available tool for the design of bisulfite primers for plants, and one of the few tools for the analysis of methylation patterns in plants. It facilitates analysis at both global and local scales, demonstrated in the examples cited in the text, allowing dissection of the genetic pathways involved in DNA methylation. Kismeth can also be used to study methylation states in different tissues and disease cells compared to a reference sequence.

Show MeSH
Related in: MedlinePlus