CpG Analyzer, a Windows-based utility program for investigation of DNA methylation

656 BioTechniques Vol. 39, No. 5 (2005) There is substantial evidence that changes in DNA methylation occur during preneoplasia, including both global changes in DNA methylation and changes in CpG dinucleotide methylation sites in specific genes (1). Aberrant DNA methylation within CpG islands is one of the earliest and more common alterations in human malignancies (2). Cytosine methylation in CpG dinucleotides has been observed to be an important control mechanism in development and differentiation (3). The bisulfite genomic sequencing technique (4) has found wide acceptance for the generation of DNA methylation status maps with singlebase resolution. This method is based on the selective deamination (induced by bisulfite treatment) of cytosine to uracil, while 5-methylcytosine residues remain unchanged. This bisulfitemodified DNA sequence is amplified by PCR and then sequenced. The uracils in the sequence are detected as thymines on PCR amplification and complement with adenines on formation of the double strand. Methylation status is obtained by the comparison of bisulfite sequence PCR products with the computer-generated bisulfite-modified sequences. Knowledge of the CpG distribution within the sequence, identifying each CpG location, and generating bisulfite-modified sequences are essential in the use of this method, while the ability to highlight CpGs in the sequence text will greatly speed up the process of sequence comparison. Some programs are available to simplify the process (see the MethDB links web page at 195.83.84.240/links. html). Methtools is a program available only on the UNIX® operating system, thus excluding Microsoft® Windows users from employing it (5). CpG Island Searcher is a web site that has a simple user interface to identify CpG islands from submitted sequences (6). However, this program does not supply the detailed CpG location information and CpGhighlighted text that are important for a DNA methylation map with single base resolution. On the Microsoft Windows platform, Anbazhagan et al. used Microsoft Excel® to identify and mark CpG islands (7). CpGs can also be analyzed and highlighted by searching for “cg” in a sequence using most word processing software. However, since the sequence text commonly used is in GenBank® flat file format and consists of line numbers, spaces, and line breaks, the searching process must be performed after these extraneous characters have been eliminated. This involves repeated use of the find and delete commands. Manual CpG analysis and sequence conversion should be avoided, since not only are these timeconsuming, but mistakes are readily introduced. Singal et al. (8,9) used Microsoft Word® macros to simplify the repetative tasks such as removing numbers, spaces, and line breaks, highlighting CpGs, and generating the bisulfite-modified sequences. However, this method does not record or generate CpG location data. Since the valuable numbers and spaces that indicate the location of CpGs are eliminated from the highlighted text, this makes it difficult to find the exact locations of the highlighted CpGs, which is important for manual inspection later, CpG Analyzer, a Windows-based utility program for investigation of DNA methylation