MethylMapper: a method for high-throughput, multilocus bisulfite sequence analysis and reporting.
Authors: Jared M Ordway; J. Bedell; Robert W. Citek; A. Nunberg; J. Jeddeloh
Year: 2005

Understanding the phenotypic contribution of epigenetic components is making DNA methylation pattern analysis more important in higher eukaryotic genomes as well as human disease. Bisulfite sequencing protocols report DNA methylation occupancy information as a positive assay output that allows methylation patterns to be elucidated from particular devel-opmental or disease states. Reported here is a new method for bisulfite sequencing project management, data analysis, and site-specific methylation test development that is designed for integration in high-throughput genomic and bioinformatics analyses.The phenotypic consequences of epigenetic alterations are being appreciated in nearly every biological system. In higher eukaryotes, DNA methylation patterns serve as a second genomic information code, an easily monitored proximate marker reflecting epigenetic cellular decisions. Most notably, in human cancer, abnormal cellular DNA methylation patterns can directly contribute to the mechanisms of tumorigenesis, most often through the induction of erroneous gene silencing (1). Recent work by many groups has demonstrated that DNA methylation abnormalities may be exploited for the development of powerful cancer diagnostic and prognostic tools (2).One tool in the arsenal of methylation monitoring is bisulfite sequencing (3). Bisulfite treatment of DNA causes the deamination of cytosine and conversion to thymine upon amplification and cloning. However, if the cytosine is methylated, it remains unchanged. In this way, subsequent sequencing of the amplified, mutagenized clones with conventional technology allows one to understand the methylation occupancy at each cytosine.Several groups have released publicly available tools for bisulfite sequencing experimental design or data analysis (4–7). Furthermore, online tools such as methBLAST (medgen.ugent.be/methblast) have become available for in silico bisulfite modification to aid in PCR primer design. However, the utility is limited in that they either only help with experimental design or with data analysis (i.e., pattern elucidation). For those that offer methylation pattern elucidation, they also require the experimenter to employ a locus-by-locus, one-gene-at-a-time approach. Given the revolution in automated sequencing and the capacity available at most genome centers, a combined design and analysis suite that affords investigators the opportunity to take advantage of a high-throughput, low-effort analysis procedure is missing. Most researchers frustratingly address this unmet need by performing each outlined step in the most labor-intensive but readily apparent manner. Typically, this involves a stepwise approach for each target, often aligning trace files by hand and manually performing the occupancy calculations. Moreover, as multiple-locus biomarkers are discovered and employed, validation of methylation patterns in a multi-locus manner from many samples will be necessary. Having an automated analysis capacity will become an even more pressing need.We have created an efficient package of PERL programs called MethylMapper that, when combined with a primer-picking program and BLASTN, simplifies the design and analysis of bisulfite experiments in a high-throughput environment. MethylMapper allows the data to self-organize to minimize mistakes and expedite simultaneous analyses of multiple loci. Furthermore, it makes data quality control as streamlined as possible. The package requires only PERL and NCBI-BLAST (www.ncbi.nlm.nih.gov/blast/download.shtml) and requires very little memory or CPU time. System requirements and CPU usage are dependent only upon the requirements of the BLASTN operation. The package is freely available for download at methylmapper.sourceforge.net.For demonstration purposes, we used MethylMapper to design primers and analyze bisulfite sequencing results for a genomic region spanning the second exon of the SLC4A3 gene on human chromosome 2 (Figure 1). Primer sequences for the analysis shown in Figure 1 are 5′-TGATTTGGGTAAGATTTTGGTTGTGAGTAG-3′ (forward) and 5′-CATCCCTAATAAACAAAACATAAAACT-3′ (reverse). Bisulfite conversion was performed with the EZ DNA Methylation Kit™ (Zymo Research, Orange, CA, USA) under the manufacturer’s recommended conditions. PCR amplification was performed under standard conditions that employed 0.2 pmol of primers, 40 ng of template in a 25-μL volume of water, to which 25 μL of FailSafe™ G 2× premix and 1 U of FailSafe Taq DNA Polymerase (EPICENTRE, Madison, WI, USA) were added. The cycling conditions employed a single 3-min incubation at 95°C, followed by 30 cycles of 95°C for 45 s, 52°C for 15 s, and 72°C for 30 s. Finally, a 10-min chase step was performed by incubating the sample at 72°C. The PCR product was purified from an agarose gel slice and cloned using the pCR2.1-TOPO TA Cloning