In my last entry, I gave a little summary of one of many techniques that are used to study DNA methylation patterns in a loci-specific fashion using the COBRA technique. This time, we’ll take a look at a high-throughput, genome-wide method for analyzing DNA methylation status using a next generation sequencing approache called bisulfite sequencing, or Bis-Seq.
Next generation sequencing has allowed researchers to glean tremendous amounts of information about their sample of interest, offering single base pair resolution and global coverage of the genome. Most next generation sequencing techniques share some common protocol components—the template DNA is immobilized on a solid support, the DNA fragment is amplified, and each clonally amplified DNA is read in parallel. This approach isn’t different for bisulfite converted DNA, rather, the only real difference is the upstream DNA processing includes the bisulfite conversion step.
This bisulfite conversion step is critical, becasue DNA methylation patterns are lost during traditional amplification. By converting the DNA pre-library construction, you can differentiate between methylated and non-methylated cytosines. Commercially available kits for bisulfite conversion now offer greater nearly 100% conversion of DNA, providing input DNA that can accurately detect methylation events in the DNA sequence. One issue, however, is that bisulfite conversion can fragment the DNA, resulting in small fragment DNA that may not be ideal for downstream sequencing techniques. Care should be taken in selecting a bisulfite conversion kit that minimally fragments the DNA and that offers a high recovery rate. Loss of DNA during the bisulfite conversion clean-up can limit the downstream possibilities.
Once you have obtained clean, converted DNA, there are several next generation sequencing techniques that can be applied. Roche offers its 454 platform that delivers read lengths of approximately 400 base pairs. The long read length makes this platform suitable for de novo genome assembly, but it comes at a cost. The 454 method is the most costly out of the methods reviewed here and has much lower throughput (about 1 Gpb per day). In contrast, Illumina and Life Technologies offer Solexa and SOLiD technologies, respectively. Both are low cost alternatives to 454 sequencing, but offer shorter reads, requiring much more bioinformatics on the back end. The Illumina Solexa HiSeq approach gives paired end read lengths of 2 x 100bp and the SOLiD 4 platform gives paired end 75 x 35bp read lengths. The Solexa platform is suitable for methylation profiling, but has a higher error rate than SOLiD, making the SOLiD platform more amenable to variant detection. Both platforms offer a throughput of about 20 Gpb per day.
As sequencing platforms evolve and the cost of sequencing continues to decrease, next generation 3rd generation sequencing platforms will be a viable option for DNA methylation analysis for many more researchers.
Resources and Reviews:
Zhang, Y. and Jeltsch, A. (2010) The Application of Next Generation Sequencing in DNA Methylation Analysis. Genes. 1, 85-101.
Gupta, R. et al. (2010). Advances in genome-wide DNA methylation Analysis. Biotechniques. Epigenetics Issue 49.
Laird, P. W. (2010) Principles and challenges of genome-wide DNA methylation analysis. Nature Reviews. 11, 191-202.
Meaburn, E. and Schulz, R. (2012). Next generation sequencing in epigenetics: Insights and Challenges. Seminars in Cell & Developmental Biology. 23, 192-199.