Reduced representation bisulfite sequencing

Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level. It combines restriction enzymes and bisulfite sequencing to enrich for areas of the genome with a high CpG content. Due to the high cost and depth of sequencing to analyze methylation status in the entire genome, Meissner et al. developed this technique in 2005 to reduce the amount of nucleotides required to sequence to 1% of the genome. The fragments that comprise the reduced genome still include the majority of promoters, as well as regions such as repeated sequences that are difficult to profile using conventional bisulfite sequencing approaches. Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level. It combines restriction enzymes and bisulfite sequencing to enrich for areas of the genome with a high CpG content. Due to the high cost and depth of sequencing to analyze methylation status in the entire genome, Meissner et al. developed this technique in 2005 to reduce the amount of nucleotides required to sequence to 1% of the genome. The fragments that comprise the reduced genome still include the majority of promoters, as well as regions such as repeated sequences that are difficult to profile using conventional bisulfite sequencing approaches. RRBS uniquely uses a specific restriction enzyme to enrich for CpGs. MspI digestion, or any restriction enzyme that recognizes CpG's and cuts them, produces only fragments with CG’s at the end. This approach enriches for CpG regions of the genome, so it can decrease the amount of sequencing required as well as decrease the cost. This technique is cost-effective especially when focusing on common CpG regions. Only a low sample concentration, between 10-300 ng, is required for accurate data analysis. This technique can be employed when there is a lack of precious sample. Another positive aspect is that fresh or live samples are not required. Formalin-fixed and paraffin-embedded inputs can also be used. In the specific protocol steps, there are also some limitations. MspI digestion covers the majority, but not all the CG regions in the genome. Some CpG’s are missed. Missing CpG’s can also occur since this protocol is only a representative sampling of the genome. Some regions thus have lower coverage. During the PCR portion of the protocol, a non-proofreading polymerase must be used as a proof-reading enzyme would stop at uracil residues found in the ssDNA template. Using a polymerase that does not proof-read can also lead to increase PCR sequencing errors. Bisulfite sequencing only converts single-stranded DNA (ssDNA). Complete bisulfite conversion requires thorough denaturation and absence of re-annealed double stranded DNA (dsDNA). Easy protocol steps have been shown to drive complete denaturation. Ensuring the usage of small fragments via shearing or digestion, fresh reagents, and sufficient denaturing time is crucial for complete denaturing Another suggested technique is to carry out the bisulfite reaction at 95 °C although DNA degradation also occurs at high temperatures. In the first hour of bisulfite reaction, it is predicted that less than 90% of the sample DNA is lost to degradation A balance between high temperature and low temperature is required to ensure complete denaturation and decreased DNA degradation. Usage of reagents, like urea, that prevent dsDNA from forming can also be employed. With contamination of dsDNA, it can be difficult to accurately computate the data. When an unconverted cytosine is observed, it is challenging to differentiate between lack of methylation and an artifact. The significance of this technique is it allows for the sequencing of methylated areas that can't be properly profiled using conventional bisulfite sequencing techniques. Current sequencing technologies are limited in regards to profiling areas of repeated sequences. This is unfortunate in regards to methylation studies, as these repeated sequences often contain methylated cytosines. This is especially limiting for studies involving profiling cancer genomes, as a loss of methylation in this repeated sequences is observed in many cancer types. RRBS eliminates the problems encountered due to these large areas of repeated sequences and thus lets these regions be more fully annotated. Aberrant methylation has been observed in cancer. In cancer, hypermethylation as well as hypomethylation has been seen in tumors. Since RRBS is highly sensitive, this technique can be used to quickly look at aberrant methylation in cancer. If samples from the patient's tumor and normal cells can be obtained, a comparison between these two cell types can be observed. A profile of the overall methylation can be produced quite rapidly. This technique can rapidly determine the overall methylation status of cancer genomes which is cost and time effective.