DNA Methylation Sequencing
The methylation of cytosines is a major epigenomic mechanism that modulates the primary genomic code. This leads to the formation of 5-methylcytosines (5mCs) at select sites of the genome. Cytosine methylation regulates gene expression and chromatin remodeling, and as a result plays important roles in many biological functions including embryonic development, cell differentiation, and stem cell pluripotency. Abnormal DNA methylation can lead to diseases, such as cancer.
DNA methylome analysis has for many years been conducted with the use of microarrays, including the widely used Illumina Infinium MethlyationEPIC BeadChips that NUSeq processes. DNA methylation sequencing is a newer technology that is usually based on bisulfite conversion to differentiate methylated vs. unmethylated cytosines. Upon treatment with bisulfite, unmethylated cytosines are converted to uracils, while 5mCs are nonreactive and retained. In the sequencing step, unmethylated cytosines are read as thymines, while methylated cytosines still as cytosines.
Based on genomic coverage, bisulfite conversion based sequencing can be conducted in NUSeq as either Whole-Genome Bisulfite Sequencing (WGBS) or Reduced Representation Bisulfite Sequencing (RRBS). WGBS costs more and the associated data analysis is much more involved. RRBS instead provides a cost effective approach to survey DNA methylation by sampling CpG-rich regions of the genome. To perform RRBS, genomic DNA is digested with a methylation-insensitive restriction enzyme, such as MspI. The digested DNA fragments are then subjected to adapter ligation, bisulfite conversion, and PCR, to generate a library for sequencing.
WGBS and RRBS DNA Methylation Sequencing Library Prep Pricing
WGBS and RRBS library prep is listed as "DNA Methyl-Seq Library Prep" on the Core Pricing page. Both are priced at $300 per sample.
DNA Methylation Sequencing
Sequencing Mode
A single-end 150 bp or paired-end 75 bp run is suggested for most cases. For run cost of such sequencing options, please check our pricing structure.
Service Request
If needed, project consultation is provided free-of-charge. DNA methylation sequencing services can be requested through NUcore. Please finish and submit our project intake form when requesting service.
Sample Submission
For library prep, 150 ng of high-quality genomic DNA is required. For DNA quantification, fluorometric-based methods, such as Qubit or PicoGreen, are preferred. Spectrophotometric-based methods, such as Nanodrop, may not be accurate.
Bioinformatics
Data analysis is provided upon request. Standard RRBS bioinformatics service includes sequencing data QC, alignment, and DNA methylation localization and quantification.
Data Storage Policy
RRBS data is stored on the NUSeq server space for one year from the date of generation. It is recommended to transfer data files to the user's own space after they are generated.
Frequently Asked Questions
What coverage should I aim for?
Based on current literature, per sample coverage should be in the range of 5x to 15x [ref]. Consistent with this, the NIH RoadMap Epigenomics Project recommends at least two replicates per condition with a combined coverage of at least 30x [ref]. This applies to both WGS and RRBS.
What percentage of a genome does RRBS survey?
RRBS covers approximately 1% of a mammalian genome. Becuase CpGs are not evenly distributed across the genome, RRBS surveys 10-15% of CpGs in the genome, making it a cost effective method for a quick survey of a methylome. RRBS to WGBS can be considered as an equivalent of WES to WGS.
For RRBS, can I use degraded genomic DNA samples such as those isolated from formalin fixed, paraffin embedded (FFPE) specimens?
Although not ideal, it is possible to generate RRBS libraries from degraded gDNA samples.
What if I am only interested in assaying one or a small number of target regions in the genome?
A method called Bisulfite Amplicon Sequencing (BSAS) [ref] can be used for targeted DNA methylation analysis. NUSeq cannot conduct bisulfite specific PCR as this is highly specific for each target region, but can build libraries and conduct sequencing for users.