DNA methylation is an epigenetic modification to cytosine bases that does not affect the primary DNA sequence but plays a critical role in the regulation of gene expression. The addition of methyl groups to DNA occurs at CpG regions by the conversion of cytosine to 5-methylcytosine. 5-methylcytosine base pairs with guanine in double stranded DNA. Regions of DNA that have higher methylation tend to be less transcriptionally active. Methylation is heritable, persisting in the germ line into the zygote.
Once studied in a locus specific manner, next-generation sequencing based methylation analysis offers complete genome-wide measurements of methylation, allowing researchers to understand the impact of methylation on gene expression. Several different library preparation applications have been developed, each offering a unique advantage for methylation analysis.
- Bisulfite-Seq is the application of next-generation sequencing on bisulfite treated genomic DNA in order to determine the methylation status at CpG sites. Bisulfite conversion, also known as bisulfite treatment is used to deaminate un-methylated cytosines in DNA to produce uracils. CpG regions that are protected by methyl groups do not convert to uracil, allowing the user to determine the locations of unmethylated cytosines and 5’ methyl-cytosines at single nucleotide resolution.
- Combined Bisulfite Restriction Analysis – is a variation of Bisulfite-Sequencing that allows quantification of the methyl status at specific loci in the genome. The process begins with bisulfite conversion of non-methylated cytosines to uracil, followed by PCR, which copies uracils into thymine residues. After PCR, the sample is digested with a restriction endonuclease, cleaving sites that were originally methylated.
- Methylated DNA immunoprecipitation (MeDIP / MeCAP) – is a large scale purification technique designed to assess the methylation status of CpG rich regions in the genome (CpG islands). MeDIP / MeCAP does not offer the single nucleotide resolution that Bisulfite-Seq does, but it allows for the measurement of the methylome in specific methyl rich regions and serves as a useful technique for comparing different cell types. The method is significantly less expensive that full genome Bisulfite-Sequencing as only rich methylated regions of the genome are targeted, reducing the amount of sequencing and coverage required. The process beings with a non-specific digestion of DNA, followed by pull down with a 5’methyl-cytidine specific antibody (MeDIP), or methyl specific capture protein (MeCAP). Methylated regions are amplified by PCR and then sequenced.
- Reduced Representation Bisulfite-Sequencing - is a high-throughput protocol that combines restriction enzyme digestion and bisulfite sequencing to enrich for areas of the genome with high CpG content. The approach reduces the number of regions that need to be sequenced to ~1% of the genome. The procedure reduces the sequencing coverage required by focusing on regions of the genome with CpG content, while allowing for a single nucleotide assessment of the methylome.
- Target Bisulfite-Seq – Allows for the targeting of selected genome regions using biotinylated probes to pull down specific bisulfite treated genomic DNA. This technique was developed as an alternative to whole genome bisulfite-sequencing and allows researchers to study promoter, enhancer and other differentially methylated regions (DMRs) of the methylome.
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Targeted bisulfite library prep services
Methyl and Bisulfite-Seq Kits
The NEXTflex Methyl-Seq kit allows the user to select for CpG rich regions using an 5’methlyC antibody or methyl capture protein. The kit utilizes “enhanced adapter ligation technology” and offers compatibility with clinical FFPE and degraded DNA samples. DNA is first fragmented, purified and ligated with non-methlyated Illumina compatible adapters. Methylated regions are then pulled down using an antibody or protein binding domain, enriching for methylated regions of the genome. The sample is then amplified, before being subjected to high throughput sequencing.
- End repair & adenylation
- Ligation using non-methlyated adapters (up to 192 available)
- 5’methly-cytosine specific antibody or protein pull down of methyl regions
- Next-generation sequencing
The NEXTflex Bisulfite-Seq kit performs reduced representation bisulfite-sequencing by first digesting all genomic material with Msp1, digesting genomic fragments at CCGG sites. Genomic material is subsequently end-repaired, adenylated and ligated with methylated, Illumina compatible adapters. The sample is then treated with bisulfite reagent, converting un-methylated cytosines to uracil, followed by PCR. This procedure allows for single nucleotide assessment of CpG rich regions while reducing the cost and time associated with performing whole genome sequencing. Simply not using the MSP1 enzyme allows the user to perform whole genome bisulfite-sequencing. 24 methylated adapters for multiplexing samples are available with this kit.
- Msp1 digestion of genomic DNA for RRBS, mechanical fragmentation for whole genome bisulfite-seq
- End repair & adenylation
- Ligation of methylated adapters (up to 24)
- Bisulfite conversion of un-methylated cytosines to uracils
- High throughput sequencing
SeqCap Epi Kits utilize an in-solution based capture method to enable enrichment of selected bisulfite treated genomic DNA. The SeqCap Epi CpG giant kit targets 84 Mb of the human genome, allowing researchers to interrogate >5.5 milllion methylation sites at single nucleotide resolution.
- The protocol begins with genomic DNA library preparation
- After the ligation step of library preparation, the sample is bisulfite treated, converting unmethlyated cytosines to uracil.
- The bisulfite treated library is hybridized to the SeqCap Epi oligo pool and magnetic beads are used to pull down the captured genomic DNA fragments
- Enriched DNA fragments are eluted from the beads, washed and the enriched fragment pool is amplified, producing sequence ready enriched targets