1. Why use the MethylMiner™ Kit instead of utilizing whole-genome bisulfite conversion and methylome sequencing?
Less starting template needed
Sequencing with MethylMiner™-enriched DNA requires ten times less DNA mass compared to whole methylome sequencing after bisulfite treatment. MethylMiner™ enrichment typically starts with as little as 1–2 micrograms of sample genomic DNA and yields 3–20% of the input DNA sample as the “methyl-CpG enriched” fraction. In contrast, a bisulfite conversion-base workflow would require a lot more starting template—approximately 5–10 micrograms.
Less sequencing coverage needed
Since the MethylMiner™ enrichment yields only a subset of all possible DNA sequences, the amount of sequencing that needs to be conducted to measure these regions at 10-fold coverage is approximately 5–30 fold less than for whole-genome sequencing. In contrast, bisulfite-based methylome sequencing is increased 3–10 fold because the bisulfite treatment is very harsh on the sample and in order to make an accurate measurement of the degree of methylation at any given cytosine residue, each cytosine position should be independently sequenced more often than without bisulfite conversion.
The cost per MethylMiner™-processed sample is reduced 5–30 fold as opposed to increased 3–10 fold for bisulfite-based methylome sequencing. If a whole genome costs $6,000 to sequence, a whole-methylome can be expected to cost $18,000–$60,000. However, a MethylMiner™-based profile of the methylation can be obtained for less than $1,200 in sequencing costs.
The costs for MethylMiner™ profiling are also scalable in the sense that a limited amount of sequencing can still yield interpretable results. This is because each sequenced fragment can be interpreted as having contained some degree of CpG methylation. As more sequencing is performed, the overall genome-wide landscape of CpG-methylation becomes more and more defined while the regions having dense methylation become more and more deeply covered.
2. What are the advantages of working with double-stranded DNA throughout the workflow compared to single-stranded DNA?
Fast and simplified workflow
The MethylMiner™ Kit enriches directly from double-stranded DNA. As such, its use can be integrated seamlessly with manufacturer-supported fragment library construction protocols. And since it is a magnetic bead-based enrichment technology, it can potentially be automated. The entire process, from DNA fragmentation through enrichment and desalting and into library construction, can be achieved in a single work day. In contrast, the use of anti-5-methylcytosine antibody requires working with single-stranded DNA and necessitates a slower, more labor intensive, and more reagent-costly workflow as compared to using the MethylMiner™ Kit.
The MethylMiner™ Kit uses about 1/5th to 1/30th the amount of reagent for adaptor ligation following enrichment compared to the antibody method. Furthermore, in order to facilitate library preparation using the antibody method, a significant mass of random primers and enzymes is required in an overnight reactionin order to reconstitute double-stranded DNA for library preparation. This adds cost and time to the process and also creates the need for an extra purification step, with expected losses in yield.
3. What are the practical way(s) that the MethylMiner™ Kit is “better” than anti-5-methylcytosine antibody for enrichment of methylated sequences? What is the evidence that its "better" sensitivity yields better data?
The MethylMiner™ Kit is demonstrably more sensitive than the antibody method, as seen by head-to-head comparisons of recovery of natural and synthetic methylated DNA molecules with qPCR assays. In these assays, the MethylMiner™ method routinely recovers 4-fold more amplifiable DNA and methylated sequences containing fewer methyl-CpG groups than the antibody method. MethylMiner™ can retrieve DNA fragments that contain as few as two methylated CpGs with the same sensitivity that the antibody-based MeDIP assay yields fragments containing 4 methylated CpGs.
Enhanced genome-wide methylation
The greater sensitivity of the MethyMiner™ Kit permits recovery of a more complex subset of genomic sequences and, hence, a better representation of the methylation that is present genome-wide within the sample. On CpG island-focused microarrays, the MethylMiner™ Kit has yielded 5 times as many high hits (S/N >10) as antibody-based MeDIP. On tiling arrays, the difference is even more pronounced. In all cases tested so far (n=6), high hits that were unique to the MethylMiner™ workflow have proven to be true positives as compared to bisulfite-sequencing.
Easy methylome profiling by serial elution
The MethylMiner™ Kit can be used to de-convolve the methylome in a manner that is impossible with anti-5methylcytosine antibody. This is because MethylMiner™- captured DNA strands can be eluted serially with changes in the salt concentration of the elution buffer.
4. The MethylMiner™ Kit cannot directly give single nucleotide resolution of the methylation pattern, so why should I use it?
The MethylMiner™ Kit is a sensitive enrichment technology that samples the CpG methylation in a minimally biased manner—thus it is an excellent discovery and characterization tool. It is readily compatible with next-generation sequencing workflows, as well as with microarray and PCR-based genome surveying technologies.
Compatible with next-generation sequencing
The resolution of methylation detection by the MethylMiner™ Kit is a function of the fragmentation of the DNA sample prior to enrichment, but can be practically stated as ~100 bp. This makes it uniquely well-matched with the short-read, high-throughput SOLiD™ System sequencing technology. In many cases, this degree of resolution may be sufficient to identify variation if genome methylation is biologically significant.
Towards single nucleotide resolution
Once differences and similarities between the methylation patterns of the samples of interest have been identified at the 100 bp resolution, it becomes much more practical to design and utilize cost-effective targeted re-sequencing strategies to determine the methylation patterns at single nucleotide resolution. Enrichment is absolutely required in order to screen the hundreds to thousands of samples that must be surveyed in the study of the complex human diseases that are the targets of DNA methylation research. In contrast, using antibody methods require and significant quantities of DNA, significant amounts of sequencing. Much of the methylation pattern is relatively invariant between samples and thus much of this investment is likely to be wasted on generating redundant information about constitutively methylated sequences.