Roche Applied Science
Technical Services Consultant
Epigenetics is the study of heritable changes in gene activity that are caused not by changes in the nucleotide sequence but through chemical modifications to DNA or histones. One such modification is DNA methylation. Methylation is an important component in the maintenance of numerous cellular processes, including embryonic development, genomic imprinting, X-chromosome inactivation and preservation of chromosome stability. As alterations in normal DNA methylation patterns have been linked to a variety of human diseases, including cancer, diabetes and cardiovascular diseases, DNA methylation has become an essential and exponentially growing area of study in both animal and plant research.
Methylation methods
Researchers have multiple options for studying DNA methylation at the genome level, including DNA microarrays and next-generation sequencing, but both suffer significant limitations. Microarrays lack the flexibility to discover new loci and provide allele-specific patterns, as genomic content is fixed and not updated often. Array probes also are susceptible to batch effects and can potentially cross-hybridize with nontarget DNA, further confounding results and requiring secondary confirmation. Finally, arrays cannot provide single-base resolution.
Whole-genome bisulfite sequencing (WGBS) does report genome-wide DNA methylation status with single-base resolution. But bisulfite treatment of DNA, which converts nonmethylated cytosine to uracil, effectively doubles the genome size, as both treated and untreated genome datasets must be collected; it also damages DNA, resulting in loss of genomic information. These artifacts make data analysis challenging. But just as significantly, as a shotgun sequencing approach, WGBS is inefficient—approximately 65% of reads fall outside of methylated regions. Consequently, the technique is cost-prohibitive for many applications.
One solution to this problem is reduced representation bisulfite sequencing (RRBS), which uses methylation-dependent restriction enzymes. By enriching for CpG-containing genomic regions, RRBS decreases both sequencing requirements and costs. But RRBS also has limitations, as sequencing coverage is limited by restriction-enzyme site distribution. The commonly used restriction enzyme Msp1 does not recognize all CpG-rich regions in the genome. And it can overlook key regions, as restriction enzymes do not always cleave their target sites in a consistent manner.
Clearly, what epigenetics researchers need is a tool that can selectively enrich likely methylated regions comprehensively and without bias. One such tool is the SeqCap Epi Enrichment System from Roche NimbleGen. Like the company’s exome-targeting SeqCap EZ system, the SeqCap Epi system uses pools of millions of oligonucleotide probes in solution to capture regions associated with a disease or phenotype, accelerating epigenetic discovery by enabling greater depths of coverage, increased sample throughput and lower costs compared with WGBS. The SeqCap Epi system shows excellent correlation with other commonly used methylation assessment methods, such as WGBS and Illumina's Infinium® HumanMethylation450 BeadChip microarray. Here are some tips for getting the most out of the system.
Probe design
To effectively identify and capture methylated targets, probes must target the different possible methylated configurations associated with any particular region of interest. Accomplishing this requires millions of oligonucleotide probes. In the case of the SeqCap Epi Enrichment System, Roche has predesigned probes targeting both strands of a bisulfite-converted genomic template, enabling researchers to obtain sequencing and methylation data from both directions on the DNA.
Custom designs are also possible for both human and nonhuman organisms, in design sizes up to 210 Mb. Researchers work directly with designers at Roche NimbleGen in creating probes for their specific, targeted areas of interest. First, genomic target coordinates are sent to Roche NimbleGen. Capture probe sequences are then calculated against the possible methylated configurations in these designated regions, and a newly designed assay is sent to the customer for confirmation. After approval, the custom probes are manufactured and sent to the customer for use.
Note that the online design tool called NimbleDesign, which customers use when creating custom, human SeqCap Exome designs, is not yet available for SeqCap Epi system users, although that could change with future updates.
SNP calling
One way targeted bisulfite sequencing can accelerate epigenetic discovery is through correct identification of single nucleotide polymorphisms (SNPs) in regions of interest. The most common type of SNP is a C-to-T conversion, which often occurs at CpG sites. This type of polymorphism can be mistaken for an nonmethylated cytosine in bisulfite sequencing, because both appear as thymine in the sequencing data.
The SeqCap Epi Enrichment System can distinguish the two events, however, as it targets both strands of DNA for capture. In that case, a true C-to-T polymorphism should be apparent, as the corresponding base pair on the other strand also would be sequenced.
Minimize sample loss
Targeted bisulfite sequencing protocols require larger amounts of input DNA than do other techniques. Additional material is needed to compensate for loss of molecular complexity during library preparation and enrichment processes and also for damage caused by the harsh bisulfite treatment. But researchers also can minimize this data loss with a simple change in workflow.
By performing the bisulfite conversion before enrichment, as in the SeqCap Epi system protocol, molecular complexity is better preserved, enabling researchers to work with lower sample input and experience higher reproducibility. Standard protocols in which bisulfite conversion occurs post-capture require 3 μg of sample. By performing the bisulfite conversion prior to capture, input can be reduced to 1 μg of sample, and the protocol can yield up to 30% improvement in key sequencing metrics, such as uniformity and reduced duplicate reads, which is reflective of improved library complexity.
Through the use of enrichment products specific for methylated regions, such as the SeqCap Epi Enrichment System, researchers can target specific regions of interest for both human and nonhuman organisms. This will enable discovery of differential methylated regions more efficiently, delivering massive time and cost savings vs. whole-genome or fixed-content sequencing approaches.
Image: Roche NimbleGen
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