June 15, 2015  |  General

Scientists Publish New Methylation Analysis Protocols Using SMRT Sequencing

Scientists from the Icahn School of Medicine at Mount Sinai and the University of Saskatchewan teamed up to develop an innovative approach to methylation analysis using Single Molecule, Real-Time (SMRT®) Sequencing. The resulting method was just published in BMC Genomics.

Lead author Yao Yang and colleagues note in the paper [“Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS)”] that existing methods for methylation analysis are limited by cost and throughput in the case of Sanger sequencing, or short read lengths with NGS technologies. Their goal was to develop a method combining long reads, high accuracy, and high throughput.

“Coupled with an optimized long-range bisulfite amplification protocol and empowered by the long read lengths of SMRT sequencing (up to ~20 kb), multiplexed SMRT bisulfite sequencing (SMRT-BS) can accurately measure CpG methylation across ~1.5 kb regions without the need for PCR amplicon subcloning,” Yang et al. write. “As a cost-effective alternative to other targeted bisulfite sequencing techniques, SMRT-BS is an efficient and highly quantitative method for DNA methylation analysis.”

The technique incorporates bisulfite conversion of DNA, followed by amplification based on targeted primers. Amplicon templates are re-amplified and barcoded for multiplexing, and then purified and sequenced prior to CpG methylation analysis. The scientists found that results from the procedure were “reproducible and highly concordant” with other methylation analysis methods, particularly as sequencing depth increased. SMRT-BS data was validated using orthogonal technologies including microarrays and short-read sequencers.

“A key component to the development of SMRT-BS was the optimization of bisulfite conversion and PCR, which resulted in amplicons up to ~1.5-2.0 kb from bisulfite-converted DNA,” the researchers write, noting that amplicons of this length “theoretically can cover ~91% of CpG islands in the human genome.”

Using long-read sequencing technology “allows for more thorough regional CpG methylation assessment and increases the capacity for studying the relationship between phased single nucleotide variants and allele-specific CpG methylation,” they report.

Yang et al. predict that this approach could be used for diagnostic methylation analysis and for confirmation of epigenome-wide association studies, in addition to the usual research applications in transcriptional regulation, human imprinting disorders, and other methylation-specific studies.

In another recent publication, entitled “CGGBP1 mitigates cytosine methylation at repetitive DNA sequences,” scientists at the Science for Life Laboratory at Uppsala University used bisulfite conversion paired with PacBio® sequencing to examine the effect of depleting the transcription factor CGGBP1 on the level of methylation in Alu and LINE repeats.

CGGBP1 is known to bind CGG-rich regions of the genome and repress transcription of Alus and LINEs, but it is not known whether this binding in turn affects methylation status.

Lead author Prasoon Agarwaal and colleagues used genome-wide amplification of Alu and LINE-1 repeats using consensus primers and PacBio sequencing to examine the extent to which an observed genome-wide increase in CpG methylation after CGGBP1 knock down was focused in these regions. Interestingly, although there was an increase in Alu methylations overall, “an inspection of the distribution of methylation frequencies indicated two different directions of methlylation change,” the scientists report. Some Alus had 12% greater methylation, while other had 8% less methylation. Methylation was also increased in LINE-1 elements. The authors note that “the possibility of bi-directional change in Alu CpG suggests that different Alu elements may be subjected to different mechanisms of CpG methylations regulation by CGGBP1,” and cite the need for follow-up studies to identify the differences between the two populations of Alu elements.

More generally, they note that while this experiment reflects an overall characterization of methylation changes, “these data give a sound platform to build upon to uncover the sequence contexts in which CGGBP1 exerts methylation regulation at specific sites.”

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