Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA

Abstract

Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.nanopore DNA sequencing | DNA methylation | DNA hydroxymethylation | nanotechnology | next generation sequencing

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

“…Second, as another area of refinement, instead of carrying out genome-wide bisulfite sequencing, one could consider the use of a more targeted approach with potential cost saving. Third, with the advent of single molecule sequencing approaches, e.g., using nanopores (37), that would allow the direct interrogation of the methylation status without bisulfite conversion, the analytic precision of the approach might be improved. In this regard, it is interesting to note that nanopore sequencing has recently been demonstrated to be applicable for analyzing maternal plasma DNA (38).…”

Recent advances of DNA sequencing via nanopore-based technologies

“…This part of study was pioneered by Gundlach's group [11,[28][29][30] at Washington University. The big advantage of MspA over a-HL lies in its much sharper constriction, which is 1.2 nm in width and 0.6 nm in length [11].…”

“…The presence of methylated and hydroxymethylated cytosines in the DNA strand could be readily distinguished by the ionic current changes when DNA passes through the pore in single-nucleotide steps (Fig. 4) [30,31]. By using mathematical algorithms, the MspA nanopore system could achieve high base calling accuracy.…”

The role of cytosine methylation on charge transport through a DNA strand

“…Current sequencing methods are mainly based on light emission 7 , which requires polymerase chain reaction (PCR) amplification. An alternative sequencing idea is to measure the transverse current through a single-stranded DNA as it translocates a nanogap or nanopore [7][8][9]38,39 . The distinguishable electronic structures of the four DNA bases result in different electrical/ionic currents, which can be used in the third generation sequencing technologies.…”

“…The main detection techniques used today involve PCR-based methods 28 , which usually require chemical reactions 29,30 to maintain the methylation feature. With the rapid development and progress in single-molecule electrical approach [31][32][33][34] , new techniques utilizing the electronic properties of DNA bases have also been used to experimentally detect the methylated strands 10,11,[35][36][37][38][39][40] . These pioneering initial works show that it may be possible to detect methylated sequence/base by direct conductance measurement.…”