Abstract
Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5’UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.
Highlights
Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function
We extended the analysis to a longer 5 kb hairpin (Supplementary Fig. 2) and found the resolution of the instrument to be 1 bp or less for molecular lengths up to 1.5 kb, with precision decreasing as a function of molecular length
In a pilot experiment with genomic DNA isolated from the human HEK cell line, we found that the CCG repeat sequence created additional spontaneous blockage events, most likely due to the formation of DNA secondary structures
Summary
Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. The ability to generate long-range genomic information has required the advent of longer read-length, singlemolecule sequencing approaches, for example using nanopores (Minion, Oxford Nanopore)[13] and zero-mode waveguides (Sequel, Pacific Biosciences)[14,15] These platforms have proven to be useful for closing gaps in short read sequencing, and in providing important long-range structural information, they lack the per-read accuracy of NGS, and to date have only characterized a very limited range of epigenetic modifications. To complement these existing systems, we are developing a universal platform for genomic and epigenomic analysis that records the complexity of information on native nucleic acid molecules of both DNA and RNA. This feature is unique among single molecule genomic technologies and allows both the mapping of multiple ligands on the same molecule (either simultaneously or sequentially), and the application of error correction techniques to improve analytical accuracy
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