Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Applications of modification detection in nanopore sequencing

Nanopore sequencing is a single molecule characterization method, allowing direct sequencing of DNA or RNA sequencing with read lengths ranging from kilobases to even megabases. Unlike traditional sequencing-by-synthesis methods, it can distinguish covalently modified nucleotides directly through their modulation of the electrolytic current. And the long reads allow for straightforward detection of structural variations, large insertions, deletions or transpositions that are often difficult to detect with short-read sequencing.

We demonstrate the power of this technique, as applied with DNA, combined with exogenous labeling, to perform an integrative, single molecule characterization of the epigenome. We used M.CviPI, a GpC methyltransferase, to label accessible chromatin in cancer and normal cell lines. This allowed us to simultaneously correlate nucleosome positioning and native CpG methylation along long (~10kb) single molecules. We investigated methods of targeted sequencing for deeper nanopore sequencing at specific genomic loci. These methods resulted in focussed coverage of long native nanopore sequencing reads, measuring single molecule methylation patterns, SVs and SNVs. Finally, we have applied native RNA sequencing to interrogate poly(A) tail lengths and modifications to RNA which may be involved in post-transcriptional regulation. RNA base modifications are detectable via modulation of the nanopore current; our initial focus is on the METTL3 motif (GGm6ACU). Poly-A tail lengths inform mRNA lifetime; we can measure these lengths from how long the molecule takes to transit the pore. With these methods we have measured gene specific and even isoform specific poly(A) tail lengths and modification signals.

Winston Timp

Biomedical Engineering
Johns Hopkins Univ.
Baltimore MD

Email: wtimp0@gmail.com