Our first journey through a nanopore

A: Length distribution histogram of basecalled reads. The wildtype sample produced more reads overall, but the majority of reads was shorter than 1,000 nt (median read length: 826). In the SL2 sample, the sequenced reads were generally longer (median read length: 1342). The difference was most pronounced for very long reads, in particular for lengths greater than 10k nt. In total, 5,543 reads were longer than 5k nt and 594 reads were longer than 10k nt. B: Spanned regions of the reference genome for the 12 longest reads (>22k nt) from both data sets. Five of these cover almost the complete genome and thus represent full viral RNA genomes, sequenced directly without a cDNA step.

We are proud to announce that our latest study is available as a preprint on bioRxiv. We applied the Oxford Nanopore Technology MinION sequencer on the Human Coronavirus 229E (HCoV-229E) and directly sequenced the viral genome. This is remarkable, as the HCoV-229E genome consists of RNA instead of DNA. With a specific MinION protocol, direct RNA sequencing is possible, which not only reduces the amount of work (and potential error sources) in the lab, but also preserves modifications of the RNA – small molecular changes on individual nucleotides that may serve a certain function in the virus life cycle. Further, the MinION sequencer is able to “read” molecules without length restriction.

In the past, modern sequencing technologies yielded read-lengths of about 300 nucleotides per scanned molecule. With MinION, the molecule of interest gets pulled through a small pore (hence nanopore) until the molecule ends – which again is only limited by the quality of your sample.

In our work, we were able to sequence one RNA molecule that nearly covered the whole HCoV-229E genome (roughly 27.000 nucleotides!). Further we analyzed all sequenced fragments for potential RNA modification and showed that the detection of these epitranscriptomic changes is possible.

We are looking forward to our next journeys through the nanopore and hope to get many new insights with this new technology. [bibtex file=https://raw.githubusercontent.com/rnajena/literature/master/webpage_literature.bib key=Viehweger:18]

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