"Flu virus finally sequenced in its native form" using nanopore technology
Fri 20th April 2018
The unique direct RNA sequencing method available with Oxford Nanopore technology has been featured in many publications, including a recent Nature Methods front cover in which the authors noted that:
"Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA."
This month, researchers posted a preprint "Complete genome direct RNA sequencing of influenza RNA virus". The abstract notes that:
"Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termi of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods."
Commentary in Nature notes that the "direct sequencing of RNA molecules such as virus genomes could help to unpick role of mysterious chemical modifications in genetic material." In the piece, one commentator noted that "Sequencing modified bases of RNA would be “a big deal” for the field... Current methods for detecting such modifications are time-consuming and expensive", and another that "nanopore sequencing could also reveal hidden diversity in RNA viruses, which is lost with other methods that cobble together much shorter stretches of genetic material to generate a genome."
Read more about how to use Oxford Nanopore's Direct RNA sequencing method, on the portable MinION, benchtop GridION or high-throughput PromethION.