PLoS ONE (Jan 2019)

Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing.

  • Andrew M Smith,
  • Miten Jain,
  • Logan Mulroney,
  • Daniel R Garalde,
  • Mark Akeson

DOI
https://doi.org/10.1371/journal.pone.0216709
Journal volume & issue
Vol. 14, no. 5
p. e0216709

Abstract

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The ribosome small subunit is expressed in all living cells. It performs numerous essential functions during translation, including formation of the initiation complex and proofreading of base-pairs between mRNA codons and tRNA anticodons. The core constituent of the small ribosomal subunit is a ~1.5 kb RNA strand in prokaryotes (16S rRNA) and a homologous ~1.8 kb RNA strand in eukaryotes (18S rRNA). Traditional sequencing-by-synthesis (SBS) of rRNA genes or rRNA cDNA copies has achieved wide use as a 'molecular chronometer' for phylogenetic studies, and as a tool for identifying infectious organisms in the clinic. However, epigenetic modifications on rRNA are erased by SBS methods. Here we describe direct MinION nanopore sequencing of individual, full-length 16S rRNA absent reverse transcription or amplification. As little as 5 picograms (~10 attomole) of purified E. coli 16S rRNA was detected in 4.5 micrograms of total human RNA. Nanopore ionic current traces that deviated from canonical patterns revealed conserved E. coli 16S rRNA 7-methylguanosine and pseudouridine modifications, and a 7-methylguanosine modification that confers aminoglycoside resistance to some pathological E. coli strains.