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Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing

Andrew M. Smith, Miten Jain, Logan Mulroney, Daniel R. Garalde, Mark Akeson
doi: https://doi.org/10.1101/132274
Andrew M. Smith
1UC Santa Cruz Genomics Institute, University of California, Santa Cruz, USA 95064
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Miten Jain
1UC Santa Cruz Genomics Institute, University of California, Santa Cruz, USA 95064
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Logan Mulroney
1UC Santa Cruz Genomics Institute, University of California, Santa Cruz, USA 95064
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Daniel R. Garalde
2Oxford Nanopore Technologies, Oxford, UK
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Mark Akeson
1UC Santa Cruz Genomics Institute, University of California, Santa Cruz, USA 95064
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Abstract

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 1, and as a tool for identifying infectious organisms in the clinic 2. 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 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 16S rRNA base modifications, and a 7-methylguanosine modification that confers aminoglycoside resistance to some pathological E. coli strains. This direct RNA sequencing technology has promise for rapid identification of microbes in the environment and in patient samples.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 29, 2017.
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Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing
Andrew M. Smith, Miten Jain, Logan Mulroney, Daniel R. Garalde, Mark Akeson
bioRxiv 132274; doi: https://doi.org/10.1101/132274
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Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing
Andrew M. Smith, Miten Jain, Logan Mulroney, Daniel R. Garalde, Mark Akeson
bioRxiv 132274; doi: https://doi.org/10.1101/132274

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