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Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing
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....
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522004/ https://www.ncbi.nlm.nih.gov/pubmed/31095620 http://dx.doi.org/10.1371/journal.pone.0216709 |
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author | Smith, Andrew M. Jain, Miten Mulroney, Logan Garalde, Daniel R. Akeson, Mark |
author_facet | Smith, Andrew M. Jain, Miten Mulroney, Logan Garalde, Daniel R. Akeson, Mark |
author_sort | Smith, Andrew M. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6522004 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-65220042019-05-31 Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing Smith, Andrew M. Jain, Miten Mulroney, Logan Garalde, Daniel R. Akeson, Mark PLoS One Research Article 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. Public Library of Science 2019-05-16 /pmc/articles/PMC6522004/ /pubmed/31095620 http://dx.doi.org/10.1371/journal.pone.0216709 Text en © 2019 Smith et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Smith, Andrew M. Jain, Miten Mulroney, Logan Garalde, Daniel R. Akeson, Mark Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing |
title | Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing |
title_full | Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing |
title_fullStr | Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing |
title_full_unstemmed | Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing |
title_short | Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing |
title_sort | reading canonical and modified nucleobases in 16s ribosomal rna using nanopore native rna sequencing |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522004/ https://www.ncbi.nlm.nih.gov/pubmed/31095620 http://dx.doi.org/10.1371/journal.pone.0216709 |
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