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 ~1.5 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.

[1]  Benedict Paten,et al.  Improved data analysis for the MinION nanopore sequencer , 2015, Nature Methods.

[2]  C. Woese,et al.  Phylogenetic structure of the prokaryotic domain: The primary kingdoms , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Niederweis,et al.  Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase , 2012, Nature Biotechnology.

[4]  Daniel R. Garalde,et al.  Highly parallel direct RNA sequencing on an array of nanopores , 2016, Nature Methods.

[5]  Chengqi Yi,et al.  Base-Resolution Mapping Reveals Distinct m1A Methylome in Nuclear- and Mitochondrial-Encoded Transcripts. , 2017, Molecular cell.

[6]  Jay Shendure,et al.  Decoding long nanopore sequencing reads of natural DNA , 2014, Nature Biotechnology.

[7]  Hugh E. Olsen,et al.  The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community , 2016, Genome Biology.

[8]  Jordan M. Eizenga,et al.  Mapping DNA Methylation with High Throughput Nanopore Sequencing , 2017, Nature Methods.

[9]  O. Elemento,et al.  Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons , 2012, Cell.

[10]  Niranjan Nagarajan,et al.  Fast and sensitive mapping of nanopore sequencing reads with GraphMap , 2016, Nature Communications.

[11]  P. Sergiev,et al.  What do we know about ribosomal RNA methylation in Escherichia coli? , 2015, Biochimie.

[12]  D. Branton,et al.  Characterization of individual polynucleotide molecules using a membrane channel. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Pelin Yilmaz,et al.  The SILVA ribosomal RNA gene database project: improved data processing and web-based tools , 2012, Nucleic Acids Res..

[14]  J. Shine,et al.  The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[15]  T. Gojobori,et al.  Dynamic evolution of translation initiation mechanisms in prokaryotes , 2010, Proceedings of the National Academy of Sciences.

[16]  Schraga Schwartz,et al.  The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution , 2017, Nature.

[17]  K. Ochi,et al.  Loss of a conserved 7‐methylguanosine modification in 16S rRNA confers low‐level streptomycin resistance in bacteria , 2007, Molecular microbiology.

[18]  Heng Li.  Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM , 2013, 1303.3997.

[19]  Aaron R. Quinlan,et al.  Poretools: a toolkit for analyzing nanopore sequence data , 2014, bioRxiv.

[20]  Chengqi Yi,et al.  Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription , 2013, Journal of Nanobiotechnology.

[21]  Harry F. Noller,et al.  Interaction of antibiotics with functional sites in 16S ribosomal RNA , 1987, Nature.

[22]  K. Schleifer,et al.  Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences , 2014, Nature Reviews Microbiology.

[23]  H. Mori,et al.  Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection , 2006, Molecular systems biology.

[24]  A. Mankin,et al.  Specific fragmentation of tRNA and rRNA at a 7-methylguanine residue in the presence of methylated carrier RNA. , 1985, European journal of biochemistry.

[25]  Mark Akeson,et al.  Automated Forward and Reverse Ratcheting of DNA in a Nanopore at Five Angstrom Precision1 , 2012, Nature Biotechnology.

[26]  J. Kowalak,et al.  The single pseudouridine residue in Escherichia coli 16S RNA is located at position 516. , 1994, Nucleic acids research.

[27]  K. Yokoyama,et al.  Plasmid-Mediated 16S rRNA Methylase in Serratia marcescens Conferring High-Level Resistance to Aminoglycosides , 2004, Antimicrobial Agents and Chemotherapy.

[28]  Y. Arakawa,et al.  Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: an update. , 2012, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[29]  K. Lieberman,et al.  Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. , 2010, Journal of the American Chemical Society.

[30]  Harry F. Noller,et al.  Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes , 1986, Cell.

[31]  David A. Eccles,et al.  MinION Analysis and Reference Consortium: Phase 1 data release and analysis , 2015, F1000Research.

[32]  E. Cundliffe,et al.  Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides. , 1987, Journal of molecular biology.

[33]  M. Akeson,et al.  Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device , 2015, Front. Bioeng. Biotechnol..

[34]  M. Kupiec,et al.  Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq , 2012, Nature.

[35]  Ji Eun Lee,et al.  De novo Identification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing , 2017, bioRxiv.

[36]  Winston Timp,et al.  Detecting DNA cytosine methylation using nanopore sequencing , 2017, Nature Methods.

[37]  Fatih Ozsolak,et al.  Single‐molecule direct RNA sequencing without cDNA synthesis , 2011, Wiley interdisciplinary reviews. RNA.

[38]  J. Clarridge,et al.  Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases , 2004, Clinical Microbiology Reviews.