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16S ribosomal RNA

16s ribosomal rna, 16s ribosomal rna sequencing
16S ribosomal RNA or 16S rRNA is the component of the 30S small subunit of a prokaryotic ribosome that binds to the Shine-Dalgarno sequence The genes coding for it are referred to as 16S rRNA gene and are used in reconstructing phylogenies, due to the slow rates of evolution of this region of the gene Carl Woese and George E Fox were two of the people who pioneered the use of 16S rRNA in phylogenetics

Multiple sequences of the 16S rRNA gene can exist within a single bacterium

Contents

  • 1 Functions
  • 2 Structure
  • 3 Universal primers
    • 31 PCR and NGS applications
    • 32 Hypervariable Regions
  • 4 16S ribosomal databases
    • 41 EzTaxon-e
    • 42 Ribosomal Database Project
    • 43 SILVA
    • 44 GreenGenes
  • 5 References
  • 6 External links

Functions

It has several functions:

  • Like the large 23S ribosomal RNA, it has a structural role, acting as a scaffold defining the positions of the ribosomal proteins
  • The 3' end contains the anti-Shine-Dalgarno sequence, which binds upstream to the AUG start codon on the mRNA The 3'-end of 16S RNA binds to the proteins S1 and S21 known to be involved in initiation of protein synthesis; RNA-protein cross-linking by AP Czernilofsky et al FEBS Lett Vol 58, pp 281–284, 1975
  • Interacts with 23S, aiding in the binding of the two ribosomal subunits 50S+30S
  • Stabilizes correct codon-anticodon pairing in the A site, via a hydrogen bond formation between the N1 atom of Adenine see image of Purine chemical structure residues 1492 and 1493 and the 2'OH group of the mRNA backbone

Structure

Universal primers

The 16S rRNA gene is used for phylogenetic studies as it is highly conserved between different species of bacteria and archaea Carl Woese pioneered this use of 16S rRNA It is suggested that 16S rRNA gene can be used as a reliable molecular clock because 16S rRNA sequences from distantly related bacterial lineages are shown to have similar functionalities Some hyperthermophilic archaea ie order Thermoproteales contain 16S rRNA gene introns that are located in highly conserved regions and can impact the annealing of "universal" primers Mitochondrial and chloroplastic rRNA are also amplified

The most common primer pair was devised by Weisburg et al and is currently referred to as 27F and 1492R; however, for some applications shorter amplicons may be necessary for example for 454 sequencing with Titanium chemistry 500-ish reads are ideal the primer pair 27F-534R covering V1 to V3 Often 8F is used rather than 27F The two primers are almost identical, but 27F has an M instead of a C AGAGTTTGATCMTGGCTCAG compared with 8F

Primer name Sequence 5'-3' Reference
8F AGA GTT TGA TCC TGG CTC AG
U1492R GGT TAC CTT GTT ACG ACT T same as above
928F TAA AAC TYA AAK GAA TTG ACG GG
336R ACT GCT GCS YCC CGT AGG AGT CT as above
1100F YAA CGA GCG CAA CCC
1100R GGG TTG CGC TCG TTG
337F GAC TCC TAC GGG AGG CWG CAG
907R CCG TCA ATT CCT TTR AGT TT
785F GGA TTA GAT ACC CTG GTA
805R GAC TAC CAG GGT ATC TAA TC
533F GTG CCA GCM GCC GCG GTA A
518R GTA TTA CCG CGG CTG CTG G
27F AGA GTT TGA TCM TGG CTC AG
1492R CGG TTA CCT TGT TAC GAC TT as above

PCR and NGS applications

In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for identification of bacteria As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid and cheap alternative to phenotypic methods of bacterial identification Although it was originally used to identify bacteria, 16S sequencing was subsequently found to be capable of reclassifying bacteria into completely new species, or even genera It has also been used to describe new species that have never been successfully cultured With Third-generation sequencing coming to many labs, simultaneous identification of thousands of 16S rRNA sequences is possible within hours, allowing metagenomic studies, for example of the gut flora

Hypervariable Regions

The bacterial 16S gene contains nine hypervariable regions V1-V9 ranging from about 30-100 base pairs long that are involved in the secondary structure of the small ribosomal subunit The degree of conservation varies widely between hypervariable regions, with more conserved regions correlating to higher-level taxonomy and less conserved regions to lower levels, such as genus and species While the entire 16S sequence allows for comparison of all hypervariable regions, at approximately 1500 base pairs long it can be prohibitively expensive for studies seeking to identify or characterize diverse bacterial communities These studies commonly utilize the Illumina platform, which produces reads at rates 50-fold and 12,000-fold less expensive than 454 pyrosequencing and Sanger sequencing, respectively While cheaper and allowing for deeper community coverage, Illumina sequencing only produces reads 75-150 base pairs long, and has no established protocol for reliably assembling the full gene in community samples Full hypervariable regions can be assembled from a single Illumina run, however, making them ideal targets for the platform

While 16S hypervariable regions can vary dramatically between bacteria, the 16S gene as a whole maintains greater length homogeneity than its Eukaryotic counterpart, which can make alignments easier Additionally, the 16S gene contains highly conserved sequences between hypervariable regions, enabling the design of universal primers that can reliably produce the same sections of the 16S sequence across different taxa Although no hypervariable region can accurately classify all bacteria from Domain to Species, some can reliably predict specific taxonomic levels Many community studies select semi-conserved hypervariable regions like the V4 for this reason, as it can provide resolution at the phylum level as accurately as the full 16S gene While lesser-conserved regions struggle to classify new species when higher order taxonomy is unknown, they are often used to detect the presence of specific pathogens In one study by Chakravorty et al in 2007, they characterized the V1-V8 regions of a variety of pathogens in order to determine which hypervariable regions would be most useful to include for disease-specific and broad assays Amongst other findings, they noted that the V3 region was best at identifying the genus for all pathogens tested, and that V6 was the most accurate at differentiating species between all CDC-watched pathogens tested, including Anthrax

While 16S hypervariable region analysis is a powerful tool for bacterial taxonomic studies, it struggles to differentiate between closely related species In the families Enterobacteriaceae, Clostridiaceae, and Peptostreptococcaceae, species can share up to 99% sequence similarity across the full 16S gene As a result, the V4 sequences can differ by only a few nucleotides, leaving reference databases unable to reliably classify these bacteria at lower taxonomic levels By limiting 16S analysis to select hypervariable regions, these studies can fail to observe differences in closely related taxa and group them into single taxonomic units, therefore underestimating the total diversity of the sample Furthermore, bacterial genomes can house multiple 16S genes, with the V1, V2, and V6 regions containing the greatest intraspecies diversity While not the most precise method of classifying bacterial species, analysis of the hypervariable regions remains one of the most useful tools available to bacterial community studies

16S ribosomal databases

The 16S rRNA gene is used as the standard for classification and identification of microbes, because it is present in most microbes and shows proper changes Type strains of 16S rRNA gene sequences for most bacteria and archaea are available on public databases such as NCBI However, the quality of the sequences found on these databases are often not validated Therefore, secondary databases that collect only 16S rRNA sequences are widely used The most frequently used databases are listed below:

EzTaxon-e

http://eztaxon-eezbiocloudnet/ The EzTaxon-e database is an extension of the original EzTaxon database It contains comprehensive 16S rRNA gene sequences of taxa with valid names as well as sequences of uncultured taxa EzTaxon-e contains complete hierarchical taxonomic structure from phylum rank to species rank for the domain of bacteria and archaea

Ribosomal Database Project

http://rdpcmemsuedu/ The Ribosomal Database Project RDP is a curated database that offers ribosome data along with related programs and services The offerings include phylogenetically ordered alignments of ribosomal RNA rRNA sequences, derived phylogenetic trees, rRNA secondary structure diagrams and various software packages for handling, analyzing and displaying alignments and trees The data are available via ftp and electronic mail Certain analytic services are also provided by the electronic mail server

SILVA

SILVA provides comprehensive, quality checked and regularly updated datasets of aligned small 16S/18S, SSU and large subunit 23S/28S, LSU ribosomal RNA rRNA sequences for all three domains of life as well as a suite of search, primer-design and alignment tools Bacteria, Archaea and Eukarya

GreenGenes

Greengenes is a quality controlled, comprehensive 16S reference database and taxonomy based on a de novo phylogeny that provides standard operational taxonomic unit sets The official home page for the site is http://greengenessecondgenomecom, and is licensed under the Creative Commons BY-SA 30 license

References

  1. ^ Schluenzen F, Tocilj A, Zarivach R, Harms J, Gluehmann M, Janell D, Bashan A, Bartels H, Agmon I, Franceschi F, Yonath A September 2000 "Structure of functionally activated small ribosomal subunit at 33 angstroms resolution" Cell 102 5: 615–23 doi:101016/S0092-86740000084-2 PMID 11007480 
  2. ^ a b Woese CR, Fox GE November 1977 "Phylogenetic structure of the prokaryotic domain: the primary kingdoms" Proceedings of the National Academy of Sciences of the United States of America 74 11: 5088–90 Bibcode:1977PNAS745088W doi:101073/pnas74115088 PMC 432104  PMID 270744 
  3. ^ Woese CR, Kandler O, Wheelis ML June 1990 "Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya" Proceedings of the National Academy of Sciences of the United States of America 87 12: 4576–9 Bibcode:1990PNAS874576W doi:101073/pnas87124576 PMC 54159  PMID 2112744 
  4. ^ Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S January 2007 "Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies" Applied and Environmental Microbiology 73 1: 278–88 doi:101128/AEM01177-06 PMC 1797146  PMID 17071787 
  5. ^ a b c Weisburg WG, Barns SM, Pelletier DA, Lane DJ January 1991 "16S ribosomal DNA amplification for phylogenetic study" Journal of Bacteriology 173 2: 697–703 doi:101128/jb1732697-7031991 PMC 207061  PMID 1987160 
  6. ^ a b Coenye T, Vandamme P November 2003 "Intragenomic heterogeneity between multiple 16S ribosomal RNA operons in sequenced bacterial genomes" FEMS Microbiology Letters 228 1: 45–9 doi:101016/S0378-10970300717-1 PMID 14612235 
  7. ^ Tsukuda, Miyuki; Kitahara, Kei; Miyazaki, Kentaro 2017-08-30 "Comparative RNA function analysis reveals high functional similarity between distantly related bacterial 16 S rRNAs" Scientific Reports 7 1 Bibcode:2017NatSR79993T doi:101038/s41598-017-10214-3 ISSN 2045-2322 
  8. ^ Jay ZJ, Inskeep WP July 2015 "The distribution, diversity, and importance of 16S rRNA gene introns in the order Thermoproteales" Biology Direct 10 35: 35 doi:101186/s13062-015-0065-6 PMC 4496867  PMID 26156036 
  9. ^ http://wwwhmpdaccorg/tools_protocolsphp#sequencing
  10. ^ Primers, 16S ribosomal DNA - François Lutzoni's Lab
  11. ^ Eden PA, Schmidt TM, Blakemore RP, Pace NR April 1991 "Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA-specific DNA" International Journal of Systematic Bacteriology 41 2: 324–5 doi:101099/00207713-41-2-324 PMID 1854644 
  12. ^ Universal Bacterial Identification by PCR and DNA Sequencing of 16S rRNA Gene PCR for Clinical Microbiology, 2010, Part 3, 209-214
  13. ^ Weidner S, Arnold W, Pühler A 1996 "Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes" PDF Appl Environ Microbiol 62 3: 766–71 PMC 167844  PMID 8975607 
  14. ^ Jiang H, Dong H, Zhang G, Yu B, Chapman LR, Fields MW June 2006 "Microbial diversity in water and sediment of Lake Chaka, an athalassohaline lake in northwestern China" Applied and Environmental Microbiology 72 6: 3832–45 doi:101128/AEM02869-05 PMC 1489620  PMID 16751487 
  15. ^ Pereira F, Carneiro J, Matthiesen R, van Asch B, Pinto N, Gusmão L, Amorim A December 2010 "Identification of species by multiplex analysis of variable-length sequences" Nucleic Acids Research 38 22: e203 doi:101093/nar/gkq865 PMC 3001097  PMID 20923781 
  16. ^ Kolbert CP, Persing DH June 1999 "Ribosomal DNA sequencing as a tool for identification of bacterial pathogens" Current Opinion in Microbiology 2 3: 299–305 doi:101016/S1369-52749980052-6 PMID 10383862 
  17. ^ Clarridge JE October 2004 "Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases" Clinical Microbiology Reviews 17 4: 840–62, table of contents doi:101128/CMR174840-8622004 PMC 523561  PMID 15489351 
  18. ^ Lu T, Stroot PG, Oerther DB July 2009 "Reverse transcription of 16S rRNA to monitor ribosome-synthesizing bacterial populations in the environment" Applied and Environmental Microbiology 75 13: 4589–98 doi:101128/AEM02970-08 PMC 2704851  PMID 19395563 
  19. ^ Brett PJ, DeShazer D, Woods DE January 1998 "Burkholderia thailandensis sp nov, a Burkholderia pseudomallei-like species" International Journal of Systematic Bacteriology 48 Pt 1 1: 317–20 doi:101099/00207713-48-1-317 PMID 9542103 
  20. ^ Schmidt TM, Relman DA 1994 "Phylogenetic identification of uncultured pathogens using ribosomal RNA sequences" Methods in Enzymology Methods in Enzymology 235: 205–22 doi:101016/0076-68799435142-2 ISBN 978-0-12-182136-4 PMID 7520119 
  21. ^ Gray JP, Herwig RP November 1996 "Phylogenetic analysis of the bacterial communities in marine sediments" Applied and Environmental Microbiology 62 11: 4049–59 PMC 168226  PMID 8899989 
  22. ^ Sanschagrin S, Yergeau E 2014 "Next-generation sequencing of 16S ribosomal RNA gene amplicons" J Vis Exp 90 doi:103791/51709 PMC 4828026  PMID 25226019 
  23. ^ Gray MW, Sankoff D, Cedergren RJ 1984 "On the evolutionary descent of organisms and organelles: a global phylogeny based on a highly conserved structural core in small subunit ribosomal RNA" Nucleic Acids Research 12 14: 5837–52 doi:101093/nar/12145837 PMC 320035  PMID 6462918 
  24. ^ a b c d Yang B, Wang Y, Qian PY March 2016 "Sensitivity and correlation of hypervariable regions in 16S rRNA genes in phylogenetic analysis" BMC Bioinformatics 17 1: 135 doi:101186/s12859-016-0992-y PMC 4802574  PMID 27000765 
  25. ^ Bartram AK, Lynch MD, Stearns JC, Moreno-Hagelsieb G, Neufeld JD June 2011 "Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end illumina reads" Applied and Environmental Microbiology 77 11: 3846–52 doi:101128/AEM02772-10 PMC 3127616  PMID 21460107 
  26. ^ a b Burke CM, Darling AE 2016-09-20 "A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq" PeerJ 4: e2492 doi:107717/peerj2492 PMC 5036073  PMID 27688981 
  27. ^ Van de Peer Y, Chapelle S, De Wachter R 1996 "A quantitative map of nucleotide substitution rates in bacterial rRNA" Nucleic Acids Research 24 17: 3381–91 doi:101093/nar/24173381 PMC 146102  PMID 8811093 
  28. ^ a b c Větrovský T, Baldrian P 2013-02-27 "The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses" PLoS One 8 2: e57923 Bibcode:2013PLoSO857923V doi:101371/journalpone0057923 PMC 3583900  PMID 23460914 
  29. ^ a b Chakravorty S, Helb D, Burday M, Connell N, Alland D May 2007 "A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria" Journal of Microbiological Methods 69 2: 330–9 doi:101016/jmimet200702005 PMC 2562909  PMID 17391789 
  30. ^ a b c Jovel J, Patterson J, Wang W, Hotte N, O'Keefe S, Mitchel T, Perry T, Kao D, Mason AL, Madsen KL, Wong GK 2016-01-01 "Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics" Frontiers in Microbiology 7: 459 doi:103389/fmicb201600459 PMC 4837688  PMID 27148170 
  31. ^ Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW October 2007 "EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences" International Journal of Systematic and Evolutionary Microbiology 57 Pt 10: 2259–61 doi:101099/ijs064915-0 PMID 17911292 
  32. ^ Larsen N, Olsen GJ, Maidak BL, McCaughey MJ, Overbeek R, Macke TJ, Marsh TL, Woese CR 1993 The ribosomal database project Nucleic Acids Res Jul 1;2113:3021-3
  33. ^ Elmar Pruesse, Christian Quast, Katrin Knittel, Bernhard M Fuchs, Wolfgang Ludwig, Jörg Peplies, Frank Oliver Glöckner 2007 Nucleic Acids Res SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB December; 3521: 7188–7196
  34. ^ DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL July 2006 "Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB" Applied and Environmental Microbiology 72 7: 5069–72 doi:101128/aem03006-05 PMC 1489311  PMID 16820507 
  35. ^ McDonald D, Price MN, Goodrich J, Nawrocki EP, DeSantis TZ, Probst A, Andersen GL, Knight R, Hugenholtz P March 2012 "An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea" The ISME Journal 6 3: 610–8 doi:101038/ismej2011139 PMC 3280142  PMID 22134646 

External links

  • University of Washington Laboratory Medicine: Molecular Diagnosis | Bacterial Sequencing
  • The Ribosomal Database Project
  • Ribosomes and Ribosomal RNA: rRNA
  • SILVA rRNA database
  • Greengenes: 16S rDNA data and tools
  • EzBioCloud

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