Skip to main content

16S-rRNA-Amplicon-Sequenzierung für Metagenomik

  • Chapter
  • First Online:
Einführung in die Bioinformatik in der Mikrobiologie

Zusammenfassung

Die 16S-rRNA-Amplicon-Sequenzierungstechnik ist eine Mikrobiomanalyse, bei der verschiedene Proben gleichzeitig mit Hilfe von Multiplexing analysiert werden. Die Ergebnisse können verwendet werden, um die mikrobielle Vielfalt auf Gattungs-, Familien-, Ordnungs-, Klassen- und Phylumebene zu bewerten. Die Auflösung ist normalerweise nicht ausreichend, um die Artenebene zu bewerten. Die verschiedenen Schritte in der bioinformatischen Analyse ermöglichen sowohl die Analyse aller Proben zusammen als auch Vergleiche zwischen Proben. Die bioinformatische Analyse konzentriert sich auf die Qualitätskontrolle der Reads, die Zusammenführung identischer Reads und die Gruppierung von Reads in operationelle taxonomische Einheiten (OTUs) mit einem Schwellenwert von 97 %. Der Schwellenwert wird vom Arten-Schwellenwert für die Klassifizierung von Arten auf der Grundlage von 16S-rRNA-Sequenzvergleichen übernommen. Die Verteilung der Reads und OTUs innerhalb und zwischen Proben kann verwendet werden, um α- und β-Diversität zu schätzen. Die relative Häufigkeit der taxonomischen Einheiten auf Gattungs-, Familien-, Ordnungs-, Klassen- und Phylumebene kann zwischen Proben verglichen werden. Diese Verteilungen können mit Hilfe der Hauptkomponentenanalyse mit Metadaten in Beziehung gesetzt werden.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 44.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 59.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Literatur

  • Anderson EL, Li W, Klitgord N, Highlander SK, Dayrit M, Seguritan V, Yooseph S, Biggs W, Venter JC, Nelson KE, Jones MB. 2016. A robust ambient temperature collection and stabilization strategy: Enabling worldwide functional studies of the human microbiome. Sci Rep. 25;6:31731.

    Article  Google Scholar 

  • Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ. 2005. At least 1 in 20 16S rRNA sequence records currently held in public repositories is esti-mated to contain substantial anomalies. Appl Environ Microbiol. 71, 7724–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 7, 335–336.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM. 2014. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42, D633–42.

    Article  CAS  PubMed  Google Scholar 

  • Danzeisen JL, Kim HB, Isaacson RE, Tu ZJ, Johnson TJ. 2011. Modulations of the chicken cecal microbiome and metagenome in response to anticoccidial and growth promoter treatment. PLoS One. 6:e27949.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Davis NM, Proctor D, Holmes SP, Relman DA, & Callahan BJ. 2017. Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. bioRxiv preprint. https://doi.org/10.1101/221499

  • DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL. 2006. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 72, 5069–5072.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Edgar RC. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461.

    Article  CAS  PubMed  Google Scholar 

  • Edgar RC. 2013. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 10:996–998.

    Article  CAS  PubMed  Google Scholar 

  • Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Faith DP. 1992. Conservation evaluation and phylogenetic diversity. Biological Conservation 64, 1–10.

    Article  Google Scholar 

  • Hamady M, Walker JJ, Harris JK, Gold NJ, Knight R. 2008. Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat Methds. 5:235–7.

    Article  CAS  Google Scholar 

  • Karst SM, Dueholm MS, McIlroy SJ, Kirkegaard RH, Nielsen PH, & Albertsen M. 2018. Retrieval of a million high-quality, full-length microbial 16S and 18S rRNA gene sequences without primer bias. Nat Biotechnol. 36:190–195.

    Article  CAS  PubMed  Google Scholar 

  • Lane DJ. 1991. 16S/23S rRNA Sequencing. In: Stackebrandt, E. and Goodfellow, M., Eds., Nucleic Acid Techniques in Bacterial Systematic, John Wiley and Sons, New York, 115–175.

    Google Scholar 

  • Langille MG, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes JA, Clemente JC, Burkepile DE, Vega Thurber RL, Knight R, Beiko RG, Huttenhower, C. 2013. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol 31, 814–821.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu Z, Lozupone C, Hamady M, Bushman FD & Knight R. 2007. Short pyrosequencing reads suffice for accurate microbial community analysis. Nucleic Acids Res. 35, e120.

    Article  PubMed  PubMed Central  Google Scholar 

  • Liu Z, DeSantis TZ, Andersen GL, Knight R. 2008. Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers. Nucleic Acids Res. 36:e120.

    Article  PubMed  PubMed Central  Google Scholar 

  • McDonald D, Price MN, Goodrich J, Nawrocki EP, DeSantis TZ, Probst A, Andersen GL, Knight R, Hugenholtz P. 2012. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6, 610–618.

    Article  CAS  PubMed  Google Scholar 

  • McKenna P, Hoffmann C, Minkah N, Aye PP, Lackner A, Liu Z, Lozupone CA, Hamady M, Knight R, Bushman FD. 2008. The macaque gut microbiome in health, lentiviral infection, and chronic enterocolitis. PLoS Pathog. 4:e20.

    Article  PubMed  PubMed Central  Google Scholar 

  • McMurdie PJ, & Holmes S. 2012. Phyloseq: a bioconductor package for handling and analysis of high-throughput phylogenetic sequence data. Pac Symp Biocomput. 12:235–46.

    Google Scholar 

  • Nossa et al. 2010. World J. Gastroenterol. 16, 4135–44.

    CAS  Google Scholar 

  • Popp et al. 2017. Biospektrum Abstractbook, p. 199, 2017.

    Google Scholar 

  • Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO. 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41:D590–6.

    Article  PubMed  PubMed Central  Google Scholar 

  • Rognes T, Flouri T, Nichols B, Quince C, Mahé F. 2016. VSEARCH: a versatile open source tool for metagenomics. PeerJ. 18:e2584.

    Article  Google Scholar 

  • Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF. 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schloss PD, Jenior ML, Koumpouras CC, Westcott SL & Highlander SK. 2016. Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system. Peer J 4:e1869.

    Article  PubMed  PubMed Central  Google Scholar 

  • Soergel DA, Dey N, Knight R & Brenner SE. 2012. Selection of primers for optimal taxonomic classification of environmental 16S rRNA gene sequences. ISME J. 6, 1440–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tang Y, Underwood A, Gielbert A, Woodward MJ, Petrovska L. 2014. Metaproteomics analysis reveals the adaptation process for the chicken gut microbiota. Appl Environ Microbiol. 80, 478–485.

    Article  PubMed  PubMed Central  Google Scholar 

  • Wagner J, Coupland P, Browne HP, Lawley TD, Francis SC, Parkhill J. 2016. Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification. BMC Microbiol. 16:274.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Henrik Christensen .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2023 Der/die Autor(en), exklusiv lizenziert an Springer Nature Switzerland AG

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Christensen, H., Andersson, A.J., Jørgensen, S.L., Vogt, J.K. (2023). 16S-rRNA-Amplicon-Sequenzierung für Metagenomik. In: Christensen, H. (eds) Einführung in die Bioinformatik in der Mikrobiologie. Springer Vieweg, Cham. https://doi.org/10.1007/978-3-031-31212-0_8

Download citation

Publish with us

Policies and ethics