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Amplicon Sequencing Introduction
This document provides an overview of 16S rRNA amplicon sequencing methodology. It discusses how environmental samples are used to extract mixed genomic DNA which is then amplified via PCR using primers targeting the 16S rRNA gene. The resulting PCR products are sequenced via next-generation sequencing. Sequence reads are processed, clustered into OTUs, and classified by matching to reference databases. Analysis of amplicon data can provide information on alpha and beta diversity of bacterial communities. While providing phylogenetic information, amplicon sequencing is limited compared to metagenomics in functional data obtained.
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Amplicon Sequencing Introduction
- 1. Amplicon Sequencing Aaron MarcSaunders Introduction To Community Systems Microbiology, Aalborg 2013 Introduction To Community Systems Microbiology, Aalborg 2013
- 2. Bacterial diversity Phylum Actinobacteria GenusTetrasphaera cultured representative Introduction To Community Systems Microbiology, Aalborg 2013
- 3. Environmental diversity DNA extraction Environmentalsample mixed genomic DNA Introduction To Community Systems Microbiology, Aalborg 2013
- 4. Environmental surveys ● Marker geneSequencing – – ● PCR product One gene fragment Metagenomics – Total DNA – All genes Introduction To Community Systems Microbiology, Aalborg 2013
- 5. Marker Genes Introduction ToCommunity Systems Microbiology, Aalborg 2013 http://fungene.cme.msu.edu/
- 6. ”Tree of Life” IntroductionrRNA gene Based on 16S To Community Systems Microbiology, Aalborg 2013
- 7. Start Stop Introduction To CommunitySystems Microbiology, Aalborg 2013
- 8. Hypervariable regions E. coli Alllife! V4 Introduction To Community Systems Microbiology, Aalborg 2013
- 9. 16S rRNA geneconservation Regions denoted ”hypervariable” http://www.bioinformatics-toolkit.o Introduction To Community Systems Microbiology, Aalborg 2013
- 10. Environmental surveys mixed genomicDNA Environmental sample PCR product 16S clone library Introduction To Community Systems Microbiology, Aalborg 2013
- 11. Introduction To CommunitySystems Microbiology, Aalborg 2013
- 12. Environmental surveys mixed genomicDNA metagenomic NGS sequencing library Environmental sample PCR product amplicon library Introduction To Community Systems Microbiology, Aalborg 2013
- 13. Amplicon vs. Metagenomics – lesscomplex ● ● – – extensive database same fragment ● – – – better coverage more samples comparable phylogenetic info PCR bias Limited phylogenetic info (genus/species level) Limited functional information Introduction To Community Systems Microbiology, Aalborg 2013
- 14. Typical results Small numberof highly abundant species and a long tail of rare species Introduction To Community Systems Microbiology, Aalborg 2013
- 15. 2. methodology Introduction ToCommunity Systems Microbiology, Aalborg 2013
- 16. 16S rRNA amplicon sequencing Sample PCR ”Short”PCR product (70-400 bp) Next-gen sequencing Sequence reads Quality screen and clustering Sequence types (OTUs) Matching to existing database Classified OTUs Introduction To Community Systems Microbiology, Aalborg 2013
- 17. 16S rRNA amplicon sequencing Sample PCR ”Short”PCR product (70-400 bp) Next-gen sequencing Sequence reads Quality screen and clustering Sequence types (OTUs) Matching to existing database Classified OTUs Introduction To Community Systems Microbiology, Aalborg 2013
- 18. Environmental surveys DNA extraction mixedgenomic DNA Environmental sample PCR product amplicon library Introduction To Community Systems Microbiology, Aalborg 2013
- 19. Hypervariable regions V4 E. coli Alllife! Introduction To Community Systems Microbiology, Aalborg 2013
- 20. PCR product 454 =400 bp Illumina = 250 bp Examples of NGS sequenced amplicons http://www.bioinformatics-toolkit.o Regions denoted ”hypervariable” Introduction To Community Systems Microbiology, Aalborg 2013
- 21. Multiplexing Normal PCR product NGS-adaptedPCR product Barcoded PCR product for NGS Introduction To Community Systems Microbiology, Aalborg 2013
- 22. Multiplexing adaptor barcode Primer sequence Amplifiedsequence …GCCATCAG GATCT CNACGCGAAGAACCTTANC NNNNNNNNNN… Sample 1 …GCCATCAG ATCAG CNACGCGAAGAACCTTANC NNNNNNNNNN… Sample 2 …GCCATCAG CACTG CNACGCGAAGAACCTTANC NNNNNNNNNN… Sample 3 …GCCATCAG CTGTG CNACGCGAAGAACCTTANC NNNNNNNNNN… Sample 4 Introduction To Community Systems Microbiology, Aalborg 2013
- 23. 16S rRNA amplicon sequencing Sample PCR ”Short”PCR product (70-400 bp) Next-gen sequencing Sequence reads Quality screen and clustering Sequence types (OTUs) Matching to existing database Classified OTUs Introduction To Community Systems Microbiology, Aalborg 2013
- 24. Bacterial Diversity Introduction ToCommunity Systems Microbiology, Aalborg 2013
- 25. Clustering Representative sequence Introduction ToCommunity Systems Microbiology, Aalborg 2013
- 26. Consider Errors! • Lowquality reads • PCR errors • Sequencing errors – Particularly homopolymers ● 454 & ion torrent! • Chimeras Introduction To Community Systems Microbiology, Aalborg 2013
- 27. 16S rRNA amplicon sequencing Sample PCR ”Short”PCR product (70-400 bp) Next-gen sequencing Sequence reads Quality screen and clustering Sequence types (OTUs) Matching to existing database Classified OTUs Introduction To Community Systems Microbiology, Aalborg 2013
- 28. Functional information • Isolates •Functional 16S rRNA work – Stable isotope probing • Metagenomics • In situ studies – – – Microscopy – eg. inclusion bodies Microautoradiography Raman or NanoSIMS Introduction To Community Systems Microbiology, Aalborg 2013
- 29. Taxonomic assignment Genus A Family GenusB Genus C Introduction To Community Systems Microbiology, Aalborg 2013
- 30. Taxonomic assignment Genus A Family GenusB Genus C Introduction To Community Systems Microbiology, Aalborg 2013
- 31. Taxonomic assignment Genus A Family GenusB Genus C Introduction To Community Systems Microbiology, Aalborg 2013
- 32. Taxonomic assignment Genus A Family GenusB Genus C Introduction To Community Systems Microbiology, Aalborg 2013
- 33. Using a ”Classifier” •Uses an existing phylogeny • Find best unambiguous match to references Introduction To Community Systems Microbiology, Aalborg 2013
- 34. Classification results Introduction ToCommunity Systems Microbiology, Aalborg 2013
- 35. Microautoradiography Radioac tive phospha te Accumulibacter Radioactive Aalborg 2013 IntroductionTo Community Systems Microbiology,acetate Other Bacteria
- 36. Metagenomics Simon McIllroy, Microthrix ISMEJ 7 (6): 1161 Introduction To Community Systems Microbiology, Aalborg 2013
- 37. Substrate specificity Accumulibacter Tetraspheara Competibacter Defluvicoccus acetate + - + + propionate + - - + Casamino acids Glu only + - - Nitrate reduction +/- +/- +/- - Determinedby FISH-MAR Introduction To Community Systems Microbiology, Aalborg 2013
- 38. Greengenes assignment species (OTU) OTU1 Class Order Betaproteobacteria Nitrosomonadales Family Genus Nitrosomonadacea Nitrosomonas e OTU2 Nitrospira Nitrospirales Nitrospiraceae Nitrospira OTU3 Betaproteobacteria Rhodocyclales Rhodocyclaceae Propionivibrio OTU4 Betaproteobacteria Gallionellales Gallionellaceae ?? OTU5 Gammaproteobacte ria ?? ?? ?? IntroductionTo Community Systems Microbiology, Aalborg 2013
- 39. midasfieldguide.org midasfieldguide.org 52 core genera 64%organisms DK 41% globally + others Introduction To Community Systems Microbiology, Aalborg 2013
- 40. MIDAS curated assignment species (OTU) Class Order Family Genus OTU1 Betaproteobacteria Nitrosomonadales Nitrosomonadaceae Nitrosomonas OTU2 Nitrospira Nitrospirales Nitrospiraceae Nitrospira OTU3 Betaproteobacteria Rhodocyclales Rhodocyclaceae Accumulibacter OTU4 Betaproteobacteria Gallionellales Gallionellaceae Nitrotoga OTU5 GammaproteobacteriaCompetibacterales Competibacteraceae Competibacter Introduction To Community Systems Microbiology, Aalborg 2013
- 41. Percent abundance Nitrite-oxidisers 0.3 genus 0.2 0.1 0 Introduction ToCommunity Systems Microbiology, Aalborg 2013
- 42. Analysis Tools ● Qiime (qiime.org) ● Mothur(mothur.org) ● R (r-project.org) – Phyloseq package (http://joey711.github.io/phyloseq/) Introduction To Community Systems Microbiology, Aalborg 2013
- 43. ● Analysis Example ● ● http://nbviewer.ipython.org/7213441 Introduction ToCommunity Systems Microbiology, Aalborg 2013
- 44. Comparing Bacterial diversityof Wastewater and activated sludge Location Sample type Replicate Aalborg East activated sludge AAE-1 Aalborg East activated sludge AAE-2 Aalborg East wastewater AAE-1 Aalborg East wastewater AAE-2 Aalborg West activated sludge AAW-1 Aalborg West activated sludge AAW-2 Aalborg West wastewater AAW-1 Aalborg West wastewater AAW-2 Hjoerring activated sludge HJO-1 Hjoerring activated sludge HJO-2 Hjoerring wastewater HJO-1 Hjoerring wastewater HJO-2 Introduction To Community Systems Microbiology, Aalborg 2013
- 45. Phylum level Classification Proteobacteria Firmicutes Bacteroidetes AS WW IntroductionTo Community Systems Microbiology, Aalborg 2013
- 46. Genus level Classification AS WW IntroductionTo Community Systems Microbiology, Aalborg 2013
- 47. Alpha Diversity • Howdiverse is the community in my sample? • Richness – How many organisms are in my sample? • Evenness – How evenly are they distributed? Introduction To Community Systems Microbiology, Aalborg 2013
- 48. Richness estimates Introduction ToCommunity Systems Microbiology, Aalborg 2013
- 49. Introduction To CommunitySystems Microbiology, Aalborg 2013
- 50. Beta Diversity • Howsimilar/different is my sample from other samples? Introduction To Community Systems Microbiology, Aalborg 2013
- 51. Clustering Introduction To CommunitySystems Microbiology, Aalborg 2013
- 52. Principal components analysis IntroductionTo Community Systems Microbiology, Aalborg 2013
- 53. Label with environmentaldata Introduction To Community Systems Microbiology, Aalborg 2013
- 54. Amplicon Sequencing • Pros: – – ”cheap”,many samples 16S: good database for classification • Cons: – – PCR and primer bias limited functional information Introduction To Community Systems Microbiology, Aalborg 2013
Editor's Notes
- #17 Roche is supposed to soon come with a 1000 bp sequencer <number>
- #22 <number>
- #23 <number>
- #24 Mantoin the concept with the large fragments and matching <number>
- #27 Quince 30-50% overestimation of OTUs <number>
- #28 Mantoin the concept with the large fragments and matching <number>
- #39 Figure: Nitrotoga, a newly-discovered genus of nitrite-oxidisers, was found to be core in EPPR plants and in >1% in some. They are therefore likely making a significant contribution to nitrite oxidiation in these plants. <number>
- #41 Figure: Nitrotoga, a newly-discovered genus of nitrite-oxidisers, was found to be core in EPPR plants and in >1% in some. They are therefore likely making a significant contribution to nitrite oxidiation in these plants. <number>
- #42 Figure: Nitrotoga, a newly-discovered genus of nitrite-oxidisers, was found to be core in EPPR plants and in >1% in some. They are therefore likely making a significant contribution to nitrite oxidiation in these plants. <number>