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Amplicon Sequencing Introduction

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Amplicon Sequencing Introduction

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A brief introduction to amplicon sequencing of the 16S rRNA gene for the analysis of microbial diversity. This talk was presented originally at the Workshop: Introduction to Systems Biology, Aalborg Denmark. 2013-10-29

A brief introduction to amplicon sequencing of the 16S rRNA gene for the analysis of microbial diversity. This talk was presented originally at the Workshop: Introduction to Systems Biology, Aalborg Denmark. 2013-10-29

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Amplicon Sequencing Introduction

  1. 1. Amplicon Sequencing Aaron Marc Saunders Introduction To Community Systems Microbiology, Aalborg 2013 Introduction To Community Systems Microbiology, Aalborg 2013
  2. 2. Bacterial diversity Phylum Actinobacteria Genus Tetrasphaera cultured representative Introduction To Community Systems Microbiology, Aalborg 2013
  3. 3. Environmental diversity DNA extraction Environmental sample mixed genomic DNA Introduction To Community Systems Microbiology, Aalborg 2013
  4. 4. Environmental surveys ● Marker gene Sequencing – – ● PCR product One gene fragment Metagenomics – Total DNA – All genes Introduction To Community Systems Microbiology, Aalborg 2013
  5. 5. Marker Genes Introduction To Community Systems Microbiology, Aalborg 2013 http://fungene.cme.msu.edu/
  6. 6. ”Tree of Life” Introduction rRNA gene Based on 16S To Community Systems Microbiology, Aalborg 2013
  7. 7. Start Stop Introduction To Community Systems Microbiology, Aalborg 2013
  8. 8. Hypervariable regions E. coli All life! V4 Introduction To Community Systems Microbiology, Aalborg 2013
  9. 9. 16S rRNA gene conservation Regions denoted ”hypervariable” http://www.bioinformatics-toolkit.o Introduction To Community Systems Microbiology, Aalborg 2013
  10. 10. Environmental surveys mixed genomic DNA Environmental sample PCR product 16S clone library Introduction To Community Systems Microbiology, Aalborg 2013
  11. 11. Introduction To Community Systems Microbiology, Aalborg 2013
  12. 12. Environmental surveys mixed genomic DNA metagenomic NGS sequencing library Environmental sample PCR product amplicon library Introduction To Community Systems Microbiology, Aalborg 2013
  13. 13. Amplicon vs. Metagenomics – less complex ● ● – – 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. 14. Typical results Small number of highly abundant species and a long tail of rare species Introduction To Community Systems Microbiology, Aalborg 2013
  15. 15. 2. methodology Introduction To Community Systems Microbiology, Aalborg 2013
  16. 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. 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. 18. Environmental surveys DNA extraction mixed genomic DNA Environmental sample PCR product amplicon library Introduction To Community Systems Microbiology, Aalborg 2013
  19. 19. Hypervariable regions V4 E. coli All life! Introduction To Community Systems Microbiology, Aalborg 2013
  20. 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. 21. Multiplexing Normal PCR product NGS-adapted PCR product Barcoded PCR product for NGS Introduction To Community Systems Microbiology, Aalborg 2013
  22. 22. Multiplexing adaptor barcode Primer sequence Amplified sequence …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. 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. 24. Bacterial Diversity Introduction To Community Systems Microbiology, Aalborg 2013
  25. 25. Clustering Representative sequence Introduction To Community Systems Microbiology, Aalborg 2013
  26. 26. Consider Errors! • Low quality reads • PCR errors • Sequencing errors – Particularly homopolymers ● 454 & ion torrent! • Chimeras Introduction To Community Systems Microbiology, Aalborg 2013
  27. 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. 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. 29. Taxonomic assignment Genus A Family Genus B Genus C Introduction To Community Systems Microbiology, Aalborg 2013
  30. 30. Taxonomic assignment Genus A Family Genus B Genus C Introduction To Community Systems Microbiology, Aalborg 2013
  31. 31. Taxonomic assignment Genus A Family Genus B Genus C Introduction To Community Systems Microbiology, Aalborg 2013
  32. 32. Taxonomic assignment Genus A Family Genus B Genus C Introduction To Community Systems Microbiology, Aalborg 2013
  33. 33. Using a ”Classifier” • Uses an existing phylogeny • Find best unambiguous match to references Introduction To Community Systems Microbiology, Aalborg 2013
  34. 34. Classification results Introduction To Community Systems Microbiology, Aalborg 2013
  35. 35. Microautoradiography Radioac tive phospha te Accumulibacter Radioactive Aalborg 2013 Introduction To Community Systems Microbiology,acetate Other Bacteria
  36. 36. Metagenomics Simon McIllroy, Microthrix ISME J 7 (6): 1161 Introduction To Community Systems Microbiology, Aalborg 2013
  37. 37. Substrate specificity Accumulibacter Tetraspheara Competibacter Defluvicoccus acetate + - + + propionate + - - + Casamino acids Glu only + - - Nitrate reduction +/- +/- +/- - Determined by FISH-MAR Introduction To Community Systems Microbiology, Aalborg 2013
  38. 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 ?? ?? ?? Introduction To Community Systems Microbiology, Aalborg 2013
  39. 39. midasfieldguide.org midasfieldguide.org 52 core genera 64% organisms DK 41% globally + others Introduction To Community Systems Microbiology, Aalborg 2013
  40. 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 Gammaproteobacteria Competibacterales Competibacteraceae Competibacter Introduction To Community Systems Microbiology, Aalborg 2013
  41. 41. Percent abundance Nitrite-oxidisers 0.3 genus 0.2 0.1 0 Introduction To Community Systems Microbiology, Aalborg 2013
  42. 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. 43. ● Analysis Example ● ● http://nbviewer.ipython.org/7213441 Introduction To Community Systems Microbiology, Aalborg 2013
  44. 44. Comparing Bacterial diversity of 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. 45. Phylum level Classification Proteobacteria Firmicutes Bacteroidetes AS WW Introduction To Community Systems Microbiology, Aalborg 2013
  46. 46. Genus level Classification AS WW Introduction To Community Systems Microbiology, Aalborg 2013
  47. 47. Alpha Diversity • How diverse 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. 48. Richness estimates Introduction To Community Systems Microbiology, Aalborg 2013
  49. 49. Introduction To Community Systems Microbiology, Aalborg 2013
  50. 50. Beta Diversity • How similar/different is my sample from other samples? Introduction To Community Systems Microbiology, Aalborg 2013
  51. 51. Clustering Introduction To Community Systems Microbiology, Aalborg 2013
  52. 52. Principal components analysis Introduction To Community Systems Microbiology, Aalborg 2013
  53. 53. Label with environmental data Introduction To Community Systems Microbiology, Aalborg 2013
  54. 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

  • Roche is supposed to soon come with a 1000 bp sequencer
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  • Mantoin the concept with the large fragments and matching
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  • Quince 30-50% overestimation of OTUs
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  • Mantoin the concept with the large fragments and matching
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  • 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>
  • 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>
  • 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>

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