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.
1. Amplicon Sequencing
Aaron Marc Saunders
Introduction To Community Systems Microbiology, Aalborg 2013
Introduction To Community Systems Microbiology, Aalborg 2013
4. Environmental surveys
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Marker gene Sequencing
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–
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PCR product
One gene fragment
Metagenomics
–
Total DNA
–
All genes
Introduction To Community Systems Microbiology, Aalborg 2013
9. 16S rRNA gene conservation
Regions denoted ”hypervariable”
http://www.bioinformatics-toolkit.o
Introduction To Community Systems Microbiology, Aalborg 2013
10. Environmental surveys
mixed genomic DNA
Environmental sample
PCR product
16S clone library
Introduction To Community Systems Microbiology, Aalborg 2013
12. Environmental surveys
mixed genomic DNA
metagenomic
NGS sequencing
library
Environmental sample
PCR product
amplicon library
Introduction To Community Systems Microbiology, Aalborg 2013
13. Amplicon vs. Metagenomics
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less complex
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●
–
–
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 number of highly abundant species and a long tail of rare species
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
33. Using a ”Classifier”
• Uses an existing phylogeny
• Find best unambiguous match to
references
Introduction To Community Systems Microbiology, Aalborg 2013
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
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
54. Amplicon Sequencing
• Pros:
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”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.
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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.
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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.
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