Amplicon Sequencing Introduction

Introduction To Community Systems Microbiology, Aalborg 2013

Amplicon Sequencing

Aaron Marc Saunders



Bacterial diversity

cultured representative

Phylum ActinobacteriaGenus Tetrasphaera


Environmental diversity

Environmental sample mixed genomic DNA

DNA extraction


Environmental surveys

● Marker gene Sequencing– PCR product

– One gene fragment

● Metagenomics– Total DNA

– All genes


Marker Genes

http://fungene.cme.msu.edu/


”Tree of Life”

Based on 16S rRNA gene


Start

Stop


Hypervariable regions

E. coli

V4

All life!


16S rRNA gene conservation

Regions denoted ”hypervariable” http://www.bioinformatics-toolkit.org

http://www.bioinformatics-toolkit.org/



Environmental sample

mixed genomic DNA

PCR product16S clone library





mixed genomic DNA

metagenomic library

PCR productamplicon library

NGS sequencing


Amplicon vs. Metagenomics

– less complex● better coverage● more samples

– extensive database– same fragment

● comparable phylogenetic info– PCR bias– Limited phylogenetic info

(genus/species level)– Limited functional information


Typical resultsSmall number of highly abundant species and a long tail of rare species


2. methodology


16S rRNA amplicon sequencing

”Short” PCR product (70-400 bp)

Sequence reads

Sequence types (OTUs)

Classified OTUs

Quality screen and clustering

Next-gen sequencing

Matching to existing database

PCR

Sample




Sequence reads


Classified OTUs


Next-gen sequencing


PCR

Sample




mixed genomic DNA

PCR productamplicon library

DNA extraction


Hypervariable regions

E. coliV4

All life!


PCR product

Examples of NGS sequenced amplicons

Regions denoted ”hypervariable” http://www.bioinformatics-toolkit.org

454 = 400 bpIllumina = 250 bp

http://www.bioinformatics-toolkit.org/


Multiplexing

Normal PCR product

NGS-adapted PCR product

Barcoded PCR product for NGS


Multiplexing

…GCCATCAG GATCT CNACGCGAAGAACCTTANC NNNNNNNNNN…

…GCCATCAG ATCAG CNACGCGAAGAACCTTANC NNNNNNNNNN…

…GCCATCAG CACTG CNACGCGAAGAACCTTANC NNNNNNNNNN…

…GCCATCAG CTGTG CNACGCGAAGAACCTTANC NNNNNNNNNN…

adaptor barcode Primer sequence Amplified sequence

Sample 1

Sample 2

Sample 3

Sample 4




Sequence reads


Classified OTUs


Next-gen sequencing


PCR

Sample


Bacterial Diversity


Clustering

Representative sequence


Consider Errors!

• Low quality reads• PCR errors• Sequencing errors

– Particularly homopolymers ● 454 & ion torrent!

• Chimeras




Sequence reads


Classified OTUs


Next-gen sequencing


PCR

Sample


Functional information

• Isolates• Functional 16S rRNA work

– Stable isotope probing

• Metagenomics• In situ studies

– Microscopy – eg. inclusion bodies– Microautoradiography– Raman or NanoSIMS


Taxonomic assignment

Genus A

Genus B

Genus C

Family



Genus A

Genus B

Genus C

Family



Genus A

Genus B

Genus C

Family



Genus A

Genus B

Genus C

Family


Using a ”Classifier”

• Uses an existing phylogeny• Find best unambiguous match to

references


Classification results

Introduction To Community Systems Microbiology, Aalborg 2013AccumulibacterOther Bacteria

Radioactive acetate

Radioactive phosphate

Microautoradiography


Metagenomics

Simon McIllroy, MicrothrixISME J 7 (6): 1161


Substrate specificity

Accumulibacter Tetraspheara Competibacter Defluvicoccus

acetate + - + +

propionate + - - +

Casamino acids

Glu only + - -

Nitrate reduction

+/- +/- +/- -

Determined by FISH-MAR


Greengenes assignment

species (OTU) Class Order Family Genus

OTU1 Betaproteobacteria Nitrosomonadales Nitrosomonadaceae

Nitrosomonas

OTU2 Nitrospira Nitrospirales Nitrospiraceae Nitrospira

OTU3 Betaproteobacteria Rhodocyclales Rhodocyclaceae Propionivibrio

OTU4 Betaproteobacteria Gallionellales Gallionellaceae ??

OTU5Gammaproteobacte

ria ?? ?? ??


midasfieldguide.orgmidasfieldguide.org

52 core genera 64% organisms DK41% globally

+ others


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


genus

Nitrite-oxidisers

0.3

0.2

0.1

0

Perc

ent

abundance


Analysis Tools

● Qiime (qiime.org)

● Mothur (mothur.org)

● R (r-project.org) – Phyloseq package

(http://joey711.github.io/phyloseq/)


● Analysis Example●

● http://nbviewer.ipython.org/7213441


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


Phylum level Classification

AS WW

ProteobacteriaFirmicutesBacteroidetes


AS WW

Genus level Classification


Alpha Diversity

• How diverse is the community in my sample?

• Richness– How many organisms are in my

sample?

• Evenness– How evenly are they distributed?


Richness estimates



Beta Diversity

• How similar/different is my sample from other samples?


Clustering


Principal components analysis


Label with environmental data


Amplicon Sequencing

• Pros:– ”cheap”, many samples– 16S: good database for classification

• Cons:– PCR and primer bias– limited functional information

Education

Amplicon Sequencing Introduction