16S AmpliconsA primer on metagenomics

Andrea Telatin and Eleonora Sattin BMR Genomics

De novo genome

Exome seq

16S Amplicons

Out of the machine

Introduction to bioinformatics

You are here

Today’s menu:!

• a biological perspective on 16S-seq

• a short primer on bioinformatic analysis

• introducing two tools:

• MEGAN (GUI)

• Qiime (pipeline)

Why?

– J. C. Venter

“whatever we look at, we are barely scratching the

surface”

Flores G. E. et al., 2011 (PLoS One)Microbial Biogeography of Public Restroom Surfaces

Key questions• Who is out there?

• What are they doing?

How?

16S Amplicon

16S AmpliconPROS!

• Ubiquitous gene

• Contains both conserved and variable regions

CONS!

• Copy number variations

• It’s only a (single) gene

metagenomics ≠ amplicon sequencing

Driving idea• Well established method

that can be used to compare different samples

• At any step we introduce bias, that have to be taken into account • Sampling (replicate or lie) • Cell breakage (are you strong enough?) • Amplification (where do your primers come from?) • Sequencing (how good is your machine?) • Analysis (database annotation?)

Sequence alignment

This is a hard example. !That is another easy example.

This is a --hard---- example. || ||||| | | ||||||||| That is another easy example.

This is a-- h-ard---- example. || ||||| | | ||||||||| That is anothe-r easy example.

This is a hard example.------ || ||||| | | That is another easy example.

Gap C

ost

1) AGT 2) AT 3) ATC

1) AGT 2) A – T 3) A TC

1) AGT 2) A T – 3) A TC

1) AGT - 2) A - T - 3) A - TC

A B C A C B B C A

INPUT

Bioinformatics analysis overview

Sequencing Reads

Pre-Processing

Denoising

OTU Picking

Taxonomical classification

Alpha/Beta Rarefaction

PCA

Sequencing output (454, Illumina, Sanger)

fastq, fasta, qual, or sff/trace files

Metadata

mapping file

Pre-processinge.g., remove primer(s), demultiplex,

quality filter

Denoise 454 Data

PyroNoise, Denoiser

Reference basedBLAST, UCLUST,

USEARCH

Pick OTUs and representative sequences

De novoe.g., UCLUST, CD-HIT, MOTHUR, USEARCH

Assign taxonomy

BLAST, RDP Classifier

Align sequences

e.g., PyNAST, INFERNAL, MUSCLE,

MAFFT

Build 'OTU table'i.e., sample by observation

matrix

Build phylogenetic treee.g., FastTree, RAxML,

ClearCut

Database Submission

(In development)

OTU (or other sample by observation) table

Phylogenetic Tree

Evolutionary relationship between OTUs

α-diversity and rarefaction

e.g., Phylogenetic Diversity, Chao1,

Observed Species

β-diversity and rarefaction

e.g., Weighted and unweighted UniFrac, Bray-

Curtis, Jaccard

Interactive visualizations

e.g., PCoA plots, distance histograms, taxonomy charts, rarefaction plots, network visualization, jackknifed hierarchical clustering.

Legend

Required step or input Optional step or input

Currently supported for marker-gene data only

(i.e., 'upstream' step)

Currently supported for general sample by observation data

(i.e., 'downstream' step)

www.QIIME.orgwww.QIIME.org

Mapping File

(Processed) Sequences

Sequencing Reads

Pre-Processing

Denoising

OTU Picking

Taxonomical classification

Alpha/Beta Rarefaction

PCA

Qiime Output Demo!http://www.bmr-genomics.it/~telatin/16S

MEGANMaking pies since 2004

Any questions?