Introduction to 16S Analysis with NGS - BMR Genomics
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Transcript of Introduction to 16S Analysis with NGS - BMR Genomics
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?