american gut project presentation at masaryk university
TRANSCRIPT
A microbe dominated world
Target and amplify the 16S/18S ribosomal RNA as a ‘fingerprint’ for different taxonomic groups.
Why 16S/18S?- gene is ubiquitous- contains conserved and variable regions
The universal nature of biochemistry. Pace NR.Proc Natl Acad Sci U S A. 2001 Jan 30;98(3):805-8.
A microbe dominated world
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http://www.g4tv.com/attackoftheshow/blog/post/717271/velociraptor-riding-a-shark/
A microbe dominated world
Lozupone and Knight PNAS. 2007 Jul 3;104(27):11436-40
A microbe dominated world
Lozupone and Knight PNAS. 2007 Jul 3;104(27):11436-40Subway image adapted from http://laughingsquid.com/the-gastrointestinal-system-represented-as-a-subway-map/
A microbe dominated world
Ley et al Nat Rev Microbiol. Oct 2008; 6(10): 776–788Subway image adapted from http://laughingsquid.com/the-gastrointestinal-system-represented-as-a-subway-map/
Hamady, 2009, PhD thesis
How “human” are we, really?
Hamady, 2009, PhD thesis
How “human” are we, really?
Hamady, 2009, PhD thesis
How “human” are we, really?
Costello et al. Science 326, 1694 (2009)
Photo credit: Steve Miller, CIRES
https://commons.wikimedia.org/wiki/File:Wallacea.png
Fierer et al. PNAS. 2010 Apr 6;107(14):6477-81
Fierer et al. PNAS. 2010 Apr 6;107(14):6477-81
Yatsunenko et al. Nature. 2012 May 9;486(7402):222-7
Yatsunenko et al. Nature. 2012 May 9;486(7402):222-7
HMP HMP
Yatsunenko et al. Nature. 2012 May 9;486(7402):222-7
Koeing et al. Proc Natl Acad Sci U S A. 2011 Mar 15;108
Video fun time!
http://microbio.me/americangut/img/mod1_main.pdf
Adapted from http://redefininghuman.comPlease see https://fundrazr.com/campaigns/aoaif/
CENSORED
Blin
din
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ization
Blin
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ization
Sequencegeneration
Data processing
Samples:
Stool, fresh, N=11
Stool, freeze-dried, N=7
Robogut N=2
Lab 1
Artificial N=2
Raw Sample TransferN = 53
Control
96 Tube Sample
Set
96 Tube Sample
Set
Lab 2
Lab 3
Lab 15
.
.
.
HMP DACCFTP Site
Site 1
.
.
.
Site 2
Site 3
Site 9
IntegratedData
Analysis
Slide credit: Dr. Amnon Amir
Variability in methods
Handling
DNA extraction kit manufacturer
Chemagen 1
MO-BIO 7
Omega BioTek 1
Promega 1
Qiagen 2
Zymo Research Corporation 1
Not reported 3
Homogenizer used?
Yes 11
No 3
Not reported 1
PCR primer set
318F/806R 1
EMP V4 515F/806R 11
Schloss 2013 2
Sequencing machine model
HiSeq 2500 1
MiSeq 13
Paired end sequencing?
Yes 13
No 1
Sequencing read length
150 4
175 1
210 1
250 6
300 2
Fraction of PhiX (range 0-0.3) 0.11 ±0.08
Fraction quality bases (range 0.57-0.96) 0.86 ±0.12
Bioinformatics
Primary QC software
QIIME 2
Trimmomatic 3
UPARSE 2
Other/custom 2
Primary OTU calling software
QIIME 4
UPARSE 5
Unsupervised OTU clustering step?
Yes 8
No 2
Taxonomic assignment strategy
Classification 3
Clustering 5
Mapping 2
OTU filtering?
Yes 3
No 7
Slide credit: Dr. Amnon Amir
Metcalf, Jessica (2014): Overview of data generation, processing and analysis using QIIME. figshare. http://dx.doi.org/10.6084/m9.figshare.902219
Understanding OTU picking
97% OTU, a “species” 3%
RepresentativeSequence
De novoOTU picking
Understanding OTU picking
RepresentativeSequence
FailedSequences
Closed reference OTU picking
Understanding OTU picking
RepresentativeSequence
NewClusters
Open reference OTU picking
Understanding OTU picking
• De novo– Single study– Poor characterization of existing taxa– Good characterization of novel taxa
• Closed-reference– Combine studies– Good characterization of existing taxa– Poor characterization of novel taxa
• Open-reference (QIIME default)– Single study– Good characterization of existing taxa– Good characterization of novel taxa
In depth discussion here in Rideout et al 2014 (https://peerj.com/articles/545/)
Alpha and beta diversity
• Alpha is within a sample
– E.g., how many species are in a sample
• Beta is between samples
– E.g., how similar are two samples
• Lots of ways to calculate these
Alpha diversity
A B
Alpha diversity
A B
Alpha diversity = 3 Alpha diversity = 4
Sample B is more diverse than sample A
Rarefaction
A B
Alpha diversity = 3Total sequences = 4
Alpha diversity = 4Total sequences = 8
Rarefy to 4 sequences
Rarefaction
A B
Rarefaction
A B
Alpha diversity = 3 Alpha diversity = 2
Sample A is more diverse than sample B
Multiple Rarefaction
adiv
# seqs1,000500
80
250
40
Higher sequencing effort will probably result in higher observed diversity
100
Multiple Rarefaction
adiv
# seqs
100
1,000500250
95
Higher sequencing effort will probably not add to observed diversity
Alpha diversity
Alpha diversity
Phylogenetic Diversity (PD)
Beta diversity
A B
C
Beta diversity
A B
C
A x B = 2
Beta diversity
A B
C
A x B = 2A x C = 2
Beta diversity
A B
C
A x B = 2A x C = 2B x C = 1
Beta diversity
American Gut ICU
• Analyses are executable, reproducible, and BSD-licensed
The American Gut Consortium
The Knight Lab
Prof. Rob Knight
Prof. Greg Caporaso
Prof. Jack Gilbert
Prof. Tim Spector
Dr. Victoria Vazquez
Prof. Paul Wischmeyer
Dr. Antonio Gonzalez
Dr. Gail Ackermann
Yoshiki Vázquez-Baeza
Dr. Jess Metcalf
Dr. Katie Amato
[email protected]: @mcdonadtGithub: @wasadewww: http://wasade.github.io
Acknowledgements
Links and questions
[email protected]: @mcdonadtGithub: @wasadewww: http://wasade.github.io
Projects of interest:• Earth Microbiome Project (http://earthmicrobiome.org)• American Gut Project (http://americangut.org)• British Gut Project (http://britishgut.org)
Key labs:• Knight Lab (http://knightlab.colorado.edu)• Caporaso Lab (http://caporasolab.us)• Spector Lab (http://www.tim-spector.co.uk/)
Education:• Coursera’s Gut Check (https://www.coursera.org/course/microbiome)• Intro. to Appl. Bioinformatics (http://caporasolab.us/An-Introduction-To-Applied-Bioinformatics/)
Useful software projects:• Quantitative Insights into Microbial Ecology (http://qiime.org)• Qiita (http://qiita.colorado.edu)*• biocore (http://github.com/biocore)• scikit-bio (http://scikit-bio.org)• EMPeror (http://emperor.colorado.edu)
These Slides:
• http://www.slideshare.net/mcdonadt/american-gut-masaryk*Soon to be opened for the general public
ICU fecal samples
American Gut fecal samples
Gevers et al. Cell Host and Microbe 2014 Mar 12;15(3)
Significant changes to protective taxa in ICU patients
Decreased in ICU patients
Unfiltered
Filtered