bacterial communities in women with bacterial … · high resolution phylogenetic analyses reveal...
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CHAIR OF HIGH PERFORMANCE COMPUTING IN THE LIFE SCIENCES
Bacterial Communities in Women with Bacterial Vaginosis:High Resolution Phylogenetic Analyses Reveal Relationshipsof Microbiota to Clinical CriteriaSeminar presentationPierre Barbera
Supervised by: Alexandros Stamatakis, Lucas Czech
KIT – University of the State of Baden-Wuerttemberg and
National Laboratory of the Helmholtz Association
www.kit.edu
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Outline1 Introduction
BasicsGoals
2 Wet Lab WorkPCR of 16S
3 Taxonomic ClassificationBuilding the Reference TreePlace Sequence on TreeTaxonomic Assignment
4 Correlation AnalysisKantorovich-RubinsteinSquash Clustering
5 Results and Summary
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 3: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/3.jpg)
Outline1 Introduction
BasicsGoals
2 Wet Lab WorkPCR of 16S
3 Taxonomic ClassificationBuilding the Reference TreePlace Sequence on TreeTaxonomic Assignment
4 Correlation AnalysisKantorovich-RubinsteinSquash Clustering
5 Results and Summary
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 4: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/4.jpg)
How are bacteria studied?
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 5: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/5.jpg)
How are bacteria studied?
Can be cultured
Can’t currently be culturedBacteria
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 6: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/6.jpg)
Metagenomics
Microbiome/Microbiota: collection of microorganisms inenvironmental niche
Metagenomics: study of collective genetic material from amicrobiome
Interactions and composition of Microbiome centrally important
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 7: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/7.jpg)
Bacterial Communities rule your life!
Human body has roughly 10 trillion cells
But it houses 10 times that many bacteria
Large part of it in the gut
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 8: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/8.jpg)
Bacterial Vaginosis (BV)
One of the most common infections of the vagina
Around 30% of women in the US are BV-positive
Cause still unknown (according to CDC)
Linked to imbalances in the microbiome of the vagina
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 9: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/9.jpg)
Central questions
Is there a core BV biome?
Can novel species be identified?
Can any synergistic relationships (between bacteria) be identified?
What is the effect of race on BV prevalence?
Can we identify correlations between microbiome compositionand clinical features (of BV)?
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 10: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/10.jpg)
Rough Workflow
Take samplefrom Patient
Isolate and amplifyimportant DNA parts PCR of 16S
Sequence the DNA
Identify whatSpecies are present
?
Establish statis-tical correlations
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 11: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/11.jpg)
Rough Workflow
Take samplefrom Patient
Isolate and amplifyimportant DNA parts PCR of 16S
Sequence the DNA
Identify whatSpecies are present
?
Establish statis-tical correlations
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 12: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/12.jpg)
Rough Workflow
Take samplefrom Patient
Isolate and amplifyimportant DNA parts PCR of 16S
Sequence the DNA
Identify whatSpecies are present
?
Establish statis-tical correlations
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 13: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/13.jpg)
Rough Workflow
Take samplefrom Patient
Isolate and amplifyimportant DNA parts PCR of 16S
Sequence the DNA
Identify whatSpecies are present
?
Establish statis-tical correlations
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 14: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/14.jpg)
Rough Workflow
Take samplefrom Patient
Isolate and amplifyimportant DNA parts PCR of 16S
Sequence the DNA
Identify whatSpecies are present
?
Establish statis-tical correlations
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 15: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/15.jpg)
Outline1 Introduction
BasicsGoals
2 Wet Lab WorkPCR of 16S
3 Taxonomic ClassificationBuilding the Reference TreePlace Sequence on TreeTaxonomic Assignment
4 Correlation AnalysisKantorovich-RubinsteinSquash Clustering
5 Results and Summary
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 16: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/16.jpg)
Sample Collection
Samples from 242 STD clinic patients (Seattle, USA)
Vaginal swabs, immediately frozen at −20 ◦C
220 samples had sufficient bacterial volume, basis for furthermethods
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 17: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/17.jpg)
Isolate and Amplify
Only a specific part of the sampled DNA, the 16S rRNA gene, isneeded
To isolate and amplify this portion the polymerase chain reactionlab technique is used
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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16S ribosomal RNA
Part of the ribosome in prokaryotes
Slow rate of evolution⇒ highly conserved between species
Very good sequence to establish phylogenies
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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Polymerase Chain Reaction (PCR)
Technique to massively multiply a certain portion of DNA
Requires Primer DNA-strands that will delimit the portion to multiply
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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Sequencing
Sequence resulting amplified samples
454 pyrosequencing
Result: many different 16S reads per sample
This is where the wet lab work ends and the bioinformatics workbegins
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 21: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/21.jpg)
Outline1 Introduction
BasicsGoals
2 Wet Lab WorkPCR of 16S
3 Taxonomic ClassificationBuilding the Reference TreePlace Sequence on TreeTaxonomic Assignment
4 Correlation AnalysisKantorovich-RubinsteinSquash Clustering
5 Results and Summary
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 22: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/22.jpg)
Preprocessing of Reads
Classify by barcodesOnly keep high quality reads that. . .
start with known barcodecontain exact match to used primerare at least 200 base pairs long (excluding primer/barcode)have sufficient quality score
Trim primer sequences and barcodes
All done in R, using R/Bioconductor package microbiome
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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Reference Tree Preparations
Take known sequences of bacteria known to reside in vaginalenvironment
Trim to 16S region, same as in samples
Perform mislabel detection, as public data is often mislabeled/wrong
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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Mislabel Detection
−3 −2 −1 0 1 2 3
•• • •• ••S
Compute pairwise distance between all sequences of a taxon
Select a primary reference sequenceS with smallest mediandistance to all others
Discard sequences that are too far from this reference sequence (bysome threshold)
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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Building the Reference Tree
Build Multiple Sequence Alignment (MSA) using cmalign
Build tree using MSA and RAxML 7.2.7, using GTR model
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 26: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/26.jpg)
Place Sequence on Tree
16S SequencesAACGTA
3(< middle >)
TTATCC
GATACA
TGATAT
?
Take sequence, find optimal placement on existing tree
Optimality meaning Bayesian posterior probability criterion
Remember where sequence was placed
Done using the pplacer tool
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 27: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/27.jpg)
Place Sequence on Tree
16S SequencesAACGTA
3(< middle >)
TTATCC
GATACA
TGATAT
?
Take sequence, find optimal placement on existing tree
Optimality meaning Bayesian posterior probability criterion
Remember where sequence was placed
Done using the pplacer tool
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 28: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/28.jpg)
Place Sequence on Tree
16S SequencesAACGTA
3(< middle >)
TTATCC
GATACA
TGATAT
?
Take sequence, find optimal placement on existing tree
Optimality meaning Bayesian posterior probability criterion
Remember where sequence was placed
Done using the pplacer tool
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 29: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/29.jpg)
Place Sequence on Tree
16S SequencesAACGTA
3(< middle >)
TTATCC
GATACA
TGATAT
?
Take sequence, find optimal placement on existing tree
Optimality meaning Bayesian posterior probability criterion
Remember where sequence was placed
Done using the pplacer tool
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 30: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/30.jpg)
Place Sequence on Tree
16S SequencesAACGTA
3(< middle >)
TTATCC
GATACA
TGATAT
?
Take sequence, find optimal placement on existing tree
Optimality meaning Bayesian posterior probability criterion
Remember where sequence was placed
Done using the pplacer tool
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 31: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/31.jpg)
Place Sequence on Tree
16S SequencesAACGTA 3(< middle >)TTATCC
GATACA
TGATAT
Take sequence, find optimal placement on existing tree
Optimality meaning Bayesian posterior probability criterion
Remember where sequence was placed
Done using the pplacer tool
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 32: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/32.jpg)
Place Sequence on Tree
16S SequencesAACGTA 3(< middle >)TTATCC
GATACA
TGATAT
?
Take sequence, find optimal placement on existing tree
Optimality meaning Bayesian posterior probability criterion
Remember where sequence was placed
Done using the pplacer tool
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 33: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/33.jpg)
Place Sequence on Tree
16S SequencesAACGTA 3(< middle >)TTATCC
GATACA
TGATAT?
Take sequence, find optimal placement on existing tree
Optimality meaning Bayesian posterior probability criterion
Remember where sequence was placed
Done using the pplacer tool
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 34: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/34.jpg)
Taxonomic Assignment
Lactobacillus
AACGTA
Taxonomic DB
Interpret asTaxonomic Identifier
AACGTA⇒ L. vaginalis
Assign mostspecific rank
Assign taxonomic labels to edges of the tree
Such that labels are as specific as possible (species, genus, familyetc.)
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 35: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/35.jpg)
Taxonomic Assignment
Lactobacillus
AACGTA
Taxonomic DB
Interpret asTaxonomic Identifier
AACGTA⇒ L. vaginalis
Assign mostspecific rank
Assign taxonomic labels to edges of the tree
Such that labels are as specific as possible (species, genus, familyetc.)
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 36: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/36.jpg)
Taxonomic Assignment
Lactobacillus
AACGTA
Taxonomic DB
Interpret asTaxonomic Identifier
AACGTA⇒ L. vaginalis
Assign mostspecific rank
Assign taxonomic labels to edges of the tree
Such that labels are as specific as possible (species, genus, familyetc.)
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 37: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/37.jpg)
Taxonomic Assignment
Lactobacillus
AACGTA
Taxonomic DB
Interpret asTaxonomic Identifier
AACGTA⇒ L. vaginalis
Assign mostspecific rank
L.va
ginali
s
Assign taxonomic labels to edges of the tree
Such that labels are as specific as possible (species, genus, familyetc.)
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 38: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/38.jpg)
Classification Result
. . .
220 virtual trees, one per sampleVirtual as in sequences are not contained on one common tree, butare associated with the reference tree by coordinates
Each representing the bacterial composition of a patients vaginalenvironment
let’s call them sample-trees
Basis for further statistical evaluation
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 39: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/39.jpg)
Outline1 Introduction
BasicsGoals
2 Wet Lab WorkPCR of 16S
3 Taxonomic ClassificationBuilding the Reference TreePlace Sequence on TreeTaxonomic Assignment
4 Correlation AnalysisKantorovich-RubinsteinSquash Clustering
5 Results and Summary
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 40: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/40.jpg)
Correlation Analysis
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 41: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/41.jpg)
What next?
Now that we have sample-trees we want compare them
In the paper this is done by assembling them into another tree, a treeof trees
To do that we need a distance metric and a way to cluster thesample-trees
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 42: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/42.jpg)
Earth-Mover Distance
Distribution 1 Distribution 2
⇒1
0
1
0
Distance between two distributions
Blue = mass
Distance is the work required to shift the mass such thatdistributions are equal
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 43: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/43.jpg)
Earth-Mover Distance
Distribution 1 Distribution 2
⇒1
0
+41
0
Distance between two distributions
Blue = mass
Distance is the work required to shift the mass such thatdistributions are equal
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
![Page 44: Bacterial Communities in Women with Bacterial … · High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre Barbera](https://reader030.vdocuments.us/reader030/viewer/2022020215/5b9a5f3609d3f2c3468d1ec3/html5/thumbnails/44.jpg)
Earth-Mover Distance
Distribution 1 Distribution 2
⇒1
0
+4 +31
0
Distance between two distributions
Blue = mass
Distance is the work required to shift the mass such thatdistributions are equal
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Earth-Mover Distance
Distribution 1 Distribution 2
⇒1
0
+4 +3 +31
0
Distance between two distributions
Blue = mass
Distance is the work required to shift the mass such thatdistributions are equal
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Earth-Mover Distance
Distribution 1 Distribution 2
⇒1
0
+4 +3 +3 +3 = 131
0
Distance between two distributions
Blue = mass
Distance is the work required to shift the mass such thatdistributions are equal
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Samples as Distribution on the Tree
0.3
0.4
0.1
0.0
0.2
Edge labels: fraction of total reads that were placed at an edge
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Phylogenetic Kantorovich-Rubinstein
Combination of earth-mover distance and trees with readdistribution
Apply earth-mover distance between the edges of two trees
Distance is minimal amount of work required to move mass tomatch other distribution
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Kantorovich-Rubinstein Visualisation
Sample 1 Sample 2
0.3
0.4
0.1
0.0
0.2 0.1
0.5
0.1
0.1
0.2
⇒
+0.2+0.1 +0.1
Distance = 0.5
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Kantorovich-Rubinstein Visualisation
Sample 1 Sample 2
0.1
0.4
0.1
0.0
0.2 0.1
0.5
0.1
0.1
0.2
⇒+0.2
+0.1 +0.1
Distance = 0.5
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Kantorovich-Rubinstein Visualisation
Sample 1 Sample 2
0.1
0.5
0.1
0.0
0.2 0.1
0.5
0.1
0.1
0.2
⇒+0.2+0.1
+0.1
Distance = 0.5
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Kantorovich-Rubinstein Visualisation
Sample 1 Sample 2
0.1
0.5
0.1
0.0
0.2 0.1
0.5
0.1
0.1
0.2
⇒+0.2+0.1 +0.1
+0.1
Distance = 0.5
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Kantorovich-Rubinstein Visualisation
Sample 1 Sample 2
0.1
0.5
0.1
0.1
0.2 0.1
0.5
0.1
0.1
0.2
⇒+0.2+0.1 +0.1 +0.1
Distance = 0.5
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Kantorovich-Rubinstein Visualisation
Sample 1 Sample 2
0.1
0.5
0.1
0.1
0.2 0.1
0.5
0.1
0.1
0.2
⇒+0.2+0.1 +0.1 +0.1
Distance = 0.5
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Reminder: Hierarchical Clustering
Pairwise Distance Matrix (PWD)
merging andbranch length assignment
A B C DA 17 21 27B 12 18C 14D
A X DA 13 27X 18D
B C
X6 6
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Reminder: Hierarchical Clustering
Pairwise Distance Matrix (PWD)
merging andbranch length assignment
A B C DA 17 21 27B 12 18C 14D
A X DA 13 27X 18D
B C
X6 6
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Reminder: Hierarchical Clustering
Pairwise Distance Matrix (PWD)
merging andbranch length assignment
A B C DA 17 21 27B 12 18C 14D
A X DA 13 27X 18D
B C
X6 6
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Squash Clustering
Building a tree of trees
Sample-trees at the tips
Pairwise distance matrix based on K-R Distance
Merge by building the average of the distributions (squashing)
Branch lengths = K-R Distance between trees at two incident nodes
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Squashing Visualised
Sample 1
Sample 2
0.1
0.5
0.1
0.2
0.1
0.3
0.4
0.1
0.0
0.2
SQUASH!
(0.4 + 0.5)/2 = 0.45
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Squashing Visualised
0.3
0.4
0.1
0.0
0.20.1
0.5
0.1
0.1
0.2SQUASH!
(0.4 + 0.5)/2 = 0.45
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Squashing Visualised
0.3
0.4
0.1
0.0
0.20.1
0.5
0.1
0.1
0.2SQUASH!
(0.4 + 0.5)/2 = 0.45
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Squashing Visualised
0.2
0.45
0.1
0.05
0.2
SQUASH!
(0.4 + 0.5)/2 = 0.45
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Squash Clustering Visualised
0.3
0.4
0.1
0.0
0.2 0.1
0.5
0.1
0.1
0.2
0.2
0.45
0.1
0.05
0.2
merge stepassign branch
lengths(K-R Distance)
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Squash Clustering Visualised
0.3
0.4
0.1
0.0
0.2 0.1
0.5
0.1
0.1
0.2
0.2
0.45
0.1
0.05
0.2
merge step
assign branchlengths
(K-R Distance)
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Squash Clustering Visualised
0.3
0.4
0.1
0.0
0.2 0.1
0.5
0.1
0.1
0.2
0.2
0.45
0.1
0.05
0.2
0.25 0.25
merge step
assign branchlengths
(K-R Distance)
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Outline1 Introduction
BasicsGoals
2 Wet Lab WorkPCR of 16S
3 Taxonomic ClassificationBuilding the Reference TreePlace Sequence on TreeTaxonomic Assignment
4 Correlation AnalysisKantorovich-RubinsteinSquash Clustering
5 Results and Summary
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Results of Clustering
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
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Results
Healthy vaginal microbiome dominated by Lactobacillus spp.
Women with BV have highly diverse vaginal microbiome
Clinical tests of BV correlate differently well to bacteria
Race appears to have influence on whether some bacteria contributeto BV
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References I
Sujatha Srinivasan et al. “Bacterial communities in women withbacterial vaginosis: High resolution phylogenetic analyses revealrelationships of microbiota to clinical criteria”. In: PLoS ONE 7.6(2012). ISSN: 19326203. DOI: 10.1371/journal.pone.0037818.
References
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Image sourcesSlide 4:
https://en.wikipedia.org/wiki/Petri_dish#/media/File:Agar_plate_with_colonies.jpg, Wikipediauser Phyzome
Slide 5:https://www.flickr.com/photos/pere/523019984, flickr user pere, with modification
Slide 10:https://en.wikipedia.org/wiki/Cotton_swab#/media/File:White_menbo.jpg, Wikipedia user Aneyhttps://commons.wikimedia.org/wiki/File:DNA_sequence.svg, Wikimedia user SjefScatterplots taken from Srinivasan et al. 2012
Slide 15:https://en.wikipedia.org/wiki/Ribosome#/media/File:Peptide_syn.png, Wikipedia user Boumphreyfr
Slide 15:https://en.wikipedia.org/wiki/Polymerase_chain_reaction#/media/File:
Polymerase_chain_reaction.svg, Wikipedia user Enzoklop
Slide 26:https://www.flickr.com/photos/darkuncle/4421756078/, flickr user darkuncle, modification by me
References
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Identifying Novel Bacteria
Take all sequenced reads from interesting bacterial order (here:Clostridiales)
Place on tree, cluster into islands with distance cutoff 0.02
Throw away islands that have reads only from one individual
Choose representative from reads arbitrarily, BLAST it to find appropriateisland label
Display islands as leaves on Ref. tree
References
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Results Novel Bacteria
11 novel bacteria identified
Less than 97% identity to known bacteria
Range: 91% to 96%
References
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Some Numbers
425775 sequence reads from 220 samples
Median read length: 225bp
Mean number of reads per subject: 1620
99.1% of reads were classified at species level
References
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Kantorovic-Rubinstein Formula
Z (P,Q) =∫
T |P(τ(y))− Q(τ(y))|λ(dy)
Z is the resulting distanceP,Q are the distributionsT is the treeτ signifies the sub tree below vertex yλ is the length measure
References
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Classifier Validation
Assemble validation set of 16S sequences belonging to Lactobacillus genus(source: RDP)
Consisting of subspecies found in human vagina
Also met previous distance metric from mislabel detection
Trim sequences to match V4 16S region
References
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Pearson Correlation Coefficient
Measure of the linear correlation (dependence) between two variables Xand Y
Gives a value between +1 and 1 inclusive
1 is total positive correlation
0 is no correlation
1 is total negative correlation
References
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p-value
Qualifies the significance of a result
Tells you whether your data rejects your null hypothesis (NH)
0 ≤ p ≤ 1
p ≤ 0.05: strong evidence against NH⇒ reject NH
p > 0.05: weak evidence against NH⇒ failed to reject NH
References
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015