CHAIR OF HIGH PERFORMANCE COMPUTING IN THE LIFE SCIENCES Bacterial Communities in Women with Bacterial Vaginosis: High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria Seminar presentation Pierre 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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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
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Outline1 Introduction
BasicsGoals
2 Wet Lab WorkPCR of 16S
3 Taxonomic ClassificationBuilding the Reference TreePlace Sequence on TreeTaxonomic Assignment
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
Results of Clustering
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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
Introduction Wet Lab Work Taxonomic Classification Correlation Analysis Results and Summary
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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
Pierre Barbera – Bacterial Communities in Women with Bacterial Vaginosis July 16, 2015
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