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Exploratory Study Rotavirus

Master's Degree Thesis
by

Rodrigo Garcia

on 19 September 2011

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Transcript of Exploratory Study Rotavirus

Exploratory study of the impact of the anti-rotavirus vaccination on the gut microbiome Master’s Degree Thesis Master in Molecular, Cell Biology and Genetics
Microbiology specialization
Director: Vicente Perez Brocal
Tutor: Andrés Moya Simarro Rodrigo García López September 20, 2011 27 million patient visits each year
Abdominal pain
Fever
Diarrhea
Vomiting 11 countries acount for 65% Vaccination Rotashield Intussusception Gut Invagination Late 1990s 1 case in every 10,000 vaccinated children Wyeth Laboratories RotaTeq Pentavalent bovine-human reassortant vaccine
Serotypes G1, G2, G3, G4 and P1[8]
Rotarix MERCK GlaxoSmithKline Monovalent attenuated human rotavirus vaccine
Protects against serotypes G1, G2, G3, G4 and G9 Microbiota & 16S rRNA Workflow & Results Discussion & Conclusion Rotavirus Double stranded RNA virus Table of Contents Microbial Niches
Bacterial, viral, archaeal and unicellular eukaryotic organisms
Important roles in ecosystem Metabolic by-products secreted by bacteria:
Provide additional nutrients
Improve the immune systems
Strengthen the lining of the gut Microbiota disequilibrium Fraune S. (2010) 16S rRNA Medini (2008) Sequencing
Technologies http://www.rightdiagnosis.com/phil/html/rotavirus/273.html http://pequebebes.com/conocer-el-rotavirus/ http://www.reoviridae.org/dsrna_virus_proteins/rotavirus%20figure.htm http://www.bebes.net/2010/12/02/enfermedades-rotavirus-infantil/ http://www.riversideonline.com/source/images/image_popup/ans7_intussusception.jpg HMP,2008 Fraune S. (2010) http://the-scientist.com/2011/08/01/helpful-bacterial-metabolites/ http://blog.malde.org/index.php/2010/09/22/exploring-16s/ http://rnajournal.cshlp.org/content/11/5/657/ Bioinformatic Tools Bigger volumes of data demand faster and more powerful computing http://www.gadg.com/2011/06/21/the-worlds-fastest-supercomputer-belongs-to-japan/ 16S rDNA Databases Analysis Programs DNA extraction Data Analysis PCR Sample Recolection 10 ml PBS
Spoonfuls = 200g 8 min
4 o C
2500 rpm Supernatant aliquoted
250, 500, 750, 1500 ul http://es.123rf.com/photo_3470751_abrir-el-vial-vac-o-eppendorf-aisladas-sobre-fondo-negro.html http://pequebebes.com/la-diarrea-en-los-ninos/ http://www.eng.wayne.edu/page.php?id=5304 http://www.psoecallosadesegura.org/?q=taxonomy/term/2&page=7 QIAGEN - QIAamp DNA Stool Mini Kit 5 min
13,000 rpm
4 o C http://www.offers4labs.com/blog/2010/11/19/eppendorf-pcr-thermocycler/ High Pure PCR Product Purification Kit (Roche) Quantification Titanium A Titanium B Sequencing 454 Genome Sequencer FLX emPCR Pyrosequencing 138,984 reads generated by the GS FLX System Software Package Workflow Picogreen (Invitrogen) PCR Kit Universal Primers for 16S rDNA V1-V2 72,364 total good quality reads recovered after quality filters and MID assignation Clustering Alignment Taxonomic
Assignation Pipeline Phylogenetic Tree Construction Alpha Diversity Beta Diversity 97% sequence similarity
3,331 Clusters Reference database contains manually curated sequences from Bacteria and Archaea The 1st sequence of each cluster was picked as a representative OTU PyNAST: Python Nearest Alignment Space Termination algorithm Each OTU was aligned to the reference alignment NaïveBayesian
RDP Classifier
97% Identity Fast Tree http://www.sciencephoto.com/images/download_lo_res.html?id=662440049 The tree was constructed with joint OTUs from all samples Approximately Maximum Likelihood Tree Rarefaction Curves constructed with Resampling methods Raw Observed Species Phylogenetic Diversity (PD) Corrected Chao1’s index Shannon’s index UniFrac metric Distance Matrix Distance Matrix Subject Clustering PCA
Jackknifing 10,000 iterations Phylum Order Class Family Sample Diversity
Bacteroidetes + Firmicutes 90% and Proteobacteria 7% at phylum level at phylum level
Similar structure could be traced up to order level

Community Comparison
No sustainable differences of RotaTeq vaccinated group and non-vaccinated group
MC3 and NC2 had abnormal composition, probably due to technical issues during amplification Issues - Uneven PCR amplification supported by:
Missing Bacteroidetes
Standard read yield
Scarce OTU diversity in abnormal samples

Recomendations:
Same subject before and after vaccination
PCR duplicates under same conditions
Barcode shuffling
Different primer selection
Usage of additional regions in the 16S rRNA gene

For future studies:
Rotarix vaccinated children
Viral diversity This work was possible thanks to: Andrés Moya Simarro Javier Diez Domingo Jorge Vázquez Castellanos Vicente Pérez Brocal Blue - RotaTeq
Red - Control Gastroenteritis 5' 3' 5' 3' l 454 SOLID SOLEXA RotaTeq Control RotaTeq Control RotaTeq Control RotaTeq Control
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