Infectious Disease Research Network Microbial Profiling Workshop Talk
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Talk given at IDRN Microbial Profiling Workshop 20th June 2011 on the 16S rRNA PhyloChip, a phylogenetic microarray developed at Lawrence Berkeley National Laboratory. The G2 chip is focused on, with mention of the G3 chip. http://idrn.org/events/upcoming/microbial.php http://www.secondgenome.com/tag/phylochip/ http://phylochip.com/phylochip.html
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Infectious Disease Research Network Microbial Profiling Workshop Talk
- 1. Use of PhyloChip to characterise microbial communities in human health and disease Mike Cox National Heart and Lung Institute, Imperial College
- 4. Species rank abundance Number of organisms Rank abundance
- 5. Species rank abundance Small number of frequently found organisms Number of organisms Lots of rare ones (rare biosphere or noise) Rank abundance
- 6. Species rank abundance Clone libraries, DGGE, TRFLP 454 16S pyrosequencing Number of organisms Illumina 16S pyrosequencing Rank abundance
- 7. Species rank abundance 16S rRNA PhyloChip – 8,600 OTUs in parallel, rather than serial Number of organisms Rank abundance
- 8. 16S rRNA PhyloChip Affymetrix microarray developed at Lawrence Berkeley National Lab Gary Andersen, Todd DeSantis and EoinBrodie BrodieEL et al 2006 AEM 72:6288–98 Based on Greengenes – curated, aligned 16S rRNA gene sequence database DeSantis, Tet al 2006 AEM 72:5069-72 G2 chip – 2002 database snapshot Detects organisms at 0.01 % total community abundance
- 10. 8 -12 PCRs per sample - over temperature gradient (48-52oC) then pooled, quantified and fragmented with DNase
- 11. Spike mix control added
- 12. 250 to 500 nghybridised to chips, one per sample
- 14. At least 11, median 22 probes per OTU
- 16. Example: Cystic Fibrosis 90 samples from age-stratified cohort 6 months to 72 years Sputum and deep throat swab Different mutations and wide range of severities Community structure associated with Lung function Mutation (∆f508 homo/heterozygous) Age Cox MJ et al. 2010 PLoS ONE 5(6): e11044
- 17. Despite detecting 1800 different OTUs (46 Phyla) amongst the samples, only three OTUs representing known CF pathogens significantly correlated with age: Pseudomonas aeruginosa (+ve) Stenotrophomonasmaltophilia (+ve) Haemophilusinfluenzae(-ve) Many more unknown OTUs or OTUs not known to contain pathogens also did
- 19. Comparisons with other 16S methods DeAngelis KM et al 2011 PLoS ONE 6(4): e19306. Several studies compared clone libraries and TRFLP directly with PhyloChip: good correspondence community structure and most abundant organisms detected. Rare taxa require more rigorous validation.
- 20. Some practicalities Depth of sampling compared to 454 On single wash station 16 samples per long day Chip cost ~$100 each (may be cheaper now) Proprietary not available off the shelf from Affymetrix, contact LBNL group for info Not MIAME compliant (can access probes via Greengenes) Validation
- 21. G3 Chip More recent database snapshot 59,000 OTUs Hierarchical probe design Commercialised 2 1 3 γ-proteobacterial probe 4 Proteobacterial probe
- 23. Acknowledgments LBNL EoinBrodie, UlasKaraoz, Kate Goldfarb, Yvette Piceno UCSF Yvonne Huang, Kei Fujimura, Nicole Slusher, Susan Lynch Elizabeth Nash/CFRI Fellowship Further info: LBNL References: Brodie EL et al 2006 AEM 72:6288–98 DeSantis, T et al 2006 AEM 72:5069-72 Cox MJ et al. 2010 PLoS ONE 5(6): e11044 DeAngelis KM et al 2011 PLoS ONE 6(4): e19306