Use of PhyloChip to characterise microbial communities in human health and disease<br />Mike Cox<br />National Heart and L...
A cautionary note…<br />
Species rank abundance<br />Number of organisms<br />Rank abundance<br />
Species rank abundance<br />Small number of frequently found organisms<br />Number of organisms<br />Lots of rare ones (ra...
Species rank abundance<br />Clone libraries, DGGE, TRFLP<br />454 16S pyrosequencing<br />Number of organisms<br />Illumin...
Species rank abundance<br />16S rRNA PhyloChip –<br />8,600 OTUs in parallel, rather than serial<br />Number of organisms<...
16S rRNA PhyloChip<br />Affymetrix microarray developed at Lawrence Berkeley National Lab<br />Gary Andersen, Todd DeSanti...
<ul><li>Lane primers 27F/1492R used (8F/1391R if Archaea as well)
8 -12 PCRs per sample - over temperature gradient (48-52oC) then pooled, quantified and fragmented with DNase
Spike mix control added
250 to 500 nghybridised to chips, one per sample
500,000 25mer probes
At least 11, median 22 probes per OTU
Chips scanned, .cel files uploaded to Greengenes where raw data is normalised and returned</li></li></ul><li>Data analysis...
Example: Cystic Fibrosis<br />90 samples from age-stratified cohort<br />6 months to 72 years<br />Sputum and deep throat ...
Despite detecting 1800 different OTUs (46 Phyla) amongst the samples, only three OTUs representing known CF pathogens sign...
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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. 1. Use of PhyloChip to characterise microbial communities in human health and disease<br />Mike Cox<br />National Heart and Lung Institute, Imperial College<br />
  2. 2. A cautionary note…<br />
  3. 3.
  4. 4. Species rank abundance<br />Number of organisms<br />Rank abundance<br />
  5. 5. Species rank abundance<br />Small number of frequently found organisms<br />Number of organisms<br />Lots of rare ones (rare biosphere or noise)<br />Rank abundance<br />
  6. 6. Species rank abundance<br />Clone libraries, DGGE, TRFLP<br />454 16S pyrosequencing<br />Number of organisms<br />Illumina 16S pyrosequencing<br />Rank abundance<br />
  7. 7. Species rank abundance<br />16S rRNA PhyloChip –<br />8,600 OTUs in parallel, rather than serial<br />Number of organisms<br />Rank abundance<br />
  8. 8. 16S rRNA PhyloChip<br />Affymetrix microarray developed at Lawrence Berkeley National Lab<br />Gary Andersen, Todd DeSantis and EoinBrodie<br />BrodieEL et al 2006 AEM 72:6288–98 <br />Based on Greengenes – curated, aligned 16S rRNA gene sequence database<br />DeSantis, Tet al 2006 AEM 72:5069-72<br />G2 chip – 2002 database snapshot<br />Detects organisms at 0.01 % total community abundance<br />
  9. 9. <ul><li>Lane primers 27F/1492R used (8F/1391R if Archaea as well)
  10. 10. 8 -12 PCRs per sample - over temperature gradient (48-52oC) then pooled, quantified and fragmented with DNase
  11. 11. Spike mix control added
  12. 12. 250 to 500 nghybridised to chips, one per sample
  13. 13. 500,000 25mer probes
  14. 14. At least 11, median 22 probes per OTU
  15. 15. Chips scanned, .cel files uploaded to Greengenes where raw data is normalised and returned</li></li></ul><li>Data analysis<br />R (various packages), Phylotrac, FastUniFrac<br />OTUs predefined - said to be sub-family level<br />Ranges from genus to species<br />
  16. 16. Example: Cystic Fibrosis<br />90 samples from age-stratified cohort<br />6 months to 72 years<br />Sputum and deep throat swab<br />Different mutations and wide range of severities<br />Community structure associated with<br />Lung function<br />Mutation (∆f508 homo/heterozygous)<br />Age<br />Cox MJ et al. 2010 PLoS ONE 5(6): e11044<br />
  17. 17. Despite detecting 1800 different OTUs (46 Phyla) amongst the samples, only three OTUs representing known CF pathogens significantly correlated with age:<br />Pseudomonas aeruginosa (+ve)<br />Stenotrophomonasmaltophilia (+ve)<br />Haemophilusinfluenzae(-ve)<br />Many more unknown OTUs or OTUs not known to contain pathogens also did<br />
  18. 18.
  19. 19. Comparisons with other 16S methods<br />DeAngelis KM et al 2011 PLoS ONE 6(4): e19306.<br />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. <br />
  20. 20. Some practicalities<br />Depth of sampling compared to 454<br />On single wash station 16 samples per long day<br />Chip cost ~$100 each (may be cheaper now)<br />Proprietary <br />not available off the shelf from Affymetrix, contact LBNL group for info<br />Not MIAME compliant (can access probes via Greengenes)<br />Validation<br />
  21. 21. G3 Chip<br />More recent database snapshot<br />59,000 OTUs<br />Hierarchical probe design<br />Commercialised<br />2<br />1<br />3 γ-proteobacterial probe<br />4 Proteobacterial probe<br />
  22. 22.
  23. 23. Acknowledgments<br />LBNL<br />EoinBrodie, UlasKaraoz, Kate Goldfarb, Yvette Piceno<br />UCSF<br />Yvonne Huang, Kei Fujimura, Nicole Slusher, Susan Lynch<br />Elizabeth Nash/CFRI Fellowship<br />Further info: LBNL<br />References:<br />Brodie EL et al 2006 AEM 72:6288–98<br />DeSantis, T et al 2006 AEM 72:5069-72<br />Cox MJ et al. 2010 PLoS ONE 5(6): e11044<br />DeAngelis KM et al 2011 PLoS ONE 6(4): e19306<br />

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