The document discusses the use of phylochip technology to characterize microbial communities in relation to human health and disease, focusing on its application in cystic fibrosis research. Despite identifying a significant number of operational taxonomic units (OTUs), the study found that only a few were strongly associated with known pathogens at varying ages. The phylochip method is compared to other sequencing methods, highlighting its efficacy in detecting community structures while noting the challenges associated with rare taxa and practical sampling considerations.

1. Use of PhyloChip to characterise microbial communities in human health and diseaseMike CoxNational Heart and Lung Institute, Imperial College

2. A cautionary note…

4. Species rank abundanceNumber of organismsRank abundance

5. Species rank abundanceSmall number of frequently found organismsNumber of organismsLots of rare ones (rare biosphere or noise)Rank abundance

6. Species rank abundanceClone libraries, DGGE, TRFLP454 16S pyrosequencingNumber of organismsIllumina 16S pyrosequencingRank abundance

7. Species rank abundance16S rRNA PhyloChip –8,600 OTUs in parallel, rather than serialNumber of organismsRank abundance

8. 16S rRNA PhyloChipAffymetrix microarray developed at Lawrence Berkeley National LabGary Andersen, Todd DeSantis and EoinBrodieBrodieEL et al 2006 AEM 72:6288–98 Based on Greengenes – curated, aligned 16S rRNA gene sequence databaseDeSantis, Tet al 2006 AEM 72:5069-72G2 chip – 2002 database snapshotDetects organisms at 0.01 % total community abundance

9. Lane primers 27F/1492R used (8F/1391R if Archaea as well)

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

13. 500,000 25mer probes

14. At least 11, median 22 probes per OTU

15. Chips scanned, .cel files uploaded to Greengenes where raw data is normalised and returnedData analysisR (various packages), Phylotrac, FastUniFracOTUs predefined - said to be sub-family levelRanges from genus to species

16. Example: Cystic Fibrosis90 samples from age-stratified cohort6 months to 72 yearsSputum and deep throat swabDifferent mutations and wide range of severitiesCommunity structure associated withLung functionMutation (∆f508 homo/heterozygous)AgeCox 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