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

1. Use of PhyloChip to characterise microbial communities in human health and disease Mike Cox National Heart and Lung Institute, Imperial College

2. A cautionary note…

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

13. 500,000 25mer probes

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