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Robustness, Reproducibility & Ecological Consistency in the Demarcation of Operational Taxonomic Units
- 1. Robustness, Reproducibility! & Ecological Consistency! in the Demarcation of Operational Taxonomic Units Sebastian Schmidt! Institute for Molecular Life Sciences! University of Zürich! sebastian.schmidt@imls.uzh.ch
- 2. A general workflow in (targeted) metagenomics ISME15, Seoul, 2014/08/29 sebastian.schmidt@imls.uzh.ch
- 3. A general workflow in (targeted) metagenomics Jean Tinguely, “Heureka”! Lake Zürich Sampling &! Sequencing “Making OTUs” ISME15, Seoul, 2014/08/29 Understanding! your data! (hopefully) sebastian.schmidt@imls.uzh.ch
- 4. Concepts ISME15, Seoul, 2014/08/29 sebastian.schmidt@imls.uzh.ch replicability! ! robustness! ! reproducibility! ! ecological consistency
- 5. Concepts ISME15, Seoul, 2014/08/29 sebastian.schmidt@imls.uzh.ch replicability! ! robustness! ! reproducibility! ! ecological consistency 42! Life, the Universe and Everything? 42! Life, the Universe and Everything?
- 6. Concepts ISME15, Seoul, 2014/08/29 sebastian.schmidt@imls.uzh.ch replicability! ! robustness! ! reproducibility! ! ecological consistency 42! Life, the Universe and Everything? 42! Life, Microbial Ecology and Everything?
- 7. Concepts ISME15, Seoul, 2014/08/29 sebastian.schmidt@imls.uzh.ch replicability! ! robustness! ! reproducibility! ! ecological consistency 42! Life, the Universe and Everything? Life, the Universe and Everything? 42!
- 8. The Human Skin Microbiome (HSM) dataset:! ! ~115,000 full-length 16S sequences! ! sampled from 21 distinct body sites! Grice et al, Science, 2009 ! clustered to 97% sequence identity ISME15, Seoul, 2014/08/29 sebastian.schmidt@imls.uzh.ch
- 9. OTU A UPARSE all methods agree (almost) 5,423 SEQ. perfectly SMALL OTUS õ4EQ PER OTU methods provide different # of “small” OTUs õTFRQFS056 OTU D 2,692 SEQ. TQMJUUJOH by Uclust OTU C 4EQ. TQMJUUJOH by CL OTU B 8,465 SEQ. MVNQJOH by SL OTUS UCLUST 3,282 OTUS CD-HIT OTUS SINGLE LINKAGE OTUS COMPLETE LINKAGE OTUS AVERAGE LINKAGE OTUS ISME15, Seoul, 2014/08/29 Schmidt et al, Environ Microbiol, in press
- 10. MORISITA-HORN 0.749 0.154 AL 0.920 -0.075 CL 0.932 -0.095 0.682 -0.051 SL HIT CD-UCLUST UPARSE AVERAGE LINKAGE COMPLETE LINKAGE SINGLE LINKAGE CD-HIT UCLUST UPARSE CHAO1 INV SIMPSON SHANNON 0.988 0.387 0.969 0.300 0.991 0.150 0.794 0.079 0.981 -0.008 0.576 0.116 0.991 -0.299 0.858 -0.261 0.966 -0.136 MORISITA-HORN 0.772 -0.099 0.928 -0.060 0.545 -0.131 0.986 0.522 0.773 0.463 0.953 0.216 MORISITA-HORN 0.749 0.154 0.922 0.087 0.551 0.167 0.973 -0.686 0.817 -0.561 0.949 -0.286 0.953 0.064 0.513 -0.358 0.358 -0.207 0.984 0.204 0.672 -0.163 0.584 0.780 0.665 0.350 0.922 0.087 PEARSON CORRELATION MORISITA-HORN 0.918 0.128 0.802 -0.194 0.805 1.511 0.855 -0.181 0.948 0.390 0.912 0.427 0.472 -0.325 0.785 2.033 0.694 -0.280 0.668 0.853 0.799 0.642 0.993 0.027 0.920 0.151 0.643 -0.158 0.881 1.347 0.884 -0.126 0.922 0.292 0.905 0.356 0.937 -0.068 0.981 -0.056 0.791 -0.209 0.838 1.734 0.862 -0.201 0.945 0.592 0.912 0.506 0.482 -0.366 0.614 -0.091 0.984 -0.095 0.764 -0.084 0.613 0.518 0.608 0.214 0.762 0.036 0.977 -0.164 0.945 0.055 0.989 -0.098 0.998 -0.071 0.558 -0.271 0.464 -0.040 0.978 -0.482 0.759 -0.009 0.584 0.219 0.574 0.063 0.552 -0.298 0.630 -0.076 0.972 -0.318 0.793 -0.064 0.569 0.317 0.570 0.134 Q Q MORISITA-HORN 0.436 -0.422 0.520 0.118 Qö 0.837 -1.829 0.617 0.117 0.559 -0.073 0.434 -0.292 0.886 -0.015 0.993 0.224 0.957 -0.020 0.974 0.202 0.995 0.079 SØRENSEN JABD CHAO1 INV SIMPSON SHANNON SØRENSEN JABD CHAO1 INV SIMPSON SHANNON SØRENSEN JABD CHAO1 INV SIMPSON SHANNON SØRENSEN JABD CHAO1 INV SIMPSON SHANNON Q SØRENSEN MORISITA-HORN JABD CHAO1 INV SIMPSON SHANNON SØRENSEN JABD B significance of mean shift red: shift towards higher values blue: shift towards lower values 0.551 0.167 0.973 -0.686 0.817 -0.561 0.949 -0.286 PEARSON CORRELATION RELATIVE SHIFT (LOG2) RELATIVE SHIFT (LOG2) PEARSON CORRELATION RELATIVE SHIFT (LOG2) Qö Q Q Q ISME15, Seoul, 2014/08/29 Schmidt et al, Environ Microbiol, in press! (data from Grice et al, Science, 2009)
- 11. 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 AVERAGE LINKAGE 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 0.5 0.6 0.7 0.8 0.9 1.0 UCLUST CD-HIT SINGLE LINKAGE COMPLETE LINKAGE AVERAGE LINKAGE COMPLETE LINKAGE SINGLE LINKAGE CD-HIT UCLUST UPARSE UPARSE ADJUSTED MUTUAL INF A ‘global’ 16S dataset! ~1.1M full-length sequences! ≥30k samples, diverse environments! ! Adjusted Mutual Information (AMI), a measure of partition similarity! ! high replicability! …when clustering twice to the exact same threshold! ! differential robustness! …to slight threshold changes Schmidt et al, Environ Microbiol,! in press
- 12. 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 AVERAGE LINKAGE 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 0.5 0.6 0.7 0.8 0.9 1.0 UCLUST CD-HIT SINGLE LINKAGE COMPLETE LINKAGE AVERAGE LINKAGE COMPLETE LINKAGE SINGLE LINKAGE CD-HIT UCLUST UPARSE UPARSE ADJUSTED MUTUAL INF A ‘global’ 16S dataset! ~1.1M full-length sequences! ≥30k samples, diverse environments! ! Adjusted Mutual Information (AMI), a measure of partition similarity! ! high replicability! …when clustering twice to the exact same threshold! ! differential robustness! …to slight threshold changes! ! differential reproducibility! pairwise similarity maxima between methods off-diagonal! comparability of results across studies? Schmidt et al, Environ Microbiol,! in press
- 13. 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 AVERAGE LINKAGE 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 0.5 0.6 0.7 0.8 0.9 1.0 UCLUST CD-HIT SINGLE LINKAGE COMPLETE LINKAGE AVERAGE LINKAGE COMPLETE LINKAGE SINGLE LINKAGE CD-HIT UCLUST UPARSE UPARSE ADJUSTED MUTUAL INF “Greengenes 97”! vs.! “SILVA 99”! AMI ~ 0.65 A ‘global’ 16S dataset! ~1.1M full-length sequences! ≥30k samples, diverse environments! ! Adjusted Mutual Information (AMI), a measure of partition similarity! ! high replicability! …when clustering twice to the exact same threshold! ! differential robustness! …to slight threshold changes! Schmidt et al, Environ Microbiol,! in press ! differential reproducibility! pairwise similarity maxima between methods off-diagonal! comparability of results across studies?
- 14. 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 AVERAGE LINKAGE 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 90 95 100 0.5 0.6 0.7 0.8 0.9 1.0 UCLUST CD-HIT SINGLE LINKAGE COMPLETE LINKAGE AVERAGE LINKAGE COMPLETE LINKAGE SINGLE LINKAGE CD-HIT UCLUST UPARSE UPARSE ADJUSTED MUTUAL INF A ~1.1M ≥ environments ! Adjusted Mutual Information (AMI) measure of partition similarity! ! high … the exact same threshold! ! differential …to slight threshold changes! ! differential pairwise similarity maxima between comparability of results across studies? Schmidt et al, Environ Microbiol,! in press But which method makes the ‘best’ OTUs?
- 15. ‘Good’ OTUs should correspond to ‘true’ bacterial lineages (‘species’)! they should comply with evolutionary theory of bacterial speciation! BUT: no unifying / commonly accepted bacterial species concept! ! ! Two main criteria for theory-compliant OTUs! phylogenetic consistency (represent monophyletic lineages)! ecological consistency (represent ecologically homogenous groups of organisms) Gevers et al., Nat Rev Microbiol, 2005! Cohan, Philos T R Soc B, 2006! Koeppel et al., PNAS, 2008! Hunt et al., Science, 2008! Fraser et al., Science, 2009! Vos, Trends Microbiol, 2011! Koeppel Wu, NAR, 2013! Preheim et al, Appl Env Microbiol, 2013! ! [and many more…] ISME15, Seoul, 2014/08/29 sebastian.schmidt@imls.uzh.ch
- 16. rumen halotolerant hypersaline pathogenic intestinal infection degradation day resistant producing gut endosymbiont deep mat thermophilic high metal activated cold milk soil environmental diverse iron diversity sediment water community marine associated acid plant sludge anaerobic field sea rhizosphere lake spring halophilic culture consortium extremely archaeon paddy pesticide activity root surface production contaminated wastewater structure degrading seawater treatment hydrothermal oil feces hot biofilm waste endophytic nodule freshwater deepsea reactor vent enrichment microbiota growth disease pathogen salt patient aerobic coastal mine host fermented culturable habitat archaeal actinomycete res pond lactic forest region clinical symbiont biodegradation temperature skin moderately antarctic methanogenic swab reveal zone ocean tract natural control bioreactor river sponge produced carbon blood fluid coral mud food shift highly leaf ice organic rock draft diet oral tree solar stream coast wild core fed low grown tidal fecal mineral flat compost saline symbiotic content saltern alkaline diseased rhizobia wound active intestine traditional sand subsurface antimicrobial fermentation effluent comb sewage condition caused product treating sulfatereducing ecology purification station hydrocarbon nitrogen coidentity degrade resistance mangrove methane polluted acidic antibiotic cultivation oxidation probiotic cultured methanogen process revealed tissue agricultural chemical heterotrophic biocontrol alkaliphilic legume denitrifying indigenous industrial correlate defense cluster heavy reduction tolerant aquifer reservoir wetland diabetic enriched chloroplast cultivated cultureindependent nitrogenfixing prolonged protease basin compound mesophilic microbiome removal formation laboratory adult anoxic petroleum termite functional aquatic association factory fresh antifungal korean terrestrial involved promoting geothermal bay black island sulfur drainage farm groundwater hydrogen ISME15, Seoul, 2014/08/29 sebastian.schmidt@imls.uzh.ch
- 17. AVERAGE LINKAGE SINGLE LINKAGE 1000 10000 100000 NUMBER OF OTUS 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 A ECOLOGICAL CONSISTENCY SCORE (ECS) COMPLETE LINKAGE UCLUST CD-HIT 97% NOMINAL SIMILARITY ISME15, Seoul, 2014/08/29 Schmidt et al, PLOS Comp Biol, 2014
- 18. AVERAGE LINKAGE SINGLE LINKAGE 1000 10000 100000 NUMBER OF OTUS 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 A ECOLOGICAL CONSISTENCY SCORE (ECS) COMPLETE LINKAGE UCLUST CD-HIT 97% NOMINAL SIMILARITY D BACTERIA, SAMPLING SITES B ARCHAEA, ECOLOGICAL TERMS 100 1000 10000 E BACTERIA, HOST TAXONOMY F 5000 4000 3000 2000 1000 1000 10000 100000 2500 2000 1500 1000 500 0 1000 10000 100000 2500 2000 1500 1000 500 BACTERIA, ENVO TERMS 1000 10000 100000 C 100 1000 10000 400 300 200 100 EUKARYA, ECOLOGICAL TERMS 700 600 500 400 300 ISME15, Seoul, 2014/08/29 Schmidt et al, PLOS Comp Biol, 2014
- 19. Conclusions ISME15, Seoul, 2014/08/29 sebastian.schmidt@imls.uzh.ch replicability! clustering was generally replicable! ! robustness! AL, CL CD-HIT were highly robust to (slightly) changing thresholds, UCLUST, UPARSE SL more sensitive! similar trends for robustness to clustering context and choice of subregion (not shown)! ! reproducibility! surprisingly discordant partitions by different methods! similarity maxima generally off-diagonal! AL and CD-HIT most similar pair! implications for reference-based OTU-binning: choice of reference clustering determines quality!! ! ecological consistency! CL provided most consistent OTU sets! implications for taxonomy and species definitions?