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A Year In the Western English Channel

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Jack Gilbert
Plymouth Marine Laboratory, UK

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A Year In the Western English Channel

  1. 1. Bacterial Diversity: A Year in the Western English Channel Jack A. Gilbert, Dawn Field, Paul Swift, Margaret Hughes, Ben Temperton, Paul Somerfield, Sue Huse, Ian Joint, etc.
  2. 2. Why look at Bacterial Diversity? • Surely it’s just stamp collecting? • Stamps are not responsible for the majority of the worlds biogeochemical cycling. • Understanding the reservoir of bacterial diversity • Base line for future change, e.g. climate, ocean acidification. • Identification of rare bacteria for the biotech harvest. • Help to resolve ecological models through identification and interrogation of trophic links.
  3. 3. What did we do? • Pyrosequencing of 16 x V6-16s-tag samples. • Current study: – 12 time points, – February – December 2007 – 11,327 to 17,339 reads per sample – 182,560 reads in total • Sample site: – Western English Channel, L4 site. – Unique historical dataset (Southward et al, 2005). – Boundary between several bodies of water, including the gulf stream. – Busiest shipping lane in the world. Gilbert et al. Env Microb., 2009
  4. 4. High Diversity • 182,560 reads in total = 17,673 “species” • Only 0.5 % found at all 12 time points – 54 % of all reads Unique A 90 % identity D 4000 1200 3500 Feb 16th Feb 16th 1000 Mar 7th Mar 7th Number of OTUs observed Number of OTUs observed 3000 Mar 26th Mar 26th 800 Apr 23rd Apr 23rd 2500 May 8th May 8th Jun 4th Jun 4th 2000 600 Jun 25th Jun 25th Jul 30th Jul 30th 1500 400 Aug 20th Aug 20th 1000 Sep 29th Sep 29th Oct 25th Oct 25th 200 500 Dec 12th Dec 12th 0 0 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Number of sequences sampled Number of sequences sampled
  5. 5. Random Re-sampling to standardise comparisons • Sampling effort was identical. • Sequencing effort varied as an artefact of pyrosequencing. • Randomly Re-sampled to smallest dataset – Daisy_Chopper v1.0 ( http://www.genomics.ceh.ac.uk/GeneSwytch/Tools.html ). – 11,327 reads. – Lost 30% of unique OTUs – 12,393 OTUs left. Gilbert et al. Env Microb., 2009
  6. 6. A word on “normalization” Taxa cannot be added when normalizing to a larger sample. Just amplifies error!
  7. 7. Random re-sampling 1400 1200 1000 Diversity index (Fisher) 800 Re-sampled bacterial species richness 600 Original bacterial species richness 400 200 0 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 Number of sequence reads Gilbert et al. Env. Microb.,
  8. 8. Rare Microbial Assemblage • Even following re-sampling: – 78% of the OTUs were time-point specific – 67% of the OTUs were singletons • Singletons diverged from other tags in the dataset by >2 base pairs – hence not sequencing errors. 14000 12000 10000 8000 Rare assemblage? Or just 6000 under-sampled? 4000 2000 0
  9. 9. Evidence for seasonality: the presence of ‘winter’, ‘spring’, ‘summer’ and ‘fall’ communities Transform: Fourth root Resemblance: S17 Bray Curtis similarity 2D Stress: 0.11 Similarity 25 5 3 7 6 1 4 11 8 2 10 9 12 Gilbert et al. Env Microb., 2009
  10. 10. Correlation to environmental parameters Rho Variables Temp Silicate PO4 Density TOC Nanoeuks Coccoliths Dinoflagels All OTUs 0.73 Gammaproteobacteria 0.75 Alphaproteobacteria 0.73 Bacteroidetes 0.72 Actinobacteria 0.63 Cyanobacteria 0.57 Betaproteobacteria 0.52 Gilbert et al. Env Microb., 2009
  11. 11. 2007-2008 2007-2008 alldomains resampled Transform: Log(X+1) Resemblance: S17 Bray Curtis similarity 2D Stress: 0.16 month Feb EMar LMar Apr May 2008 2008 correlates with Temperature, EJun LJun nutrients and density! Jul Aug Sep Oct Dec 2007
  12. 12. Densisty and Salinity in 2008 1028 1026 1024 1022 Density-2007 Denisty-2008 1020 1018 1016 1 2 3 4 5 6 7 8 9 10 11 12
  13. 13. Top 10 most abundant bacterial phyla Alphaproteobacteria 4000 Bacteroidetes 3500 Gammaproteobacteria Organelle Effect of depressed Cyanobacteria salinity? Unknow n 3000 Unknow n bacteria Betaproteobacteria 2500 Actinobacteria Deferribacteres 2000 1500 1000 500 0 16 07 26 23 25 30 20 29 25 12 17 21 06 02 23 21 08 08 04 20 05 20 22 27 2_ 6_ 7_ 8_ 3_ 4_ 5_ 6_ 2_ 3_ 3_ 4_ 5_ 6_ 9_ 0_ 2_ 2_ 3_ 6_ 7_ 8_ 9_ 0_ _0 _0 _0 _0 _0 _0 _0 _0 _0 _0 _0 _1 _1 _0 _0 _0 _0 _0 _0 _0 _0 _0 _1 _1 07 07 07 07 07 07 07 07 07 07 07 07 08 08 08 08 08 08 08 08 08 08 08 08 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
  14. 14. Conclusions • Large sequencing effort but no plateau on rarefaction. • Majority of unique species are rare • Bacterial diversity can be linked to environment. • Inter-annual trends exist • Climate can affect microbial diversity. Gilbert et al. Env Microb., 2009
  15. 15. New Study – GS-flx Titanium • Seasonal Diel cycling • Metagenomics, Metatranscriptomics and 16S- tag sequencing. • No rRNA removal for metatranscriptomics – Check for GenomiPHI exclusion theory. • 1.5 million transcripts (250 Mbp) • 5.75 million gene fragments (1.9 Billion bp)
  16. 16. AllDomains L4_diel-resampled 16S tag diversity analysis Transform: Log(X+1) Resemblance: S17 Bray Curtis similarity 2008_04_22_Np 2D Stress: 0.04 Month Jan Aug Apr 2008_04_22_Dp 2008_01_28_D 2008_04_22_D 2008_01_28_Np 2008_08_27_Np 2008_08_27_Dp 2008_08_28_Dp 2008_08_28_Np 2008_01_28_Dp
  17. 17. AllDomains L4_diel-resampled 16S tag diversity analysis Transform: Log(X+1) Resemblance: S17 Bray Curtis similarity 2008_04_22_Np 2D Stress: 0.04 Month Jan Aug Apr 2008_04_22_Dp 2008_01_28_D 2008_04_22_D 2008_01_28_Np 2008_08_27_Np 2008_08_27_Dp 2008_08_28_Dp 2008_08_28_Np 2008_01_28_Dp
  18. 18. AllDomains L4_diel-resampled 16S tag diversity analysis Transform: Log(X+1) Resemblance: S17 Bray Curtis similarity 2008_04_22_Np 2D Stress: 0.04 Month Jan Aug Apr 2008_04_22_Dp 2008_01_28_D 2008_04_22_D 2008_01_28_Np 2008_08_27_Np 2008_08_27_Dp 2008_08_28_Dp 2008_08_28_Np 2008_01_28_Dp
  19. 19. Western English Channel http://www.westernchannelobservatory.org.uk/ • One of the longest time series in the world (Southward et al., 2005) • Recently added 7 years worth of bacterial 16S-rDNA pyrosequencing tag data: • 1 million 16S rDNA sequences!! • Within next six months: • >30 Million pyrosequencing reads – metagenomic and metatranscriptomic • ~12 billion base pairs of data!!
  20. 20. NERC - PG&P for funding the Aquatic Microbial Metagenomics consortium CEH – Mark Bailey for additional funding SEED – Rob Edwards, Folker Meyer and the MG- RAST group Metascience needs JCVI – Doug Rusch and team EMBL-EBI - Susanna-Assunta Sansone, Philippe Rocca-Serra a Metacommunity CAMERA – Ying Huang, Weizhong Li, Paul Gilna, Adam Godzik NEBC – Dawn Field, Bela Tiwari, Tim Booth Liverpool-MGF – Neil Hall, Margaret Hughes, Kevin Ashelford PML – Ian Joint - for being a great mentor.

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