Judging the Relevance and worth of ideas part 2.pptx
A Year In the Western English Channel
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. 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. 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. 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. 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. A word on “normalization”
Taxa cannot be added when normalizing to a
larger sample. Just amplifies error!
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. 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. 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. 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. 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
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. 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)
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. 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.