Probes can be designed in an evolutionary hierarchy

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Probes can be designed in an evolutionary
hierarchy
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Probes can be designed to be highly redundant to
increase the certainly of identification
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The match between clone counts and hybridization
intensity
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Genomics terminology

• Shotgun cloning undirected cloning effort where
  the entire sample is cloned and sequenced
• Contig - assembled continuous sequence derived
  from sequence reads from a single clone
• Scaffold assembled sequence reads derived from
  multiple overlapping clones
• nX coverage mean number of times a region was
  sequenced from independent clones
• Mini-scaffold scaffold assembled only by paired
  ends of overlapping contigs (approx. 1X coverage)

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Environmental GenomicsEd Delong

• Shotgun cloning of Megabase fragments from marine
  environments
• Probe for those with rRNA gene of interest
• Sequence and use bioinformatics to infer function
• Used to connect diverse psbA (photosystem II)
  genes to known 16S sequence groups

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Shotgun cloning and assembly of enviromental
genomes

• Tyson et al 2004, Nature 42837-43
• Venter et al 2004, Science 30466-74.

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Tyson et al. 2004. Iron Mt. study

• Pink biofilm growing at pH 0.87, which was known
  to be composed of 6 rRNA types
• 103,462 sequence reads from shotgun clones
  provided 10X coverage for two species
  (Leptospirillum III and Ferroplasma II), and 3X
  coverage for Leptospirillum II.
• Very low polymorphism in Leptospirillum III
  interpreted as evidence for a single strain
• Higher polymorphism (2.2) in Ferroplasma II
  interpreted as evidence for 3 strains which show
  evidence of past recombination
• A single nitrogen fixer was found (Leptospirillum
  III

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An aside about rRNA

• 16S rRNA sequences of Fer I isolate and assembled
  Fer II strains differ by less than 1.
• The assembled genomes of Fer I and FerII differ
  by more than 22 even though gene order and
  content appear to be conserved.

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Tyson et al. FISH image of biofilm
Yellow (red green) Leptospirillum Green
(Eubacterial) Blue (Archaea) predominantly
Ferroplasma
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Some numbers from the Venter et al. study

• From 200L of filtered sea water, 1.66 million
  sequence reads were derived.
• 246 mbp were assembled into 64,398 scaffolds
  ranging from 826 bp to 2.6 Mbp
• 170 mbp of miniscaffolds and unpaired reads
• 1.2 million protein-coding genes (10X more than
  previously in protein database)
• 69,901 conserved open reading frames with no
  assignable function
• 60,000 16S sequences, 148 of which are at least
  3 different from previously known sequence

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Summary of genes found in the Sargasso sea survey
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Assembly problems

• Most abundant genomes are over-represented
• Assembled genomes are composites of different
  individuals (particularly genomes with lower
  coverage)

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Estimates of species diversity

• At least 300 species/sample assuming homologous
  sequences that are greater than gt6 are from
  different species
• Using models based on a poisson distribution and
  3 different coverage models, estimates of species
  for the whole study range from 1800 to 47,000.
• A minimum of 12X greater sequence effort would be
  needed to sample 95 of the unique sequence

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Population level findings

• Scaffolds with 14X coverage contain about 1
  SNP/10,000 bases, and also contain inserted phage
  sequences
• SAR 11- like (a previously characterize 16S type)
  sequences are abundant but are very polymorphic

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Venter et al. 2004 evidence the the composite
genome represents a population
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Other interesting findings

• Species distributions are patchy even in the
  ocean (e.g. Burkholderia and Sewanella abunance
  in sample 1 but not 2)
• Clear copy bias in rRNA gene sequences in favor
  of beta and gamma proteobacteria, which typically
  have two or more gene copies
• Huge diversity of rhodopsin sequences, possible
  non-chlorophyll light harvesting?

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Estimates of abundance of major groups based on
different gene families
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Rhodopsin tree showing the novelty of the
Sargasso sea samples (SAR)
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Advantages and disadvantages of environmental
genomics

• Avoids PCR and all the inherent biases
• Not dependent on rRNA
• Lots of new genes and new information about who
  has them

• Currently way too expensive for mere mortals
• Not very efficient if structure and activity are
  the main questions

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Hybridization to rRNA for identification and
quantification

• Extract total RNA from sample (no amplification
  or cloning!)
• Spot it on a filter
• Probe it with oligonucliotide probes

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From MacKay et al. 2002
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From MacKay et al. 2002
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Landeweert et al 2003

• Wanted to measure competition between two
  mycorrhizal fungi Suillus Paxillus
• Setup pot inoculations with pine trees and either
  fungus or both fungi together
• Measured total mycelium, PLFAs
• Amplified ITS region with basidiomycete specific
  primers
• Compared via DGGE, clone counts, real-time PCR
  quantification

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Mycelium (white) of Suillus in co- culture with
pine
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DGGE gel of amplified basidiomycete ITS from soil
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Conclusions

• DGGE, Clone counts, and real-time quantification
  agree that Suillus ITS increases as Paxillus
  decreases
• What have we gained by real-time?
• Quantification of the template, rather than the
  amplicons