Probes can be designed in an evolutionary hierarchy

Probes can be designed to be highly redundant to increase the certainly of identification

The match between clone counts and hybridization intensity

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)

“Environmental Genomics”Ed 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

Shotgun cloning and assembly of enviromental genomes

• Tyson et al 2004, Nature 428:37-43

• Venter et al 2004, Science 304:66-74.

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

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.

Tyson et al. FISH image of biofilm

Yellow (red + green) Leptospirillum

Green (Eubacterial)

Blue (Archaea) predominantly Ferroplasma

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

Summary of genes found in the Sargasso sea survey

Assembly problems

• Most abundant genomes are over-represented

• Assembled genomes are composites of different individuals (particularly genomes with lower coverage)

Estimates of species diversity

• At least 300 species/sample assuming homologous sequences that are greater than >6% 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

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

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

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?

Estimates of abundance of major groups based on different gene families

Rhodopsin tree showing the novelty of the Sargasso sea samples (SAR)

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

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

From MacKay et al. 2002

From MacKay et al. 2002

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

Mycelium (white) of Suillus in co- culture with pine

DGGE gel of amplified basidiomycete ITS from soil

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