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relative frequency

% tot biomass

Mycorrhizal Diversity in an Abies concolor Forest

Fungal species in ranked order

Work by Antonio IzzoBased on 36 soil cores from a total of 9 plotscontained within a 2.5 hectare region

Assumptions of clone and sequence approaches

• No extraction bias

• No amplification bias

• No cloning biases

• Sequences retrieved were from living organisms

• Cloning and PCR artifacts are unimportant

• Phylogenetic placement is predictive of functional attributes

Chimera formation via partial extensions

partial extension

denature

Heterologous partial fragments anneal

Extension creates chimara

Further rounds of amplificationcreate many copies

Assumptions of clone and sequence approaches

• No extraction bias

• No amplification bias

• No cloning biases

• Sequences retrieved were from living organisms

• Cloning and PCR artifacts are unimportant

• Phylogenetic placement is predictive of functional attributes

What can we say about unique sequences?

Giovannoni et al. 1996

Achenbach and Coates 2000

photosynthetic

Fe reducing, obligate anarobe

Non-Fe reducing, facultative anarobe

From Achenbach and Coates 2000

*

*

Advantages and Disadvantages of Sequence approach to community analysis

• Still is one of the best ways to identify a pool of total unknowns

• Produces an imperfect quantitative picture

• Doesn’t tell you much about function

• Cost and effort limit the number of replicate samples

O’Brien et al. 2005

O’Brien et al. 2005

O’Brien et al. 2005

• Amplify portion of rDNA gene using a primer with a 5’ GC clamp

• Load pool of amplicons onto denaturing gradient gel

• Slightly different products are separated by sequence differences that cause different levels of partial denaturation.

DGGE - Denaturing gradient gel electrophoresis& TGGE - temperature gradient gel electrophoresis

From Ward et al 1998. Mol. Biol. Rev

DGGE gel From Ward et al 1998. Mol. Biol. Rev

denature

extension

annealing of primers

extension

denature

Reannealing of strands

heteroduplex

homoduplex

Heteroduplex formation: a feature of all PCR reaction with complex mixtures of similar products

Amplify pool of sequences with one of the primers labeled

Digest with a restriction enzyme

A B C

B

A

C

Each ampliconproduces a singledetected fragment

T-RFLP (terminal restriction fragment length polymorphism)

An example of t-RFLP data from a very simple community

T-RFLP analysis & gel

Moeseneder et al 1999

How can t-RFLP analysis separate as many 16S sequences as DGGE?

Because many of the differences are based on indels rather than base substitutions in restriction sites

What can’t you do with t-RFLP that you could do with DGGE or TGGE?

Retrieve the entire sequence by cutting the fragment out of the gel and sequencing it

SSCP - Single stranded conformational polymorphisms

• Amplify target (100-600 bp), with one of the primers phosphorylated

• Digest products with Lambda exonuclease (only phosphorylated strand is digested)

• Separate remaining single-stranded products on non-denaturing gel

• Migration of fragments due to conformation rather than size

SSCP gel from soil microbial communitySchmalenberger & Tebbe Mol. Ecol. 2003 12:251-262

Excised bands were cloned and sequenced and found to be complex pools of sequences

Comparison of finger printing methodsMethod Unique advantages Unique Disadvantages

DGGE, TGGE Can excise, clone and sequence bands

Specialized gel or equipment needed, heteroduplex bands

Results difficult to reproduce between labs

T-RFLP Can be run on an automated sequencer

Highly reproducible size estimates

Can not excise and sequence bands easily, may not be very useful for protein coding sequences

SSCP Can excise, clone and sequence bands

Single fragments may have multiple conformers

Results difficult to reproduce between labs

For higher resolution the same methods canbe applied to the spacer region, but no database exists!

• Size typically 540-950 bp• Specific primers allow fungal sequences to

be easily amplified from complex environments

• Usually highly variable between species groups

• Variation is often rich in IDELs

The ITS (internal transcribed spacer) region in fungi