Work by Antonio Izzo Based on 36 soil cores from a total of 9 plots contained 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

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

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 Mol. Biol. Rev

DGGE gel From Ward et al Mol. Biol. Rev

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

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 ( 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 community Schmalenberger & Tebbe Mol. Ecol : Excised bands were cloned and sequenced and found to be complex pools of sequences

Comparison of finger printing methods MethodUnique advantagesUnique Disadvantages DGGE, TGGECan excise, clone and sequence bands Specialized gel or equipment needed, heteroduplex bands Results difficult to reproduce between labs T-RFLPCan 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 SSCPCan excise, clone and sequence bands Single fragments may have multiple conformers Results difficult to reproduce between labs

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

Size typically 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