2. Work by Antonio Izzo Based on 36 soil cores from a total of 9 plots contained within a 2.5 hectare region

3. 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

4. Chimera formation via partial extensions

5. 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

6. What can we say about unique sequences? Giovannoni et al. 1996

7. Achenbach and Coates 2000 photosynthetic Fe reducing, obligate anarobe Non-Fe reducing, facultative anarobe

8. From Achenbach and Coates 2000 * *

9. 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

11. O’Brien et al. 2005

14. 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

15. From Ward et al 1998. Mol. Biol. Rev

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

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

18. T-RFLP (terminal restriction fragment length polymorphism)

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

20. T-RFLP analysis & gel

21. Moeseneder et al 1999

22. 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

23. 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

24. SSCP gel from soil microbial community Schmalenberger & Tebbe Mol. Ecol. 2003 12:251-262 Excised bands were cloned and sequenced and found to be complex pools of sequences

25. 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

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

27. 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