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

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

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

8. From Achenbach and Coates 2000 * *

9. 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 Advantages and Disadvantages of Sequence approach to community analysis

11. O’Brien et al. 2005

12. O’Brien et al. 2005

13. O’Brien et al. 2005

14. DGGE - Denaturing gradient gel electrophoresis & TGGE - temperature gradient gel electrophoresis • 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.

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

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

27. The ITS (internal transcribed spacer) region in fungi • 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