Analysis of Microbial Community Structure - PowerPoint PPT Presentation

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Analysis of Microbial Community Structure

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Analysis of. Microbial Community Structure. Historical ... Bands may be cut out and DNA cloned for phylogenetic analysis. Larger bands more information. ... – PowerPoint PPT presentation

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Title: Analysis of Microbial Community Structure


1
Analysis of Microbial Community Structure
  • Historical Perspective on Microbial Diversity
  • Assessing Diversity Alone
  • Diversity With Phylogeny
  • Diversity of Function

2
Historical Perspective on Microbial Diversity
  • Knowledge is limited by our tools.
  • Cultivation missed about 99.9 of no. estimates.
  • Both biomass and diversity underestimated.
  • Molecular techniques and phylogeny
  • Independent of cultivation
  • Unknowns can be grouped with knowns

3
Study Diversity Independent of Cultivation
  • Extract DNA from sample (or not?)
  • PCR Amplification of 16SrDNA
  • Clone all amplicons
  • Screen clones for differences (e.g. ARDRA).
  • Document richness and evenness of clones.
  • Preliminary phylogeny
  • Sequence clones of interest.
  • Perform complete phylogenetic analysis
    identify known or prior documented unknown
    strains.

4
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5
Ligate PCR amplicon into a cloning plasmid
transform host bacterium isolate recombinant
plasmids for sequencing of inserted 16SrDNA.
6
Screen Clones
  • ARDRA is an RFLP on the 16SrDNA amplified by PCR
  • RFLP restriction fragment length polymorphism
  • ARDRA has been used
  • - post-cloning
  • pre-cloning
  • isolates or communities
  • For screening clones with 16SrDNA, enzymes that
    separate plasmid DNA are used for clearer results.

7
Preliminary Phylogenetic Analysis
  • Fingerprints (banding patterns) can be converted
    to analog data based on presence or absence of
    bands of different sizes.
  • Pairwise comparison of all clone fingerprints
    will yield a similarity (distance) matrix.
  • Phylogenic tree can be computed from a cluster
    analysis of the matrix.

8
Complete Analysis 16SDNA Sequencing
1) Dideoxynucleotides stop synthesis at different
sites different size fragments made for each
sequence position.
2) Each ddNTP has a fluorescent label for easy
specific detection as its separated by HPLC or
electrophoresis.
9
Quick Assessment of DiversityOne band One
bug (?)(little phylogeny information)
  • RISA (ribosomal intergenic spacer analysis)
  • Often get overlapping bands (on band gt 1 bug)
  • Phylogenetic information limited by 16SrDNA
    overlap
  • ARDRA (amplified ribosomal DNA restriction
    analysis)
  • Good for identification of isolates esp. with
    multiple restriction enzymes.
  • Too many bands makes it hard to interpret mixed
    populations.
  • T-RFLP (terminal restriction fragment
    polymorphism)
  • Steps like ARDRA, but terminal 3 end of gene is
    fluorescent
  • Multiple restriction enz. Give best results
    maybe used to query RDP.

10
DGGE
  • Denaturing Gradient Gel Electrophoresis
  • Separated DNA of same size based on sequence
    differences.
  • Different sequences behave differently at
    different amounts of denaturing chemical (or
    heat see TGGE)
  • At some point 16SrRNA DNA strands completely
    separates.
  • Complete separation of PCR amplicon is hindered
    by GC-clamp added to one of the PCR primers.

11
Gradient Perpendicular to Electrophoresis to
Optimize Run
12
Gradient Parallel for Analysis
  • Conditions change mostly due to size of
    amplicon.
  • May be applied at different taxa (groups)
  • Bands may be cut out and DNA cloned for
    phylogenetic analysis.
  • Larger bands more information.

13
Diversity Metrics
  • Shannon-Weaver
  • Simpson
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