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Cottrell, M. T., L. A. Waldner, L. Yu, and D. L. Kirchman. 2005. Bacterial diversity of metagenomic and PCR libraries from the Delaware River. Environmental Microbiology. 2005. 7(12): 1883-1895. • What is a metagenomic clone library? • What is the utility of metagenomic analysis? • What is a 16SrDNA clone library? • What is FISH? • Do metagenomic libraries (key to link function to taxa for non-culturables) represent similar diversity as PCR library and FISH? • Why study bacterial diversity in rivers? Metagenomic clone fosmid library • What’s a fosmid? • Gentle DNA extraction to avoid excessive shearing. • Ligate 40 kb fragments, package with phage proteins, infect host bacterium, and select for clones. • Screen clones for 16SrDNA by PCR and DGGE, bands are reamplified and sequenced (160 bp). 16SrDNA PCR Library • Take same DNA extract as above and PCR amplify 16SrDNA. • Clone 16SrDNA amplicons in plasmid vector and transform host bacterium. • Screen clones by amplified ribosomal DNA restriction analysis (ARDRA); same as RFLP on 16SrDNA amplicons. • Representative clone for each unique fingerprint was sequenced (~1400 bp). FISH • Filter paraformaldehyde fixed river bacteria onto polycarbonate filter. • Treat cells on filter with hybridization buffer with Cy3 labeled oligonucleotide probe; then wash, dehydrate, and mount on slide with DAPI stain. Data Analysis • Identify 16SrDNA clone sequences: – BLASTN database similarity search – ARB software Tool • Alignment and phylograms with ARB tools. • Library coverage comparison (LIBSHUFF). • Checked for heteroduplex artifacts in the PCR library via reconditioning. Results & Discussion • How did coverage over evolutionary distance compare between the two library types (Fig 4)? • Were heteroduplexes a large problem for the PCR library? • Which was method gave more diversity? • What were the differences in composition of major taxa between methods (Table 1)? Results & Discussion ARCHAEA LUCA • Cytophaga-like Bacteria? • Beta-Proteobacteria? • Actinobacteria? EUCARYA Cytophaga-like bacteria? • Polysaccharide degradation! • Flavobacteriales • Chitinophaga • Flectobacillus β-Proteobacteria? • Broad freshwater distribution. • Chemoautotrophic bacteria are well represented. • Rhodoferax Actinobacteria? • High G+C Content Gram Positive • Mostly Sporichtya • DNA extraction problems with Gram+? • No strong terrestrial “signal” (e.g. no low Firmicutes). Conclusions • What was learned about bacterial diversity in large rivers? • How well did the metagenomic library represent the bacterial community? • Any weaknesses in sampling or experimental design?