Terminal Restriction Fragment Length Polymorphism for Soil Microbial Community Fingerprinting
Terminal restriction fragment length polymorphism (TRFLP) fingerprinting of 16S rRNA genes is a popular technique for analyzing bacterial communities. This review considers the technical aspects of deriving a community fingerprint with the goal of improving TRFLP as a tool for community ecologists, particularly those working in soil science. Analysis parameters are reviewed for samples run with GeneScan and the capillary system Peak Scanner software of ABI Prism genetic analysis units. Analysis parameters should be chosen using principles that preserve community composition and define inherent community variability, ensuring that methodology does not bias the distribution of an unknown population structure. Choices for fragment sizing include sizing algorithm, sizing accuracy, peak detection, split peak correction, peak smoothing options, minimum peak half width, baseline setting, and analysis range. Results indicate that a standard protocol should include choices of local southern sizing and use of a 35 relative fluorescence unit baseline with light smoothing. A size calling accuracy of 0.998 (for GeneScan gels) is recommended for each internal lane standard, based on the principle that the extent of linearity across the size standard selection is the given limit for an analysis range. Analysis baseline is a choice that in combination with heavy peak smoothing can halve the number of fragments detected. Heavy smoothing will increase the number of replicate polymerase chain reaction samples necessary to represent all fragments of a soil community DNA and it is proposed that using heavy smoothing will produce a biased community profile and loss of population information. Choices during profile and alignment editing are also discussed. Generated profiles should be edited to ensure a minimum integrity of one base pair resolution between consecutive fragments. Results indicate that profile editing, for a specified analysis range of 80 to 827 base pairs, can decrease the linear relationship between total fluorescence and fragment number and thus increase independence between abundance and species richness.Please view the pdf by using the Full Text (PDF) link under 'View' to the left.
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