Phylogenetic Analysis of Terminal Restriction Fragments (tRFs)
TReFID is a tool for analyzing terminal restriction fragments of multiple restriction enzyme digests. It allows to create "species lists" on the basis of tRFLP data from complex microbial communities. An impression on how it works is given by figures 1 through 5, below. [AEM paper on TReFID] [Download TReFID]
Currently databases for three genes are available (included in the download packages, see below):
Note: The TReFID software has no web interface; all required components - including databases for the three genes - are included in these packages.
In case of nifH and nosZ all sequences published in GenBank (Jan 2004 and Feb 2005, respectively) were tested for TReFID. Only sequences containing the binding site for the fluorochrome-labelled PCR primers used in tRFLP analyses were included in the databases. These were the primers nifH-F (aaa ggY ggW atc ggY aaR tcc acc ac), binding near the 5' end of nifH, and nosZ-R (tcc atg tgc agN gcR tgg cag aa), binding close to the 3' terminus of nosZ. Because the nifH and nosZ genes have no motifs comparably highly conserved as some regions in the SSU gene, and because of the limited sequence information, primers targetting these genes are "less universal" than established SSU primers like 8F, 27F, or 63F. As a consequence, the primers nifH-F and nosZ-R allow to exploit only about half the available sequence information for the respective gene. However, this proportion would not have been larger using alternative primers. Concerning the SSU gene, about 40,000 out of 150,000 GenBank sequences published in Feb 2005 were tested. Of these, about 21,000 sequences contained in the TReFID SSU database (version B) included the binding site for 63F, the primer used for tRFLP. Database overview
Note: all three databases are included in the ZIP packages above and need not be downloaded separately. This is for future updates or new databases. New databases can be added by copying the unzipped .TReFID.org files into the TReFID\source subdirectory. Existing databases can be altered and extended with user-specific data in any text editor.
The figures below are taken from the original publication on TReFID (C Rösch & H Bothe. 2005. Appl. Environ. Microbiol. 71(4) 2026-2035). To obtain high quality images of these figures click on the respective image. For figure legends and detailed background information follow the links.