REPK: an analytical web server to select restriction endonucleases for terminal restriction fragment length polymorphism analysis

W58–W62 Nucleic Acids Research, 2007, Vol. 35, Web Server issue doi:10.1093/nar/gkm384 REPK: an analytical web server to select restriction endonucleases for terminal restriction fragment length polymorphism analysis Roy Eric Collins and Gabrielle Rocap* School of Oceanography, University of Washington, Seattle WA, USA Received January 31, 2007; Revised April 18, 2007; Accepted April 30, 2007 ABSTRACT INTRODUCTION Terminal restriction fragment length polymorphism (T-RFLP) analysis is a microbial fingerprinting technique capable of discriminating microbial communities quickly and relatively inexpensively (1–3). T-RFLP is increasingly used in high-throughput studies of microbial communities in combination with or even in lieu of clone library analysis (4,5). Briefly, the method involves PCR amplification of a gene of interest (often 16S rRNA genes) with fluorescent dye-labeled primers, followed by multiple single restriction digests done in parallel. The resulting fragments are then separated by capillary electrophoresis with an internal size standard to determine the lengths of the terminal (fluorescently labeled) fragments. Each distinct terminal restriction fragment is considered an operational taxonomic unit (OTU), thus the choice of *To whom correspondence should be addressed. Tel: 206 685 9994; Fax: 206 685 6651; Email: ß 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widespread technique for rapidly fingerprinting microbial communities. Users of T-RFLP frequently overlook the resolving power of well-chosen restriction endonucleases and often fail to report how they chose their enzymes. REPK (Restriction Endonuclease Picker) assists in the rational choice of restriction endonucleases for T-RFLP by finding sets of four restriction endonucleases that together uniquely differentiate userdesignated sequence groups. With REPK, users can provide their own sequences (of any gene, not just 16S rRNA), specify the taxonomic rank of interest and choose from a number of filtering options to further narrow down the enzyme selection. Bug tracking is provided, and the source code is open and accessible under the GNU Public License v.2, at http://code.google.com/p/repk. The web server is available without access restrictions at http:// rocaplab.ocean.washington.edu/tools/repk. restriction enzymes can impact the number of OTUs observed in each sample and the calculation of diversity statistics. When analyzing uncharacterized and very diverse bacterial communities, sufficient community discrimination can often be accomplished with multiple randomlychosen tetrameric restriction enzymes (6). However, a brief review of the literature indicates that there is still no standard in even this simplified case. We examined 26 papers (1–5,7–26) that were published between 1997 and 2007 and used T-RFLP. Of those papers, 38% used universal bacterial primers combined with a single restriction enzyme, but the choice of enzyme was not consistent. MspI was used most frequently (four studies), followed by TaqI (two studies), and one study each used AluI, CfoI, HhaI and HaeIII. Overall, only three of the 26 papers included a rationalization of enzyme selection (1,2,17). An alternate approach to T-RFLP can be taken if the microbial community has been characterized (by clone library analysis or by prediction from previous studies) or if a particular taxonomic group is being targeted with specific primers. In this case, a more reasoned choice of restriction enzymes can be conducted. In particular, specific species or microbial taxa of interest to the researcher—particularly closely related taxa that may share some restriction sites—can often be differentiated if the proper restriction enzymes are selected. There are, however, few resources available to narrow down the selection process. Over 600 Type II restriction enzymes are commercially available, accounting for 262 distinct specificities (27). Existing computer programs for assisting in the choice of restriction enzymes include TAPTRFLP (28), MiCA Enzyme Resolving Power Analysis (http://mica.ibest.uidaho.edu) and TRF-CUT (29). These programs perform in silico restriction digestions of a predefined sequence database or user-provided sequences, but these results must still be manually examined to determine which enzymes are best suited to discriminate that set of sequences. CLEAVER (30), a stand alone program, provides the above features as well as the ability Nucleic Acids Research, 2007, Vol. 35, Web Server issue W59 to assign sequences to taxonomic groups at multiple levels and to search for enzymes that cut one group but not another group. However, it is limited to comparing only two groups at once. Restriction Endonuclease Picker (REPK) addresses this gap by finding enzymes that are able to discriminate an unlimited number of userdesignated sequence groups on the basis of their terminal restriction fragment lengths. If no single enzyme can discriminate all groups, REPK reports sets of four restriction enzymes that together are able to differentiate the groups of interest. An important component of REPK is this ability to specify the taxonomic rank of sequences to be differentiated, which is particularly useful in the case where a diverse microbial community has been characterized by clone library analysis or there is an existing database of several subgroups of sequences that amplify with the same specific primers. Finally, users can define their own custom enzymes if they are not included in the standard list. The default (all standard enzymes) was used for the example in Figure 1. For computational efficiency isoschizomers are grouped by cleavage site. The final output is refined by setting several options. Some of these, the minimum and maximum allowable fragment lengths and the maximum difference in size between two fragments that will still be considered the ‘same’ fragment, will be dependent on the specifications and resolving power of particular capillary electrophoresis systems. Users can also set the minimum threshold for the number of groups each enzyme must be able to discriminate on its own (the enzyme stringency), and the number of groups allowed to remain undifferentiated in the case that no ‘perfect’ enzyme groups are discovered. SITE USAGE AND EXAMPLES A complete manual and example input files are provided on the REPK website (http:// rocaplab.ocean.washington. edu/tools/repk). The example shown in Figure 1 was prepared using REPK v. 1.0, with the following operating parameters (also the defaults): example sequence file (alignment5.txt), all commercially available Type IIP enzymes (REBASE Versio (...truncated)