The ProReg XL Tool (Profile Regrouping Excel Tool) is a new tool suite designed to rapidly regroup a large number of identical electrophoretic profiles. This tool suite is coded in Visual Basic Application for Microsoft Excel, and thus requires this spreadsheet software to operate. It was designed for use with a new screening strategy of clones from an rrs (16S rDNA) clone library, but it may also be helpful in other electrophoretic applications. ProReg XL Tool is organized in different steps where the user has the capability—in addition to regrouping electrophoretic profiles—to control gel quality, determine signal attenuation, and draw pie charts.
Gel electrophoresis is widely employed in biology to separate and characterize biomolecules, and it is used in proteomic and molecular biology applications. Since an electrophoretic profile is characteristic of a given sample, comparing profiles makes it possible to classify and select a set of samples. Commercial software is available to the scientific community for regrouping electrophoretic profiles by different methods associated to a similarity or a distance matrix generally based on Dice's coefficient (1,2). Profile regrouping abilities of some software packages have been compared and the results published (3,4,5,6). However, in these packages, regrouping results were often limited to a dendrogram representation that remains poorly readable for a large collection of profiles. Furthermore, they are generally expensive.
Our laboratory recently developed a new technique for screening a large number of clones from rrs (16S rDNA) clone libraries. Such a process is sometimes referred to as dereplication (7,8). In brief, this new technique, clone screening by denaturing gradient (CSbyDG), permits one to obtain a specific electrophoretic pattern for each inserted 16S rDNA gene. Regrouping the identical electrophoretic profiles enables limitation of the number of clones necessary to sequence. However, such a regrouping is impossible to accomplish with the naked eye and requires the help of a computer. Due to the limitations of existing software, which specializes in profile regrouping, we needed a software that could rapidly and strictly regroup an important number of profiles.
To address these needs, we developed ProReg XL Tool (Profile Regrouping Excel Tool), a new, easy-to-use tool suite designed for regrouping identical electrophoretic profiles from a large collection of gels. The gels may come from many diverse methods such as random amplification polymorphism DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), allozymes, field inversion gel electrophoresis (FIGE), pulsed field gel electrophoresis (PFGE), or CSbyDG, for example. The application is coded in Visual Basic for Microsoft Excel (Microsoft Corp., Redmond, WA, USA) and is provided online freely to the community for nonprofit research purposes. Regrouping is achieved with a score matrix and multiple-pass strategy. Beyond regrouping, ProReg XL Tool can also do several other data analyses that are particularly helpful for environmental studies. It can control the quality of the gels according to statistical analysis of profile occurrence calculated for each gel and for all gels. It can also define the signal attenuation of new profiles, allowing selection of the best sample number to analyze for the best results. In addition, ProReg XL Tool can draw pie-graphs of the regrouping results with the group designation given by the user.Material and methods System and programming language
The development of ProReg XL Tool was performed on a PC with a 2.6 GHz Intel Pentium IV processor and 512 GB dual-channel SDRAM memory running Microsoft Windows XP (Microsoft Corp.). Since ProReg XL Tool is rich in tables (input data and output results) and in many kinds of graphs, ProReg XL Tool has been developed in Visual Basic for Microsoft Excel, since Excel provides an easy-to-use spreadsheet software with advanced graphical capacities. Consequently, the user must have this spreadsheet software installed in order to operate ProReg XL Tool. ProReg XL Tool is available online for download at www.enitab.fr/recherche/lmba/proreg.html.Regrouping algorithm
The regrouping algorithm is schematized in Figure 1.Figure 1.
Before any calculation, migration distances are put in an increasing order. For each profile, bands k are numbered from 1 to n. A score matrix is then built. Given two electrophoretic profiles A and B with NA and NB bands, respectively, if NA is equal to NB, then the score matrix coordinate (A,B) is filled with score S(A,B). If NA and NB are different, no score S(A,B) is computed and the profiles A and B will be considered to belong to two different groups. Score S(A,B) is defined as the maximum absolute deviation observed between all migration distances of band couples k(A,B) and thus can be written as followed: