Graphical representation of ribosomal RNA probe accessibility data using ARB software package

The introduction and use of comparative sequence analysis of appropriate marker genes as a powerful tool in taxonomy has substantially contributed to the rapid growth of molecular sequence databases such as EMBL [1], GenBank [2], and ribosomal RNA (rRNA) databases [3–5]. Evidently, molecular phylogenetic analyses have greatly influenced the restructuring of systematics especially in the case of prokaryotes. Nowadays, identification and classification at the species and higher taxonomic levels mainly relies on a genotypic approach, typically involving an analysis of small, and to a lesser extent, large ribosomal RNA gene (rRNA) structures. The backbone of the current taxonomy of the prokaryotes is almost exclusively based upon a phylogenetic network derived from comparative sequence analysis of the small subunit rRNAs and respective phylogenetic marker genes [6]. As 'living fossils', these molecules at least roughly reflect the evolutionary history of the respective organisms. The mosaic-like primary structures comprising highly variable to highly conserved or invariant regions provide diagnostic information for different levels of phylogenetic relationship. Consequently, this information can be used to identify oligonucleotide target regions unique to phylogenetic entities, for use as taxon-specific hybridization probes or PCR primers. Depending on the target site such oligonucleotide probes or probe combinations can be designed for phylogenetic groupings as diverse as bacterial species or an entire phylum.

Ever since the fluorescence in situ hybridization (FISH) technique became an integral part of the rRNA approach to microbial ecology and evolution [7], rRNA-targeted oligonucleotide probes have evolved into a widely used tool for the direct, cultivation-independent identification and enumeration of individual microbial cells or specific groups of bacteria in simple to complex natural environments. In this regard, a good probe design and careful further evaluation in silico plays a crucial role to ensure sensitivity and specificity of a potential probe in its practical application. Besides uniqueness of the target sequence, number, character and position of diagnostic residues, comprehensiveness with respect to the inclusion of members of the desired target group (taxon) and exclusion of non-members along with a target molecule or region accessibility in the real hybridization experiment, have to be taken into consideration. Recently, data on in situ accessibility of rRNA targets in several microorganisms have become available [8–11].

Since biology is a highly visual science, there is a general demand for tools to visualise the variety of biological knowledge as diagrams, illustrations, two-dimensional and three-dimensional reconstructions, and other types of graphical formats. Hence, the visualization of molecular data in an interactive and intuitive graphical user interface ideally will serve as third eye for a molecular biologist. In this paper, we describe how the ARB software package [3] provides a workbench for designing, evaluation and visualization of oligonucleotide probes in more intuitive way, using interactive graphical user interface to visually examine characteristics and criteria of target regions.

As the demand for oligonucleotide probes that can identify and quantify bacteria by nucleic acid hybridization is permanently increasing, in silico evaluation and visualization of such probes and targets are necessary, particularly, when used for FISH experiments. Target accessibility is among the crucial criteria to be evaluated with respect to experimental success of the respective probe based identification and detection system [7–12]. To facilitate this evaluation procedure, new functionalities were added to the ARB software package providing a more intuitive graphical environment. As an example, oligonucleotide probes were designed for the enterobacteria group represented by 947 database entries. The 5'-UGGAGGGGGAUAACUACU-3' probe was selected from the list of potential probes and evaluated against the background of the full dataset of complete and partial small subunit rRNA sequences. The selected probe perfectly matches the respective target of 497 members of the enterobacteria group (Figure 1). The same probe has been visualized in all the screenshots presented in the paper.

Although a phylogenetic probe is primarily judged in terms of its taxonomic range to identify the members of its intended target taxon to the exclusion of non-target bacteria, for a practical consideration it must also fulfil certain other criteria with respect to its applicability depending on the particular hybridization format. In case of the fluorescence in situ hybridization approach the results of the accessibility studies conducted by Fuchs and co-workers on the 16S and 23S rRNA of Escherichia coli and other organisms are among such criteria. They showed that some regions of E. coli ribosome are virtually inaccessible for oligonucleotide probes when FISH is performed [8, 9]. They proposed a color code assigned to six intensity classes of in situ hybridization signals. Within the ARB program, these classes are coded in respective SAIs (so called Sequence Associated Information) and optionally visualized as background colors of the sequences in primary structure (ARB_Edit4), secondary structure (SEC_Edit), and probe visualization windows (PROBE Match) of ARB. All the displays produced by the ARB software are interconnected and any changes in one window are automatically updated in other windows as well. Simultaneous visualization and evaluation of oligonucleotide probes in different levels allows the user to look carefully and closely into the proposed probe candidates in silico, before carrying out further in situ or in vivo studies. More importantly, the user can perform a variety of sequence related operations such as importing sequence data, aligning, treeing, designing, evaluation and visualization of probes, performing statistical calculations and many other functions using interoperating and user friendly tools controlled from a common graphical platform within the ARB software package.

Visualization of potential probe candidates and the sequence associated information (SAI) such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ target accessibility patterns, is possible at three different levels: the local alignment (PROBE Match tool), global alignment (ARB Primary Structure Editor) and secondary structure levels (Secondary Structure Editor).

Visualization of SAI in probe match window

Visualization of probe candidates in a local alignment along with additional sequence associated information (SAI) can be managed with the PROBE Match SAI window. The neighboring region up to nine nucleotides on either terminus of the potential probe target is retrieved from the database. A local alignment of the extracted rRNA sequence is established and displayed along with the respective unique identifier such as ARB short_name, accession number, or any other underlying database fields (eg., Full Name, Group) (Figure 2, 3, 4). The user can select any information that is associated with the sequences (SAI) such as secondary structure masks (Figure 2) or any statistical calculations performed on the sequence level like sequence consensus, positional variability using parsimony method (Figure 3) or any other user defined models, filters or statistics as well as in situ accessibility maps for visualization (Figure 4). Different background colors can be assigned to characters and values or character groups and ranges of values of the particular SAIs, respectively. Optionally, the real characters or values contained in such SAIs can directly be visualized below the individual sequences. This offers a researcher a deeper insight in to the proposed oligonucleotide probe targets for careful examination of probe candidates in silico before making any decision on the selection of probe.

Visualization of SAI in ARB primary structure editor

On the global alignment level, the user selected oligonucleotide probe is visualized in different background colors in the primary structure editor window of ARB [3]. The primary structure editor (Figure 5) of ARB displays multiple sequence alignments generated by the respective ARB software tools [3] of the selected sequences from the underlying database in the user-defined colors and symbols.

As already described for the local alignment level, any type of SAI can be visualized by the user defined background colors for the individual alignment columns. Customized color selections can be assigned to the different types of SAIs mentioned before. By scrolling the mouse or the use of ARB search tools, the user gets an easy access to the information for any range or the selection of sequences. In the context of probe evaluation for in situ hybridization experiments, mapping of experimentally derived in situ accessibility patterns onto the primary structures of interest certainly provides valuable support to the users for probe evaluation. Part of a multiple 16S rRNA sequence alignment is shown in the figure 5. The brightness classes defined for 16S rRNA structural model of Methanosaeta sedula [11] are mapped on the aligned sequences and indicated by background colors according to Behrens et al [11].

The evaluation of proposed probe target position with respect to higher-order rRNA structure is of more importance especially when probes are intended to be used for in situ hybridizations [7–12]. Albeit there have been several software programs developed for the design of rRNA targeted oligonucleotide probes [18, 19], the criteria taken to design the probes are generally restricted to certain parameters such as size, nucleotide composition, specificity definition, and the general hybridisation behavior. None of the software described [18, 19] takes into account the special requirements of rRNA targeted probes that are destined for FISH applications which is, the structure dependant probe accessibility of the ribosomal RNA. This feature has been developed and implemented in ARB. Using this tool, in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. By identifying and excluding the probes targeting sites with a poor accessibility the number of time consuming empirical tests can be reduced.

The entire ARB software and the periodic updates of well aligned and annotated ribosomal RNA databases are made freely available for the scientific community via World Wide Web [13]. Currently, the ARB Software is available for PCs running LINUX operating systems and SUN SOLARIS systems.