This tutorial will take the reader through the analytical functions of the Ribosomal Database Project. The tutorial assumes that the reader has basic computer skills as well as at least an advanced undergraduate standing in molecular biology and evolution. The window in which the tutorial appears will open when the Begin Tutorial button is clicked. The window from which you are now reading will be used by the tutorial pages to present the analytical functions of the RDP. Hence if you select Sequence Match from the tutorial Table of Contents (TOC), the window containing the tutorial text will advance to the selected section and the main browser window (this window) will advance to the Sequence Match page of the RDP. Navigation buttons are provided on the top/bottom of pages within the tutorial. The following introductory pages describe sequence analysis at the RDP.

Before we begin the tutorial, let s look at the structure of the RDP. The RDP was initially designed as a repository of rRNA sequences for use by investigators interested in phylogeny as well as rRNA structure and function. To that end, rRNA sequences were deposited along with annotation that included the source of the sequence and references to the literature. The sequences are currently stored in flat text files that can be queried in many different ways. More importantly, the sequences are curated by the RDP, meaning that when a new sequence is added to the RDP, it is aligned to all of the sequences in the current database release and the references are checked. The alignment is based on models of secondary and tertiary structure of rRNA derived from chemical, physical, and comparative sequence analyses (see Figure 1). Keep in mind that no alignment is perfect (see below).

Figure 1. Secondary structure model for E. coli 16S

Because sequences are added to databases on a daily basis, there are two files maintained by the RDP, one containing aligned sequences and one containing newly deposited sequences that have yet to be aligned. The unaligned sequences are included in the RDP analysis functions wherever possible because they represent a significant collection of information. In some instances however, the nature of the query demands that only the aligned sequence database is addressed. The general structure of the RDP, presented in Figure 2, depicts a user addressing the database through the provided analytical functions. The two diagramed queries address the RDP through either the aligned database (eg. Sequence_Align function), or the complete database including both the aligned and unaligned databases (eg. Sequence_Match function).

Figure 2. The structure of the RDP at Michigan State

All analytical functions of the RDP are accessed by clicking the Online Analyses button on the RDP homepage (Figure 3). Doing so takes the user to a page that lists the analytical functions available online.

Figure 3. Entering the Analyses Page of the RDP

Online Analyses functions currently available at the RDP are graphically listed in Figure 3. The first page of Online Analyses presents a list of functions that is hypertext-active with buttons linking the user to detailed descriptions of each function (the info buttons) as well as to the work page of each analysis function. In addition, the database within the RDP that is accessed for each analysis function is indicated. As described above, some functions address the aligned database and others require only unaligned sequences. In the latter case, all sequences currently stored in the database, including both aligned as well as those that have yet to be brought into alignment, would be queried. The analysis functions are described briefly below. Probe Match searches the complete database for sequences that match a user-provided oligonucleotide sequence (probe). Sequence Match searches the complete database for the sequences that most closely match a user-provided sequence. Sequence Aligner searches the aligned database for sequences that most closely match a user-provided sequence and aligns the submitted sequence with the identified closely related sequences. Similarity Matrix searches the aligned database for sequences that most closely match a user-provided sequence; aligns the submitted sequence with related sequences; creates a mask or filter that eliminates all ambiguous positions, and calculates a similarity matrix based on "identity" of characters at the unambiguous positions. Chimera Check attempts to find a "breakpoint" at which the inferred phylogenies from the regions on each side of the point are different in a user-provided sequence. Alignment Slices permits the user to specify a region of the 16S rRNA alignment to download. T-RFLP constructs a similarity matrix for T-RFLP profiles provided by the user. TAP-TRFLP this function allows the user to perform an in silico digestion of the entire database and determine the size of terminal restriction fragments generated through a user-provided primer sequence and a user-selected restriction enzyme(s). Sub-Trees this function constructs a graphic java display of phylogenetic trees provided by either the RDP (17 provided) or the user. Sequence Selection this function enables the user to select specific sequences or entire phylogenetic groups to download. Phylip Interface NEW! this function allows the user to construct UPGMA or Neighbor Joining trees through a graphic interface with Phylip 3.5c. Both user-supplied sequences and RDP-selected sequences can be incorporated into the analysis. Matrices and trees can be downloaded in several formats.

Figure 4. Flow chart of RDP Tutorial

The order of analysis functions presented in this tutorial is designed to reflect a typical scenario of investigators in the field. Frequently a microorganism is isolated and its rDNA is cloned and sequenced. The investigator then connects to the RDP and attempts to compare his/her sequence with sequences in the database in order to identify the closest phylogenetic relative. This working scenario will guide us through the analytical functions of the RDP. The series of steps in this scenario are presented in Figure 4. Note that the first analysis function discussed is Chimera Check. This is the logical starting point in as much as an initial triage that eliminates chimeras will conserve time and energy. The remaining analysis functions are described in the order presented in Figure 4.

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