TaxMan: a server to trim rRNA reference databases and inspect taxonomic coverage

Abstract

Amplicon sequencing of the hypervariable regions of the small subunit ribosomal RNA gene is a widely accepted method for identifying the members of complex bacterial communities. Several rRNA gene sequence reference databases can be used to assign taxonomic names to the sequencing reads using BLAST, USEARCH, GAST or the RDP classifier. Next-generation sequencing methods produce ample reads, but they are short, currently ∼100-450 nt (depending on the technology), as compared to the full rRNA gene of ∼1550 nt. It is important, therefore, to select the right rRNA gene region for sequencing. The primers should amplify the species of interest and the hypervariable regions should differentiate their taxonomy. Here, we introduce TaxMan: a web-based tool that trims reference sequences based on user-selected primer pairs and returns an assessment of the primer specificity by taxa. It allows interactive plotting of taxa, both amplified and missed in silico by the primers used. Additionally, using the trimmed sequences improves the speed of sequence matching algorithms. The smaller database greatly improves run times (up to 98%) and memory usage, not only of similarity searching (BLAST), but also of chimera checking (UCHIME) and of clustering the reads (UCLUST). TaxMan is available at http://www.ibi.vu.nl/programs/taxmanwww/.

Figure 1.

Partial tree view of the amplicons based on the CORE database. For each node, it shows the number of sequences targeted by the given primers, followed by number in the original reference as well as the percentage. The data used for the tree (except the percentages) is downloadable as the tab-delimited lineage file.

Figure 2.

An example of pie plots for the amplicons (CORE database). The distribution of sub-categories within three taxonomic levels, shown as the chart titles, is plotted. The percentage threshold is 0 for all plots. The top panel series is obtained by clicking on Bacteria (Root pie) and Actinobacteria (Bacteria pie). Clicking a pie slice or legend label will produce the next chart and hide the legend of the previous one (except the legend of the Root pie). The bottom panel series of charts is similar, but for the phylum Actinobacteria a plot of differences, indicated by the pink header, is shown. Here, the data refers to the number of sequences missed by the amplicons as compared with the reference data. For the class Actinobacteridae, 46 out of 110 sequences are missing (see legend). The ‘100%’ in the Actinobacteridae pie slice illustrates that all missed sequences in the phylum Actinobacteria belong to the Actinobacteridae class. For Coriobacteridae, no sequences are missing (indicated by 0/9 in the legend). When hovering over a ‘legend’ label, always the number of sequences that are targeted is displayed in the pie (Actinobacteridae; cnt: 64/110). Therefore, this information is the same for both types of pies for Actinobacteria.