PrimerProspector: de novo design and taxonomic analysis of barcoded polymerase chain reaction primers

Abstract

Motivation: PCR amplification of DNA is a key preliminary step in many applications of high-throughput sequencing technologies, yet design of novel barcoded primers and taxonomic analysis of novel or existing primers remains a challenging task.

Results: PrimerProspector is an open-source software package that allows researchers to develop new primers from collections of sequences and to evaluate existing primers in the context of taxonomic data.

Availability: PrimerProspector is open-source software available at http://pprospector.sourceforge.net

Contact: rob.knight@colorado.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

Fig. 1.

Taxonomic coverage summary of the 515f/806r 16S SSU rRNA primer pair at the phylum level for (A) archaea, (B) eukarya and (C) bacteria. The y-axes represent percent coverage and the value on top of each bar is the total number of reference sequences in each taxon. In this analysis, the reference sequences were derived from the Silva database, and filtered at 97% sequence identity with uclust (Edgar, 2010). Archaeal and bacterial sequences shorter than 1450 bases, and eukaryotic sequences less that 1800 bases, were excluded from the reference set. As illustrated, this primer pair is nearly universal for archaeal and bacterial 16S but is generally poor for eukaryotic (notably metazoan) 18S sequences. This plot and additional PrimerProspector analyses informed the decision to use this primer pair in Caporaso et al. (2010), Bates et al. (2010) and G.Bergmann et al. (2010). Comparisons with the unoptimized primer pair and with an alternative popular pair (27f/338r) are shown as Supplementary Figures S1 and S2, respectively.