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Evaluation of primer pairs using TestPrime 1.0

TestPrime allows you to evaluate the performance of primer pairs by running an in silico PCR on the SILVA databases. From the results of the PCR, TestPrime computes coverages for each taxonomic group in all of the taxonomies offered by SILVA. These coverages can then be inspected in our taxonomy browser, making it easy to quickly identify strengths and weaknesses of a particular pair of primers.

Please be aware that an in silico evaluation doesn’t guarantee good lab results. Please consider SILVA TestPrime as a first step for selecting the most appropriate primer pair for specific applications.

Features

  • Combined evaluation of forward and reverse primers
  • Configurable stringency parameters
    • number of mismatches
    • enforced "perfect match" zone at 3' end of primer
  • Coverage summaries for all taxonomic units in SILVA
  • Graphical result presentation with taxonomy drill-down
  • Result file download in CSV format

Overview

TestPrime relies on the "ARB PT server" to run the in silico PCR. First, both primers are resolved into sets of "wobble"-free oligos. Each olligo is then analyzed by the PT server according to the configured stringency parameters. The results are merged and cleaned and the sequences sorted into to three groups: match, mismatch and nodata. The last group contains all sequences for which no clear decision could be made. Most commonly, this is the case when the sequence in question does not cover the primer match position. The coverage values are then computed for each taxonomic unit by dividing the number of matched sequences in that group by the number of matched or mismatched sequences. Additionally, the relative proportions of match, mismatch and nodata and the relative proportions of mismatches and "nodata" for the forward and/or reverse primer are computed to help determining whether the forward or the reverse primer was the cause for high mismatch or "nodata" rates.

Documentation

Opens internal link in current windowTestPrime Tutorial

Reference

Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M. and Glöckner, F.O. (2012) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Opens external link in new windowNucleic Acids Res. 2013 Jan 1;41(1):e1. doi: 10.1093/nar/gks808