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Primer Design and Sequence Statistics for Phylogenetic DNA Datasets


Choosing and designing primers based on available DNA sequence data and statistical contrasting of domains or structural features is a common routine among molecular biologists. Currently available, free software tools were found to lack desirable features related to these tasks. This was the motivation for developing a new program, SeqState. SeqState locates regions that remain to be sequenced in phylogenetic DNA datasets, evaluates user-provided primers and selects primers best suited to fill gaps in the sequences. If the primers provided by the user are unsuitable, new primers are designed. Primers can be loaded from a primer database, be supplied as part of the alignment or be entered manually. The position of internal primers is automatically localised in the loaded data file. Primers can be edited, and changes and new primers can be saved to the database. Primer sheets allow the user to view internal dimers, complements to a second primer, mismatches to all loaded sequences, and other primer characteristics. Calculation of various sequence statistics can be requested for the whole dataset or parts thereof (character sets), with standard errors estimated by bootstrapping. Insertion-deletion events can be evaluated statistically and encoded for subsequent phylogenetic analysis according to several published coding principles.

Availability: SeqState runs on all major computer platforms and is downloadable for free from, together with documentation and sample data files, or can be requested from the author.

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This work has resulted from the need to frequently design internal primers during studies financed by the Deutsche Forschungsgemeinschaft (DFG), grant number Bo 1815/1 to T. Borsch (DFG project ‘Systematics of Amaranthaceae and evolution of pollen characters’). Many thanks to Professor Barthlott for his constant support and to D. Quandt, C. Löhne, A. Worberg and T. Borsch, as well as to anonymous reviewers, for helpful comments and testing of the program.

The author has no conflicts of interest that are directly relevant to the content of this article.

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Correspondence to Kai Müller.

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Müller, K. SeqState. Appl-Bioinformatics 4, 65–69 (2005).

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  • Sequence Statistic
  • Internal Primer
  • Java Virtual Machine
  • Parsimony Informative Site
  • Provide Confidence Interval