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SeqState Description

Primer design, sequence statistics, and indel coding according to various schemes

Although many other features have been added since 2003, one of the main purposes of SeqState remains automated primer design. For this, the alignment (NEXUS format; non-interleaved; see sample files) is screened for aligned internal primers, which also may be loaded from and saved to resource files. External primers (upstream and downstream of the alignment) can be specified as well. In each sequence, SeqState searches for stretches of missing data ("?"), which may contain, start with, or end with indel gap characters ("-") resulting from insertion of gaps in multiple sequences simultaneously. For each region of missing data, all primers are evaluated in terms of distance to the region and fit to the matching part of the sequence to be completed. Degeneracy of primers as well as ambiguity code in the template sequence is correctly interpreted, and mismatches in the head region receive particular attention.

For each section of absent data, the best primers are provided in the output. If no suitable primers are found, SeqState screens the sequence adjacent to the missing nucleotides (subtracting "-" and jumping over ambiguities) and suggests new primers for synthesis. To select the best primers, the program evaluates nucleotide composition of head and tail region, maximum length of primer dimer complements, primer specificity and annealing temperature, as well as the percentage of other sequences in the alignment to which the primer fits. SeqState allows specification and saving of user assumptions (e.g., the lengths of primer reads which strongly depend on the sequencer used).

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