Fig. 2

Testing and validation of PrimerView. a Performance testing of PrimerView. Files containing different numbers of sequences (10, 20, 50, 100, 200, 400, 1000, 20,284 – obtained from WormBase ftp site: ftp://ftp.wormbase.org/pub/wormbase/) were provided as input to PrimerView and ran using default settings to generate PNG file primer distribution outputs. Fitting the relationship between sequence number and run-time with a quadratic equation yields an R² value of 1.00. b Comparison of primer T _m values between PrimerView with Primer3 [10]. To perform these tests only the calcTm subroutine within PrimerView was called for 400 primers designed using Primer3 [10]. A robust correlation (R² value of 0.97) was observed between each method, both of which employ nearest-neighbour parameter sets. c Specificity and orientation of primers returned using PrimerView was tested using the MFE-Primer 2.0 in silico PCR tool (http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/) [11, 12]. Sample output from one test showing the sequence file (NC_003284.7) in FASTA format, and displaying all sequence matches in the database that lie between and include the primer pair (Seq2 and Seq1). The FASTA header details the region in the database as well as the primers; the body of the FASTA sequence is capitalized in areas where the primer sequence matches the database sequence and in lower-case elsewhere