To validate amplification specificity and to check for the presence of non-specific amplicons, qPCR assays were performed using 66 randomly selected genes from thale cress, oilseed rape, rice (Oryza sativa), sweet orange (Citrus sinensis), silkworm (Bombyx mori) and zebrafish (Danio rerio). In these qPCR experiments, only the best primer pair for each gene from qPrimerDB was selected, as summarized in Supplementary Table S2 . To ensure the reliability of experimental results, the setup and validation of qPCR were performed, as prescribed in the MIQE guidelines (26 (link)). Based on the results of melting curve analysis and gel electrophoresis, all selected primer pairs were demonstrated to be of high specificity and amplification efficiency (Supplementary Figure S1 ).
In addition, to test the specificity and accuracy of the primer design workflow used in qPrimerDB, we collected 454 and 137 qPCR primer pairs for A. thaliana and B. mori, respectively, from 55 previously published studies (Supplementary Table S3 ). Using the validation and classification steps in the qPrimerDB workflow, the specificity and classification of all collected primers were determined. The results showed that the ratios of gene-specific primers were 75.87% for A. thaliana and 98.23% for B. mori in qPrimerDB, whereas values collected from the published studies were only 71.09 and 79.65%, respectively (Supplementary Table S4 ). These assays established that the automatic primer design workflow developed in qPrimerDB generated high quality gene-specific qPCR primers, whose specificity and consistency were superior to those of manually designed primers.
We also compared the qPCR primers for H. sapiens generated with MRPrimer and qPrimerDB workflows. For both two methods, only the best pre-commutated primers were retained and used for specificity and classification comparison. The results showed that a total of 14 500 MRPrimer and 17 632 qPrimerDB qPCR primers were gene-specific, and the ratio of level 1 primer in qPrimerDB was 50.36%, much higher than that in MRPrimer (13.14%) (Supplementary Table S5 ), indicating that the qPrimerDB workflow might well be one of the most effective gene-specific qPCR primer design tools, suitable for automatic primer design for newly sequenced genomes.
In addition, to test the specificity and accuracy of the primer design workflow used in qPrimerDB, we collected 454 and 137 qPCR primer pairs for A. thaliana and B. mori, respectively, from 55 previously published studies (
We also compared the qPCR primers for H. sapiens generated with MRPrimer and qPrimerDB workflows. For both two methods, only the best pre-commutated primers were retained and used for specificity and classification comparison. The results showed that a total of 14 500 MRPrimer and 17 632 qPrimerDB qPCR primers were gene-specific, and the ratio of level 1 primer in qPrimerDB was 50.36%, much higher than that in MRPrimer (13.14%) (