A practical workflow for forensic species identification using direct sequencing of real-time PCR products

Background Forensic scientists are often required to identify species of unknown biological samples. Although methods based on sequencing of DNA barcode regions are the gold standard for species identification in single-source forensic samples, they are cumbersome to implement as routine work in forensic laboratories that perform many tests, including human DNA typing. We have developed a species identification workflow that incorporates direct sequencing with real-time PCR products (real-time PCR–direct sequencing) as the technical trick for easy testing in forensic practice. Method and results Following our workflow, DNA samples from vertebrates, such as mammals, amphibians, reptiles, birds, and fish, were subjected to species identification using vertebrate universal primers targeting each of the four DNA barcode regions. In real-time PCR melting curve analysis, humans and animals (nonhuman) could be differentiated by comparing melting temperatures, and subsequent real-time PCR–direct sequencing contributed to simplified sequencing. Searches against public DNA databases using the obtained sequences were compatible with the origin of the samples, indicating that this method might be used to identify animal species at the genus level. Furthermore, this workflow was effective in actual casework, which provided rapid test results according to the needs of the investigating agencies. Conclusions The species identification workflow will simply sequence as much as possible and can be integrated into routine forensic practice. The real-time PCR–direct sequencing used in this workflow might be beneficial not only for species identification but also for DNA sequencing by using the Sanger method for a variety of life sciences.