Developmental validation of SpeID: A pyrosequencing-based assay for species identification.

In crime scenes, biological exhibits are often human in origin, yet biological stains from other fauna may also be present at a crime scene, creating confusion during an investigation. Furthermore, identifying the source of a biological sample can be critical during an investigation. To identify the presence of biological material from non-human sources, it is common to use genetic markers within mitochondrial DNA such as cytochrome b, 16S rRNA, and 12S rRNA genes. This process usually requires DNA sequencing, a process that is neither quick nor easy. In general, a faster, more standardized method for species identification from tissue and body fluids is desirable.For this reason, we have developed a vertebrate specific real-time quantitation method that is followed by an automated pyrosequencing-based procedure that sequences a short fragment within the 12S rRNA gene. Using no more than 35 bases, the assay can distinguish between 32 different species commonly found in and around a household with a turnaround time of 6 h from extraction to sequencing. -Using this procedure, up to 48 samples can be run at a time without the need for expensive reagents or bioinformatic skills.