toaSTR: A web application for forensic STR genotyping by massively parallel sequencing

Massively parallel sequencing (MPS) is emerging within the forensic community as a promising technique for high-resolution short tandem repeat (STR) genotyping, discovering both length and sequence polymorphisms. Conversely, the application of MPS to routine casework poses new challenges to the DNA analyst in view of the complex sequence data that is generated with this technology. We developed the web application toaSTR to help forensic experts work with MPS data simply and efficiently. An intuitive graphical user interface guides through the STR genotyping workflow. This versatile software handles data from various popular MPS platforms and supports both commercial and in-house multiplex PCR kits. Users can define locus-specific stutter thresholds and create custom sets of STR markers to be analyzed. toaSTR's innovative sequence-based stutter model predicts and identifies common stutter variants. The algorithm automatically differentiates biological (iso-)alleles from stutter and other artefacts to assist the interpretation of mixed samples. toaSTR features a comprehensive data visualization with interactive diagrams and a dynamic tabular overview of sequence observations. The software provides an interface to biostatistics tools and enables PDF result export in compliance with the sequence notation recommended by the International Society for Forensic Genetics (ISFG). An initial compatibility and concordance study confirmed the software's independent functionality and precise allele calling with data of different MPS platforms, STR amplification kits, and library preparation chemistries. Discussion of genotyping results for single source and mixed samples demonstrates toaSTR's advantages and includes suggestions for future MPS software development. The beta version of toaSTR is freely accessible at www.toastr.online.