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


      • toaSTR is a novel online tool for simple and efficient STR genotyping with MPS data.
      • Flexible configuration supports both custom and commercial STR kits.
      • Stutter prediction and automatic allele calling help to interpret results.
      • Universal usability was demonstrated with data from various MPS platforms.


      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


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Forensic Science International: Genetics
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Gettings K.B.
        • Kiesler K.M.
        • Faith S.A.
        • Montano E.
        • Baker C.H.
        • Young B.A.
        • Guerrieri R.A.
        • Vallone P.M.
        Sequence variation of 22 autosomal STR loci detected by next generation sequencing.
        Forensic Sci. Int. Genet. 2016; 21: 15-21
        • Novroski N.M.M.
        • King J.L.
        • Churchill J.D.
        • Seah L.H.
        • Budowle B.
        Characterization of genetic sequence variation of 58 STR loci in four major population groups.
        Forensic Sci. Int. Genet. 2016; 25: 214-226
        • Churchill J.D.
        • Schmedes S.E.
        • King J.L.
        • Budowle B.
        Evaluation of the Illumina® Beta Version ForenSeqTM DNA Signature Prep Kit for use in genetic profiling.
        Forensic Sci. Int. Genet. 2016; 20: 20-29
        • Zeng X.
        • King J.
        • Hermanson S.
        • Patel J.
        • Storts D.R.
        • Budowle B.
        An evaluation of the PowerSeqTM Auto System: a multiplex short tandem repeat marker kit compatible with massively parallel sequencing.
        Forensic Sci. Int. Genet. 2015; 19: 172-179
        • Guo F.
        • Zhou Y.
        • Liu F.
        • Yu J.
        • Song H.
        • Shen H.
        • Zhao B.
        • Jia F.
        • Hou G.
        • Jiang X.
        Evaluation of the Early Access STR Kit v1 on the Ion Torrent PGMTM platform.
        Forensic Sci. Int. Genet. 2016; 23: 111-120
        • Wang Z.
        • Zhou D.
        • Wang H.
        • Jia Z.
        • Liu J.
        • Qian X.
        • Li C.
        • Hou Y.
        Massively parallel sequencing of 32 forensic markers using the Precision ID GlobalFilerTM NGS STR Panel and the Ion PGMTM System.
        Forensic Sci. Int. Genet. 2017; 31: 126-134
        • Young B.
        • King J.L.
        • Budowle B.
        • Armogida L.
        A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis.
        PLoS One. 2017; 12
        • Bragg L.M.
        • Stone G.
        • Butler M.K.
        • Hugenholtz P.
        • Tyson G.W.
        Shining a light on dark sequencing: characterising errors in ion torrent PGM data.
        PLoS Comput. Biol. 2013; 9e1003031
        • Schirmer M.
        • Ijaz U.Z.
        • D’Amore R.
        • Hall N.
        • Sloan W.T.
        • Quince C.
        Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform.
        Nucleic Acids Res. 2015; 43 (e37–e37)
        • Hoogenboom J.
        • van der Gaag K.J.
        • de Leeuw R.H.
        • Sijen T.
        • de Knijff P.
        • Laros J.F.J.
        FDSTools: a software package for analysis of massively parallel sequencing data with the ability to recognise and correct STR stutter and other PCR or sequencing noise.
        Forensic Sci. Int. Genet. 2017; 27: 27-40
        • Børsting C.
        • Morling N.
        Next generation sequencing and its applications in forensic genetics.
        Forensic Sci. Int. Genet. 2015; 18: 78-89
        • Alonso A.
        • Müller P.
        • Roewer L.
        • Willuweit S.
        • Budowle B.
        • Parson W.
        European survey on forensic applications of massively parallel sequencing.
        Forensic Sci. Int. Genet. 2017; 29: e23-e25
        • Illumina
        ForenSeqTM Universal Analysis Software Guide.
        2016 (Accessed 27 November 2017)
      1. Applied Biosystems, HID STR Genotyper Plugin User Guide.
        2017 (Accessed 27 November 2017)
        • Gymrek M.
        • Golan D.
        • Rosset S.
        • Erlich Y.
        lobSTR: a short tandem repeat profiler for personal genomes.
        Genome Res. 2012;
        • Woerner A.E.
        • King J.L.
        • Budowle B.
        Fast STR allele identification with STRait Razor 3.0.
        Forensic Sci. Int. Genet. 2017; 30: 18-23
        • Liu Y.-Y.
        • Harbison S.
        A review of bioinformatic methods for forensic DNA analyses.
        Forensic Sci. Int. Genet. 2018; 33: 117-128
        • Magoč T.
        • Salzberg S.L.
        FLASH: fast length adjustment of short reads to improve genome assemblies.
        Bioinformatics. 2011; 27: 2957-2963
        • Bushnell B.
        • Rood J.
        • Singer E.
        BBMerge – accurate paired shotgun read merging via overlap.
        PLoS One. 2017; 12e0185056
        • Gettings K.B.
        • Aponte R.A.
        • Vallone P.M.
        • Butler J.M.
        STR allele sequence variation: current knowledge and future issues.
        Forensic Sci. Int. Genet. 2015; 18: 118-130
        • Walsh P.S.
        • Fildes N.J.
        • Reynolds R.
        Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus VWA.
        Nucleic Acids Res. 1996; 24: 2807-2812
        • Klintschar M.
        • Wiegand P.
        Polymerase slippage in relation to the uniformity of tetrameric repeat stretches.
        Forensic Sci. Int. 2003; 135: 163-166
        • Brookes C.
        • Bright J.-A.
        • Harbison S.
        • Buckleton J.
        Characterising stutter in forensic STR multiplexes.
        Forensic Sci. Int. Genet. 2012; 6: 58-63
        • Bright J.-A.
        • Stevenson K.E.
        • Coble M.D.
        • Hill C.R.
        • Curran J.M.
        • Buckleton J.S.
        Characterising the STR locus D6S1043 and examination of its effect on stutter rates.
        Forensic Sci. Int. Genet. 2014; 8: 20-23
        • van der Gaag K.J.
        • de Leeuw R.H.
        • Hoogenboom J.
        • Patel J.
        • Storts D.R.
        • Laros J.F.J.
        • de Knijff P.
        Massively parallel sequencing of short tandem repeats—population data and mixture analysis results for the PowerSeqTM system.
        Forensic Sci. Int. Genet. 2016; 24: 86-96
        • Faith S.A.
        • Scheible M.
        Analyzing Data from Next Generation Sequencers Using the PowerSeq® Auto/Mito/Y System.
        2016 (Accessed 7 August 2017)
        • Parson W.
        • Ballard D.
        • Budowle B.
        • Butler J.M.
        • Gettings K.B.
        • Gill P.
        • Gusmão L.
        • Hares D.R.
        • Irwin J.A.
        • King J.L.
        • de Knijff P.
        • Morling N.
        • Prinz M.
        • Schneider P.M.
        • Neste C.V.
        • Willuweit S.
        • Phillips C.
        Massively parallel sequencing of Forensic STRs: Considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements.
        Forensic Sci. Int. Genet. 2016; 22: 54-63
        • Zeng X.
        • King J.L.
        • Budowle B.
        Investigation of the STR loci noise distributions of PowerSeqTM Auto System.
        Croat. Med. J. 2017; 58: 214-221