International Journal of Legal Medicine

, Volume 131, Issue 1, pp 73–86 | Cite as

A preliminary assessment of the ForenSeq™ FGx System: next generation sequencing of an STR and SNP multiplex

  • Ashley L. Silvia
  • Nathan Shugarts
  • Jenifer Smith
Original Article


The ForenSeq™ FGx System (Illumina, San Diego, CA) was initially evaluated in concordance with SWGDAM guidelines for internal validation to determine the quality of the system’s components: the ForenSeq™ DNA Signature Prep Kit reagents, the MiSeq FGx™ instrument, and the ForenSeq™ Universal Analysis Software, for the analysis of targeted, forensically informative single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). This multiplex consisted of STRs (autosomal, X, and Y) and SNPs (identity, ancestry, and phenotypic) that were run using one preparation process. Overall, the ForenSeq™ FGx System performed as well as the traditional capillary electrophoresis-based method in producing usable profile information, along with additional information that could aid in investigative leads. The MiSeq FGx™ System was validated using DNA samples in studies testing reproducibility, repeatability, concordance, sensitivity, and mock case single donor samples. Overall, genotyping results for STRs and SNPs were concordant with the profiles generated from conventional STR analysis using Identifiler and SNPs typed by 23andMe analysis. Genotypes of the ForenSeq™ aSNPs were used to evaluate biogeographical ancestry estimations using ForenSeq™ Universal Analysis Software, FROG-kb database (KIDD aiSNP 55 panel), and 23andMe. The system was shown to provide reproducible genotypes and reliable results were obtained at levels as low as 50 pg. All mock case samples were concordant with the donor profile. The results support consideration of the ForenSeq™ FGx System as an acceptable alternative to current STR and SNP analysis, pending formal developmental and internal validation studies.


Forensic MiSeq FGx™ STR analysis SNP analysis 



The authors wish to thank Carey Davis, Joe Valaro, Cydne Holt, Dan Gheba, and Steven Lee along with the many scientists and corporate leaders from Illumina for their support and suggestions on project design, providing the instrument, and providing the reagents. We would also like to thank the Forensic Science Department at the Pennsylvania State University and the many participants who provided samples for the study. Finally, we thank the reviewers of this manuscript for their thoughtful comments and suggestions. The views and opinions expressed are those of the authors and may not reflect the views of the Pennsylvania State University.

Compliance with ethical standards

Informed consent was obtained from all individual participants included in the study.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Supplementary material

414_2016_1457_MOESM1_ESM.docx (305 kb)
ESM 1 (DOCX 305 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Ashley L. Silvia
    • 1
  • Nathan Shugarts
    • 1
  • Jenifer Smith
    • 1
  1. 1.Forensic Science ProgramThe Pennsylvania State UniversityUniversity ParkUSA

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