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Forensic Sci Int Genet. 2018 May;34:105-115. doi: 10.1016/j.fsigen.2018.01.002. Epub 2018 Jan 31.

Body fluid identification using a targeted mRNA massively parallel sequencing approach - results of a EUROFORGEN/EDNAP collaborative exercise.

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Zurich Institute of Forensic Medicine, University of Zurich, Switzerland. Electronic address:
Zurich Institute of Forensic Medicine, University of Zurich, Switzerland.
National Center for Forensic Science, University of Central Florida, Orlando, USA.
Carabinieri Scientific Department of Rome- Genetic Unit, Rome, Italy.
Malopolska Centre of Biotechnology of the Jagiellonian University, Gronostajowa st. 7A, 30-387, Krakow, Poland.
National Institute of Legal Medicine and Forensic Sciences, Portugal.
Orchid Cellmark Ltd., Abingdon, UK.
National Institute of Standards and Technology, Material Measurement Laboratory, Gaithersburg, MD, United States.
Department of Pharmacy and Forensic Science, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, UK.
Institute of Legal Medicine, Medical Faculty, University of Cologne, Germany.
Department of Forensic Biology, Oslo University Hospital, Oslo, Norway.
Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
Department of Genetic Identification, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands.
Laboratoire de Police Scientifique de Lyon, Institut National de Police Scientifique, F-69134, Ecully, France.
Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain.
Institute of Legal Medicine, Medical University of Innsbruck, Austria; Forensic Science Program, Pennsylvania State University, PA, USA.
Institute of Legal Medicine, University of Münster, Germany.
Gerhard-Domagk-Institute of Pathology, University Hospital Münster, Germany.
Division of Biological Traces, Netherlands Forensic Institute, P.O. Box 24044, 2490 AA, The Hague, The Netherlands.
Institute of Legal Medicine, Medical University of Innsbruck, Austria.
National Center for Forensic Science, University of Central Florida, Orlando, USA; Department of Chemistry, University of Central Florida, PO Box 162366, Orlando, FL, 32816-2366, USA.


In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants' own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.


Body fluid identification; Forensic science; Massively parallel sequencing; mRNA profiling

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