Analyzing degraded DNA and challenging samples using the ForenSeq™ DNA Signature Prep kit |
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| Institution: | 1. Office of Chief Medical Examiner, Department of Forensic Biology, New York, NY 10016, USA;2. Imperial College London, National Heart and Lung Institute (NHLI), London SW3 6LR, UK;1. Department of Forensic Biology, Oslo University Hospital, Norway;2. Zurich Institute of Forensic Medicine, University of Zurich, Zurich, Switzerland;3. University of Oslo, Faculty of Medicine, Oslo, Norway;1. U.S. National Institute of Standards and Technology, Biomolecular Measurement Division, 100 Bureau Drive, Gaithersburg, MD, 20899, USA;2. King’s Forensics, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London, UK;3. Institute of Legal Medicine, Medical University of Innsbruck, Austria;4. Center for Human Identification, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX, 76107, USA;5. Forensic Science Program, The Pennsylvania State University, USA;6. Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Spain;1. Genomics Core Facility, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, 08003 Barcelona, Catalonia, Spain;2. Division of Biological Traces, Netherlands Forensic Institute, Laan van Ypenburg 6, 2497 GB, The Hague, The Netherlands;3. Institut de Biologia Evolutiva (UPF-CSIC), Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, 08003 Barcelona, Catalonia, Spain;1. Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd. Fort Worth, TX 76107, USA;2. Molecular Anthropology Laboratory, Department of Anthropology, University of California, One Shields Avenue, Davis, CA 95616, USA;3. Graduate Group in Forensic Science, University of California, One Shields Avenue, Davis, CA 95616, USA;4. Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia;1. Forensic Science Program, School of Chemical Sciences, University of Auckland, New Zealand;2. Institute of Environmental Science and Research Limited, Private Bag 92021, Auckland, New Zealand;1. Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd. Fort Worth, TX 76107, USA;2. Department of Chemistry Malaysia Kuching, Ministry of Science, Technology and Innovation (MOSTI) Malaysia;3. Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia |
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| Abstract: | Typing short tandem repeats (STRs) is the basis for human identification in current forensic testing. The standard method uses capillary electrophoresis (CE) to separate amplicons by length and fluorescent labeling. In recent years new methods, including massively parallel sequencing (MPS), have been developed which increased the discriminative power of STRs through sequencing. MPS also offers the opportunity to test more genetic markers in a run than is possible with standard CE technology. Verogen’s ForenSeq™ DNA Signature Prep kit includes over 150 genetic markers STRs and single nucleotide polymorphisms (SNPs)]. Further, MPS separation depends on sequences rather than lengths; therefore, amplicons can be small or even of the same lengths. These improvements are advantageous when testing challenging forensic samples that could be severely degraded.This study tested the ForenSeq™ DNA Signature Prep kit in repeated experimental runs on series of degraded DNA samples, ranging from mild to severe degradation, as well as 24 mock case-type samples, derived from bones, blood cards, and teeth. Despite passing the quality metrics, positive controls (2800 M) showed drop-outs at some loci, mostly SNPs. Sequencing DNA samples repeatedly in two experimental runs as well as sequencing one pooled library in triplicate led to the assumption that spurious alleles of the Y-STRs in this study were not a result of sequencing artifacts but could be due to sequence structures (e.g. duplications, palindromes) of the Y-chromosome and/or might be accumulated during library preparation.Two sets of serially degraded DNA samples revealed that dropped-out loci were primarily loci with long amplicons as well as low read numbers (coverage), e.g. PentaE, DXS8378, and rs1736442. STRs started to drop out at degradation indices (DIs) > 4. However, severely degraded DNA (DI: 44) still resulted in 90% of the 20 CODIS loci, while only 35% were obtained using Promega’s PowerPlex® Fusion kit, a current standard CE kit. Mock case-type samples confirmed these results. ForenSeq™ DNA Signature Prep kit demonstrated that it can be successfully used on degraded DNA samples. This study may be helpful for other laboratories assessing and validating MPS technologies. |
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| Keywords: | Massively parallel sequencing Short tandem repeats Degraded DNA Challenging samples |
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