Short tandem repeat typing on the 454 platform: Strategies and considerations for targeted sequencing of common forensic markers |
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| Affiliation: | 1. American Registry of Pathology, Camden, DE, United States;2. Armed Forces DNA Identification Laboratory, Armed Forces Medical Examiner System, Dover AFB, DE, United States;1. Instituto de Pesquisa de DNA Forense—Polícia Civil do Distrito Federal, Brasília, Brazil;2. Secretaria Nacional de Segurança Pública do Ministério da Justiça, SENASP/MJ, Brasília, Brazil,;3. Laboratório de Diagnóstico por DNA (LDD), Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro, Brazil;4. Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal;5. Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal;6. Department Forensic Genetics, Institute of Legal Medicine and Forensic Sciences Charité-Universitätsmedizin, Berlin, Germany;1. Division of Legal Medicine, Faculty of Medicine, Tottori University, Yonago 683-8503, Japan;2. Department of Legal Medicine, Kansai Medical University, Hirakata, Japan;3. Department of Legal Medicine and Bioethics, Nagoya University School of Medicine, Nagoya, Japan;4. Department of Forensic Medicine, Fukuoka University School of Medicine, Fukuoka, Japan;5. Department of Pathobiological Science and Technology, Faculty of Medicine, Tottori University, Yonago, Japan;6. Department of Forensic Medicine, Yamagata University School of Medicine, Yamagata, Japan;7. Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan;1. Key Laboratory of Forensic Genetics, Institute of Forensic Science, Ministry of Public Security, Beijing 100038, PR China;2. School of Forensic Medicine, Shanxi Medical University, Taiyuan 030001, PR China;3. The First Research Institute of the Ministry of Public Security, Beijing 100048, PR China;1. Section of Forensic Genetics, Department of Forensic Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark;2. Thermo Fisher Scientific, South San Francisco, CA, USA |
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| Abstract: | To investigate the feasibility of next generation sequencing technology (NGS) for the multiplex detection and sequence production of short tandem repeats (STRs) from degraded and low DNA quantity samples, standard polymerase chain reaction amplification methods were used to enrich for commonly employed STR markers. Samples were amplified with two multiplexing strategies: a multiplex containing thirteen miniSTR markers and a series of multiplexes containing four miniSTR markers each. Each sample multiplex was barcoded with a sample-specific multiplex identifier for subsequent parallel tagged sequencing on the GS Junior System (454 Life Sciences, a Roche company, Branford, CT). Sequencing results from over fifty DNA extracts representing both pristine samples and low-quality evidentiary specimens reflected known genotypes and were consistent across multiple extracts and/or amplifications of the same sample. Furthermore, the NGS data revealed sequence information not available with standard capillary electrophoresis-based detection alone. For the population samples tested, a total of 152 single nucleotide polymorphisms or insertions/deletions were identified in over 935 recovered alleles, averaging one polymorphism for every six recovered alleles. For three of the loci, the sequence information doubled the number of alleles detected via traditional STR typing by fragment analysis. In addition, twenty-eight of these variants were only seen once within our dataset, highlighting the potential for discrimination among individuals. These additional data are likely to be particularly valuable in missing persons and disaster victim identification cases for which only partial profiles may be recovered and/or only distant kin are available as references. And, considering the opportunity to target only small amplicons with NGS, this type of STR typing will allow for greater information recovery from challenging casework samples. While our results highlight the potential of new technologies for recovering discriminatory genetic information from evidentiary specimens, our data also reveal the complexities of NGS-based STR typing, both in terms of the laboratory assays themselves as well as the downstream data processing and analysis. |
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| Keywords: | 454 sequencing Short tandem repeat Next generation sequencing |
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