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      Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling

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          Abstract

          Nanopore sequencing for forensic short tandem repeats (STR) genotyping comes with the advantages associated with massively parallel sequencing (MPS) without the need for a high up-front device cost, but genotyping is inaccurate, partially due to the occurrence of homopolymers in STR loci. The goal of this study was to apply the latest progress in nanopore sequencing by Oxford Nanopore Technologies in the field of STR genotyping. The experiments were performed using the state of the art R9.4 flow cell and the most recent R10 flow cell, which was specifically designed to improve consensus accuracy of homopolymers. Two single-contributor samples and one mixture sample were genotyped using Illumina sequencing, Nanopore R9.4 sequencing, and Nanopore R10 sequencing. The accuracy of genotyping was comparable for both types of flow cells, although the R10 flow cell provided improved data quality for loci characterized by the presence of homopolymers. We identify locus-dependent characteristics hindering accurate STR genotyping, providing insights for the design of a panel of STR loci suited for nanopore sequencing. Repeat number, the number of different reference alleles for the locus, repeat pattern complexity, flanking region complexity, and the presence of homopolymers are identified as unfavorable locus characteristics. For single-contributor samples and for a limited set of the commonly used STR loci, nanopore sequencing could be applied. However, the technology is not mature enough yet for implementation in routine forensic workflows.

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          Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM

          Heng Li (2013)
          Summary: BWA-MEM is a new alignment algorithm for aligning sequence reads or long query sequences against a large reference genome such as human. It automatically chooses between local and end-to-end alignments, supports paired-end reads and performs chimeric alignment. The algorithm is robust to sequencing errors and applicable to a wide range of sequence lengths from 70bp to a few megabases. For mapping 100bp sequences, BWA-MEM shows better performance than several state-of-art read aligners to date. Availability and implementation: BWA-MEM is implemented as a component of BWA, which is available at http://github.com/lh3/bwa. Contact: hengli@broadinstitute.org
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            Massively parallel sequencing techniques for forensics: A review

            Abstract DNA sequencing, starting with Sanger's chain termination method in 1977 and evolving into the next generation sequencing (NGS) techniques of today that employ massively parallel sequencing (MPS), has become essential in application areas such as biotechnology, virology, and medical diagnostics. Reflected by the growing number of articles published over the last 2–3 years, these techniques have also gained attention in the forensic field. This review contains a brief description of first, second, and third generation sequencing techniques, and focuses on the recent developments in human DNA analysis applicable in the forensic field. Relevance to the forensic analysis is that besides generation of standard STR‐profiles, DNA repeats can also be sequenced to look for polymorphisms. Furthermore, additional SNPs can be sequenced to acquire information on ancestry, paternity or phenotype. The current MPS systems are also very helpful in cases where only a limited amount of DNA or highly degraded DNA has been secured from a crime scene. If enough autosomal DNA is not present, mitochondrial DNA can be sequenced for maternal lineage analysis. These developments clearly demonstrate that the use of NGS will grow into an indispensable tool for forensic science.
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              Microfluidic Devices for Forensic DNA Analysis: A Review

              Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10–20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook.
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                Author and article information

                Journal
                Genes (Basel)
                Genes (Basel)
                genes
                Genes
                MDPI
                2073-4425
                01 April 2020
                April 2020
                : 11
                : 4
                : 381
                Affiliations
                [1 ]Laboratory of Pharmaceutical Biotechnology, Ghent University, 9000 Gent, Belgium; Olivier.Tytgat@ 123456UGent.be (O.T.); Yannick.Gansemans@ 123456UGent.be (Y.G.); Jana.Weymaere@ 123456UGent.be (J.W.); Kaat.Rubben@ 123456UGent.be (K.R.); Dieter.Deforce@ 123456UGent.be (D.D.)
                [2 ]Department of Life Science Technologies, Imec, 3001 Leuven, Belgium
                Author notes
                [* ]Correspondence: Filip.VanNieuwerburgh@ 123456UGent.be ; Tel.: +32-9264-8048
                [†]

                These authors contributed equally.

                Author information
                https://orcid.org/0000-0002-1788-5963
                https://orcid.org/0000-0001-5018-044X
                https://orcid.org/0000-0002-8213-1973
                https://orcid.org/0000-0002-7452-2544
                Article
                genes-11-00381
                10.3390/genes11040381
                7230633
                32244632
                f60bc0b0-93fe-4d32-931a-4a476f147966
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 09 March 2020
                : 31 March 2020
                Categories
                Article

                forensic,dna fingerprinting,short tandem repeats,oxford nanopore sequencing,massively parallel sequencing

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