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Science & Justice : Journal of the... Sep 2022Recognizing and interpreting mixtures are challenges that occur frequently in forensic casework. Therefore, any new analysis methods that are implemented must handle the...
Recognizing and interpreting mixtures are challenges that occur frequently in forensic casework. Therefore, any new analysis methods that are implemented must handle the challenges of mixed forensic samples. Next generation sequencing offers advantages over capillary electrophoresis in amplicon multiplexing and degraded sample analysis; however, advantages with mixed samples rely heavily on the advancement of user-friendly analysis software. This research analyzed samples with the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx® and compared them with the GlobalFiler™ STR Kit for capillary electrophoresis. Metrics tested for both chemistries included concordance, limits of detection, and mixture analysis. Data analysis for mixture samples was completed with the MixtureAce™ plug-in and ArmedXpert™ software. Next generation sequencing offered distinct advantages in limits of detection and isoallele heterozygosity but suffered from increased variability in stutter and allele count ratios compared to capillary electrophoresis.
Topics: Humans; DNA Fingerprinting; Microsatellite Repeats; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Reproducibility of Results; Sequence Analysis, DNA; DNA
PubMed: 36336448
DOI: 10.1016/j.scijus.2022.07.008 -
International Journal of Legal Medicine Jan 2023Microbiome studies have contributed to many fields, such as healthcare and medicine; however, these studies are relatively limited in forensics. Microbiome analyses can...
Microbiome studies have contributed to many fields, such as healthcare and medicine; however, these studies are relatively limited in forensics. Microbiome analyses can provide information, such as geolocation and ancestry information, when short tandem repeat (STR) profiling fails. In this study, methods for DNA extraction and sampling from the skin and saliva were optimized for the construction of a Korean Forensic Microbiome Database (KFMD). DNA yields were estimated using four DNA extraction kits, including two automated kits (Maxwell® FSC DNA IQ™ Casework Kit and PrepFiler™ Forensic DNA Extraction Kit, updated) and two manual kits (QIAamp DNA Mini Kit and QIAamp DNA Micro Kit) commonly used in forensic DNA profiling laboratories. Next-generation sequencing of the 16S rRNA V4 region was performed to analyze microbial communities in samples. The Bacterial Transport Swab with Liquid Media (NobleBio), two cotton swabs (PoongSung and Puritan), and nylon-flocked swabs (NobleBio and COPAN) were tested for DNA recovery. The PrepFiler and Maxwell kits showed the highest yields of 3.884 ng/μL and 23.767 ng/μL from the scalp and saliva, respectively. With respect to DNA recovery, nylon-flocked swabs performed better than cotton swabs. The relative abundances of taxa sorted by DNA extraction kits were similar contributions; however, with significant differences in community composition between scalp and saliva samples. Lawsonella and Veillonella were the most abundant genera in the two sample types. Thus, the Maxwell® FSC DNA IQ™ Casework Kit and nylon-flocked swab (NobleBio) were optimal for DNA extraction and collection in microbiome analyses.
Topics: Humans; Saliva; Nylons; RNA, Ribosomal, 16S; DNA Fingerprinting; DNA, Ribosomal
PubMed: 36416962
DOI: 10.1007/s00414-022-02919-6 -
Frontiers in Bioscience (Landmark... Sep 2022As we continually reflect on the wars of the 20th century, identification of the remains of victims takes an increasingly prominent position in ongoing research.... (Review)
Review
As we continually reflect on the wars of the 20th century, identification of the remains of victims takes an increasingly prominent position in ongoing research. Existing work on the identification of human remains from 20th century wars primarily covers the determination of phenotypic characteristics, kinship and geographic origins, supporting the establishment of genetic information databases. Compared with standard forensic methods, DNA analyses have revealed greater effectiveness. The process of DNA analysis includes DNA extraction, genetic marker testing and data analysis. Protocols from ancient DNA research can be applied to degraded remains, and next-generation sequencing (NGS) techniques can compensate for shortcomings in the most commonly-used PCR-capillary electrophoresis typing. As it stands, wide-ranging inter-governmental and inter-institutional collaboration is necessary in order to set up NGS-based public databases, and thereby promote the identification of human remains and archaeological forensics.
Topics: Body Remains; DNA Fingerprinting; DNA, Ancient; Genetic Markers; High-Throughput Nucleotide Sequencing; Humans; Microsatellite Repeats
PubMed: 36224018
DOI: 10.31083/j.fbl2709271 -
Journal of Forensic Sciences Jul 2023Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence-related DNA extracts can degrade DNA or inhibit PCR as applied...
Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence-related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification (real-time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an "inhibition study" and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in-house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000-fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR-based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin.
Topics: Humans; Tin; DNA Fingerprinting; Microsatellite Repeats; Real-Time Polymerase Chain Reaction; DNA; Metals
PubMed: 37326229
DOI: 10.1111/1556-4029.15312 -
Forensic Science International. Genetics Jan 2018DNA evidence sample processing typically involves DNA extraction, quantification, and STR amplification; however, DNA loss can occur at both the DNA extraction and... (Review)
Review
DNA evidence sample processing typically involves DNA extraction, quantification, and STR amplification; however, DNA loss can occur at both the DNA extraction and quantification steps, which is not ideal for forensic evidence containing low levels of DNA. Direct PCR amplification of forensic unknown samples has been suggested as a means to circumvent extraction and quantification, thereby retaining the DNA typically lost during those procedures. Direct PCR amplification is a method in which a sample is added directly to an amplification reaction without being subjected to prior DNA extraction, purification, or quantification. It allows for maximum quantities of DNA to be targeted, minimizes opportunities for error and contamination, and reduces the time and monetary resources required to process samples, although data analysis may take longer as the increased DNA detection sensitivity of direct PCR may lead to more instances of complex mixtures. ISO 17025 accredited laboratories have successfully implemented direct PCR for limited purposes (e.g., high-throughput databanking analysis), and recent studies indicate that direct PCR can be an effective method for processing low-yield evidence samples. Despite its benefits, direct PCR has yet to be widely implemented across laboratories for the processing of evidentiary items. While forensic DNA laboratories are always interested in new methods that will maximize the quantity and quality of genetic information obtained from evidentiary items, there is often a lag between the advent of useful methodologies and their integration into laboratories. Delayed implementation of direct PCR of evidentiary items can be attributed to a variety of factors, including regulatory guidelines that prevent laboratories from omitting the quantification step when processing forensic unknown samples, as is the case in the United States, and, more broadly, a reluctance to validate a technique that is not widely used for evidence samples. The advantages of direct PCR of forensic evidentiary samples justify a re-examination of the factors that have delayed widespread implementation of this method and of the evidence supporting its use. In this review, the current and potential future uses of direct PCR in forensic DNA laboratories are summarized.
Topics: DNA; DNA Fingerprinting; Humans; Microsatellite Repeats; Polymerase Chain Reaction; Touch
PubMed: 29059581
DOI: 10.1016/j.fsigen.2017.10.005 -
Forensic Science International Jan 2019
Topics: Bias; DNA; DNA Contamination; DNA Fingerprinting; Databases, Nucleic Acid; Forensic Genetics; Humans; Touch
PubMed: 30579629
DOI: 10.1016/j.forsciint.2018.12.003 -
Methods in Molecular Biology (Clifton,... 2021Retrotransposable elements (RTEs) are highly common mobile genetic elements that are composed of several classes and make up the majority of eukaryotic genomes. The...
Retrotransposable elements (RTEs) are highly common mobile genetic elements that are composed of several classes and make up the majority of eukaryotic genomes. The "copy-out and paste-in" life cycle of replicative transposition in these dispersive and ubiquitous RTEs leads to new genome insertions without excision of the original element. RTEs are important drivers of species diversity; they exhibit great variety in structure, size, and mechanisms of transposition, making them important putative components in genome evolution. Accordingly, various applications have been developed to explore the polymorphisms in RTE insertion patterns. These applications include conventional or anchored polymerase chain reaction (PCR) and quantitative or digital PCR with primers designed for the 5' or 3' junction. Marker systems exploiting these PCR methods can be easily developed and are inexpensively used in the absence of extensive genome sequence data. The main inter-repeat amplification polymorphism techniques include inter-retrotransposon amplified polymorphism (IRAP), retrotransposon microsatellite amplified polymorphism (REMAP), and Inter-Primer Binding Site (iPBS) for PCR amplification with a single or two primers.
Topics: DNA Fingerprinting; DNA Primers; Genetic Markers; Genetic Variation; Microsatellite Repeats; Polymerase Chain Reaction; Polymorphism, Genetic; Retroelements
PubMed: 33301099
DOI: 10.1007/978-1-0716-0997-2_15 -
Science & Justice : Journal of the... May 2020Typing short tandem repeats (STRs) is the basis for human identification in current forensic testing. The standard method uses capillary electrophoresis (CE) to separate...
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.
Topics: DNA; DNA Fingerprinting; Genetic Markers; High-Throughput Nucleotide Sequencing; Humans; Microsatellite Repeats; Polymorphism, Single Nucleotide; Sequence Analysis, DNA
PubMed: 32381241
DOI: 10.1016/j.scijus.2019.11.004 -
Journal of Forensic Sciences Jul 2022Bone cells are a suitable substrate for DNA analysis if required to identify the person from whom a sample was taken. Osteocytes, the most abundant cell type in bone,...
Bone cells are a suitable substrate for DNA analysis if required to identify the person from whom a sample was taken. Osteocytes, the most abundant cell type in bone, are embedded within mineralized bone matrix. To release DNA from osteocytes for subsequent analyses, either demineralization of the mineral matrix or an overnight incubation is routinely carried out. In this study, we report on a simplified and rapid approach to analyze preserved bone samples that omits this lengthy decalcification process. Nine tibial bone samples were processed to release matrix-free bone cells after fragmentation without the use of liquid nitrogen. Cell morphology was assessed by microscopy at 220× magnification following staining with Diamond Nucleic Acid Dye. Based on the presence of stained nuclei, samples were processed either using a DNA extraction process or by a semi-direct PCR process. The analysis of the quantity and quality of DNA isolated by both methods was carried out by real-time PCR and STR profiling to assess inhibition of PCR and DNA degradation. All samples resulted in informative STR profiles with minimal indication of inhibitors. These results demonstrate a potential approach of STR profiling from matrix-free bone cells within 8 hours without decalcification and DNA extraction.
Topics: DNA; DNA Fingerprinting; Humans; Microsatellite Repeats; Osteocytes; Real-Time Polymerase Chain Reaction
PubMed: 35324000
DOI: 10.1111/1556-4029.15033 -
Scientific Reports Dec 2023DNA analysis-based identification is by far the gold standard in forensic genetics and it should be performed in every case involving human remains or unidentified...
DNA analysis-based identification is by far the gold standard in forensic genetics and it should be performed in every case involving human remains or unidentified bodies. Bones and teeth are the preferred source of human DNA for genetic analysis. However, there are cases where the nature of the proceedings and historical significance prevent the disruption of skeletal structure. The remains may also be heavily degraded. In such situations, forensic geneticists seek alternative sources of human DNA. Teeth calculus has proven to be a viable source of DNA for identification purposes. The aim of this study was to assess the concentration of human DNA in teeth calculus and evaluate the usefulness of teeth calculus as a DNA source in the identification process. Teeth calculus was collected from skeletons exhumed between 2021 and 2022 by the PBGOT (Polish Genetic Database of Victims of Totalitarianism) team from the former Stalag IID prisoner-of-war camp in Stargard. Genetic analyses included the determination of autosomal and Y-STR markers. The total concentration of human DNA was also evaluated in samples from teeth calculus and teeth taken from the same individuals. The pilot study included 22 skeletons with a sufficient amount of calculus for isolation (specified in the protocol). Samples were taken from the largest areas of calculus deposited on lingual surfaces of mandibular incisors. The prepared samples underwent DNA extraction. Our study demonstrated that teeth calculus is a source of human DNA for remains from the World War II period. The obtained DNA concentration allowed for the determination of STR markers. It was shown that teeth calculus contains human DNA in an amount suitable for preliminary identification analyses.
Topics: Humans; Dental Calculus; Pilot Projects; DNA Fingerprinting; Microsatellite Repeats; DNA; Incisor
PubMed: 38066060
DOI: 10.1038/s41598-023-48761-7