Aportaciones y avances de la genética forense en los sucesos con víctimas múltiples / Contributions and advances of forensic genetics in mass fatality incidents

La identificación de los afectados por un suceso con víctimas múltiples es una prioridad por razones humanitarias y legales. La genética forense juega un importante papel en estas situaciones que, por su complejidad, a menudo se convierten en un reto para los distintos profesionales implicados. El establecimiento de guías y recomendaciones facilita el seguimiento de protocolos estandarizados que permiten garantizar la fiabilidad del resultado final de la identificación. Así mismo, los avances en la genética forense contribuyen a agilizar la respuesta, aportando nuevas estrategias de análisis y herramientas de tipo bioinformático. Con este artículo, se pretende ofrecer una visión general de cómo la genética forense y sus avances pueden contribuir en estas situaciones, así como algunas claves para entender la labor de los laboratorios de genética forense en la identificación de cadáveres en sucesos con víctimas múltiples. (AU)

Disaster victim identification is crucial for humanitarian and legal reasons. Forensic genetics plays an important role in these situations which often become a challenge for the different professionals involved due to their complexity. The establishment of guidelines and recommendations makes it easier to follow standardized protocols that make it possible to guarantee the reliability of the identification final result. Likewise, advances in forensic genetics contribute to speeding up the response, providing new analysis strategies and bioinformatic tools. This article aims to provide an overview of how forensic genetics and its advances can contribute in these situations, as well as some keys to understanding the work of forensic genetics laboratories in the identification of corpses in events with multiple victims. (AU)

Genealogía forense. Implicaciones sociales, éticas, legales y científicas / Forensic genealogy. Social, ethical, legal and scientific implications

A pesar del avance de las técnicas investigativas de Genética Forense, todavía existen gran cantidad de casos criminales no resueltos y en muchos casos inabordables por su antigüedad. Recientemente hemos asistido al nacimiento de una nueva disciplina forense, la genealogía forense o genealogía genética investigativa, que permite que muchos de estos casos puedan ser resueltos satisfactoriamente combinando la tecnología de análisis de ADN y las herramientas de búsquedas genealógicas.La potencialidad de esta nueva estrategia investigativa es evidente, de la misma manera que lo son sus riesgos. Hemos de mantener un equilibrio entre la privacidad personal y los intereses de las personas afectadas por un lado y la seguridad pública, el bien de la comunidad y la resolución y esclarecimiento de hechos delictivos por el otro, siendo necesario un debate social, legal y científico que clarifique todos estos aspectos y posibilite una adecuada regulación legal de estas prácticas. (AU)

Despite the advances in investigative techniques in Forensic Genetics, there are still a large number of unsolved criminal cases. We have recently seen the birth of a new forensic discipline called forensic genealogy or investigative genetic genealogy, which allows to us resolve satisfactorily many of these cold cases combining DNA analysis technology with genealogical search tools.The potential and risks of this new research strategy are manifest. We must maintain a balance between personal privacy and personal interests on the one hand and on the other, public security, the good of the community and the resolution and clarification of criminal acts, requiring a social, legal and scientific debate that clarifies all these aspects and enables adequate legal regulation of these practices. (AU)

Validation of the Verogen ForenSeq™ DNA Signature Prep kit/Primer Mix B for phenotypic and biogeographical ancestry predictions using the Micro MiSeq® Flow Cells.

In anticipation of offering phenotypic and biogeographical ancestry predictions to help resolve cases, the Verogen ForenSeq™ DNA Signature Prep kit/Primer Mix B was evaluated in the context of Micro MiSeq® Flow Cells. These flow cells were determined as the best format for a quick turnaround time response and cost effective approach compared to standard flow cells. The phenotype informative SNPs (piSNPs) and ancestry informative SNPs (aiSNPs) were thoroughly examined through sensitivity, reproducibility and repeatability, concordance, robustness (mock casework) and low level DNA mixture studies purposely selecting individuals with different phenotypes (hair and eye color) when possible and different biogeographical ancestry. SNP locus-specific interpretation thresholds were established for the Universal Analysis Software (UAS) based on surviving alleles and SNP predictor rank to minimize false homozygous genotypes and maximize the information that can be derived from an unknown sample. Dropin alleles' intensity determined an appropriate threshold to minimize false heterozygous SNP genotypes. The selection of inappropriate interpretation thresholds was shown to have major consequences on phenotypic predictions. A 3.2% and 4.8% minor DNA component contribution to a DNA mixture had no impact on ancestry predictions whereas a 9.1% contribution did. The multi-locus SNP genotypes generated using the ForenSeq™ DNA Signature Prep kit/Primer Mix B were shown to be reliable, reproducible, concordant and resulted in predictions that were also reliable, reproducible and concordant based on the limited number of donors (N = 19) used in this study.

Evaluation of the AGCU Expressmarker 30 Kit composed of 31 loci for forensic application.

With the widespread use of STR in identification of individuals, paternity testing, as well as population genetics, many commercially robust and validated STR multiplex kits were developed. The AGCU Expressmarker 30 Kit is a new autosomal STR system that contains 29 autosomal STR loci (D3S1358, vWA, D1S1656, CSF1PO, D8S1132, D19S253, D3S3045, D8S1179, D21S11, D16S539, TPOX, D6S477, Penta D, D2S441, D5S818, TH01, FGA, D15S659, D22S1045, D19S433, D13S317, D7S820, D6S1043, D10S1435, D10S1248, D2S1338, D18S51, D12S391, and Penta E), one insertion/deletion polymorphic marker on the Y chromosome (Y indel), and the amelogenin locus. A series of validation studies were performed in this context according to the guidelines of "Validation Guidelines for Forensic DNA Analysis Methods". The sensitivity study showed that a full profile was observed with template DNA as low as 40 pg. In the stability study, all STR profiles were obtained at concentrations of humic acid up to 800 ng/µL, hematin up to 250 µM, and tannic acid up to 200 ng/µL. The mixture study demonstrated that all of the minor alleles could be called at ratios from 1:1-29:1 when the total DNA was 2 ng. In the population study, the total discrimination power for three population (Sichuan-Han, Gansu-Hui, and Guangxi-Zhuang) were above 0.9999999999999999999999999999999992, 0.999999999999999999999999999999998 and 0.999999999999999999999999999999994 as well as the cumulative probability of paternity exclusion were 0.999999999999953, 0.999999999999178, and 0.999999999999611 respectively. These results demonstrated that the AGCU Expressmarker 30 Kit is a useful tool for analyzing both forensic casework and database samples.

Developmental validations of a self-developed 39 AIM-InDel panel and its forensic efficiency evaluations in the Shaanxi Han population.

Most of insertion/deletion polymorphisms are diallelic molecular markers characterized as small amplicon sizes, high inter-population diversities, and low mutation rates, which make them the promising genetic markers in biogeographic ancestor inference field. The developmental validations of a 39 ancestry informative marker-insertion/deletion (AIM-InDel) panel and the genetic polymorphic investigations of this panel were performed in the Shaanxi Han population of China. The developmental validation included the optimizations of PCR-related indicators, repeatability, reproducibility, precision, accuracy, sensitivity, species specificity, stability of the panel, and the abilities in analyzing degraded, casework, and mixture samples, and the present results demonstrated that this 39 AIM-InDel panel was robust, sensitive, and accurate. For the population diversity analyses, the combined discrimination power value of 38 AIM-InDel loci except for rs36038238 locus was 0.999999999931257, indicating that this novel panel was highly polymorphic, biogeographic informative, and could be also used in individual identifications in the Shaanxi Han population.

Developmental validation of VeriFiler™ Plus PCR Amplification Kit: A 6-dye multiplex assay designed for casework samples.

The VeriFiler™ Plus PCR Amplification Kit is a 6-dye multiplex assay that simultaneously amplifies a set of 23 autosomal markers (D3S1358, vWA, D16S539, CSF1PO, D6S1043, D8S1179, D21S11, D18S51, D5S818, D2S441, D19S433, FGA, D10S1248, D22S1045, D1S1656, D13S317, D7S820, Penta E, Penta D, TH01, D12S391, D2S1338, and TPOX), a quality indicator system, and two sex-identification markers. Combined, the markers satisfy the requirements of the Chinese National autosomal DNA database as well as expanded CODIS (Combined DNA Index System). The VeriFiler Plus kit was developed with an improved Master Mix which incorporates the brighter TED™ dye, and accommodates a higher sample loading volume thus allowing for increased sensitivity and enabling maximum information recovery from challenging casework samples including touch, degraded, and inhibited samples. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit's robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

Resizing reaction volumes for the ForenSeq™ DNA Signature Prep kit library preparation.

The introduction of next generation sequencing (NGS; also known as massively parallel sequencing) technology in the field of forensic genetics has been welcomed by the scientific community, above all because it complements the weaknesses of capillary electrophoresis (CE) in the analysis of genetic markers, such as single nucleotide polymorphism (SNP) typing. However, one of the main obstacles to its adoption does not seem to be the cost of the instrumentation, but rather the cost of the NGS library preparation kits. With the aim of reducing the cost of library preparation without compromising the quality of the results, we tried to scale down reaction volumes for the first two polymerase chain reactions in the amplification and enrichment phases of the targeted loci of library preparation using the ForenSeq™ DNA Signature Prep kit. We used 1 µL templated DNA input to a concentration of 1 ng/µL, instead of the 5 µL at 0.2 ng/µL recommended by the manufacturer. Our findings indicate that reduction of the library preparation volume using the ForenSeq™ DNA Signature Prep kit did not interfere with the quality and reproducibility of the DNA profiles obtained and can help lower the overall cost of NGS.

Evaluation of the efficiency of Isohelix™ and Rayon swabs for recovery of DNA from metal surfaces.

PURPOSE: We investigated the recovery and extraction efficiency of DNA from three metal surfaces (brass, copper, steel) relevant to forensic casework, and plastic (control) using two different swabbing systems; Rayon and Isohelix™ swabs, with sterile water and isopropyl alcohol respectively, as the wetting solutions. METHODS: Twenty nanograms of human genomic DNA were applied directly to Isohelix™ and Rayon swabs; and to the metal and plastic substrates. All substrates were left to dry for 24 h, followed by single wet swabbing and extraction with the DNA IQ™ System. DNA extracts were quantified using real time quantitative PCR assays with SYBR green chemistry. RESULTS: DNA was extracted from directly seeded Isohelix™ swabs with a high efficiency of 98%, indicating effective DNA-release from the swab into the extraction buffer. In contrast, only 58% of input DNA was recovered from seeded Rayon swabs, indicating higher DNA retention by these swabs. Isohelix™ swabs recovered 32 - 53% of DNA from metal surfaces, whilst the Rayon swabs recovered 11-29%. DNA recovery was lowest from copper and highest from brass. Interestingly, Rayon swabs appeared to collect more DNA from the plastic surface than Isohelix™ swabs, however, due to the lower release of DNA from Rayon swabs they returned less DNA overall following extraction than Isohelix™ swabs. CONCLUSION: These results demonstrate that DNA samples deposited on metal surfaces can be more efficiently recovered using Isohelix™ swabs wetted with isopropyl alcohol than Rayon swabs wetted with sterile water, although recovery is affected by the substrate type.

A confirmatory test for sperm in sexual assault samples using a microfluidic-integrated cell phone imaging system.

Rapid and efficient processing of sexual assault evidence to accelerate forensic investigation and decrease casework backlogs is urgently needed. Therefore, the standardized protocols currently used in forensic laboratories can benefit from continued innovation to handle the increasing number and complexity of samples being submitted to forensic labs. To our knowledge, there is currently no available rapid and portable forensic screening technology based on a confirmatory test for sperm identification in a sexual assault kit. Here, we present a novel forensic sample screening tool, i.e., a microchip integrated with a portable cell phone imaging platform that records and processes images for further investigation and storage. The platform (i) precisely and rapidly screens swab samples (<15 min after sample preparation on-chip); (ii) selectively captures sperm from mock sexual assault samples using a novel and previously published SLeX-based surface chemistry treatment (iii) separates non-sperm contents (epithelial cells and debris in this case) out of the channel by flow prior to imaging; (iv) captures cell phone images on a portable cellphone-integrated imaging platform, (v) quantitatively differentiates sperm cells from epithelial cells, using a morphology detection code that leverages Laplacian of Gaussian and Hough gradient transform methods; (vi) is sensitive within a forensic cut-off (>95% accuracy) compared to the manual counts; (vii) provides a cost-effective and timely solution to a problem which in the past has taken a great deal of time; and (viii) handles small volumes of sample (20 µL). This integration of the cellphone imaging platform and cell recognition algorithms with disposable microchips can be a new direction toward a direct visual test to screen and differentiate sperm from epithelial cell types in forensic samples for a crime laboratory scenario. With further development, this integrated platform could assist a sexual assault nurse examiner (SANE) in a hospital or sexual assault treatment center facility to flag sperm-containing samples prior to further downstream testing.

Assessment of autosomal and male DNA extracted from casework samples using Casework Direct Kit, Custom and Maxwell 16 System DNA IQ Casework Pro Kit for autosomal-STR and Y-STR profiling.

Short repetitive regions in autosomal and Y chromosomes known as short tandem repeats (STRs) are currently used for DNA profiling in crime investigations. However, DNA profiling requires a sufficient quality and quantity of DNA template, which is often not obtained from trace evidence or degraded biological samples collected at the scene of a crime. Here, we assessed autosomal and male DNA components extracted from crime scene and mock casework samples using the Casework Direct Kit, Custom and compared the results against those obtained by extraction of matching samples using well-established Maxwell 16 System DNA IQ Casework Pro Kit. The quantity and quality of extracted DNA obtained using both Casework Direct Kit, Custom and Maxwell 16 System DNA IQ Casework Pro Kit were analyzed using PowerQuant Systems followed by autosomal and Y-chromosome STR profiling using GlobalFiler Express PCR Amplification Kit and PowerPlex Y23 System, respectively. Our results showed that the Casework Direct Kit and Maxwell 16 DNA IQ Casework Pro Kit have more or less equal capacity to extract inhibitor free DNA, but that the latter produces slightly better quality and more DNA template and subsequently higher numbers of STR allele calls for autosomal and Y-STR analyses. Nonetheless, the Casework Direct Kit, Custom is the quicker and cheaper option for extraction of good, clean DNA from high content material and might best be used for extraction of reference samples. Such reference DNA samples typically come from buccal swabs or freshly drawn blood. So, in general, they can confidently be expected to have a high nucleic acid content and to be inhibitor-free.

Silicon µPCR Chip for Forensic STR Profiling with Hybeacon Probe Melting Curves.

The demand to perform forensic DNA profiling outside of centralized laboratories and on the crime scene is increasing. Several criminal investigations would benefit tremendously from having DNA based information available in the first hours rather than days or weeks. However, due to the complexity and time-consuming nature of standard DNA fingerprinting methods, rapid and automated analyses are hard to achieve. We here demonstrate the implementation of an alternative DNA fingerprinting method in a single microchip. By combining PCR amplification and HyBeacon melting assays in a silicon Lab-on-a-chip (LoC), a significant step towards rapid on-site DNA fingerprinting is taken. The small form factor of a LoC reduces reagent consumption and increases portability. Additional miniaturization is achieved through an integrated heating element covering 24 parallel micro-reactors with a reaction volume of 0.14 µl each. The high level of parallelization allows the simultaneous analysis of 4 short tandem repeat (STR) loci and the amelogenin gender marker commonly included in forensic DNA analysis. A reference and crime scene sample can be analyzed simultaneously for direct comparison. Importantly, by using industry-standard semiconductor manufacturing processes, mass manufacturability can be guaranteed. Following assay design and optimization, complete 5-loci profiles could be robustly generated on-chip that are on par with those obtained using conventional benchtop real-time PCR thermal cyclers. Together, our results are an important step towards the development of commercial, mass-produced, portable devices for on-site testing in forensic DNA analysis.

DNA extraction from soil by EZ1 advanced XL (Qiagen).

EZ1 Advanced XL (Qiagen) is an instrument designed to purify automatically nucleic acids from a wide variety of samples: different protocols for DNA extraction from forensic samples are pre-loaded into special EZ1 Advanced XL DNA Investigator Cards. The present study focuses on DNA extraction from biological samples (blood, saliva, sperm) mixed with 3 different types of soils (loam, silt, sandy), using a modified version of the Manufacturer standard protocol. In order to create several traces in the soil, we used biological samples (blood, saliva, sperm) from known donors. Quantification data, reliability and trends in STRs typing success rates using two different commercial multiplexes were evaluated. EZ1 modified DNA extraction protocol allows to recover DNA free of inhibitors and in good quantity for downstream applications.

Forensic characterization of Brazilian regional populations through massive parallel sequencing of 124 SNPs included in HID ion Ampliseq Identity Panel.

Use of Massive Parallel Sequencing (MPS) techniques has been investigated by forensic community aiming introduction of such methods in routine forensic casework analyses. Interesting features presented by MPS include high-throughput, ability to simultaneous genotyping of significant number of samples and forensic markers, workflow automation, among others. Emergence of single nucleotide polymorphism (SNP) as forensic relevant markers was facilitated in this process, since concurrent typing of larger marker sets is necessary for obtaining same levels of individual discrimination provided by other marker categories. In this context, HID Ion Ampliseq Identity Panel is a commercial solution with forensic purposes comprising simultaneous analysis of 90 highly informative autosomal SNPs and 34 Y -chromosome superior clade SNPs for male lineage haplotyping. SNP typing can be obtained with smaller amplicons, and this panel was designed for efficient processing of critical or challenging forensic samples. In this work, a sample of 432 individuals from all five Brazilian geopolitical regions was evaluated with this panel, in order to access feasibility of this panel use in a national basis. Results obtained for all five regions, including forensic parameters, show that this marker set can be efficiently employed for Brazilian nationals in human identification or kinship determination applications, due to high levels of genetic discriminative information content displayed by Brazilians. Interpopulation comparison studies were executed among Brazilian regional populations and 26 worldwide populations, in order to access genetic stratification occurrence. Some levels of population structure were identified, and impact on database design was discussed. Y-chromosome haplotyping of Brazilian samples revealed high levels of European ancestry in Brazilian male lineages, and utility of haplotyping in real forensic casework is addressed. Finally, genotyping and sequencing efficiency with this panel were addressed, as an effort to appraise the adequacy of this panel use in Brazilian national forensic demands.

Familial DNA searching- an emerging forensic investigative tool.

In recent years, jurisdictions across the United States have expressed a growing interest in aiding criminal investigations through the use of familial DNA searching (FDS)- a forensic technique to identify family members through DNA databases. The National Survey of CODIS Laboratories surveyed U.S. CODIS laboratories about their perceptions, policies, and practices related to FDS. In total, 103 crime labs completed the survey (77% response rate). Labs in 11 states reported using FDS, while labs in 24 states reported using a similar-but distinct- practice of partial matching. Although the majority of labs had positive perceptions about the ability of FDS to assist investigations, labs also reported a number of concerns and challenges with implementing FDS. Respondents reported using either practice a limited amount with modest numbers of convictions resulting from both FDS and partial matching. The article reports on varying practices related to official policies, training, eligibility, the software search, lineage testing, requirements for releasing information, and subsequent investigative work. Finally, the article discusses what can be learned from this survey, accompanying limitations, and implications for decision-makers considering using FDS.

Utility of ForenSeq™ DNA Signature Prep Kit in the research of pairwise 2nd-degree kinship identification.

The scope of forensic kinship analysis is being extended to more distant or complex relationships. However, current methods and standards in this field do not meet the needs of casework. The next-generation sequencing (NGS) technology may hold an advantage in this field to traditional methods due to its strong power to get much more genetic information. To evaluate the effectiveness of NGS to identify the 2nd-degree kinship pairs, DNA samples of 227 individuals from 49 Hebei Han pedigrees were tested by Goldeneye™ 20A kit using capillary electrophoresis (CE) to confirm the relationships within each pedigree, and those of 111 individuals within 97 confirmed grandparent-grandchild or avuncular pairs were analyzed by ForenSeq™ DNA Signature Prep Kit using MiSeq® FGx™ DNA sequencing platform. We calculated the likelihood ratio (LR) based on ITO method and the identical by state (IBS) score of 97 kinship pairs and compared with those of 97 unrelated pairs. According to the results summarized and analyzed by Fisher discriminant analysis and leave-one-out cross-validation (LOOCV) method, ITO method showed higher accuracy than IBS method, even with less information. Therefore, we proposed a recommendation of the thresholds for pairwise 2nd-degree kinship identification for Hebei Han population based on ITO method. When using ITO method based on 94 SNPs and the length information of 27 autosomal STRs, cumulative likelihood ratio (CLR) > 1 and CLR < 0.1 are recommended as the thresholds of confirming and excluding, respectively. The accuracy applying such thresholds is greater than 95%, indicating the promising application value of NGS in this field and providing a direction for further kinship identification strategy selection. Further studies are needed to get the population genetic data of loci contained in the kit based on all sequence information including flanking regions to make full use of the NGS data to improve the accuracy of kinship analysis.

"The acid test"-validation of the ParaDNA® Body Fluid ID Test for routine forensic casework.

The identification of the cellular origin and composition of crime scene-related traces can provide crucial insight into a crime scene reconstruction. In the last decade, especially mRNA-based body fluid and tissue identification (BFI) has been vigorously examined. Besides capillary electrophoretic (CE) and real-time quantitative PCR (RT-qPCR)-based approaches for mRNA detection, melt curve analysis bears potential as a simple-to-use method for BFI. The ParaDNA® Body Fluid ID Test relies on HyBeacon® probes and was developed as a rapid test for mRNA-based BFI of six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual, and peripheral blood. The herein presented work was performed as an "acid test" of the system and should clarify whether the approach matches the requirements of forensic routine casework in German police departments. Tested samples consisted of single source as well as of mixed samples.

Developmental validation of the ParaDNA® Body Fluid ID System-A rapid multiplex mRNA-profiling system for the forensic identification of body fluids.

Identifying the biological origin of forensic traces can provide crucial evidence to aid criminal investigations. Current forensic practice for the identification of body fluids mostly uses protein-based presumptive tests. Such tests cannot identify all of the forensically relevant fluids and have issues of cross-reactivity. More recently, messenger RNA methods have been developed that have expanded the range of body fluids that can be positively identified. However, these methods are slow and require expert scientists to run the processes and interpret the results. The ParaDNA® Body Fluid ID System has been designed to provide a simple, fast and robust way to identify forensically relevant body fluids in a lab or field-deployable manner. The system can analyse and detect mRNA targets for six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual blood and peripheral blood. Utilising the ParaDNA Sample Collector and instruments, minimal training is required to enable both forensic scientists and non-specialist personnel to analyse biological samples directly, without prior sample processing, in approximately 90 min. The developmental validation studies described here were designed to address requirements of end users, based on the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Body Fluid ID System on a range of mock evidence items. The data collected demonstrate that the Body Fluid ID System can automatically determine the presence of specific body fluid mRNA markers in single-source and mixed samples on multiple substrate types and body fluids could be identified with as little as 0.05ng total RNA and 1µl of the relevant fluid. Results can either be used to support confirmation of source from previously obtained STR DNA profiling results or to improve sample success rates by making better informed evidential submissions.

Evaluation of the precision ID mtDNA whole genome panel on two massively parallel sequencing systems.

Sequencing whole mitochondrial genomes by capillary electrophoresis is a costly and time/labor-intensive endeavor. Many of the previous Sanger sequencing-based approaches generated amplicons that were several kilobases in length; lengths that are likely not amenable for most forensic applications. However, with the advent of massively parallel sequencing (MPS) short-amplicon multiplexes covering the entire mitochondrial genome can be sequenced relatively easily and rapidly. Recently, the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific by Applied Biosystems™) has been introduced. This panel is composed of 162 amplicons (in two multiplexes) that are considerably smaller in length (∼163bp) and thus are more amenable to analyzing challenged samples. This panel was evaluated on both the Ion S5™ System (Thermo Fisher Scientific) and the MiSeq™ FGx Desktop Sequencer (Illumina). A script was developed to extract phased haplotypes associated with these amplicons. Levels of read-depth were compared across sequencing pools and between sequencing technologies and haplotype concordances were assessed. Given modest thresholds on read depth, the haplotypes identified by either technology were consistent. Nuclear mitochondrial sequences (Numts) were also inferred, and the effect of different mapping strategies commonly used to filter out Numts were contrasted. Some Numts are co-amplified with this amplification kit, and while the choice of reference sequence can mitigate some of these effects, some data from the mitochondrial genome were lost in the process in this study. This study demonstrates that the Ion and MiSeq platforms provide consistent haplotype estimation of the whole mitochondrial genome, thus providing further support for the reliability and validity of the Precision ID mtDNA Whole Genome Panel.

In-field collection and preservation of decomposing human tissues to facilitate rapid purification and STR typing.

Short tandem repeats (STR) are currently the gold standard in human identification for forensic casework purposes, and successful STR typing is dependent on sufficient quantity and quality DNA. In the aftermath of a mass disaster and some forensic cases, human remains are recovered for identification in various stages of decomposition, and ideally these remains are transported to a refrigerated facility in order to halt the decomposition process and preserve the integrity of DNA within the tissue. However, in situations where refrigeration is not available (e.g., after a mass disaster or in rural forensic casework), remains continue to be exposed to environmental insults after collection, causing further DNA damage and degradation. Therefore, successful STR typing is dependent on the time of collection and preservation of the DNA sample. This study aims to test two simple in-field collection and preservation methods for decomposing human tissues that are subsequently stored at room temperature for up to six months either in a tissue preservative solution (modified TENT buffer) or on an FTA® Elute Card. In addition, these collection and preservation methods were tested for their ability to facilitate more direct and faster processing of DNA from preserved tissues or DNA leached into the surrounding TENT preservative solution for STR typing. Pre-PCR methods tested in this study include a quick lysis of FTA® Elute Cards, silica-based purification (QIAquick®), enzyme-based extractions (PDQeX), and simple dilution of liquid preservative. The traditional DNA analysis pipeline, which includes DNA extraction and quantification, will be compared to an alternate direct PCR method, thereby allowing the elimination of these two time-consuming and costly steps. The results indicate that modified TENT preservative and FTA® Elute Cards both preserved DNA from relatively fresh tissue for up to six months at room temperature. However, mostly partial profiles were produced from decomposed tissues (day 6 - day 14 in this study) when stored for up to six months compared to when tissues were processed immediately following collection. Overall, the modified TENT preservative produced higher DNA concentrations and more successful STR results than FTA® Elute Cards. In addition, a rapid DNA extraction platform (PDQeX) generated the most successful STR typing results from the decomposed tissues stored in TENT for up to six months at room temperature. The direct PCR method used in this study generated comparable STR results to the traditional DNA analysis approach, warranting further investigation of direct PCR methods for forensic casework type samples.