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We describe an adaption of Bright et al.'s work modeling peak height variability in CE-DNA profiles to the modeling of allelic aSTR (autosomal short tandem repeats) read counts from NGS-DNA profiles, specifically for profiles generated from the ForenSeq™ DNA Signature Prep Kit, DNA Primer Mix B. Bright et al.'s model consists of three key components within the estimation of total allelic product-template, locus-specific amplification efficiencies, and degradation. In this work, we investigated the two mass parameters-template and locus-specific amplification efficiencies-and used MLE (maximum likelihood estimation) and MCMC (Markov chain Monte Carlo) methods to obtain point estimates to calculate the total allelic product. The expected read counts for alleles were then calculated after proportioning some of the expected stutter product from the total allelic product. Due to preferential amplicon selection introduced by the sample purification beads, degradation is difficult to model from the aSTR outputs alone. Improved modeling of the locus-specific amplification efficiencies may mask the effects of degradation. Whilst this model could be improved by introducing locus specific variances in addition to locus specific priors, our results demonstrate the suitability of adapting Bright et al.'s allele peak height model for NGS-DNA profiles. This model could be incorporated into continuous probabilistic interpretation approaches for mixed DNA profiles.

There has been an increase in the number of laboratories and researchers adopting new sequencing technologies, known as next-generation sequencing (NGS). An understanding of the behaviour of NGS DNA profiles is needed to enable for the development of probabilistic genotyping methods for the interpretation of such profiles. In this work, we investigate NGS analyte signal variation, specifically heterozygous balance and stutter variability from profiles generated using the ForenSeq™ DNA Signature Prep Kit, DNA Primer Mix B. We also investigate additivity of analyte signals in NGS profiles for overlapping allelic and stutter signals originating from the same or different contributors. We describe models that can be used to inform a continuous method for the interpretation of DNA profiling data.

Massively parallel sequencing (MPS) provides forensic DNA laboratories an option to overcome the limitations associated with CE and current STR assays. Verogen's MPS ForenseqTM DNA Signature kit concomitantly amplifies 27 autosomal, 7 X-, and 24 Y-STRs. In addition, 94 identity, 56 ancestry, and 22 phenotypic-informative SNPs are included for a total of over 200 markers in one multiplex. An internal validation of this platform was conducted using reference samples to investigate whether the Forenseq™ DNA Signature Prep kit, specifically primer panel B, has the capability to provide consistent and accurate typing/sequencing data. The data presented in this report is limited to that corresponding to autosomal and Y-STRs. Results suggest that the system can consistently generate accurate genotyping data when up to 40 high-quality, high-quantity (i.e. 1 ng) single source samples are pooled into a sequencing reaction. The results generated were used to determine appropriate analysis parameters and thresholds for streamlined data interpretation of reference samples.

The ANDE 6C Rapid DNA system could offer a potential alternative for the processing of calcified and soft tissue samples, often encountered in mass disaster scenarios. While originally designed for single source buccal swabs, interest in the performance of these instruments when using other types of single source samples continues to grow. To enhance the recovery of otherwise lesser quality samples, the manufacturer developed the investigative biochip, an alternative to the NDIS approved Arrestee biochip for reference sample buccal swabs. This study explores the viability of using the ANDE 6C system and the investigative biochip to process soft and calcified tissue, and uses conventional sample processing to contrast the results. Though the success rate obtained using the instrument's expert system was lower than expected - 0% muscle, 11% ribs, and 50% teeth -, the ANDE 6C offers an advantage over conventional calcified tissue processing in terms of turn-around time and processing complexity. If robust analysis parameters can be established to allow the evaluation of the generated data by a qualified analyst on a third party software platform, the use of the ANDE 6C and investigative biochip could be a suitable alternative for currently employed procedures. However, as is the case with conventional DNA typing, the quantity, age, type of biological material and quality of the exemplars could all play a role in the success of the ANDE 6C typing process. In addition, it appears as if the calcified tissue pre-processing protocol that provides the better opportunity for the ANDE 6C success is not appropriate to be carried out in the field or by non-trained personnel as special equipment as well as a certain level of exe expertise and technique is necessary. Nevertheless, disaster victim and unidentified human remain samples could be processed in a laboratory setting using the Rapid DNA ANDE 6C platform provided sufficient material is available to conduct a second, 'rescue' sample processing if necessary.

Rapid DNA platforms are fully integrated systems capable of producing and analyzing short tandem repeat (STR) profiles from reference sample buccal swabs in less than two hours. The technology requires minimal user interaction and experience making it possible for high quality profiles to be generated outside an accredited laboratory. The automated production of point of collection reference STR profiles could eliminate the time delay for shipment and analysis of arrestee samples at centralized laboratories. Furthermore, point of collection analysis would allow searching against profiles from unsolved crimes during the normal booking process once the infrastructure to immediately search the Combined DNA Index System (CODIS) database from the booking station is established. The DNAscan/ANDE™ Rapid DNA Analysis™ System developed by Network Biosystems was evaluated for robustness and reliability in the production of high quality reference STR profiles for database enrollment and searching applications. A total of 193 reference samples were assessed for concordance of the CODIS 13 loci. Studies to evaluate contamination, reproducibility, precision, stutter, peak height ratio, noise and sensitivity were also performed. The system proved to be robust, consistent and dependable. Results indicated an overall success rate of 75% for the 13 CODIS core loci and more importantly no incorrect calls were identified. The DNAscan/ANDE™ could be confidently used without human interaction in both laboratory and non-laboratory settings to generate reference profiles.

Copper wires are a common component of improvised explosive devices (IEDs) and are increasingly found in forensic evidence. When processed for DNA, ions from the metal can be co-extracted with the genetic material, and potentially cause complications during the analysis procedure. It was determined in this project that copper ions do have adverse effects on DNA processing by either interacting directly with the DNA or affecting the electrophoretic mobility of the molecule. It was also found that these negative effects could be improved by utilizing a purification method capable of reducing the amount of copper carry-over into the sample.

Three commercially available integrated rapid DNA instruments were tested as a part of a rapid DNA maturity assessment in July of 2018. The assessment was conducted with sets of blinded single-source reference samples provided to participants for testing on the individual rapid platforms within their laboratories. The data were returned to the National Institute of Standards and Technology (NIST) for review and analysis. Both FBI-defined automated review (Rapid DNA Analysis) and manual review (Modified Rapid DNA Analysis) of the datasets were conducted to assess the success of genotyping the 20 Combined DNA Index System (CODIS) core STR loci and full profiles generated by the instruments. Genotype results from the multiple platforms, participating laboratories, and STR typing chemistries were combined into a single analysis. The Rapid DNA Analysis resulted in a success rate of 80% for full profiles (85% for the 20 CODIS core loci) with automated analysis. Modified Rapid DNA Analysis resulted in a success rate of 90% for both the CODIS 20 core loci and full profiles (all attempted loci per chemistry). An analysis of the peak height ratios demonstrated that 95% of all heterozygous alleles were above 59% heterozygote balance. For base-pair sizing precision, the precision was below the standard 0.5 bp deviation for both the ANDE 6C System and the RapidHIT 200.

Allele distributions for twenty-three autosomal short tandem repeat (STR) loci – D1S1656, D2S441, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, CSF1PO, FGA, Penta D, Penta E, SE33, TH01, TPOX and vWA – were determined in Caucasians, Southwestern Hispanics, Southeastern Hispanics, African Americans, Bahamians, Jamaicans, Trinidadians, Chamorros, Filipinos, Apaches, and Navajos. The data are included in the FBI PopStats software for calculating statistical estimates of DNA typing results and cover the expanded CODIS Core STR Loci required of U.S. laboratories that participate in the National DNA Index System (NDIS).

Rapid DNA technology will provide law enforcement booking personnel the ability to process and query reference samples obtained from qualifying arrestees against unsolved crimes of special concern in real-time, potentially enhancing the speed of an investigation. The technology could also be utilized within the laboratory setting to expedite the processing of reference samples submitted with a case. Validation studies have found that when used with reference samples, the ANDE 6C Rapid DNA technology generates successful profiles that are concordant to orthogonal system generated data ∼85 % of the time. The success rate, however, appears to be contingent on the use of the ANDE swab. During this study a comparison between ANDE and conventional cotton swab results was performed to establish if there was a difference in success rate between the two substrates. The results demonstrate a substantial decrease in expert system passing profiles when conventional swabs were used. For those instances, a modified Rapid DNA protocol could be developed to aid in the recovery of the data.

Rapid DNA platforms are fully integrated systems capable of producing and analyzing short tandem repeat (STR) profiles from reference sample buccal swabs in less than two hours. The technology requires minimal user interaction and experience making it possible for high quality profiles to be generated outside an accredited laboratory. The automated production of point of collection reference STR profiles could eliminate the time delay for shipment and analysis of arrestee samples at centralized laboratories. Furthermore, point of collection analysis would allow searching against profiles from unsolved crimes during the normal booking process once the infrastructure to immediately search the Combined DNA Index System (CODIS) database from the booking station is established. The DNAscan/ANDE™ Rapid DNA Analysis™ System developed by Network Biosystems was evaluated for robustness and reliability in the production of high quality reference STR profiles for database enrollment and searching applications. A total of 193 reference samples were assessed for concordance of the CODIS 13 loci. Studies to evaluate contamination, reproducibility, precision, stutter, peak height ratio, noise and sensitivity were also performed. The system proved to be robust, consistent and dependable. Results indicated an overall success rate of 75% for the 13 CODIS core loci and more importantly no incorrect calls were identified. The DNAscan/ANDE™ could be confidently used without human interaction in both laboratory and non-laboratory settings to generate reference profiles.

Electronic DNA genotype data corresponding to eleven FBI relevant populations, typed with the expanded STR chemistries, are presented. Such data may prove valuable for population genetic studies and other forensic relevant inquiries. Keywords: FBI populations, Genotypes, Expanded core loci

The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the α, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood.

The measure of quality in DNA sample processing starts with an effective nucleic acid isolation procedure. Most problems with DNA sample typing can be attributed to low quantity DNA and/or to the presence of inhibitors in the sample. Therefore, establishing which isolation method is best at removing potential inhibitors may help overcome some of the problems analysts encounter by providing useful information in the determination of the optimal approach for any given sample. Direct analysis in real time (DART) mass spectrometry was used in this study to investigate the ability of different extraction methods to remove PCR inhibitors. Methods investigated included both liquid/liquid (phenol-chloroform) and solid phase based robotic procedures, (PrepFiler™ and EZ1 chemistries). Following extraction, samples were analyzed by DART in order to determine the level of remaining inhibitors and then quantified and amplified to determine the effect any remaining inhibitor had on the overall results. The data suggests that organic extraction methods result in detrimental amounts of phenol carryover while automated methods may produce carry-over of bile salts and other chemicals that preferentially bind the solid phase matrix. Both of these effects can have a negative impact in downstream sample processing and genotyping by PCR.

The combination of soil's ubiquity and its intrinsic abiotic and biotic information can contribute greatly to the forensic field. Although there are physical and chemical characterization methods of soil comparison for forensic purposes, these require a level of expertise not always encountered in crime laboratories. We hypothesized that soil microbial community profiling could be used to discriminate between soil types by providing biological fingerprints that confer uniqueness. Three of the six Miami-Dade soil types were randomly selected and sampled. We compared the microbial metagenome profiles generated using amplicon length heterogeneity-polymerase chain reaction analysis of the 16S rRNA genes with inductively coupled plasma optical emission spectroscopy analysis of 13 elements (Al, B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Si, and Zn) that are commonly encountered in soils. Bray-Curtis similarity index and analysis of similarity were performed on all data to establish differences within sites, among sites, and across two seasons. These data matrices were used to group samples that shared similar community patterns using nonmetric multidimensional scaling analysis. We concluded that while chemical characterization could provide some differentiation between soils, microbial metagenome profiling was better able to discriminate between the soil types and had a high degree of reproducibility, therefore proving to be a potential tool for forensic soil comparisons.

Though shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, DNA testing has historically focused on the recovery of just about 600 base pairs of the mitochondrial DNA control region. Here, we describe our success in recovering complete mitochondrial genome (mtGenome) data (∼16,569bp) from single shed hairs. By employing massively parallel sequencing (MPS), we demonstrate that particular hair samples yield DNA sufficient in quantity and quality to produce 2-3kb mtGenome amplicons and that entire mtGenome data can be recovered from hair extracts even without PCR enrichment. Most importantly, we describe a small amplicon multiplex assay comprised of sixty-two primer sets that can be routinely applied to the compromised hair samples typically encountered in forensic casework. In all samples tested here, the MPS data recovered using any one of the three methods were consistent with the control Sanger sequence data developed from high quality known specimens. Given the recently demonstrated value of complete mtGenome data in terms of discrimination power among randomly sampled individuals, the possibility of recovering mtGenome data from the most compromised and limited evidentiary material is likely to vastly increase the utility of mtDNA testing for hair evidence.

The placement of cadavers in shallow, clandestine graves may alter the microbial and geochemical composition of the underlying and adjacent soils. Using amplicon length heterogeneity-PCR (LH-PCR) the microbial community changes in these soils can be assessed. In this investigation, nine different grave sites were examined over a period of 16weeks. The results indicated that measurable changes occurred in the soil bacterial community during the decomposition process. In this study, amplicons corresponding to anaerobic bacteria, not indigenous to the soil, were shown to produce differences between grave sites and control soils. Among the bacteria linked to these amplicons are those that are most often part of the commensal flora of the intestines, mouth and skin. In addition, over the 16week sampling interval, the level of indicator organisms (i.e., nitrogen fixing bacteria) dropped as the body decomposed and after four weeks of environmental exposure they began to increase again; thus differences in the abundance of nitrogen fixing bacteria were also found to contribute to the variation between controls and grave soils. These results were verified using primers that specifically targeted the nifH gene coding for nitrogenase reductase. LH-PCR provides a fast, robust and reproducible method to measure microbial changes in soil and could be used to determine potential cadaveric contact in a given area. The results obtained with this method could ultimately provide leads to investigators in criminal or missing person scenarios and allow for further analysis using human specific DNA assays to establish the identity of the buried body.

Sulfidic, anoxic sediments of the moderately hypersaline Salton Sea contain gradients in salinity and carbon that potentially structure the sedimentary microbial community. We investigated the abundance, community structure, and diversity of Bacteria and Archaea along these gradients to further distinguish the ecologies of these domains outside their established physiological range. Quantitative PCR was used to enumerate 16S rRNA gene abundances of Bacteria , Archaea , and Crenarchaeota . Community structure and diversity were evaluated by terminal restriction fragment length polymorphism (T-RFLP), quantitative analysis of gene (16S rRNA) frequencies of dominant microorganisms, and cloning and sequencing of 16S rRNA. Archaea were numerically dominant at all depths and exhibited a lesser response to environmental gradients than that of Bacteria . The relative abundance of Crenarchaeota was low (0.4 to 22%) at all depths but increased with decreased carbon content and increased salinity. Salinity structured the bacterial community but exerted no significant control on archaeal community structure, which was weakly correlated with total carbon. Partial sequencing of archaeal 16S rRNA genes retrieved from three sediment depths revealed diverse communities of Euryarchaeota and Crenarchaeota , many of which were affiliated with groups previously described from marine sediments. The abundance of these groups across all depths suggests that many putative marine archaeal groups can tolerate elevated salinity (5.0 to 11.8% [wt/vol]) and persist under the anaerobic conditions present in Salton Sea sediments. The differential response of archaeal and bacterial communities to salinity and carbon patterns is consistent with the hypothesis that adaptations to energy stress and availability distinguish the ecologies of these domains.

Ocular Adnexal Lymphomas (OALs) are the most common tumors of the eye, the majority being extranodal mucosa-associated lymphoid tissue (MALT) lymphomas. Association with Chlamydia psittaci was described in some geographic areas. OAL response to antibiotic therapy was reported in cases not harboring Chlamydia psittaci DNA, suggesting that other bacterial infection might be implicated in the pathogenesis. We examined 49 MALT OALs for bacterial DNA using two distinct polymerase chain reaction techniques based on universal bacterial primers. No bacterial DNA that could be implicated in OAL pathogenesis was detected, suggesting that bacterial infection is not associated with OAL in South Florida.