Evaluation of metal ions and DNA recovery from the surface of fired and unfired brass ammunition to improve STR profiling.

Interest in recovering DNA from the surface of ammunition evidence for genotyping has increased over the past few years. Numerous studies have examined a variety of methods to maximize DNA recovery from these types of challenging samples, but successful DNA profiling has been inconsistent. Low amounts of DNA and PCR inhibition due to metal ions have been suggested as the leading causes of poor results; however, no study quantitatively examined the presence of metal ions at various stages of the DNA analysis workflow from DNA collection through to amplification. In this study, the effectiveness of six different DNA collection and purification methods commonly used by forensic laboratories to process brass ammunition for DNA evidence was investigated. The amount of copper, zinc, and other metals co-recovered from fired and unfired brass casings during DNA collection (using numerous soaking, swabbing, and direct PCR protocols) was quantified via Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). This same panel of metals was subsequently quantified after DNA lysis and purification steps. Results demonstrated that low amounts of DNA, DNA damage, and degradation are more detrimental to STR typing results than PCR inhibition, as metal ions were successfully removed by all DNA purification methods tested. Furthermore, the use of metal ion chelators increased the amount of DNA recovered and number of reportable STR alleles. This research informs the forensic community on the most effective way to collect and process trace amounts of biological material from brass ammunition and similar evidence.

Quantitative color analysis of burned bone to predict DNA quantity, quality, and genotyping success.

Badly burned skeletal remains are commonly submitted to forensic laboratories for victim identification via DNA analysis methods. Burned skeletal remains present many challenges for DNA analysis as they can contain low amounts of DNA which can also be damaged and degraded, resulting in partial or no STR profiles. Therefore, a simple, but effective screening method that identifies which samples may provide the most successful STR or mtDNA typing results for identification would enable forensic laboratories to save time, money, and resources. One metric that can be used and a screening method is the color of burned bone, as bone color changes with exposure to fire as temperature and length of exposure increase. This research developed a quantitative screening method based on the surface color of burned bone. The different visual bone colors (light brown, dark brown, black, gray, and white) were quantified using the Commission on Illumination L*a*b color space. These values were then compared to DNA yield, STR, and mtDNA profile completeness to identify whether the L*a*b values can predict genotyping success. A Bayesian network was constructed to determine the probability of STR typing success, given a set of L*a*b values. Results demonstrated that samples with an a* value greater than or equal to one and b* value greater than eight (light brown and dark brown burned samples) were the most predictive of STR typing success and mtDNA typing success. A decision tree for processing burned bones was constructed based on the color value thresholds.

Genotyping strategies for tissues fixed with various embalming fluids for human identification, databasing, and traceability.

Within anatomical willed body programs and skeletal collections, whole bodies and their disassociated limbs and organs are identified and tracked. However, if these tracking mechanisms fail, DNA recovered from the formalin-fixed tissues/organs could provide an additional layer of quality assurance. Embalming fluids preserve biological tissues; however, they also damage, fragment, and cross-link DNA and protein molecules. This project investigated the success of STR-typing from various soft tissue and bone samples that were fixed with embalming solutions with a range of formaldehyde concentrations. Formalin-fixed samples dissected from five cadavers, including skin, muscle, bone, heart, and kidney were used in Phase 1 of this study. In Phase 2, an additional 57 tissue samples from various embalmed organs and body parts were collected to demonstrate long-term fixation and direct applicability within a body donor program. DNA was extracted from the samples using the QIAamp® FFPE Tissue Kit (QIAGEN), quantified with the Investigator® QuantiPlex® Pro RGQ qPCR Kit (QIAGEN), and amplified using the Investigator® 24plex and 26plex QS Kits and the Investigator® DIPplex Kit (QIAGEN). The results show the DNA was severely damaged, degraded, and often in low amounts (after one year post-embalming). Sampling from skin and muscle tissues embalmed with ~2.5%-5% formaldehyde solutions appears to be the best strategy for identification, while also maintaining the preservation of the tissues. The results of this project can provide informative data when determining which genotyping strategy may be best suited for the identification, re-association, and establishment of a database for the provenance of formalin-fixed human remains.

The Sexome - A proof of concept study into microbial transfer between heterosexual couples after sexual intercourse.

The detection and recovery of male DNA post-assault is important in sexual assault investigations, particularly where an offender is unknown to the victim. The collection of DNA evidence often occurs when the female victim undergoes a forensic medical assessment. Analysis regularly results in mixed autosomal DNA profiles with both victim and perpetrator DNA, often making it difficult to interpret a male profile suitable for DNA database searching. While short tandem repeat (STR) profiling of the male Y-chromosome is often used to overcome this challenge, successful identification of an individual can be hindered by the paternal inheritance pattern of Y-STRs and small Y-STR databases. Human microbiome research has suggested that a person's microbial diversity is unique. Therefore microbiome analysis using Massively Parallel Sequencing (MPS) could serve as a useful adjunct method of perpetrator identification. This study aimed to identify bacteria taxa that were unique to each participant and compare the bacterial communities found on their genitals both pre- and post-coitus. Samples were collected from six male-female sexual partner pairs. Volunteers were asked to self-collect low vaginal (females) and penis shaft and glans (males) samples before and after intercourse. Samples were extracted using the PureLink™ Microbiome DNA Purification Kit. Extracted DNA underwent library preparation using primers targeting the V3-V4 hypervariable regions of the bacterial 16S rRNA gene (∼450 bp). Libraries were sequenced on the Illumina MiSeq® platform. From the sequence data derived, statistical analysis was performed to investigate if bacteria sequences could be used to infer contact between each male-female pairing. Unique bacterial signatures were detected in low frequencies (<1%) in male and female participants pre-coitus. The data indicated a significant disruption to microbial diversity post-coitus in all samples. A transfer of the female microbiome during intercourse was most significant. As expected, one couple who did not use a barrier contraceptive yielded the most microbial transfer and disruption to diversity demonstrating a proof-of-concept in the utility of microbiome interrogation for sexual assault cases. Further genomic analysis is needed to confirm species and subspecies classification of bacteria that may produce a unique microbial profile that could then be used to identify a specific individual.

Optimization of InnoXtract™ extraction and purification system for DNA extraction from skeletal samples.

The InnoXtract™ extraction and purification system is a purification method designed for DNA extraction from low-template samples, specifically rootless hair shafts. Its ability to successfully capture highly fragmented DNA suggests its suitability for use with other challenging sample types, including skeletal remains. However, the lysis and digestion parameters required modifications to successfully optimize the method for this sample type. A two-part digestion was developed utilizing a homebrew digestion buffer (0.5 M EDTA, 0.05% Tween 20, and 100 mM NaCl) and a supplemental lysis with the Hair Digestion Buffer included in the InnoXtract™ kit. Additionally, the magnetic bead volume was modified to improve DNA recovery from these challenging samples. With the altered protocol, the quality and quantity of DNA recovered from InnoXtract™ extracts were comparable to another commercial skeletal extraction method (PrepFiler™ BTA). This modified extraction method successfully purified sufficient amounts of quality DNA from a variety of skeletal samples to produce complete STR profiles. Successful STR typing from surface decomposition, burned, cremated, buried, and embalmed remains indicates the potential of this new method for challenging human identification and missing-person cases.

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes.

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.

Preliminary Investigation of the Effect of Maceration Procedures on Bone Metabolome and Lipidome.

The study of post-mortem changes is a crucial component of forensic investigation. Human forensic taphonomic facilities (HFTFs) are the only institutions allowing the design and execution of controlled human decomposition experiments. When bodies are skeletonized, bones are normally stored in skeletal collections and used for anthropological studies. However, HFTFs apply chemical and/or thermal treatments to the remains prior bone long-term storage. These treatments are believed to alter heavily the original biochemical and molecular signature of bone material. The present study aims to evaluate the effect of these procedures on the bone metabolome and lipidome by using an animal bone model. Three intact bovine tibiae were processed using three protocols routinely applied at HFTFs, and their three counterparts were used as non-treated controls. Bone powder samples were subjected to biphasic extraction and both metabolites and lipids were analysed via liquid chromatography tandem mass-spectrometry. Results showed severe reductions in the abundances of both metabolites and lipids, and the presence of contamination introduced by cleaning agents. Despite the preliminary nature of the study, we demonstrated that the biochemical profile of bone is heavily affected by the maceration procedures. Ideally, these treatments should be avoided, or replaced by minimally invasive procedures agreed across HFTFs.

Assessment of the ForenSeq mtDNA control region kit and comparison of orthogonal technologies.

The ForenSeq® mtDNA Control Region Kit, MiSeq FGx®, and Universal Analysis Software (UAS) were assessed to better define the performance and limitations of the system with forensically relevant samples to provide data for its transition into practice. A total of six MiSeq FGx sequencing runs of ForenSeq mtDNA Control Region kit, three runs of additional orthogonal sequencing chemistries, and Sanger sequencing results for 14 samples were used to test for concordance. Sensitivity, reproducibility, mixture detection studies, as well as studies to measure the performance of amplification and sequencing controls were performed. The use and reliability of the UAS for data analysis was also examined. With a variety of sample types and controls representing many mitochondrial haplotypes, the recently developed mtDNA Control Region Kit, with the MiSeq FGx and UAS, was found to be fit for purpose as reliable, reproducible, and robust. Sensitivity down to 1 pg of input genomic DNA was demonstrated, which allows the system to offer low limits of detection for better interrogation of potential heteroplasmy in samples. Concerns for implementing next generation sequencing (NGS) for mtDNA in laboratories were addressed in this research, including initial template quantification and confirmation of haplotypes generated by UAS software regarding length-based polymorphisms. To improve performance with forensic samples, laboratories could implement mitochondrial-specific qPCR assays for quantification and perform the optional manual normalization protocol. Additional optimization on sample multiplexing can provide methods that either increase sensitivity or cost efficiency of the assay.

Collection and storage of DVI samples with microFLOQ® Direct swabs for direct amplification.

The rapid identification of decomposing human remains is a crucial component of disaster victim identification (DVI), often occurring in remote areas without access to laboratory or storage facilities. Due to the ease of collection and amenability to storage in harsh conditions, swabs may be used to collect DNA from decomposing remains as an alternative to sampling tissue or bone. Direct amplification could further streamline the process and reduce costs. This study investigated the efficacy of direct amplification of DVI samples using microFLOQ® Direct swabs and the QIAGEN Investigator QS GO! Kit. A comparison of performance between direct amplification and traditional methods was made to assess whether direct amplification offered an improvement to traditional methods. DNA was collected by swabbing the muscle of a decomposing human cadaver using three swab types (ADS Genetics 4N6FLOQSwabs®, NADS Genetics 4N6FLOQSwabs®, and the microFLOQ® Direct swab). Traditional swabs (4N6FLOQSwabs®) were extracted and quantified, while a direct amplification strategy was used with the microFLOQ® Direct swabs coupled with the Investigator 24Plex GO! Kit. Processing of the microFLOQ® Direct swabs were optimized and a hybrid strategy that used 4N6FLOQSwabs® to collect and store DNA before swabbing or "subsampling" the 4N6FLOQSwabs® for processing with microFLOQ® Direct swabs was developed. This hybrid strategy allowed for rapid processing without the consumption of the original sample. Traditional and direct PCR methods were comparable up to day 10 of decomposition depending on the sample location and for up to 3 months of storage at room temperature. This research indicated that microFLOQ® Direct swabs in conjunction with the Investigator 24Plex GO! Kit can be used to facilitate rapid direct processing of DNA from decomposing human remains.

The performance of quality controls in the Investigator® Quantiplex® Pro RGQ and Investigator® 24plex STR kits with a variety of forensic samples.

Forensic DNA laboratories process database reference samples on FTA® cards or buccal swabs, which commonly contain adequate amounts of quality DNA resulting in full STR profiles and high first-pass rates. However, some reference samples and many forensic casework samples are exposed to a variety of insults that may lead to low quantities of DNA, DNA degradation, DNA mixtures, and/or PCR inhibition, posing a challenge to downstream genotyping success. The inclusion of multiple amplification targets and internal PCR controls (IPCs) in DNA quantification kits, and quality sensors within STR amplification kits can aid in the accurate interpretation of sample/profile quality, and guide more efficient rework strategies when needed. In order to assess the effectiveness of these quality systems we subjected database-type samples (buccal swabs and blood or saliva on FTA® cards), mock casework samples (low-template, degraded, inhibited, DNA mixtures), and authentic post-coital samples to various challenging conditions. Concordance between the quality flags in the Investigator® Quantiplex® Pro RGQ kit (QIAGEN), the QS markers in QIAGEN's Investigator® 24plex QS kit, and overall STR profile quality was evaluated for all casework-type samples. To assess the value of the QS markers in the Investigator® 24plex QS and GO! STR kits, samples with partial or failed STR profiles were reworked based on the quality of the electropherogram first with the QS markers redacted, and second in conjunction with the QS markers. Results from each of the rework approaches were compared to determine which strategy, if any, improved the STR profile quality and the number of reportable alleles. The QS markers in the 24plex STR kits correctly confirmed sample quality in 99.9% of databasing samples and 98% of mock casework samples. Quality flags during DNA quantification were concordant with downstream STR profiles for the majority (77%) of the mock casework samples. Additionally, when samples with partial STR profiles were reworked, more loci were obtained for 80% of the samples regardless of the rework strategy used. However, the most notable improvement in STR completeness was observed in inhibited samples that were reworked based on the information provided by the STR quality sensors, with an average increase of 56% reportable alleles.

Efficacy of "touch" DNA recovery and room-temperature storage from assault rifle magazines.

Crimes committed with assault rifles are becoming increasingly prevalent in the United States. In the absence of other evidence, DNA analysis can often provide informative leads. Unfortunately, any DNA transferred to rifle components left behind at a crime scene is likely to be low in quantity and/or quality. Furthermore, collected evidence is unlikely to be processed immediately and may require storage. Long-term storage can subject DNA to damage and degradation, which ultimately affects DNA profile interpretation and may prevent the identification of potential suspects. This study assessed the ability of a new swab storage device, the SwabSaver®, to preserve "touch" DNA from AR-15 magazine rifles using three different collection devices. Three volunteers loaded bullet cartridges into plastic polymer and aluminum AR-15 magazines. DNA was collected with traditional cotton swabs, layered cotton paper swabs, or nylon-flocked swabs. Collection devices were then stored at room-temperature for up to two months in either the SwabSaver® device or an empty centrifuge tube. The results suggest that substrate and swab type had less of an effect on profile completeness than storage type. Furthermore, SwabSaver® storage yielded DNA quantities comparable to "touch" DNA extracted after 24 h.