Preliminary Study: DNA Transfer and Persistence on Non-Porous Surfaces Submerged in Spring Water.

Submerged items are often thought to lack evidentiary value. However, previous studies have shown the ability to recover DNA from submerged porous items for upwards of six weeks. The crevices or interweaving fibers in porous items are thought to protect DNA from being washed away. It is hypothesized that, because non-porous surfaces do not have the same traits that might aid in DNA retention, then DNA quantities and the number of donor alleles recovered would decrease over longer submersion periods. Additionally, it is hypothesized that DNA quantity and the number of alleles would be negatively affected by flow conditions. Neat saliva of known DNA quantity was applied to glass slides and exposed to stagnant and flowing spring water to observe the effects on both DNA quantity and STR detection. Results supported that DNA deposited onto glass and subsequently submerged in water experienced a decrease in DNA quantity over time, yet submersion did not have as strong of a negative effect on the detected amplification product. Additionally, an increase in DNA quantity and detected amplification product from designated blank slides (no initial DNA added) could indicate the possibility of DNA transfer.

The persistence and stabilization of auxiliary genes in the human skin virome.

BACKGROUND: The human skin contains a diverse microbiome that provides protective functions against environmental pathogens. Studies have demonstrated that bacteriophages modulate bacterial community composition and facilitate the transfer of host-specific genes, potentially influencing host cellular functions. However, little is known about the human skin virome and its role in human health. Especially, how viral-host relationships influence skin microbiome structure and function is poorly understood. RESULTS: Population dynamics and genetic diversity of bacteriophage communities in viral metagenomic data collected from three anatomical skin locations from 60 subjects at five different time points revealed that cutaneous bacteriophage populations are mainly composed of tailed Caudovirales phages that carry auxiliary genes to help improve metabolic remodeling to increase bacterial host fitness through antimicrobial resistance. Sequence variation in the MRSA associated antimicrobial resistance gene, erm(C) was evaluated using targeted sequencing to further confirm the presence of antimicrobial resistance genes in the human virome and to demonstrate how functionality of such genes may influence persistence and in turn stabilization of bacterial host and their functions. CONCLUSIONS: This large temporal study of human skin associated viruses indicates that the human skin virome is associated with auxiliary metabolic genes and antimicrobial resistance genes to help increase bacterial host fitness.

Internal Validation of MaSTR™ Probabilistic Genotyping Software for the Interpretation of 2-5 Person Mixed DNA Profiles.

Mixed human deoxyribonucleic acid (DNA) samples present one of the most challenging pieces of evidence that a forensic analyst can encounter. When multiple contributors, stochastic amplification, and allele drop-out further complicate the mixture profile, interpretation by hand becomes unreliable and statistical analysis problematic. Probabilistic genotyping software has provided a tool to address complex mixture interpretation and provide likelihood ratios for defined sets of propositions. The MaSTR™ software is a fully continuous probabilistic system that considers a wide range of STR profile data to provide likelihood ratios on DNA mixtures. Mixtures with two to five contributors and a range of component ratios and allele peak heights were created to test the validity of MaSTR™ with data similar to real casework. Over 280 different mixed DNA profiles were used to perform more than 2600 analyses using different sets of propositions and numbers of contributors. The results of the analyses demonstrated that MaSTR™ provided accurate and precise statistical data on DNA mixtures with up to five contributors, including minor contributors with stochastic amplification effects. Tests for both Type I and Type II errors were performed. The findings in this study support that MaSTR™ is a robust tool that meets the current standards for probabilistic genotyping.

Genome Sequence of Feline Papillomavirus Strain P20 Assembled from Metagenomic Data from the Skin of a House Cat Owner.

A feline papillomavirus genome was assembled from metagenomic sequencing data collected from the skin of a house cat owner. The circular genome of strain P20 is 8,069 bp in length, has a GC content of 54.38%, and displays genome organization typical of feline papillomaviruses. The genome exhibits approximately 75% sequence similarity to other feline papillomavirus genomes.

The application of the skin virome for human identification.

The use of skin virome offers a unique approach for human identification purposes in instances where a viable and statistically relevant human DNA profile is unavailable. The skin virome may act as an alternative DNA profile and/or an additional form of probative genetic material. To date, no study has attempted to investigate the human virome over a time series across various physical locations of the body to identify its diagnostic potential as a tool for human identification. For this study, we set out to evaluate the stability, diversity, and individualization of the human skin virome. An additional goal was to identify putative viral signatures that can be used in conjunction with traditional forensic STR loci. In order to accomplish this, human viral metagenomes were collected and sequenced from 42 individuals at three anatomical locations (left hand, right hand, and scalp) across multiple collection periods over a 6-month window of time. Assembly dependent and independent bioinformatic approaches, along with a database centered assessment of viral identification, resulted in three sets of stable putative viral markers. In total, with the three sets combined, we identified 59 viral biomarker regions, consisting of viral species and uncharacterized viral genome assemblies, that were stable over the sampling period. Additionally, we found the abundance profiles of these 59 viral biomarkers, based on presence or absence, to be significantly different across subjects (P < 0.001). Here we demonstrate that not only is the human virome applicable to be used for human identification, but we have identified many viral signatures that can putatively be used for forensic applications, thus providing a foundation to the novel field of forensic virology.

Assessment of PowerPlex® Fusion 5C's ability to type degraded DNA.

DNA samples collected at crime scenes are often degraded so this research focused on the ability of the Promega PowerPlex® Fusion 5C amplification kit to type both naturally and artificially degraded DNA. DNA was degraded naturally by placing equal volumes of blood on white fabric that was stored either inside, outside in a shaded area, or outside in direct sunlight. Samples were then collected every 10 days for 60 days and the DNA extracted (QIAamp® DNA Investigator). Artificially degraded samples were created by exposing extracted DNA to either UV light or 95 °C heat for varying times. DNA was also degraded artificially by placing blood samples into a 50% bleach solution for varying times prior to extraction. Following sample treatment, standard forensic DNA analysis was performed including quantification (Investigator® Quantiplex) and amplification (PowerPlex® Fusion 5C). Separation and detection were performed on an ABI 3130xl capillary electrophoresis unit and analysis was performed using GeneMapper ID v3.2.1. While the time and shade samples showed similar amounts of degradation, the samples exposed to direct sun showed more degradation. The artificially degraded samples showed more signs of degradation such as reduced overall peak height and peak height imbalance at heterozygous loci. There were also some cases where an allele that was known to be in the profile exhibited total dropout. Although there were some instances of both allelic dropout and heterozygote peak imbalance, PowerPlex® Fusion was able to reliably type degraded DNA as all alleles detected were consistent with the known donor profile. The results show that PowerPlex® Fusion is a robust kit capable of handling forensically challenged samples.

A preliminary assessment of the effect of PreCR™ DNA repair treatment on mixture ratios in two person mixtures.

In this study, DNA extracted from known buccal samples was combined into two component mixture samples. These were subjected to UV exposure prior to their amplification with the Promega PowerPlex® 16HS amplification kit, and subsequent capillary electrophoresis on the ABI 3130xl instrument. Damaged samples were subjected to enzymatic repair treatment and retested to assess the amount of repair. Data showed that there is fidelity associated with the application with profile concordance after its use, and a corresponding increase in the amount of recovered alleles post damage. Results also showed changes in the stochastic relationship between mixture components that appear to be induced by the repair process itself. The mixture ratios of DNA samples were altered from an approximate original 1:3 ratio, to a ratio of 1:2 or greater. This variation can have a significant effect regarding the ability to reliably de-convolute DNA mixtures that have been subjected to the repair process.

The Potential of Cosmetic Applicators as a Source of DNA for Forensic Analysis.

Personal products, such as toothbrushes, have been used as both known reference and evidentiary samples for forensic DNA analysis. This study examined the viability of a broad selection of cosmetic applicators for use as targets for human DNA extraction and short tandem repeat (STR) analysis using standard polymerase chain reaction (PCR) conditions. Applicator types included eyeliner smudgers, pencils and crayons, eye shadow sponges, mascara wands, concealer wands, face makeup sponges, pads and brushes, lipsticks and balms, and lip gloss wands. The quantity and quality of DNA extracted from each type of applicator were examined by assessing the number of loci successfully amplified and the peak balance of the heterozygous alleles in each full STR profile. While degraded DNA, stochastic amplification, and PCR inhibition were observed for some items, full STR profiles were developed for 14 of 76 applicators. The face makeup sponge applicators yielded the highest proportional number of full STR profiles (4/7).

Evaluation of methods to improve the extraction and recovery of DNA from cotton swabs for forensic analysis.

Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol's incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations.

Validation of male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex.

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.

Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex.

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.