Understanding the behavior of stutter through the sequencing of STR alleles

This work explores the influence of several variables on stutter formation across sequenced autosomal STR loci (simple, compound, and complex motifs) and different alleles within each locus. The variables are sequence variations within the repeating motifs and flanking region [1,2]; longest uninterrupted stretch (LUS) [3]; parental allele length [3]; and base pair content and length value of each repeating motif from which the stutter has generated [3,4]. Over six hundred unrelated individuals from different populations were amplified with the prototype PowerSeq 46GY System and sequenced on the Illumina MiSeq platform. Raw FASTQ files were analyzed with STRait Razor v3 [5]. Stutter ratio was calculated for motifs that exhibited stutter using the ratio of the observed coverage of the stutter sequence at (N-1) position to the observed coverage of the allelic sequence. Understanding the behavior (abundance, reproducibility, sequence context) of non-allelic artifacts will help in establishing probabilistic models for the prediction of stutter rate and interpretation of sequence-based STR profiles.     

Family reunification through DNA analysis of survivors of the greatest natural disaster in Colombia in Armero-tolima (1985)

On November 13, 1985 at 9:20 p.m., the Nevado del Ruiz Volcano erupted (A Glacier Snowy Volcano 17457 fts above sea level). The lahar, melting ice and landslide devastated the town of Armero located 45 kilometers away in a matter of minutes, in the Tolima department of Colombia. More than 25,000 people died or were reported missing. This tragedy is the largest natural disaster to date in Colombia. The Armando Armero Foundation is a Non-Governmental Organization (NGO) that brings together survivors of this tragedy. This NGO is making efforts to reunite families in association with Servicios Médicos Yunis Turbay y Cia. Different strategies are used to search and find matches in our database, including documents and records, autosomic STR, Y chromosome STR and mtDNA analysis. Thus far, four families have been reunited in this process.     

DNA on the inside door handles of entrance doors

DNA from door handles on entry doors could provide a clue as to who last left the scene. However, after years of extensive research on DNA transfer and persistence it can be considered common knowledge that general claims like "the last who touched leaves the most DNA" do not hold true. But who's DNA do we find on door handles that are usually used several times per day by the inhabitants? To assess this question, we sampled inside door handles from real-life burglaries and at the same time collected reference samples from all the inhabitants, to determine if we can detect any (major) profiles from non-inhabitants. We also searched to evaluate how often we detect DNA from the person who last touched the door handle as a (major) contributor. Only small amounts of DNA were recovered from the handles, originating most often, but not always, from inhabitants or even the last inhabitant touching the handle.     

A multifaceted,multijurisdictional, multiagency,and multidisciplinary approach to investigating unidentified and missing persons cases in Australia

Currently, there are approximately 750 unidentified human remains and 2500 long-term missing persons in Australia. The Australian Federal Police National DNA Program for Unidentified and Missing Persons (Program) is using a multifaceted, multijurisdictional, multiagency, and multidisciplinary approach in a dedicated effort to identify these unknown deceased persons, scientifically link them to known missing persons, and provide answers to their families. The nationally coordinated Program provides its police, forensic, and coronial stakeholders with a suite of contemporary forensic technologies, databases, and experts to forensically examine the skeletonised remains and recover post-mortem data for comparison to the available ante-mortem data for each missing person. Through a number of physical and virtual public outreach activities, families with missing relatives have been encouraged to provide vital ante-mortem forensic information, records, and samples to aid the identification process. To date, this unique Program has assisted to resolve a number of unidentified and missing persons cases from both historical and contemporary contexts, using a combination of genetic and non-genetic techniques, and local and national databases. The centralisation of Program capabilities, expertise, and resources to conduct this type of unique and challenging casework is proving to be the most effective and efficient way to generate investigative leads, identify human remains, and resolve long-term missing persons cases in Australia.     

DNA-based identification of big cats and traditional Chinese medicine artifacts in the Czech Republic

The aim of this study is to provide an overview of ongoing research on and the development of identification tools for big cats (Panthera tigris, Panthera leo, Panthera pardus, …). The set of tools includes a species-specific RTPCR quantitation system (nuclear and mitochondrial), STR multiplexes, a rapid system for big cat species determination, and a database solution.     

Experimental long-distance haplotyping of OCA2-HERC2 variants

The regulatory HERC2 SNP, rs12913832, is strongly associated with blue and brown eye colour. However, eye colour in heterozygous rs12913832 individuals is observed to vary greatly. Missense mutations in OCA2, such as rs1800407 and rs74653330, are associated with lighter eye colour in some but not all heterozygous rs12913832 individuals. Determining the physical linkage of these variants might help to further explain eye colour variation. So far, experimental haplotyping of these variants has been challenging because the genomic distance between them (∼ 135 kb) exceeds the fragment lengths produced by commonly used DNA isolation kits. The aim for this study was to explore novel methods for long distance haplotyping to assess associations between OCA2-HERC2 haplotypes and eye colour. DNA was isolated from frozen blood samples collected from Norwegians that are known to be heterozygous for both HERC2 rs12913832 and OCA2 SNPs, either rs1800407 (n = 23) or rs74653330 (n = 17), using the newly commercially available Monarch® HMW (heigh molecular weight) DNA Extraction Kit (New England BioLabs_inc). We successfully isolated DNA fragments up to 210 kb, which were long enough to haplotype OCA2-HERC2 loci by droplet digital PCR (ddPCR). Three haplotypes were observed in the study population: rs12913832:A-rs1800407:T in 22/23 individuals, rs12913832:A-rs1800407:C in 1/23 individuals and rs12913832:A-rs74653330:T in 16/16 individuals. As expected, all individuals with the rs12913832:A-rs74653330:T haplotype had intermediate to blue eye colour. However, the rs12913832:A-rs1800407:T haplotype was observed in both blue and brown-eyed individuals, suggesting more research is needed.     

Evaluation of DNA Extraction Methods for Processing Fingerprint Powder-Coated Forensic Evidence

In unison, fingerprinting and DNA analysis have played a pivotal role in forensic investigations. Fingerprint powders that are available on the market can come in a range of colors and with specific properties. This study evaluated the efficiency of DNA extraction from samples coated with 3 brands of fingerprint powders: Lightning, Sirchie, and SupraNano, covering a range of colors and properties. A total of 23 fingerprint powders were tested using the Chelex, Promega DNA IQ™, and Applied Biosystems™ PrepFiler™ DNA extraction protocols. The DNA IQ™ and PrepFiler™ methods extracted higher yields of DNA in comparison to Chelex, which also accounted for better quality of PowerPlex x00AE; 21 DNA profiles recovered. There were no signs of degradation or inhibition in the quantification data, indicating that samples returning low DNA yield was due to interference during DNA extraction and not PCR inhibition. DNA profiles were recovered from the majority of fingerprint powders with only a single powder, Sirchie Magnetic Silver, failing to produce a profile using any of the methods tested. A link was observed between the DNA extraction chemistry, fingerprint powder property, that is, nonmagnetic, magnetic and aqueous, and the brand of fingerprint powder. Overall, the DNA IQ™ method was favorable for nonmagnetic fingerprint powders, while magnetic fingerprint powders produced more DNA profiles when extracted with the PrepFiler™ chemistry. This study highlights the importance of screening DNA extraction chemistries for the type of fingerprint powder used, as there is not a single DNA extraction method that suits all fingerprint powder brands and properties.     

Detection and analyses of latent DNA

Latent DNA detection has the potential to transform aspects of DNA collection at scenes and from items. In the absence of being able to visualise the location of cellular material, all collection of samples at crime scenes is currently performed blind. With the advent of the application of a nucleic acid staining dye, the DNA within skin cells (commonly called keratinocytes and corneocytes) can be visualised. Diamond Dye fluoresces when it binds to the backbone of DNA. This fluorescence can be recorded using a simple mini-microscope allowing the location and number of cells to be recorded. The potential to visualise cells on a wide range of substrates opens the possibility to target sample collection and to triage samples for further analyses to only those containing DNA. Diamond Dye has been found to be safe at the concentration used, inexpensive, available commercially, easy to apply, is highly sensitive, and does not inhibit further analyses such as PCR. This work presented at the ISFG congress gives an overview of the current developments on using DNA staining dyes to record the number of cells present on a wide range of substrates. It is essential to firstly understand the composition of cellular material deposited by touch, where it originates and the relative composition of corneocytes and cell-free DNA. Insight into the origins of touch DNA will be presented along with the staining of nuclei using a range of dyes to show corneocyte degradation. The presentation will cover how DNA binding dyes can be used to effectively triage sample collection, monitor cell collection using different swabs and tapes.     

Probabilistic SNP genotyping at low DNA concentrations

We present a statistical method for biallelic SNP genotyping that reduces the risk of wrong SNP calls and gives fewer no-calls. The method uses a symmetric multinomial logistic regression model with an intuitive graphical interpretation. Its probabilistic nature gives the user control over the accepted risk through the estimated genotype probabilities. We compared the performance of our method with the HID SNP Genotyper v.4.3.1 plug-in (HSG) (Thermo Fisher Scientific) and the additional criteria of the University of Copenhagen (UCPH) through a series of six DNA dilutions from 500 pg to 16 pg DNA. The HSG method made wrong calls from 62.5 pg DNA and below, while the UCPH method made wrong calls at 16 pg DNA. Our method allowed SNP genotyping of 16 pg DNA without making wrong calls. Depending on the DNA dilution, our method also reduced the number of no-calls by 70–96 % compared to UCPH method and 59–69 % compared to the HSG method. Our method can be used for any biallelic genotyping.     

Comparison of the M-Vac® Wet-Vacuum-Based Collection Method to a Wet-Swabbing Method for DNA Recovery on Diluted Bloodstained Substrates*†‡

A wet-vacuum-based collection method with the M-Vac^® was compared to a wet-swabbing collection method by examining the recovery of diluted blood on 22 substrates of varying porosity. The wet-vacuum method yielded more total nuclear DNA than wet-swabbing on 18 porous substrates, recovering on average 12 times more DNA. However, both methods yielded comparable amounts of total DNA on two porous and two nonporous substrates. In no instance did wet-swabbing significantly recover more DNA. The wet-vacuum method also successfully collected additional DNA on previously swabbed substrates. Mitochondrial DNA yields were assessed, and outcomes were generally similar to the nuclear DNA outcomes described above. Results demonstrate that wet-vacuuming may serve as an alternative collection method to swabbing on difficult porous substrates and could potentially recover additional DNA on previously swabbed substrates. However, swabbing remains the preferred collection method on substrates with visible stains and/or nonporous surfaces for reasons of convenience, simplicity, and lower cost relative to the wet-vacuum method.