Base specific variation rates at mtDNA positions 16093 and 16183 in human hairs.

Small variations between haplotypes detected in different tissues from the same individual have been previously described. These differences complicate the interpretation of mtDNA results in real forensic casework. mtDNA haplotypes detected in hair strands collected at the crime scene have to be frequently compared with haplotypes of reference samples (buccal swabs) from victims or suspects. Nucleotide position 16093 is a well-known hot spot where differences can accumulate between different tissues of the same individual. Intra individual variation was also detected at positions 16182 and 16183 in haplotypes showing an uninterrupted HV1 poly-C stretch (with 16189C). In order to better characterize the type of variation in these positions between buccal cells and hair strands from the same individual, we have performed Sanger sequencing in 25-28 hair strands (411 in total) from 15 individuals showing either an uninterrupted HV1 polyC-stretch (16189C) or 16093C/Y in their buccal cells. The results have been evaluated by also taking into account our previous results published in [19]. We have found that no variation among hair strands was detected in individuals showing T16093 in buccal cells, while variation in hair strands (T16093, 16093C and 16093Y) were detected in individuals showing 16093C or 16093Y in buccal cells. Regarding nucleotide positions 16182 and 16183 in combination with an uninterrupted polyC-stretch, no variation was detected in hairs from individuals showing A16182 16183C in their buccal cells. In contrast, individuals A16182 A16183 showed hair strands with A16182 16183 M and A16182 16183C. And finally, individuals with 16182C 16183C showed some variation in a small amount of their hair strands (some hairs with 16182 M 16183C). These results can be relevant for forensic practitioners when comparing reference samples with hair strands, which is the type of sample most tested by using mtDNA analysis in forensic casework.

Developmental validation of the ANDE™ rapid DNA system with FlexPlex™ assay for arrestee and reference buccal swab processing and database searching.

A developmental validation was performed to demonstrate reliability, reproducibility and robustness of the ANDE System with the FlexPlex assay, including an integrated Expert System, across a number of laboratories and buccal sample variations. Previously, the related DNAscan™/ANDE 4C Rapid DNA System using the PowerPlex®16 assay and integrated Expert System Software received NDIS approval in March 2016. The enhanced ANDE instrument, referred to as ANDE 6C, and the accompanying 6-dye, 27-locus STR assay, referred to as FlexPlex, have been developed to be compatible with all widely used global loci, including the expanded set of the CODIS core 20 loci. Six forensic and research laboratories participated in the FlexPlex Rapid DNA developmental validation experiments, testing a total of 2045 swabs, including those obtained from 1387 unique individuals. The goal of this extensive and comprehensive validation was to thoroughly evaluate and document the ANDE System and its internal Expert System to reliably genotype reference buccal swab samples in a manner compliant with the FBI's Quality Assurance Standards and the NDIS Operational Procedures. The ANDE System, including automated Expert System analysis, generated reproducible and concordant results for buccal swabs when testing various instruments at different laboratories by a number of different operators. When testing a number of non-human DNAs, including oral bacteria, the ANDE System and FlexPlex assay demonstrated limited cross-reactivity. Potential PCR inhibitors were evaluated as part of the validation and no inhibition was detected. Reproducible and concordant profiles were generated from buccal swab samples collected with a limit of detection appropriate for buccal swab collections from arrestees. The precision and resolution of the System met industry standards for detection of microvariants and single base resolution. The integrated Expert System appropriately demonstrated the ability to correctly pass or fail profiles for CODIS upload without human review. During this comprehensive developmental validation, the ANDE System successfully interpreted over 2000 samples tested with over 99.99% concordant alleles. The data package described herein led to the ANDE System with the FlexPlex assay receiving NDIS approval in June 2018.

Organic extraction of bone lysates improves DNA purification with silica beads.

In our standard protocol for DNA extraction from skeletal remains of unidentified bodies, bone lysates resulting from decalcification and Proteinase K treatment were purified with the DNA IQ™ Casework Pro Kit for Maxwell®16 automate (Promega, WI). Despite its success in the majority of cases, the DNA purification with paramagnetic silica beads failed in some challenging samples. This failure in DNA recovery was often associated with filter clogging during the required volume reduction of the lysate to enable loading on the automate. Two modifications to the standard method were tested for a more efficient filtering and purification. Adding collagenase to the lysate reduced the filter lead time but did not enhance DNA yield, while organic extraction of the crude lysate solved the filter clogging and resulted in successful DNA purification. The modified method in which a phenol treated lysate was loaded on the automate resulted in successful STR-profiling of the skeletal remains of all 13 unidentified bodies tested, which showed a wide variety in post mortem interval and preservation conditions. The variation in DNA yield between the 28 samples tested showed the importance of bone type selection and multiple sampling in successful STR-profiling of skeletal remains. Despite the disadvantages inherent to phenol, the organic extraction of crude bone lysates enhanced the efficiency of DNA purification with paramagnetic silica beads. The combined method of organic extraction and purification with silica beads resulted in STR-profiling of challenging bone samples.

Hairy matters: MtDNA quantity and sequence variation along and among human head hairs.

Hairs from the same donor have been found to differ in mtDNA sequence within and among themselves and from other tissues, which impacts interpretation of results obtained in a forensic setting. However, little is known on the magnitude of this phenomenon and published data on systematic studies are scarce. We addressed this issue by generating mtDNA control region (CR) profiles of >450 hair fragments from 21 donors by Sanger-type sequencing (STS). To mirror forensic scenarios, we compared hair haplotypes from the same donors to each other, to the corresponding buccal swab reference haplotypes and analyzed several fragments of individual hairs. We also investigated the effects of hair color, donor sex and age, mtDNA haplogroup and chemical treatment on mtDNA quantity, amplification success and variation. We observed a wide range of individual CR sequence variation. The reference haplotype was the only or most common (≥75%) hair haplotype for most donors. However, in two individuals, the reference haplotype was only found in about a third of the investigated hairs, mainly due to differences at highly variable positions. Similarly, most hairs revealed the reference haplotype along their entire length, however, about a fifth of the hairs contained up to 71% of segments with deviant haplotypes, independent of the longitudinal position. Variation affected numerous positions, typically restricted to the individual hair and in most cases heteroplasmic, but also fixed (i.e. homoplasmic) substitutions were observed. While existing forensic mtDNA interpretation guidelines were found still sufficient for all comparisons to reference haplotypes, some comparisons between hairs from the same donor could yield false exclusions when those guidelines are strictly followed. This study pinpoints the special care required when interpreting mtDNA results from hair in forensic casework.

Comparison of performance of genetics 4N6 FLOQSwabs™ with or without surfactant to rayon swabs.

The collection of traces is the first step in the process of forensic genetics analysis. Currently, several different techniques are used (eg. gauze). Nevertheless, swabbing appears to be the most common of these. In a second step, the sampling devices should allow the use of preliminary tests in combination with an immunological confirmatory test (e.g. Hexagon Obti or Hemdirect). Our previous study shows that sampling with Genetics 4N6FLOQswabs™ coated with surfactant reduces by a factor of at least 100 the detection threshold of blood using two immunological tests. The aim of this work was to compare the ability to recover blood trace and the compatibility with immunological confirmatory test of various Genetics 4N6FLOQswabs™ nylon flocked swabs with or without surfactant. The results obtain in this study show that Genetics 4N6FLOQswabs™ not coated with surfactant and Human DNA free FLOQswabs™ were suitable for the used in combination with immunological blood detection tests. Nevertheless, the Genetics 4N6FLOQswabs™ not surfactant coated give a better blood trace recovery.

Nylon flocked swab severely reduces Hexagon Obti sensibility.

Hexagon Obti immunological blood test and flocked swab are widely used in forensic laboratories. Nevertheless, up to now, no compatibility tests have been published between sampling with the ethylene oxide treated flocked swab and the Hexagon Obti blood detection strip. In this study, we investigated this compatibility. Our work shows that sampling with ethylene oxide treated flocked swab reduces by a factor of at least 100 the detection threshold of blood using the Hexagon Obti immunological test.

Efficiency of a novel forensic room-temperature DNA storage medium.

The success of forensic genetics has led to considerable numbers of DNA samples that must be stored. Thus, the ability to preserve the integrity of forensic samples is essential. The possibility of retesting these samples after many years should be guaranteed. DNA storage typically requires the use of freezers. Recently, a new method that enables DNA to be stored at room temperature was developed. This technology is based on the principles of anhydrobiosis and thus permits room-temperature storage of DNA. This study evaluates the ability of this technology to preserve DNA samples mimicking true mixture casework samples for long periods of time. Mixed human DNA from 2 or 3 persons and at low concentrations was dried and stored for a period ranging from 6 months to 2 years in the presence of a desiccant. The quality of the stored DNA was evaluated based on quantitative peak height results from Short Tandem Repeat (STR) genotyping and the number of observed alleles. Furthermore, we determined whether this matrix has a potential inhibitory or enhancing effect on the PCR genotyping reactions. In our previous work, we demonstrated the considerable potential of this new technology. The present study complements our previous work. Our results show that after 2 years of aging at room temperature, there is a decrease in the number of observed alleles and in the peak height of these alleles.

Forensic interlaboratory evaluation of the ForFLUID kit for vaginal fluids identification.

Identification of vaginal fluids is an important step in the process of sexual assaults confirmation. Advances in both microbiology and molecular biology defined technical approaches allowing the discrimination of body fluids. These protocols are based on the identification of specific bacterial communities by microfloraDNA (mfDNA) amplification. A multiplex real time-PCR assay (ForFLUID kit) has been developed for identifying biological fluids and for discrimination among vaginal, oral and fecal samples. In order to test its efficacy and reliability of the assay in the identification of vaginal fluids, an interlaboratory evaluation has been performed on homogeneous vaginal swabs. All the involved laboratories were able to correctly recognize all the vaginal swabs, and no false positives were identified when the assay was applied on non-vaginal samples. The assay represents an useful molecular tool that can be easily adopted by forensic geneticists involved in vaginal fluid identification.

Evaluation of novel forensic DNA storage methodologies.

An issue in forensic sciences is the secure storage of extracted DNA. Most of the time, DNA is frozen at -20°C or -80°C. Recently, new room temperature DNA storage technologies have been developed based on anhydrobiosis. Two products use this technology: Qiasafe (Qiagen) and Gentegra (Genvault). In this study we focused on the recent Gentegra product and initiated a comparison versus -20°C and Qiasafe storage. We compared the quantity and quality of DNA stored using anhydrobiosis technology against DNA stored at -20°C, by performing STR profiling after short term storage. Furthermore, we studied the quantity and integrity of DNA after long term storage. Our results prove the high potential of this technology but it seems to be extraction dependent. Phenol/chloroform extracted DNA could be stored using the Gentegra matrix for more than 6 months without any obvious degradation. However, DNA extracted using magnetic beads could not be safely stored over the same period. Adaptations are therefore required to store this kind of samples.