Short Tandem Repeat Profiles Research Articles

High polymorphism detected by massively parallel sequencing of autosomal STRs using old blood samples from a Chinese Han population

The development of massively parallel sequencing (MPS) has quickly changed forensic short tandem repeat (STR) genotyping. By providing detailed sequence information, MPS technology may be used as an alternative or additional method to overcome the limitations of capillary electrophoresis-based STR profiling. Most current NGS processes are labour-intensive with regard to library preparation and require high-quality DNA template. In this study, a 16-plex STR typing system (SeqType®R16) was used to achieve direct library preparation without DNA extraction and adaptor ligation. The efficiency of this system was tested in 601 individuals, including 593 old blood samples from the Chinese Han population and eight positive controls. It took approximately 4 hours for library preparation, including blood direct multiplex PCR (1.5 hours), mixing of the product (15 minutes), single tube purification (2 hours) and quantification (15 minutes). The results showed that MPS presented a broader allele range and higher discrimination power. Except for FGA and D19S433, the allele number almost doubled or more than doubled at all complex STR loci and simple STR loci, including D13S317, D16S539, D5S818, and D7S820. The range of discrimination power increased from 0.8008–0.9572 to 0.8401–0.9753, and the culminated matching probability decreased from 1.7 × 10−15 to 1.1 × 10−17.

Authentication Analysis of MT-4 Cells Distributed by the National Institutes of Health AIDS Reagent Program.

The MT-4 human T-cell line expresses HTLV-1 Tax and is permissive for replication of an HIV-1 gp41 mutant lacking the cytoplasmic tail. MT-4 cells (lot 150048), distributed by the NIH AIDS Reagent Program (NIH-ARP), were found to be Tax deficient and unable to host replication of the gp41-truncated HIV-1 mutant. These findings, together with short tandem repeat profiling, established that lot 150048 are not bona fide MT-4 cells.

Internal validation of GlobalFilerTM kit using reduced reaction volume

For approximately two decades, Microsatellites or Short Tandem Repeat (STR) markers have been the backbone for human identification testing in the forensic field. The expansion of STR multiplex kits can improve global sharing of STR profiles, rapid DNA typing, and DNA typing Flores et al. (2014). GlobalFiler™ kit is highly sensitive and has extremely high power of discrimination. An internal validation of GlobalFiler™ Kit was carried out to test the robustness of this kit through a range of internal validation studies including half volume reaction, reproducibility, sensitivity, specificity, stability, mixture, analytical threshold, sensitivity & stochastic threshold, heterozygous balance & stutter threshold studies in accordance with SGWDAM guidelines Scientific Working Group on DNA . Reference and real casework samples previously tested using other autosomal STR kits from forensic cases were used during the validation. It was demonstrated that the GlobalFiler™ kit has enhanced discrimination power, is robust and extremely useful for forensic casework.

An evaluation of the SureID 23comp Human Identification Kit for kinship testing

Short tandem repeat (STR) profiling has been routinely used in kinship testing since the introduction of commercial kits in the mid-1990s. While 15 to 23 STR loci normally give definitive results in simple kinship testing, additional loci are sometimes required to resolve complex cases. The SureID 23comp Human Identification Kit, recently released by Health Gene Technologies (China), multiplexes amelogenin and 22 autosomal STRs, 17 of which are non-CODIS STRs. This enables the profiling of 38–40 loci when used in conjunction with widely used commercial kits. In this study, the kit was evaluated for kinship applications as a supplementary STR kit following the minimum criteria for validation recommended by the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) using 500 samples. Performance was comparable with other commercial kits demonstrating: repeatability and reproducibility; precision (maximum s.d. 0.1048 nt); accuracy, all alleles were within ±0.41 nt compared to the actual sizes; heterozygous peak balances at all loci >68%; stutter ratios ranged from 3.8% to 16.15%; full profiles were generated with 125 pg DNA (95.12% of alleles at 62 pg),; and we found 100% concordance over 5 common STRs with the GlobalFiler kit.

Single Cell Analysis of Circulating Endothelial Cells in Allogeneic Hematopoietic Stem Cell Transplant; To Whom Do They Belong: Host or Donor?

Background. We recently reported that Circulating Endothelial Cell (CEC) count changes represent a promising marker to monitor endothelial damage in patients undergoing allogeneic hematopoietic stem cell transplant (allo-HSCT), potentially becoming a valuable tool in the diagnostic definition of GVHD. Besides confirming an increase of CEC counts at GVHD onset, we repeatedly documented at time of engraftment statistically significant higher numbers of CEC in patients who will not manifest GVHD in comparison to patients in which GVHD will be diagnosed (Transplantation 2014,98:706-12; Bone Marrow Transplantation 2017,52:1637-42; Scientific Reports 2019,9:1-12). Recent knowledges in organ transplant pointed out that endothelial cells from the grafted organ, besides being a continuous source of alloantigens, can downregulate alloreactivity exerting tolerogenic responses. By inference to the allo-HSCT field, it could be envisaged that presence of donor CEC could induce protective effects on alloreactivity. Methods. We planned a study to test the hypothesis that at time of engraftment, CEC present in peripheral blood (PB), besides coming from cells shedding from patient vasculature, could partly belong to donor, originating from the cellular graft. Therefore, in an exploratory set, we performed FISH analysis on flowcytometry-sorted CEC (CD45neg/CD34bright/CD146pos, Lyotube #623920, BD Biosciences) (n=3) and on whole PB derived culture-expanded CEC (n=3) (EGM-2 BulletKit, Lonza), obtained at engraftment in sex-mismatched allo-HSCT. In the confirmatory set (n=15), single CEC were recovered from PB, at engraftment (T1) and at 90 days (T2) after allo-HSCT, through the DEPArrayTM technology (Menarini Silicon Biosystems), after preliminary bulk separation step carried out with the CellSearch® System. Single recovered CEC was whole genome amplified (Ampli1™ WGA Kit) and short tandem repeat (STR) profile determined (Ampli 1TM STR kit) on each single CEC. To confirm host/donor origin, single CEC STR profile was compared to that determined on patient and donor cells before allo-HSCT. Moreover, donor CEC presence was evaluated by CISH analysis on formaline fixed and paraffin-embedded biopsy sections obtained at least three months after sex mismatched allo-HSCT. Results. By positive findings of the exploratory set, we proved, at the single cell level in the confirmatory set, the presence of donor CEC at engraftment (T1) in 4 out of 15 patients (Table 1). Of them, 2 did not manifested GVHD, despite a GVHD risk score of 2, and the other 2 presented GVHD grade I. On the contrary, among the 10 patients in whom no donor CEC were detected, 6 experienced GVHD grade II-III, while 4 did not manifested GVHD, despite a 1-3 GVHD risk score. Conclusions. Our data represent the proof of principle that donor CEC may flow in host PB early on from hematopoietic recovery and seldom persist thereafter at steady-state conditions, being potentially embedded in host vascular wall. These puzzling findings suggest that neovascularization takes place in parallel with hematopoietic engraftment and could provide further clues on shedding light on tissue tolerance in the context of GVHD, opening up paradoxical scenarios on the protective role potentially played by donor CEC. Disclosures Fontana: Menarini Silicon Biosystem: Employment. Rotta:BD Biosciences Italia: Employment. Manaresi:Menarini SIlicon Biosystem: Employment, Membership on an entity's Board of Directors or advisory committees.

Loss of Heterozygosity in the Short Tandem Repeat (STR) Loci Found in Tumor DNA of De Novo Diagnosed ALL Patients As a Factor Predicting Poor Outcome

Introduction: Loss of heterozygosity (LOH) was described for many malignancies including leukemia. Using chromosomal microarray analysis (CMA) we have shown preliminary (Risinskaya et al. HemaSphere, 2019, V.3, No S1, P.782) that LOH in STR loci in tumor cells with the normal karyotype represents the loss of extended chromosomal areas and could be associated with uniparental disomy of the entire chromosomal arm or the entire chromosome. Uniparental disomy results not only in the elimination of the wild type allele, but also may lead to the duplication of the mutant allele of certain candidate tumor gene(s). Retrospective analysis of archival samples have shown that in most cases STR loci absent at the relapse of disease were already lost at the onset of disease. We noted LOH at STR loci is especially frequent for relapsed ALL patients and could be used as prognostic factor for possible poor clinical outcome. The aim: To identify LOH in the blast cells of the patients with ALL at the onset of disease and to analyze therapy outcome relative to the patients without LOH. We expect LOH in the de novo ALL to be an unfavorable factor, and those patients possibly should be treated like high-risk group. Patients and Methods: This study includes a comparative analysis of the STR profiles of the DNA of tumor and normal cells from bone marrow samples on a cohort of 32 patients with de novo diagnosed Ph-negative ALL undergoing treatment according to the "RALL-2016" regimen at the National Research Center for Hematology (Moscow, Russia). Inclusion criteria: de novo diagnosed Ph-negative acute lymphoblastic leukemia (ALL) patients, 18-55 years old, intermediate risk group without MLL translocation t(4;11)(q21;q23), treatment by RALL-2016 protocol. Exclusion criteria: other diagnosis, adult patients older than 55 years, MLL translocation t(4;11)(q21;q23), pretreatment or treatment by other protocol. The presence of blast cells in bone marrow samples was confirmed morphologically. DNA was isolated from patient bone marrow samples taken at different disease stages. Control DNA samples were taken from blood of patients in complete remission and/or from buccal epithelium (buccal swab). STR-profiles were assessed by PCR with COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on ABI3130 Genetic Analyzer. The data processing was accomplished using GeneMapper v.4-0 software. A multivariate survival analysis was used to assess the independent impact of the LOH as risk factor against the background of the abnormal tumor karyotype as standard factor for this cohort of patients. We have chosen Failure Free Survival (FFS) as primary endpoint for short-time follow-up and small patient sample size. Death from any reasons, relapse and second leukemia were chosen as failure events, survival time interval starts from begin of treatment. SAS V9/4 was used for statistical analysis of the data. Results: Of the 32 patients six were found with LOH in the certain STR loci (19%). Of these six, two were resistant to therapy and died from disease progression. One patient with LOH is currently in relapse and one patient with multiple LOH has a second leukemia (Fig.1a). Only two from six patients are "Failure Free" now, whereas only three patients from 26 in group without LOH died (two from infectious complications and one from GvHD after BMT). P value < 0,05 (Fig.1b). Conclusions: We have found statistically significant association of clinical failures with the LOH in STR loci measured at the onset of ALL. We assume that LOH is an unfavorable prognostic factor for de novo ALL patients and could be used for risk stratification and choice of adequate therapy implying allogeneic hematopoietic stem cells transplantation or modern innovative therapy approaches. Disclosures No relevant conflicts of interest to declare.

Inter and intra-individual variation in skeletal DNA preservation in buried remains

Our ability to identify skeletal remains often relies on the quality and quantity of DNA extracted from bone and teeth. Current research on buried remains has been retrospective, and no study to our knowledge has comprehensively assessed both intra-individual and inter-individual variation in human skeletal DNA from all representative skeletal element types recovered from a burial. Three individuals were interred together in a single grave for four years. Following disinterment, skeletal DNA was extracted, quantified, and GlobalFiler™ results were produced from 49 bones per skeleton, representing all bone types. Multiple sites per bone were also tested to determine intra-bone variability. Co-extracted bacterial and fungal DNA were quantified to determine microbial loads in the bones. Results show that the small, cancellous bones of the feet outperformed other bones in terms of DNA yield, measured as nanograms per gram of bone powder, and short tandem repeat (STR) profile completeness. The cuneiforms, in particular, had consistently high human DNA yields for all three individuals. DNA yield varied by individual and depth within the grave, with the shallowest individual demonstrating the highest DNA yields While the feet exhibited the greatest variation in DNA yield across bone type and sampling site, they also demonstrated some of the highest DNA yields and the most complete STR profiles, evoking a re-evaluation of their use for skeletal DNA sampling and analysis.

Investigation of the efficiency of whole genome amplification prior to short tandem repeat analysis using degraded DNA

Short tandem repeat (STR) analysis is prone to failure because DNA is frequently degraded by various environmental factors. Increasing the number of starting templates may improve success in STR profiling. One approach to increase the number of DNA templates is whole genome amplification (WGA); however, few studies have demonstrated that WGA can be used on degraded DNA samples in forensics. Therefore, we performed a PCR-based WGA termed “modified improved primer extension preamplification” (mIPEP) prior to STR analysis of degraded DNA, since this method was less affected by DNA quantity and quality than other approaches are.Saliva from four volunteers was dried onto filter papers. These samples were irradiated with UVA light (365 nm) for 3, 7, 14, and 30 days. The mIPEP method was initiated using 5, 0.5, and 0.05 ng of DNA after DNA extraction. Following mIPEP, STR analysis was performed using the AmpFlSTR Identifiler PCR Amplification Kit. This study was approved by the ethics committee at Tokyo Women’s Medical University.The number of detectable STR loci in 0.05 ng DNA sample was greater in those prepared by mIPEP than those prepared without mIPEP, when no radiation was applied. After 3 days of irradiation, the number of detectable STR loci in 0.5 ng DNA sample was smaller in those treated with mIPEP than in samples without mIPEP. There was no significant difference between the number of detectable STR loci with and without mIPEP after 7 and 14 days of irradiation. However, after 30 days of irradiation, the number of detectable STR loci in 5 ng DNA samples prepared with mIPEP was greater than in those without mIPEP. Hence, mIPEP was not necessary for STR loci detection in moderately degraded samples, but it did improve the success of STR profiling in severely degraded samples.

GENYOi005-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) carrying a p.Thr196Ala variant.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patients.

Assessment of autosomal and male DNA extracted from casework samples using Casework Direct Kit, Custom and Maxwell 16 System DNA IQ Casework Pro Kit for autosomal-STR and Y-STR profiling

Short repetitive regions in autosomal and Y chromosomes known as short tandem repeats (STRs) are currently used for DNA profiling in crime investigations. However, DNA profiling requires a sufficient quality and quantity of DNA template, which is often not obtained from trace evidence or degraded biological samples collected at the scene of a crime. Here, we assessed autosomal and male DNA components extracted from crime scene and mock casework samples using the Casework Direct Kit, Custom and compared the results against those obtained by extraction of matching samples using well-established Maxwell 16 System DNA IQ Casework Pro Kit. The quantity and quality of extracted DNA obtained using both Casework Direct Kit, Custom and Maxwell 16 System DNA IQ Casework Pro Kit were analyzed using PowerQuant Systems followed by autosomal and Y-chromosome STR profiling using GlobalFiler Express PCR Amplification Kit and PowerPlex Y23 System, respectively. Our results showed that the Casework Direct Kit and Maxwell 16 DNA IQ Casework Pro Kit have more or less equal capacity to extract inhibitor free DNA, but that the latter produces slightly better quality and more DNA template and subsequently higher numbers of STR allele calls for autosomal and Y-STR analyses. Nonetheless, the Casework Direct Kit, Custom is the quicker and cheaper option for extraction of good, clean DNA from high content material and might best be used for extraction of reference samples. Such reference DNA samples typically come from buccal swabs or freshly drawn blood. So, in general, they can confidently be expected to have a high nucleic acid content and to be inhibitor-free.

Is it reliable to use paraffin embedded tumor tissues as biological evidence in parentage cases?

Short tandem repeats (STR) are reliable markers used for forensic identification and paternity testing. In some cases, histological paraffin blocks containing malignant cells can be used for STR typing in identification and paternity. DNA profiling and result interpretation with these samples often becomes a challenging job. The malignant tissues may show preferential amplification, loss of heterozygosity, and microsatellite instability.Due to the suspected paternity case, two paraffin blocks were sent for DNA analysis by Court of Justice. Pathology report of the paraffin blocks have shown that they contain lung cancer tissues taken with bronchoscopy. After extracting DNA with silica based method from the samples taken from different parts of these two blocks, 21 autosomal STR loci and 17 gonosomal STR loci were studied.When we have compared the STR profiles of son and his alleged father, Y-chromosomal STR loci matched fully. But for 2 of the 21 autosomal STR loci were found mismatches in father-son comparing. These two mismatched loci have been homozygous in alleged father. Moreover, 10 of 21 autosomal loci have shown homozygosity, and at one locus D16S539 has three alleles. While increased homozygosity in FFPE sample of alleged father has increased the paternity index, mismatched loci have lessened it in our model with mutations but without dropout.These results suggest that great care and proper statistical model should be taken in the evaluation of the DNA typing results obtained from clinical cancerous specimens, in particular when no other reference samples containing normal tissue are available.

Performance of EuroForMix deconvolution on PowerPlex® Fusion 6C profiles

Deconvolving mixed autosomal short tandem repeat profiles can be useful for database searching, profile comparisons and weight of evidence evaluations. This study describes results for the Top Marginal deconvolution within EuroForMix. Ratio To Next values were defined above which the major contributor’s genotype in PowerPlex® Fusion 6C profiles was predicted correctly and can be used as thresholds for storage in a national DNA database.

Automated estimation of the number of contributors in autosomal short tandem repeat profiles using a machine learning approach

The number of contributors (NOC) to (complex) autosomal STR profiles cannot be determined with absolute certainty due to complicating factors such as allele sharing and allelic drop-out. The precision of NOC estimations can be improved by increasing the number of (highly polymorphic) markers, the use of massively parallel sequencing instead of capillary electrophoresis, and/or using more profile information than only the allele counts.In this study, we focussed on machine learning approaches in order to make maximum use of the profile information. To this end, a set of 590 PowerPlex® Fusion 6C profiles with one up to five contributors were generated from a total of 1174 different donors. This set varied for the template amount of DNA, mixture proportion, levels of allele sharing, allelic drop-out and degradation. The dataset contained labels with known NOC and was split into a training, test and hold-out set. The training set was used to optimize ten different algorithms with selection of profile characteristics. Per profile, over 250 characteristics, denoted ‘features’, were calculated. These features were based on allele counts, peak heights and allele frequencies. The features that were most related to the NOC were selected based on partial correlation using the training set. Next, the performance of each model (=combination of features plus algorithm) was examined using the test set. A random forest classifier with 19 features, denoted the ‘RFC19-model’ showed best performance and was selected for further validation. Results showed improved accuracy compared to the conventional maximum allele count approach and an in-house nC-tool based on the total allele count. The method is extremely fast and regarded useful for application in forensic casework.

DNA Testing Reveals the Putative Identity of JB55, a 19th Century Vampire Buried in Griswold, Connecticut.

In 1990 in Griswold, Connecticut, archaeologists excavated a burial found in a “skull and crossbones” orientation. The lid of the 19th century coffin had brass tacks that spelled “JB55”, the initials of the person lying there and age at death. JB55 had evidence of chronic pulmonary infection, perhaps tuberculosis. It is possible that JB55 was deemed a vampire due to his disease, and therefore had to be “killed” by mutilating his corpse. In an attempt to reveal the identity of JB55, DNA testing was performed. Ancestry informative single nucleotide polymorphism (SNP) analysis using the Precision ID Ancestry Panel indicated European ancestry. A full Y-chromosomal short tandem repeat (Y-STR) profile was obtained, belonging to haplogroup R1b. When the Y-STR profile was searched in the publicly accessible FamilyTreeDNA R1b Project website, the two closest matches had the surname “Barber”. A search of historical records led to a death notice mentioning John Barber, whose son Nathan Barber was buried in Griswold in 1826. The description of Nathan Barber closely fits the burial of “NB13,” found near JB55. By applying modern forensic DNA tools to a historical mystery, the identity of JB55 as John Barber, the 19th century Connecticut vampire, has been revealed.

An assessment of preserved DNA in decomposed biological materials by using forensic DNA profiling

BackgroundDNA profiling for the victim identification in several forensic situations is mostly challenging due to a high degree of degradation or due to a high level of intermingling of remains. There are many intrinsic and extrinsic factors involved in the postmortem decay of tissues. Forensic DNA analysts come across a variety of samples ranging from stain samples to visceral material to bone or teeth. The amount and quality of DNA recovery are highly variable/challenging in dealing such forensic evidence.Material and methodsIn the present study, two different types of postmortem tissues (brain and prostate/uterus) along with teeth of 20 naturally decomposed human bodies were analysed. DNA extraction has been performed using the phenol-chloroform extraction method. Further, the extracted samples have been subjected for quantitative analysis using RT-PCR. Samples were amplified using the PowerPlex® 21(PP 21) multiplex system and were genotyped using capillary electrophoresis in 3500XL Genetic Analyzer.ResultsA perfect short tandem repeat (STR) profile has been generated from all the teeth samples. In most of the cases, brain tissue samples also provided an optimal profile. In contrast, prostate tissue samples gave partial amplification at almost all the samples. These findings were further evaluated by DNA profile quality measures for the studied samples.ConclusionThe present study may provide a platform to effectively evaluate forensic samples that contain small or degraded DNA template. The multiplex autosomal STR loci PP 21 system used in the study is efficient enough for typing such degraded samples with high sensitivity and discrimination power.

Aged fingerprints for DNA profile: First report of successful typing

Fingerprint detection and Short Tandem Repeat (STR) typing are two approaches used for identification of individuals. The main goal of forensic laboratories is the development of a standardized protocol to obtain an STR profile from latent fingerprints, by typing the DNA transferred onto touched objects. The results obtained in this field derive from studies conducted under controlled laboratory conditions. Here, for the first time, we report two different profiles obtained by DNA purified by latent fingerprints enhanced with dactyloscopic powders at a crime scene, 14 years previously. DNA extraction phase was optimized to improve removal of powder and automatically conducted. Despite the low concentration of purified DNA, it was not degraded. Even if quality of the profile is influenced by several factors such as the method of acquisition and storage conditions of the fingerprint, results obtained are adequately informative and could be uploaded to the CODIS database.

A Unique Panel of Patient-Derived Cutaneous Squamous Cell Carcinoma Cell Lines Provides a Preclinical Pathway for Therapeutic Testing.

Background: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. Methods: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. Results: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. Conclusions: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation.

Oral gram-positive bacterial DNA-based identification of saliva from highly degraded samples

Analyzing degraded evidence is an important challenge in forensic casework. Saliva remaining at a crime scene may deteriorate, due to various factors, making it difficult to identify. This study aims to clarify the efficacy of oral gram-positive and -negative bacterial DNA-based identification of saliva for analyzing highly degraded samples. Saliva samples were subjected to three different degradation treatments (heat denaturation: 40–80 °C in wet conditions; microbial decomposition: 1–5 days in humid soil; and ultraviolet (UV) irradiation: 0.01–1 J/cm2). We compared saliva markers’ detectability from the degraded samples—oral gram-positive bacterial DNA (Streptococcus salivarius and Streptococcus oralis), oral gram-negative bacterial DNA (Veillonella atypica and Prevotella maculosa) and salivary α-amylase. Oral bacterial DNA was detected using a melting curve analysis following real-time PCR. The efficacy of short tandem repeats (STR) and mitochondrial DNA (mtDNA) analyses were also compared. All oral bacterial DNA were detected with specific melting peaks from the heat-denatured samples, while neither catalytic nor immunochromatographic tests detected salivary α-amylase from the heat (80 °C) samples. The gram-positive bacterial DNA (S. salivarius and S. oralis) was detected from the microbial degradation (1–5 days) samples. In contrast, the gram-negative bacterial DNA (V. atypica and P. maculosa) and salivary α-amylase were not detected from samples treated for more than two days. UV exposure made bacterial DNA-based saliva identification difficult in a dose–dependent manner; however, UV irradiation did not influence protein-based saliva tests using salivary α-amylase as an indicator. As a result of STR and mtDNA typing, partial or null STR profiles were generated from the severely degraded (microbial (2–5 days) and UV (0.1–1 J/cm2) degradation) samples, but full mtDNA profiles were obtained from all degraded samples. The forensic applicability of bacterial DNA test evaluated, using mock case samples, indicates that the oral gram-positive bacterial DNA was more resistant to degradation than the other markers. We conclude that the oral gram-positive bacterial DNA-based examination could be useful for identifying saliva from severely environmentally-exposed forensic samples as well as mtDNA typing.

DNAxs/DNAStatistX: Development and validation of a software suite for the data management and probabilistic interpretation of DNA profiles

The data management, interpretation and comparison of sets of DNA profiles can be complex, time-consuming and error-prone when performed manually. This, combined with the growing numbers of genetic markers in forensic identification systems calls for expert systems that can automatically compare genotyping results within (large) sets of DNA profiles and assist in profile interpretation. To that aim, we developed a user-friendly software program or DNA eXpert System that is denoted DNAxs. This software includes features to view, infer and match autosomal short tandem repeat profiles with connectivity to up and downstream software programs. Furthermore, DNAxs has imbedded the ‘DNAStatistX’ module, a statistical library that contains a probabilistic algorithm to calculate likelihood ratios (LRs). This algorithm is largely based on the source code of the quantitative probabilistic genotyping system EuroForMix [1]. The statistical library, DNAStatistX, supports parallel computing which can be delegated to a computer cluster and enables automated queuing of requested LR calculations. DNAStatistX is written in Java and is accessible separately or via DNAxs. Using true and non-contributors to DNA profiles with up to four contributors, the DNAStatistX accuracy and precision were assessed by comparing the DNAStatistX results to those of EuroForMix. Results were the same up to rare differences that could be attributed to the different optimizers used in both software programs. Implementation of dye specific detection thresholds resulted in larger likelihood values and thus a better explanation of the data used in this study. Furthermore, processing time, robustness of DNAStatistX results and the circumstances under which model validations failed were examined. Finally, guidelines for application of the software are shared as an example. The DNAxs software is future-proof as it applies a modular approach by which novel functionalities can be incorporated.

Interlaboratory study to validate a STR profiling method for intraspecies identification of mouse cell lines.

The Consortium for Mouse Cell Line Authentication was formed to validate Short Tandem Repeat (STR) markers for intraspecies identification of mouse cell lines. The STR profiling method is a multiplex polymerase chain reaction (PCR) assay comprised of primers targeting 19 mouse STR markers and two human STR markers (for interspecies contamination screening). The goals of the Consortium were to perform an interlaboratory study to–(1) validate the mouse STR markers to uniquely identify mouse cell lines (intraspecies identification), (2) to provide a public database of mouse cell lines with the National Institute of Standards and Technology (NIST)-validated mouse STR profiles, and (3) to publish the results of the interlaboratory study. The interlaboratory study was an international effort that consisted of 12 participating laboratories representing institutions from academia, industry, biological resource centers, and government. The study was based on 50 of the most commonly used mouse cell lines obtained from the American Type Culture Collection (ATCC). Of the 50 mouse cell lines, 18 had unique STR profiles that were 100% concordant (match) among all Consortium laboratory members, and the remaining 32 cell lines had discordance that was resolved readily and led to improvement of the assay. The discordance was due to low signal and interpretation issues involving artifacts and genotyping errors. Although the total number of discordant STR profiles was relatively high in this study, the percent of labs agreeing on allele calls among the discordant samples was above 92%. The STR profiles, including electropherogram images, for NIST-validated mouse cell lines will be published on the NCBI BioSample Database (https://www.ncbi.nlm.nih.gov/biosample/). Overall, the interlaboratory study showed that the multiplex PCR method using 18 of the 19 mouse STR markers is capable of discriminating at the intraspecies level between mouse cell lines. Further studies are ongoing to refine the assay including (1) development of an allelic ladder for improving the accuracy of allele calling and (2) integration of stutter filters to identify true stutter.