Forensic DNA Profiling Research Articles

TigerBase: A DNA registration system to enhance enforcement and compliance testing of captive tiger facilities

The illegal trade in tigers (Panthera tigris) and their derivatives, such as bones, teeth and pelts, is a major threat to the species’ long-term persistence. As wild tiger populations have dwindled, a large proportion of trafficked tiger products now derive from captive breeding facilities found throughout Asia. Moreover, wild tigers have been poached and laundered into captive facilities, then falsely designated as captive-bred. The establishment of a DNA registration system is recognized as a key tool to monitor compliance of captive facilities, support tiger trade investigations and improve prosecution outcomes. Here, we present a standardised wildlife forensic DNA profiling system for captive tigers called TigerBase. TigerBase has been developed in four South-East Asia countries with captive tiger facilities: Malaysia, Vietnam, Thailand and Lao PDR. TigerBase DNA profile data is based on 60 single nucleotide polymorphism (SNP) markers, genotyped using two different TaqMan®-based approaches: OpenArray® chip (capable of genotyping 60 SNPs for 48 samples in a single chip), and singleplex TaqMan® assays (capable of genotyping one SNP for one sample per reaction). Of the 60 SNPs, 53 are autosomal nuclear markers, suitable for individualisation and parentage applications, two are sex-linked markers, suitable for sexing, and five are mtDNA markers, suitable for maternal subspecies identification. We conducted a series of validation experiments to investigate the reliability and limitations of these SNP genotyping platforms. We found that the OpenArray® chip platform is more appropriate for generating reference data given its greater throughput, while the singleplex TaqMan® assays are more appropriate for genotyping lower quality casework samples, given their higher sensitivity and throughput flexibility. Only 19 autosomal nuclear markers were validated as singleplex TaqMan® assays, which generally provides ample power for individualisation analysis (probability of identity among siblings was <6.9 ×10−4), but may lack power for specific parentage questions, such as determining parentage of an offspring when one of the parent’s genotypes is missing. Further, we have developed pipelines to support standardised SNP calling and decrease the chance of genotyping errors through the use of analytical workflows and synthetic positive controls. We expect the implementation of TigerBase will enhance enforcement of tiger trafficking cases and encourage compliance among captive tiger facilities, together contributing to combatting the illegal tiger trade.

Integration of Bite Mark Microbiome Analysis with Forensic DNA Profiling: Advancements, Challenges, and Synergistic Approaches.

Bite mark analysis plays a pivotal role in forensic investigations, by helping to identify suspects and establish links between individuals and crime scenes. However, traditional bite mark methodologies face significant challenges due to issues with reliability and subjectivity. Recent advances in microbiome analysis, which involves identifying and characterizing the microbial communities found in bite marks, have led to the emergence of a promising tool for forensic investigations. The integration of microbiome analysis with conventional DNA profiling enables more accurate interpretation of bite mark evidence in forensic investigations. This review provides an in-depth look at the integration of bite mark microbiome analysis with forensic DNA profiling. It also addresses the challenges and strategies involved in microbiome-based bite mark analysis for forensic purposes.

The Efficiency of the Alu Insertion Sequence in Discrimination Among some Individuals

Background: The Alu element is a widely distributed short interspersed nuclear element (SINE) in the human genome and has important applications in forensic science. The current study focused on assessing the effectiveness of Alu insertion polymorphism in forensic DNA profiling to identifying samples of some individuals living in the Babylon Governorate. Materials and methods: DNA was extracted From frozen blood samples (60) individuals were collected from Babylon - Al-Hilla Governorate from 8/8/2022 until 8/9/2022. Results: purified then a PCR technique Alu insertions were A(2q21.1)(111-115bp) 0.75, Alu deletion (0.25), B(8q23.1) (0.375) (0.625) C(13q34) (0.5)(0.5), D(15q23) (0.775) (0.225), E(16q23.3) (0.692) (0.308) and F(19q13.12)(0.616)(0.384) respectively.The alleles with Alu insertions at (2q21.1) were the most prevalent, whereas the Alu insertions at (8q23.1) were the least common. The similarity coefficient among individuals varied from 0.4 to 1, based on the proportion of genetic relatedness between them. Conclusions: Alu elements have efficacy in families’ discrimination it can be used more than one site, set of Alu insertion sequences should be used to accurate results.

Non-Manipulable Things? Maintaining a Techno-Judicial Imaginary on Sealed Biological Samples in the French Criminal Justice

This article investigates the maintenance of sealed biological samples in the context of the French criminal justice. It extends maintenance studies to judicial objects and introduces the topic of maintenance in legal materiality studies. Our contribution to these two bodies of literature is to deepen understanding of the epistemic and material politics of maintenance in a judicial context where the issue of authenticity of objects is crucial because of their legal liability. Sealed biological samples, which are associated to a techno-judicial imaginary on cold cases, exacerbate this issue of authenticity. Once sealed, biological samples used to produce forensic DNA profiles are stored for a period of 40 years at the Central Service for the Preservation of Biological Samples – Service Central de Préservation des Prélèvements Biologiques (SCPPB), a dedicated facility, and interventions are no longer authorised on them. What then does it entail to maintain these objects which are non-manipulable, at least to some extent? What are the ‘sciences of maintenance’ mobilised and generated for this purpose, if they exist at all? This article addresses these questions, drawing on the archives of the technical committee of the Fichier National Automatisé des Empreintes Génétiques (FNAEG)—the French national forensic database used by national police—from 1998 to 2016 and on a six-hour interview with the lieutenant-colonel head of the SCPPB. It shows how sealed biological samples raise intertwined debates on their very nature and on the competences and responsibilities of genetic experts, the SCPPB and the judicial authorities on the maintenance of their technical quality and their judicial potential. It examines the knowledges and practices of maintenance of these objects, which are distributed all along the process from their manufacturing on the crime scenes to their preservation at the SCPPB. Finally, it contributes analysis of the temporality of maintenance and the objects to be maintained. By and large, this article offers reflections on the subtle ways of maintenance in the material ordering of the judicial world.

InDEL instability in two different tumoral tissues and its forensic significance.

There may be cases where malignant tumor samples can be used for forensic DNA profiling studies. STRs are the first systems preferred in forensic science laboratories for identification purposes. However, genetic instability in tumoral tissues causes STR polymorphism to change, leading to erroneous results. On the other hand, insertion/deletion polymorphism (InDels) are used as genetic markers in forensic science, as they have features that make both STR and SNPs preferable. Although previous studies approved that STR instability is observed in many different tumors, there are only a few studies that have displayed the instability of InDels in tumoral tissues before. In this study, it was aimed to determine whether instability is observed in formalin-fixed paraffin-embedded breast and thyroid tumoral tissues at 36plex InDel Panel. A total of 47 cases, 26 of which were diagnosed as breast cancer and 21 as thyroid cancer, were included in the study. In 21 of 26 (80.76%) breast cancers mutational changes were observed, however only 6 of 21 (28.57%) thyroid carcinoma cases displayed instability.Moreover, in these six cases, mutations were detected at only 1 or 2 loci. The most common change in both tissues was loss of heterozygosity. These findings suggest that paraffin embedded tissues of thyroid tumor can be used in cases of forensic genetic identification, however paraffin embedded breast cancer tissues should be examined with care. In conclusion, low InDel mutation rates compared to STR instability, make InDel analysis from paraffin blocks suitable for forensic genetic identification. However, researchers should keep in mind that there may be differences between the profiles of the tumoral tissues taken as reference and the actual case. In addition, by incorporating additional markers such as SNPs and microhaplotypes with low mutation rates into the study alongside Indels, researchers can significantly enhance the discrimination power in identification processes.

Forensic DNA Profiling: Legal and Ethical Considerations

Forensic DNA analysis is vital in criminal investigations, posing intricate legal and ethical dilemmas. This emphasises the need to balance DNA technology benefits with individual rights, prioritizing privacy, consent, and ethical practices. Stringent regulation of DNA data management is essential to prevent misuse and ensure privacy protection. Advances like probabilistic genotyping software improve interpretation accuracy, addressing complexities in mixture analysis. Responsible DNA use also requires navigating emerging technologies like next-generation sequencing while upholding ethical standards. Establishing transparency, accuracy, and ethical conduct throughout the analysis process is crucial for building public trust. Regulatory oversight and accountability mechanisms are necessary to address errors and maintain fairness in DNA data management. Through continuous dialogue and adherence to ethical standards, the forensic community can uphold scientific integrity and preserve public confidence in DNA analysis.

Deconvoluting multi-person biological mixtures and accurate characterization and identification of separated contributors using non-targeted single-cell DNA sequencing

The genetic characterization and identification of individuals who contributed to biological mixtures are complex and mostly unresolved tasks. These tasks are relevant in various fields, particularly in forensic investigations, which frequently encounters crime scene stains generated by more than one person. Currently, forensic mixture deconvolution is mostly performed subsequent to forensic DNA profiling at the level of the mixed DNA profiles, which comes with several limitations. Some previous studies attempted at separating single cells prior to forensic DNA profiling. However, these approaches are biased at selection of the cells and, due to their targeted DNA analysis on low template DNA, provide incomplete and unreliable forensic DNA profiles. We recently demonstrated the feasibility of performing mixture deconvolution prior to forensic DNA profiling through the utilization of a non-targeted single-cell transcriptome sequencing (scRNA-seq). In addition to individual-specific mixture deconvolution, this approach also allowed accurate characterisation of biological sex, biogeographic ancestry and individual identification of the separated mixture contributors. However, RNA has the forensic disadvantage of being prone to degradation, and sequencing RNA - focussing on coding regions - limits the number of single nucleotide polymorphisms (SNPs) utilized for genetic mixture deconvolution, characterization, and identification. These limitations can be overcome by performing single-cell sequencing on the level of DNA instead of RNA. Here, for the first time, we applied non-targeted single-cell DNA sequencing (scDNA-seq) by applying the scATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) technique to address the challenges of mixture deconvolution in the forensic context. We demonstrated that scATAC-seq, together with our recently developed De-goulash data analysis pipeline, is capable of deconvoluting complex blood mixtures of five individuals from both sexes with varying biogeographic ancestries. We further showed that our approach achieved correct genetic characterization of the biological sex and the biogeographic ancestry of each of the separated mixture contributors and established their identity. Furthermore, by analysing in-silico generated scATAC-seq data mixtures, we demonstrated successful individual-specific mixture deconvolution of i) highly complex mixtures of 11 individuals, ii) balanced mixtures containing as few as 20 cells (10 per each individual), and iii) imbalanced mixtures with a ratio as low as 1:80. Overall, our proof-of-principle study demonstrates the general feasibility of scDNA-seq in general, and scATAC-seq in particular, for mixture deconvolution, genetic characterization and individual identification of the separated mixture contributors. Furthermore, it shows that compared to scRNA-seq, scDNA-seq detects more SNPs from fewer cells, providing higher sensitivity, that is valuable in forensic genetics.

The present and future of forensic genetics

In this article, we discuss the current state and future directions in the field of forensic genetics. The DNA analysis of biological traces found at a crime scene, which was first used in a criminal investigation in 1987, did revolutionize forensic science. Over the past three decades, significant advances have been made in the recognition capacity, speed, and sensitivity of DNA profiling methods, as well as in their capability of typing increasingly complex patterns. Creation of DNA databases of criminals and crime scenes, as well as population allele frequencies, allows suspects to be identified from crime scene samples and DNA evidence to be statistically processed to verify its reliability. At present, it has become possible to identify even single cells left at a crime scene and to successfully analyze ancient human remains. Forensic DNA profiling can be used to identify not only individuals known to the investigating authorities. Experts are increasingly applying new genetic markers that can expand the scope of DNA profiling methods. Modern developments enable extraction of new types of forensically significant information from biological traces, e.g., using molecular approaches to searching for individuals previously unknown to investigators. New methods have been proposed to identify the relationship between the donors of forensic samples and the crimes committed. Modern advances in the decoding of the human genome, as well as the availability of genome-wide analysis and sequencing techniques, pave the way for new forensic DNA tools capable of enhancing the quality of forensic science in the near future.

Review: Wildlife forensic genetics-Biological evidence, DNA markers, analytical approaches, and challenges.

Wildlife-related crimes are the second most prevalent lawbreaking offense globally. This illicit trade encompasses hunting, breeding and trafficking. Besides diminishing many species and their habitats and ecosystems, hindering the economic development of local communities that depend on them, undermining the rule of law and financing terrorism, various cross-species transmissions (zoonoses) of pathogens, including COVID-19, can be attributed to wildlife crimes. Wildlife forensics applies interdisciplinary scientific analyses to support law enforcement in investigating wildlife crimes. Its main objectives are to identify the taxonomic species in question, determine if a crime has been committed, link a suspect to the crime and support the conviction and prosecution of the perpetrator. This article reviews wildlife crime and its implications, wildlife forensic science investigation, common forms of wildlife biological evidence, including DNA, wildlife DNA techniques and challenges in wildlife forensic genetics. The article also reviews the contributions of genetic markers such as short tandem repeat (STR) and mitochondrial DNA (mtDNA) markers, which provide the probative genetic data representing the bulk of DNA evidence for solving wildlife crime. This review provides an overview of wildlife DNA databases, which are critical for searching and matching forensic DNA profiles and sequences and establishing how frequent forensic DNA profiles and sequences are in a particular population or geographic region. As such, this review will contain an in-depth analysis of the current status of wildlife forensic genetics, and it will be of general interest to wildlife and conservation biologists, law enforcement officers, and academics interested in combating crimes against wildlife using animal forensic DNA methods.

Salmon sperm DNA increases sample recovery from cotton swabs

Forensic samples with low DNA template amounts are difficult to analyze and interpret. There is a large body of research demonstrating that adding carrier nucleic acid to storage tubes, solid phase extractions, or filtering devices can improve yields of target DNA. However, the addition of carrier nucleic acid to sampling substrates, like cotton swabs, has not yet been attempted. In this proof-of-concept study, carrier nucleic acids in the form of either Poly (A) RNA or salmon sperm DNA were spotted onto cotton swabs, followed by human genomic DNA, to determine if introducing the carrier prior to sample collection would increase recovery from the swabs post-extraction. Extracts were also evaluated to determine whether adding the carrier nucleic acids to human DNA would interfere with downstream forensic DNA analysis processes such as real-time PCR quantitation, PCR amplification of STR loci, or capillary electrophoresis. The RNA carrier did not improve human sample recovery from cotton swabs. The extraction efficiency of human DNA from cotton swabs was increased when the DNA carrier was applied to the swabs prior to sample deposition, and the scale of the increase depended on the amount of carrier DNA used. When applying the salmon sperm DNA carrier to cotton swabs, with each increase from no carrier to 0.001–1–10 µg, human DNA recovery went from ∼29 % to ∼50 % to ∼75 % to ∼100 %. Additionally, no inhibitory effects from the carrier DNA were observed post-extraction with quantitation or in the DNA profile after amplification. Therefore, salmon sperm DNA carrier will increase human DNA yield from cotton swabs without negative effects on downstream forensic DNA profiling methods, with the optimal carrier amount being 10 µg.

"Total Human DNA Sampling" – Forensic DNA profiles from large areas

Employed for the first time in 1986, DNA profiling is nowadays established as one of the most widely used forensic techniques worldwide. However, until today, no efficient sampling technique existed to collect DNA from human skin cells from a large area, not to say from the floor of an entire room. This has been extremely unfortunate, as there is enormous forensic potential in these DNA traces from the ground to provide clues as to who has been present at a particular location, i.e. at the crime scene. By desquamation, humans loose several millions of skin cells per day, everywhere they stand, sit or walk; and they can do little about it. We developed a fast and simple method by which we can make use of all those lost skin cells. We use a vacuum cleaner equipped with a specialized filter cartridge to sample the ground. Fragmentation of the filter membrane and subsequent parallel processing of the filter fragments, using a modified Chelex® 100 extraction protocol, significantly reduce the complexity of the dust mixture. In this way, a large number of interpretable major contributor DNA profiles can be generated from individuals who have been present on the sampled surface. Overall, at least 38 % of the generated DNA profiles from all sampled test areas fulfilled the criteria for submission of single major contributor profiles to the Swiss DNA database. As demonstrated through a mock crime scene scenario simulating an indoor stabbing event, the perpetrator's DNA could be found on the floor even after a very short stay in the room of less than one minute. Furthermore, already the first application of the method at a real crime scene led to relevant case information for the police. Given its large investigative potential, we recommend Total Human DNA Sampling as a helpful complemental forensic tool to conventional DNA trace collection in major crimes.

Exploring rare differences in mitochondrial genome between MZ twins using Ion Torrent semiconductor sequencing

Monozygotic (MZ) twins are considered to be genetically identical in that they have the same genomic DNA sequences in theory, and thus cannot be differentiated using forensic standard STR-based DNA profiling. However, a recent study employed deep sequencing to explore extremely rare mutations in the nuclear genome and reported that the mutation analysis could be applied to differentiate between MZ twins. Compared with the nuclear genome, the mitochondrial DNA (mtDNA) exhibits higher mutation rates due to fewer DNA repair mechanisms in the mitochondrial genome (mtGenome) and the lack of proofreading capability of the mtDNA polymerase. In a previous study, we used Illumina ultra-deep sequencing to describe point heteroplasmy (PHP) and nucleotide variant of the mtGenomes in venous blood samples of MZ twins. In the present study, we characterized minor differences of the mtGenomes in three tissue samples from seven sets of MZ twins using Ion Torrent semiconductor sequencing (Thermo Fisher Ion S5 XL system) and commercialized mtGenome sequencing kit (Precision ID mtDNA Whole Genome Panel). PHP was observed in blood samples from one set of MZ twins and in saliva samples from two sets of twins, but it presented in hair shaft samples from all seven sets of MZ twins. Overall, the coding region of the mtGenome exhibits more PHPs than the control region. The results of this study have further attested the competence of mtGenome sequencing in differentiating between MZ twins, and that among the three kinds of samples tested, hair shaft is more likely to accumulate minor differences in the mtGenomes of MZ twins.

Cost-Effective Next Generation Sequencing-Based STR Typing with Improved Analysis of Minor, Degraded and Inhibitor-Containing DNA Samples

Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts.

Applications of X-Chromosome Short Tandem Repeats for Human Identification: A Review

The forensic DNA profiling technique has tremendously contributed to forensic human identification, an important aspect in forensic investigations. In instances whereby comparison samples are unavailable, utilization of short tandem repeats of X chromosome (X-STRs) may prove useful to resolve complex kinship investigations involving missing persons and mass disasters. Despite such evidential values, the use of X-STRs during investigations remains scarce in many Southeast Asian countries including Malaysia, requiring concerted efforts for establishing forensic statistical support for its diverse populations (especially the admixture populations), standardizing core loci and procedure, improving the knowledge among practitioners as well as developing suitable standard operating procedure for incorporating X-STRs analysis in the overall DNA profiling framework. Hence, this review paper aims to highlight the developments, applications and population data of X-STRs, as well as its challenges and future insights for forensic casework.

Optimization of DNA extraction and sampling methods for successful forensic microbiome analyses of the skin and saliva.

Microbiome studies have contributed to many fields, such as healthcare and medicine; however, these studies are relatively limited in forensics. Microbiome analyses can provide information, such as geolocation and ancestry information, when short tandem repeat (STR) profiling fails. In this study, methods for DNA extraction and sampling from the skin and saliva were optimized for the construction of a Korean Forensic Microbiome Database (KFMD). DNA yields were estimated using four DNA extraction kits, including two automated kits (Maxwell® FSC DNA IQ™ Casework Kit and PrepFiler™ Forensic DNA Extraction Kit, updated) and two manual kits (QIAamp DNA Mini Kit and QIAamp DNA Micro Kit) commonly used in forensic DNA profiling laboratories. Next-generation sequencing of the 16S rRNA V4 region was performed to analyze microbial communities in samples. The Bacterial Transport Swab with Liquid Media (NobleBio), two cotton swabs (PoongSung and Puritan), and nylon-flocked swabs (NobleBio and COPAN) were tested for DNA recovery. The PrepFiler and Maxwell kits showed the highest yields of 3.884ng/μL and 23.767ng/μL from the scalp and saliva, respectively. With respect to DNA recovery, nylon-flocked swabs performed better than cotton swabs. The relative abundances of taxa sorted by DNA extraction kits were similar contributions; however, with significant differences in community composition between scalp and saliva samples. Lawsonella and Veillonella were the most abundant genera in the two sample types. Thus, the Maxwell® FSC DNA IQ™ Casework Kit and nylon-flocked swab (NobleBio) were optimal for DNA extraction and collection in microbiome analyses.

Developmental validation of STRmix™ NGS, a probabilistic genotyping tool for the interpretation of autosomal STRs from forensic profiles generated using NGS

We describe the developmental validation of the probabilistic genotyping software – STRmix™ NGS – developed for the interpretation of forensic DNA profiles containing autosomal STRs generated using next generation sequencing (NGS) also known as massively parallel sequencing (MPS) technologies. Developmental validation was carried out in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) Guidelines for the Validation of Probabilistic Genotyping Systems and the International Society for Forensic Genetics (ISFG) recommendations and included sensitivity and specificity testing, accuracy, precision, and the interpretation of case-types samples. The results of developmental validation demonstrate the appropriateness of the software for the interpretation of profiles developed using NGS technology.

The effect of freezing, thawing and long-term storage on forensic DNA extracts

Following forensic DNA profiling (extraction, quantification and STR typing) the remaining extract is generally stored frozen. Our routine at the Swedish National Forensic Centre is to immediately after analysis freeze the sample. If a subsequent reanalysis is needed the sample is thawed and then refrozen. In this study the effects of freezing and thawing as well as long-term storage of DNA extracts in refrigerator or freezer have been investigated. The following sample types were extracted: two levels of blood and saliva, saliva on cigarette filter paper, saliva on cotton swabs and a combination of saliva and semen to mimic samples from sexual assaults. All extraction methods used were Chelex-based, DNA quantification was performed using PowerQuant System and STR profiling with PowerPlex ESX 16 Fast System. The study was divided into three parts: 1) freezing and thawing the extracts up to ten times, 2) storage in refrigerator or freezer up to four weeks and 3) long-term storage in refrigerator or freezer for 3, 6, 9, 12 and 35 months. Generally, the quantification and STR typing results show no indication of degradation after repeated freezing and thawing or long-term storage in refrigerator or freezer.

New Perspectives for Whole Genome Amplification in Forensic STR Analysis.

Modern PCR-based analytical techniques have reached sensitivity levels that allow for obtaining complete forensic DNA profiles from even tiny traces containing genomic DNA amounts as small as 125 pg. Yet these techniques have reached their limits when it comes to the analysis of traces such as fingerprints or single cells. One suggestion to overcome these limits has been the usage of whole genome amplification (WGA) methods. These methods aim at increasing the copy number of genomic DNA and by this means generate more template DNA for subsequent analyses. Their application in forensic contexts has so far remained mostly an academic exercise, and results have not shown significant improvements and even have raised additional analytical problems. Until very recently, based on these disappointments, the forensic application of WGA seems to have largely been abandoned. In the meantime, however, novel improved methods are pointing towards a perspective for WGA in specific forensic applications. This review article tries to summarize current knowledge about WGA in forensics and suggests the forensic analysis of single-donor bioparticles and of single cells as promising applications.

Development and validation of a fast and automated DNA identification line

The importance of DNA evidence for gaining investigative leads demands a fast workflow for forensic DNA profiling performed in large volumes. Therefore, we developed software solutions for automated DNA profile analysis, contamination check, major donor inference, DNA database (DDB) comparison and reporting of the conclusions. This represents the Fast DNA IDentification Line (FIDL) and this study describes its development, validation and implementation in criminal casework at the authors’ institute. This first implementation regards single donor profiles and major contributors to mixtures. The validation included testing of the software components on their own and examination of the performance of different DDB search strategies. Furthermore, end-to-end testing was performed under three conditions: (1) testing of scenarios that can occur in DNA casework practice, (2) tests using three months of previous casework data, and (3) testing in a casework production environment in parallel to standard casework practices. The same DNA database candidates were retrieved by this automated line as by the manual workflow. The data flow was correct, results were reproducible and robust, results requiring manual analysis were correctly flagged, and reported results were as expected. Overall, we found FIDL valid for use in casework practice in our institute. The results from FIDL are automatically reported within three working days from receiving the trace sample. This includes the time needed for registration of the case, DNA extraction, quantification, polymerase chain reaction and capillary electrophoresis. FIDL itself takes less than two hours from intake of the raw CE data to reporting. Reported conclusions are one of five options: (1) candidate retrieved from DDB, (2) no candidate retrieved from DDB, (3) high evidential value with regards to reference within the case, (4) results require examination of expert, or (5) insufficient amount of DNA obtained to generate a DNA profile. In our current process, the automated report is sent within three working days and a complete report, with confirmation of the FIDL results, and signed by a reporting officer is sent at a later time. The signed report may include additional analyses regarding e.g. minor contributors. The automated report with first case results is quickly available to the police enabling them to act upon the DNA results prior to receiving the full DNA report. This line enables a uniform and efficient manner of handling large numbers of traces and cases and provides high value investigative leads in the early stages of the investigation.

Independent development and validation of a novel six-color fluorescence multiplex panel including 61 diallelic DIPs and 2 miniSTRs for forensic degradation sample.

Current forensic DNA profiles are obtained based on analyses of PCR product sizes or DNA sequence polymorphisms. Sometimes routine forensic analysis using short tandem repeat (STR) generates unsuccessful DNA testing result if the biological sample encountered is excessively degraded and low-template DNA. Herein, a new six-color fluorescence labeling system, including 59 autosomal diallelic deletion or insertion polymorphisms (DIPs), 2 miniSTRs, 2 Y-chromosome DIPs, and 1 Amelogenin gene with the amplicon sizes of less than 200bp, was self-developed. According to the validation guidelines for DNA analysis methods formulated by the Scientific Working Group on DNA Analysis Methods, the validation studies have also been carried out for the multiplex system. This novel panel possessed the features of strong stability, high sensitivity, and good specificity, which was especially suitable for the forensic degraded and mixed sample detections. The cumulative power of exclusion and cumulative matching probability of the system were 0.9999978 and 9.833E-28, respectively, in Han Chinese in Hunan, China. Moreover, this system will be an effective new tool that can be independently applied to forensic personal identification and paternity testing in the populations from the East Asia region, even from the South Asia, America, and Europe regions. The system can also contribute to population phylogenetic affinity and genetic structure analyses among different populations.