Forensic DNA Laboratories Research Articles

Review of SNP assays for disaster victim identification: Cost, time, and performance information for decision-makers.

In mass disaster events, forensic DNA laboratories may be called upon to quickly pivot their operations toward identifying bodies and reuniting remains with family members. Ideally, laboratories have considered this possibility in advance and have a plan in place. Compared with traditional short tandem repeat (STR) typing, single nucleotide polymorphisms (SNPs) may be better suited to these disaster victim identification (DVI) scenarios due to their small genomic target size, resulting in an improved success rate in degraded DNA samples. As the landscape of technology has shifted toward DNA sequencing, many forensic laboratories now have benchtop instruments available for massively parallel sequencing (MPS), facilitating this operational pivot from routine forensic STR casework to DVI SNP typing. Herein, we present the commercially available SNP sequencing assays amenable to DVI, we use data simulations to explore the potential for kinship prediction from SNP panels of varying sizes, and we give an example DVI scenario as context for presenting the matrix of considerations: kinship predictive potential, cost, and throughput of current SNP assay options. This information is intended to assist laboratories in choosing a SNP system for disaster preparedness.

Advancements in differentiation between sperm cells and epithelial cells for efficient forensic DNA analysis in sexual assault cases.

Most of the sexual assault casework samples are of mixed sources. Forensic DNA laboratories are always in the requirement of a precise technique for the efficient separation of sperm and non-sperm DNA from mixed samples. Since the introduction of the differential extraction technique in 1985, it has seen significant advancements in the form of either chemicals used or modification of incubation times. Several automated and semi-automated techniques have also adopted the fundamentals of conventional differential extraction techniques. However, lengthy incubation, several manual steps, and carryover over non-sperm material in sperm fraction are some of the major limitations of this technique. Advanced cell separation techniques have shown huge promise in separating sperm cells from a mixture based on their size, shape, composition, and membrane structure and antigens present on sperm membranes. Such advanced techniques such as DEParray, ADE, FACS, LCM, HOT and their respective pros and cons have been discussed in this article. As current-day forensic techniques should be as per the line of Olympic slogan i.e., faster, higher, stronger, the advanced cell separation techniques show a huge potential to be implemented in the casework samples.

An inter-laboratory comparison of probabilistic genotyping parameters and evaluation of performance on DNA mixtures from different laboratories

Probabilistic genotyping (PG) is becoming the preferred standard for evidence interpretation, amongst forensic DNA laboratories, especially those in the United States. Various groups have expressed concern about reliability of PG systems, especially for mixtures beyond two contributors. Studies involving interlaboratory testing of known mixtures have been identified as ways to evaluate the reliability of PG systems. Reliability means different things in different contexts. However, it suffices here to think about it as a mixture of precision and accuracy. We might also consider whether a system is prone to producing misleading results – for example large likelihood ratios (LRs) when the POI is truly not a contributor, or small LRs when the POI is a truly a contributor. In this paper we show that the PG system STRmix™ is relatively unaffected by differences in parameter settings. That is, a DNA mixture that is analyzed in different laboratories using STRmix™ will result in different LRs, but less than 0.05% of these LRs would result in a different, or misleading conclusion as long as the LR is greater than 50. For the purposes of this study, we define LRs assigned using different parameters for the same mixtures as similar if the LR of the true POI is greater than the LRs generated for 99.9% of the general population. These findings are based on an interlaboratory study involving eight laboratories that provided twenty known DNA mixtures of two to four contributors and their individual laboratory STRmix™ parameters. The eight sets of laboratory parameters included differences in STR kits and PCR cycles as well as the peak, stutter, and locus specific amplification efficiency variances.

Application of bioluminescence assay to assess PCR carryover contamination in forensic DNA laboratories

In forensic DNA testing, PCR-based multilocus short tandem repeat (STR) profiling kits, which have high sensitivity and discriminatory power, are generally used to analyze autosomal and Y-chromosomal DNA profiles. Forensic DNA laboratories require strict quality control for DNA testing, as contamination during analyses leads to incorrect interpretation of DNA profiles. Here, we aimed to apply bioluminescence assay to detect and monitor residual PCR products on laboratory work area and equipment surfaces by targeting dATP in the PCR product and allelic ladder marker. Two commercially available bioluminescence assay kits (CheckLite HS Plus and UltraSnap™) were examined for their sensitivity after confirming their reactivity to dATP. In the assay using CheckLite HS Plus, the lower detectable sample volumes were calculated as 10 pl of PCR product of GlobalFiler and PowerPlex Fusion and 1 pl of PCR product of Yfiler Plus and the allelic ladder marker of GlobalFiler, whereas those in the assay using UltraSnap™ were calculated as 1 nl of PCR product and allelic ladder marker. The sample volumes of these kits were lower than those detected through electrophoresis. Thus, the sensitivity of these kits was sufficient to control PCR carryover contamination in the post-PCR areas. Furthermore, residual PCR products in the post-PCR areas were continuously monitored using a bioluminescence assay. The results showed that the bioluminescence values increased after handling PCR samples for electrophoresis and decreased after decontamination. Therefore, we concluded that the bioluminescence assay is useful for assessing PCR carryover contamination in post-PCR processes in forensic DNA laboratories.

Evaluation of Storage Conditions and the Effect on DNA from Forensic Evidence Objects Retrieved from Lake Water.

DNA analysis of traces from commonly found objects like knives, smartphones, tapes and garbage bags related to crime in aquatic environments is challenging for forensic DNA laboratories. The amount of recovered DNA may be affected by the water environment, time in the water, method for recovery, transport and storage routines of the objects before the objects arrive in the laboratory. The present study evaluated the effect of four storage conditions on the DNA retrieved from bloodstains, touch DNA, fingerprints and hairs, initially deposited on knives, smartphones, packing tapes, duct tapes and garbage bags, and submerged in lake water for three time periods. After retrieval, the objects were stored either through air-drying at room temperature, freezing at -30 °C, in nitrogen gas or in lake water. The results showed that the submersion time strongly influenced the amount and degradation of DNA, especially after the longest submersion time (21 days). A significant variation was observed in success for STR profiling, while mtDNA profiling was less affected by the submersion time interval and storage conditions. This study illustrates that retrieval from water as soon as possible and immediate storage through air-drying or freezing before DNA analysis is beneficial for the outcome of DNA profiling in crime scene investigations.

DNA identification of monozygotic twins

This study details the differentiation of identical twins based on single mutational base differences. There were three pairs of male monozygotic (MZ) twins in this study. DNA samples from blood, a buccal swab or saliva from each individual were all initially genotyped using 22 autosomal STR and 27 Y-STR loci. Preliminary screening confirmed there were no differences in the STR data between each pair of MZ twins. Whole Genome Sequence (WGS) data were generated from DNA extracted from the three body fluids from each individual. Kinship coefficients with 0.4254, 0.4557 and 0.4543 from 3 twins were generated based on WGS data to further confirm that their relationship was that of MZ twins. The fastq data generated by the Illumina Hiseq 2000 between MZ twins were then treated as “normal” as opposed to “tumor” using commercially available software tools to identify mutational single base changes. Sanger DNA sequencing confirmed there were 1, 5 and 9 single base changes found in WGS data from each of the three MZ twin sets. There was individual variation in the mutational base changes when comparing data from the three body fluids. The methods used in this study to differentiate MZ twins based on WGS data can readily be performed in many operational forensic DNA laboratories using user friendly software.

Rapid genotyping of 32 insertion/deletion panel for human identification using fluorogenic probes-based multiplex real-time PCR

BackgroundInsertion and deletion (InDel) polymorphisms have considerable potential in the field of forensic genetics because of their low mutation rate and small amplicons. At present, InDel polymorphisms detection based on the technique of capillary electrophoresis is the main technique used in forensic DNA laboratory. However, this method is complicated and time-consuming, and is not suitable for rapid on-site paternity and personal identification. Next-generation sequencing analysis of InDels polymorphisms requires expensive instruments, large upfront reagent and supply costs, computational requirements and complex bioinformatics, increased the time to obtain results. Thus, there is an urgent need to establish a method to provide reliable, rapid, sensitive and economical genotyping for InDels. MethodA rapid InDels (32 InDels) panel was established using fluorogenic probes-based multiplex real-time PCR with microfluidic test cartridge and portable real-time PCR instrument. Then, we performed several validation studies including concordance, accuracy, sensitivity, stability, species specificity. ResultsIt showed that the complete genotypes could be obtained from ≥100 pg of input DNA and from a series of challenging samples with high accuracy and specificity within 90 min. ConclusionThis method provides a rapid and cost-effective solution for InDels genotyping and personal identification in portable format.

Combining artificial neural network classification with fully continuous probabilistic genotyping to remove the need for an analytical threshold and electropherogram reading

Standard processing of electrophoretic data within a forensic DNA laboratory is for one (or two) analysts to designate peaks as either artefactual or non-artefactual in a process commonly referred to as profile ‘reading’. Recently, FaSTR™ DNA has been developed to use artificial neural networks to automatically classify fluorescence within an electropherogram as baseline, allele, stutter or pull-up. These classifications are based on probabilities assigned to each timepoint (scan) within the electropherogram. Instead of using the probabilities to assign fluorescence into a category they can be used directly in the profile analysis. This has a number of advantages; increased objectivity in DNA profile processing, the removal for the need for analysts to read profiles, the removal for the need of an analytical threshold. Models within STRmix™ were extended to incorporate the peak label probabilities assigned by FaSTR™ DNA. The performance of the model extensions was tested on a DNA mixture dataset, comprising 2–4 person samples. This dataset was processed in a ‘standard’ manner using an analytical threshold of 50rfu, analyst peak designations and STRmix™ V2.9 models. The same dataset was then processed in an automated manner using no analytical threshold, no analysts reading the profile and using the STRmix™ models extended to incorporate peak label probabilities. Both datasets were compared to the known DNA donors and a set of non-donors. The result between the two processes was a very close performance, but with a large efficiency gain in the 0rfu process. Utilising peak label probabilities opens up the possibility for a range of workflow process efficiency gains, but beyond this allows full use of all data within an electropherogram.

Direct STR profiling from laundered bloodstains: an investigation of different factors of laundering.

Bloodstains on fabrics may be washed or cleaned to eliminate incriminating evidence. These actions reduce the chances of obtaining an interpretable DNA profile. Previous studies have shown that conventional short-tandem repeat (STR) typing is affected by various factors associated with washing or laundering of stains. Here, we aim to increase the chances of obtaining interpretable STR profiles from laundered bloodstains using direct PCR. Preliminary investigations showed direct STR typing resulted in more alleles compared to conventional STR typing. We then further investigated the following factors with direct STR typing: fabric type (cotton, polyester, and denim), washing method (hand-washing and machine-washing), type of detergents (powder and liquid), washing temperature (cold to 90°C), pretreatment agents (sodium hypochlorite and hydrogen peroxide), and the number of washes (one, three, and five). Direct PCR could be successfully used for STR typing from laundered bloodstains with very high success rates. Among the three fabric types, only denim negatively affected direct STR typing, while laundering of bloodstains on cotton and polyester had a negligible effect as mostly full profiles were obtained. Multiple washes resulted in a decrease in both the numbers of alleles and peak heights. Surprisingly, washing method, type of detergent, washing temperature, and pretreatment agents only had minimal to no effect on STR profile quality. Due to the robustness and sensitivity of direct STR typing from laundered bloodstains, the method could be beneficial for violent crime investigations in forensic DNA laboratories worldwide.

Short tandem repeat (STR) instability in the oral mucosa of patients submitted to fixed orthodontic therapy: a limitation of STR profile quality for human identification.

The purpose of this study was to evaluate changes in short tandem repeat (STR) profile quality before and after fixed orthodontic therapy. Samples of oral epithelial cells were obtained from 28 volunteers who had an indication for orthodontic treatment. The samples were collected before and three months after starting orthodontic treatment with fixed appliances. DNA extraction and integrity were evaluated by electrophoresis, and STR profiles were obtained by polymerase chain reaction amplification and STR typing via capillary electrophoresis. DNA electrophoresis showed a higher proportion (7/28, 25%) of DNA degradation in the samples collected after fixed orthodontic treatment compared to those obtained before starting orthodontic therapy (3/28, 11%), however, changes in DNA were not significant (p=0.289). In concordance all STR profiles showed complete genotyping; however, imbalances in the size of heterozygotes and in the signal were detected in 25% of STR profiles after orthodontic therapy. Moreover, STR instability was demonstrated by an increase in stutter bands detected in 60% of the DNA profiles after treatment and a spurious allele of the D195433 marker was found in one sample after treatment. The STR profiles of samples obtained from the oral cavity with orthodontic appliances should be interpreted with caution. STR instability increases the incidence of artifacts that could compromise the quality of the results of tests performed in forensic DNA laboratories.

Sequence variations, flanking region mutations, and allele frequency at 31 autosomal STRs in the central Indian population by next generation sequencing (NGS)

Capillary electrophoresis-based analysis does not reflect the exact allele number variation at the STR loci due to the non-availability of the data on sequence variation in the repeat region and the SNPs in flanking regions. Herein, this study reports the length-based and sequence-based allelic data of 138 central Indian individuals at 31 autosomal STR loci by NGS. The sequence data at each allele was compared to the reference hg19 sequence. The length-based allelic results were found in concordance with the CE-based results. 20 out of 31 autosomal STR loci showed an increase in the number of alleles by the presence of sequence variation and/or SNPs in the flanking regions. The highest gain in the heterozygosity and allele numbers was observed in D5S2800, D1S1656, D16S539, D5S818, and vWA. rs25768 (A/G) at D5S818 was found to be the most frequent SNP in the studied population. Allele no. 15 of D3S1358, allele no. 19 of D2S1338, and allele no. 22 of D12S391 showed 5 isoalleles each with the same size and with different intervening sequences. Length-based determination of the alleles showed Penta E to be the most useful marker in the central Indian population among 31 STRs studied; however, sequence-based analysis advocated D2S1338 to be the most useful marker in terms of various forensic parameters. Population genetics analysis showed a shared genetic ancestry of the studied population with other Indian populations. This first-ever study to the best of our knowledge on sequence-based STR analysis in the central Indian population is expected to prove the use of NGS in forensic case-work and in forensic DNA laboratories.

Evaluation of diallelic STR markers with inter-population allelic database for their usefulness in paternity trios in the central Indian population

Background Most of the forensic DNA laboratories have migrated to new generation STR kits of 6 dye chemistry with more than twenty autosomal STRs. The population-specific database of such STR markers is lacking in many regions. Aim To evaluate the effect of the inter-population database in 100 paternity trios with no inconsistencies at 23 STRs. Subjects and methods 100 paternity trios were evaluated considering inter-population allelic frequency values for calculation of Combined Paternity Index (CPI) and Probability of Paternity (POP). Results No significant variation (p < 0.05) among the allele frequencies at the interpopulation level was observed. The number of obligate alleles and the likelihood of transferring obligate alleles from the putative father showed a positive correlation (p < 0.005) with Power of Discrimination (PD), Polymorphic Information Content (PIC), Power of Exclusion (PE), Paternity Index (PI), Observed and Expected Heterozygosity (Ho and He), and a statistically significant negative correlation (p < 0.005) with Matching Probability (Pm). The average Combined Paternity Index (CPI) and Probability of Paternity (POP) did not show any statistically significant difference (p < 0.05) at the interpopulation level. Conclusion The allelic database showed no effect on the CPI and POP in 100 paternity trios. This suggests no urgent need for using population-specific databases for statistical evaluation of paternity trios without inconsistencies.

The performance of quality controls in the Investigator® Quantiplex® Pro RGQ and Investigator® 24plex STR kits with a variety of forensic samples

Forensic DNA laboratories process database reference samples on FTA® cards or buccal swabs, which commonly contain adequate amounts of quality DNA resulting in full STR profiles and high first-pass rates. However, some reference samples and many forensic casework samples are exposed to a variety of insults that may lead to low quantities of DNA, DNA degradation, DNA mixtures, and/or PCR inhibition, posing a challenge to downstream genotyping success. The inclusion of multiple amplification targets and internal PCR controls (IPCs) in DNA quantification kits, and quality sensors within STR amplification kits can aid in the accurate interpretation of sample/profile quality, and guide more efficient rework strategies when needed. In order to assess the effectiveness of these quality systems we subjected database-type samples (buccal swabs and blood or saliva on FTA® cards), mock casework samples (low-template, degraded, inhibited, DNA mixtures), and authentic post-coital samples to various challenging conditions. Concordance between the quality flags in the Investigator® Quantiplex® Pro RGQ kit (QIAGEN), the QS markers in QIAGEN’s Investigator® 24plex QS kit, and overall STR profile quality was evaluated for all casework-type samples. To assess the value of the QS markers in the Investigator® 24plex QS and GO! STR kits, samples with partial or failed STR profiles were reworked based on the quality of the electropherogram first with the QS markers redacted, and second in conjunction with the QS markers. Results from each of the rework approaches were compared to determine which strategy, if any, improved the STR profile quality and the number of reportable alleles. The QS markers in the 24plex STR kits correctly confirmed sample quality in 99.9% of databasing samples and 98% of mock casework samples. Quality flags during DNA quantification were concordant with downstream STR profiles for the majority (77%) of the mock casework samples. Additionally, when samples with partial STR profiles were reworked, more loci were obtained for 80% of the samples regardless of the rework strategy used. However, the most notable improvement in STR completeness was observed in inhibited samples that were reworked based on the information provided by the STR quality sensors, with an average increase of 56% reportable alleles.

Challenges with co-amplification of microbial DNA in interpretation of STR profiles obtained from human skeletal remains

STR-based DNA analysis is still the main tool for human DNA identification in most forensic DNA laboratories. DNA typing of aged human skeletal elements faces well-known interpretation challenges characteristic of degraded and low copy number DNA samples. Analyzing tens of thousands of human bone and teeth samples, we found that the occasional presence of artefactual peaks of presumed microbial origin adds another layer of complexity to the interpretation of STR profiles. In this paper, we present our approach and suggest guidelines for identifying and distinguishing non-human peaks, developed over the last 18 years. Additionally, we report a compendium of artefact peaks of presumed microbial origin recorded in human STR profiles obtained from bone and teeth samples, originating from Iraq, Chile, Maldives, Brazil and Western Balkans. Our experience has shown that these artefacts are not uncommon in bone STR testing, suggesting the possibility of occurrence in other forensic contexts, particularly trace DNA samples. Raising awareness among the forensic DNA community and accounting for this phenomenon is important for accurate STR interpretation.

Isolation and Amplification of Touch DNA from Portable Computer

Analysis of touched DNA from crime scenes is fundamental in forensic DNA laboratories. Many factors affect the recovery of DNA from touched surfaces and then affect the quality of the final results. The aim of this work is studying the possibility of recovery suitable amount of DNA from ltouched portable computer. The computer was cleaned with 10% Bleach then touch and DNA collected for extraction by Organic method and two STR regions D5S818 (115-163bp) and FGA (308-464bp) were amplified. The results showed that it is possible to isolate a proper amount of DNA from touched portable computer where it was amplified and then analyzed by Agarose gel electrophoresis. The conclusion is that portable computer is suitable source for forensic analysis.

An integrated system for forensic DNA testing of sexual assault cases in the Philippines

In the Philippines, more than 7000 cases of sexual assault are reported annually. DNA technology is a powerful tool in identifying assailants. However, it is not routinely used in sexual assault investigations due to insufficient government support to cover the high cost of DNA testing and the absence of a national system for sample collection, handling, storage, and DNA testing of biological evidence. In itself, the nature of sexual assault samples containing DNA mixtures presents challenges to laboratory methods and interpretation of results. The sample recovered from the victim may only contain trace amounts of the assailant’s DNA, may have degraded due to prolonged storage in ambient conditions which is warm and humid in the tropics, or contaminated with inhibitors, such as in anal swabs. Hence, a closer evaluation of the processes of evidence collection and DNA testing is needed to increase the likelihood of success in generating conclusive results. In this paper, we propose an integrated system for DNA testing of biological samples collected from sexual assault victims considering the limitations of resources and the prevailing warm climate. Recommendations in this work should provide basis for formulating national guidelines for DNA analysis in aid of criminal investigations. The proposed scheme can be adopted by forensic DNA laboratories in the Philippines and in other countries facing similar challenges.

Assessment of QIAGEN\u2122 Investigator\xae 24plex GO! kit workflow for autosomal STR profiling of forensic reference samples

BackgroundDNA profiling has proven to be a valuable technique for identification of individuals in crime. Currently, the technique targets several short tandem repeat (STR) regions in human genome. However, increasing number of samples submitted for STR analysis may lead to delays due to the limited number of experienced analysts who might be available at any given moment and the time taken to complete lengthy DNA profiling procedures. This study was conducted to test the specificity, repeatability, reproducibility and robustness of Investigator® 24plex GO! kit for genotyping of reference samples submitted to the Royal Malaysian Police Forensic DNA Laboratory for DNA database.Material and methodsIn this study, Investigator® 24plex GO! kit was used to directly amplify STR loci from buccal swab cell of reference samples that had previously been STR typed using GlobalFiler™ Express kit. Capillary electrophoresis was carried out on a 3500xL Genetic Analyser using POP-4® Polymer. Amplified products were assigned to particular STR alleles using the GeneMapper ID-X version 1.4 software.ResultsOur study shows that STR profiles generated using Investigator® 24plex GO! gave concordance results with those previously obtained using the GlobalFiler™ Express kit. In addition, quality sensors included in the kit are of particular importance for determining the effectiveness of the PCR reaction and help to indicate the nature and quantity of DNA template for PCR amplification.ConclusionThe Investigator® 24plex GO! kit is reliable for STR typing of reference samples.

Recent trends and developments in forensic DNA extraction

AbstractThe extraction of DNA is a fairly standard procedure routinely undertaken in biological academic and research settings. In a forensic DNA context, DNA extraction is the crucial first step toward the generation of DNA profiles used to support convictions or exonerations in the criminal justice system. However, it is often complicated by the additional challenge of yielding sufficient quantities of clean DNA that is free from environmental inhibitors, from a wide spectrum of sample types which may contain limited quantities of biological material. While improvements in methods and technology over the decades have sped up and simplified the process of DNA extraction, several issues remain to be fully addressed—such as the incomplete separation of spermic DNA from sperm‐epithelial cell mixtures, increasing demands for ever‐faster DNA results, or even on‐scene DNA processing, and processing of difficult samples such as skeletal remains or samples contaminated by Chemical, Biological, or Radiological (CBR) agents. These challenges are often compounded by high work volumes and limited quantities of starting material. As such, this review focuses on recent trends and developments in four areas of forensic DNA extraction: (a) analysis of sexual assault evidence which often represents a significant portion of a forensic DNA laboratory's casework, (b) use of portable rapid DNA instruments which has gained much traction among law enforcement in recent times, (c) DNA extraction from difficult and CBR‐contaminated samples which, albeit rare, would be essential in a DNA laboratory's toolbox in the event of an incident, and (d) bypassing DNA extraction altogether.This article is categorized under: Forensic Biology &gt; Forensic DNA Technologies

Updated population genetic data of 15 autosomal STR loci in a Shandong Han population from East China and genetic relationships among 26 Chinese populations

Background The population genetics investigation of STR loci in specific populations is the basic premise for forensic practice, and the AmpFlSTRTM IdentifilerTM Plus kit is still widely used in most forensic DNA laboratories. Aim To obtain more reliable and accurate population genetic data of 15 autosomal STRs in the Shandong Han population and explore the genetic relationship with 25 neighbouring Chinese populations. Subjects and methods A total of 5356 unrelated Chinese Han individuals from Shandong Province were genotyped using the IdentifilerTM Plus Kit. Allele frequencies and corresponding forensic statistical parameters were calculated. Population comparisons were further explored through the neighbour-joining tree and multidimensional scaling analysis. Results A total of 217 alleles were calculated, the allele frequencies spanned from 0.0001 to 0.5340. The combined discrimination power (CDP) and the combined probability of paternity (CPE) of 15 STR loci were 0.99999999999999998755 and 0.999994524, respectively. Population comparisons demonstrated that Shandong Han have genetic homogeneity with most Sino-Tibetan populations. Conclusions Our study updates the Shandong Han population database with a large sample size. These 15 STR loci in the Shandong Han population are more polymorphic and discriminatory than shown in previous data, which could be more applicable to personal identification and paternity testing, as well other population genetics studies.

A work in progress - accreditation of forensic DNA laboratories as a part of the ,,European Forensic Science Area 2020 (EFSA 2020)” concept

As 2020 is approaching, various stakeholders and EU Member States are currently implementing some elements of the EFSA2020 concept. EFSA2020 offers Europe the chance to raise the quality level of its forensic science and in doing so, improve the public’s confidence in the administration of justice. However, there is plenty of opportunity for further progress in creating the common forensic area. The paper will focus on accreditation of forensic DNA laboratories in V4 countries.