A fast analysis system for forensic DNA reference samples

On January 1st, 2006, the Swedish legislation on obtaining DNA reference samples from suspects and the recording of DNA profiles in databases was changed. As a result the number of samples analysed at the Swedish National Laboratory of Forensic Science (SKL) increased from about 4500 in 2005 to more than 25,000 in 2006. To meet this challenge, SKL launched a new analysis system to create an unbroken chain, from sampling to incorporation of a profile in the national DNA database and subsequent automatic generation of digitally signed hit reports. The system integrates logistics, digital data transfer, new functions in LIMS (ForumDNA Version 4, Ida Infront AB) and laboratory automation. Buccal swab samples are secured on a FTA^® card attached to an identity form, which is barcoded with a unique sample ID. After sampling, the police officer sends a digital request to SKL. The sample is automatically registered in LIMS and processed on delivery. The resulting DNA profiles are automatically classified according to quality using a custom-made expert system. Building the evaluation around mathematical rules makes it reproducible, standardised and minimises manual work and clerk errors. All samples are run in duplicate and the two profiles are compared within LIMS before incorporation in the database. In the first year of operation, the median time for completion of an analysis was 3 days, measured from delivery of the sample to incorporation of the profile in the national DNA database. In spite of the dramatic increase in the number of reference samples there was no backlog.     

The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives

This paper presents an overview of the organisation and the results of the collaborative exercises (CE) of the European DNA Profiling (EDNAP) Group's mitochondrial DNA population database project (EMPOP). The aim of the collaborative exercises was to determine whether uniformity of mtDNA sequencing results could be achieved among different laboratories. These were asked to sequence either the complete mtDNA control region or the two hypervariable regions HVI (16024-16365) and HVII (73-340) from DNA extracts, buccal swabs or bloodstains, proceeding in accordance with the protocol and strategies used in each individual laboratory. The results of the collaborative exercises were employed to identify possible sources of errors that could arise during the analysis and interpretation of mtDNA profiles. These findings were taken as a basis to tentatively make suitable arrangements for the construction of a high quality mtDNA database. One hundred fifty mtDNA profiles were submitted to the evaluating laboratory, and disaccording profiles were classified into four groups corresponding to the source of error: clerical errors, sample mix-ups, contaminations and discrepancies with respect to the mtDNA nomenclature. Overall, 14 disaccording haplotypes (16 individual errors) were observed. The errors included 10 clerical errors, 3 interpretation problems, 2 cases of sample mix-up and 1 case of point heteroplasmic mixture, where the 2 sequencing reactions brought inconsistent base calls. This corresponds to an error rate of 10.7% in a virtual mtDNA database consisting of the collaborative exercise results. However, this estimate is still conservative compared to conclusions drawn by authors of meanwhile numerous publications critically reviewing published mtDNA population databases. Our results and earlier published concerns strongly emphasize the need for appropriate safety regulations when mtDNA profiles are compiled for database purposes in order to accomplish the high standard required for mtDNA databases that are used in the forensic context.     

How to avoid driving DNA caseworkers crazy: CaseSolver,an expert system to investigate complex crime scenes

DNA analyses can be used for both investigative (crime scene-focused), or evaluative (suspect-focused) reporting. Investigative, DNA-led exploration of serious crimes always involves the comparison of hundreds of biological samples submitted by the authorities for analysis. Crime stain comparisons include both evidence to evidence profiles and reference to evidence profiles. When many complex DNA results (mixtures, low template LT-DNA samples) are involved in the investigation of a crime, the manual comparison of DNA profiles is very time-consuming and prone to manual errors. In addition, if the person of interest is a minor contributor, the classical approach of performing searches of national DNA databases is problematic because it is realistically restricted to clear major contributors and the occurrence of masking and drop-out means that there will not be a definitive DNA profile to perform the search with.CaseSolver is an open source expert system that automates analysis of complex cases. It does this by three sequential steps: a) simple allele comparison b) likelihood ratio (LR) based on a qualitative model (forensim) c) LR based on a quantitative model (EuroForMix). The software generates a list of potential match candidates, ranked according to the LRs, which can be exported as a report. The software can also identify contributors from small or large databases (e.g., staff database or 1 mill. individuals). In addition, an informative graphical network plot is generated that easily identifies contributors in common to multiple stains. Here we describe recent improvements made to the software in version v1.5.0, made in response to user requirements during intensive casework usage.     

Mini-STRs: A powerful tool to identify genetic profiles in samples with small amounts of DNA

Degraded human remains and crime scene evidences with small amounts of DNA typically reveal incomplete or null genetic profiles when using standard (large) STR amplicons. The technology of mini-STRs, using reduced-size STR amplicons, can help to recover information from these samples. In our Forensic Genetic Service several genetic profiles were obtained or completed using MiniFiler kit (Applied Biosystems) increasing the success rate in sample typing. In all studied cases no inconsistencies were found between profiles obtained with MiniFiler and Identifiler, suggesting that this mini-STR kit can be used to include low copy number (LCN) evidence profiles in STR databases.     

A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpFlSTR^® Idenitfiler^® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.     

Performance Testing of a Semi-Automatic Card Punch System,Using Direct STR Profiling of DNA from Blood Samples on FTA™ Cards

The 1.2 mm Electric Coring Tool (e-Core™) was developed to increase the throughput of FTA^™ sample collection cards used during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots. Direct short tandem repeat (STR) DNA profiling was used to compare samples taken by the e-Core tool with those taken by the manual micro-punch. The performance of the e-Core device was evaluated using a commercially available PowerPlex™ 18D STR System. In addition, an analysis was performed that investigated the potential carryover of DNA via the e-Core punch from one FTA disc to another. This contamination study was carried out using Applied Biosystems AmpflSTR™ Identifiler™ Direct PCR Amplification kits. The e-Core instrument does not contaminate FTA discs when a cleaning punch is used following excision of discs containing samples and generates STR profiles that are comparable to those generated by the manual micro-punch.     

DNA Purification Cell Lysis and Wash Step Modifications for Low-Template DNA Sample Processing,

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low-template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re-extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low-template DNA sample processing are not enough to justify the risk associated with additional manipulation.     

Internal Validation of RapidHIT®ID ACE Sample Cartridge and Assessment of the EXT Sample Cartridge*†

A new rapid DNA solution, the RapidHIT^®ID, can accommodate two different sample cartridges, ACE, for the analysis of a single swab and EXT, for the analysis of DNA extracts. An efficient internal validation designed for low‐throughput rapid DNA is described. An evaluation of the EXT sample cartridge is also described. Each cartridge generated profiles with sufficient data quality to meet CODIS eligibility in fewer than 120 min. The results exhibited 100% correlation when compared to conventional DNA typing methods. Precision, reproducibility, stochastic, mixture, and contamination experiments produced expected results. Sensitivity of the ACE sample cartridge was acceptable for buccal swab analysis. The sensitivity of the EXT sample cartridge is discussed. The ACE validation and the EXT evaluation utilized a minimalist, cost‐saving, efficient design to generate a validated RapidHIT^®ID instrument capable of producing genetic profiles from both extracted forensic DNA samples and buccal swab samples within 120 min.     

Optimization and validation of a fully automated silica-coated magnetic beads purification technology in forensics

Automated procedures for forensic DNA analyses are essential not only for large-throughput sample preparation, but are also needed to avoid errors during routine sample preparation. The most critical stage in PCR-based forensic analysis is DNA isolation, which should yield as much highly purified DNA as possible. The extraction method used consists of pre-treatment of stains and samples, cell lysis using chaotropic reagents, binding of the DNA to silica-coated magnetic particles, followed by elution of the DNA. Our work focuses mainly on sample preparation, obtaining the maximum possible amount of biological material from forensic samples, and the following cell lysis, to create a simple standardized lysis protocol suitable for nearly all forensic material. After optimization and validation, the M-48 BioRobot((R)) workstation has been used for more than 20,000 routine lab samples. There has been no evidence of cross contamination. Resulting DNA from as small as three nuclear cells yield reliable complete STR amplification profiles. The DNA remains stable after 2 years of storage.     

Development of a simulation model to assess the impact of contamination in casework using STRs

Because contamination is usually tube-specific, negative controls cannot give assurance that an associated batch of extracted casework material is contaminant-free. However, it is possible to use them to predict the level of overall (undetected) contamination that is processed by an operational DNA unit. A MATLAB-based program was used to combine results of negative controls with actual casework DNA profiles to assess the probability that laboratory contaminants will give rise to reportable profiles (along with their likelihood ratios). Using data from an operational DNA unit as an example, it was demonstrated that the risk is inextricably linked to guidelines used to interpret DNA profiles. We have demonstrated how computer-based models can predict the levels of contamination expected in the process and, in addition, how the process can be made more robust by changing reporting guidelines. There is a need to compare DNA profiles against staff and plasticware elimination databases in order to determine sources of contamination. The likeliest outcome of a contamination event is false exclusion.     

The best possible result from the minimum available

In accordance with the Italian DNA legislation (DPR 7 April 2016, n. 87) a number of markers lower than seven are not considered usable for inclusion in the Italian forensic DNA database. For this reason, if the forensic DNA analysis performed in our laboratory do not provide acceptable results for a number greater than or equal to seven, the profile is not indicated in the final report. Thus, having indications about the possible success of an analysis before executing it, is a crucial point in the validation process of the accreditated method used in our laboratory.To achieve this goal, the quantification of extracts before typing plays a fundamental role. Especially when touched objects need to be examined tens or hundreds of nanograms may be present, but also very few or no cell can be present on the object. As such, quantification of every sample can ensure the maximum efficiency and prevent repeat analyses, over-amplified samples or completely useless examination.Quantifiler® Trio DNA quantification kit was validated in our laboratory according the guidelines approved by the ENFSI and always used before STR amplification of forensic casework DNA samples. Our attention has focused in particular on the definition of a minimum threshold at which it is useless to carry out DNA typing defining correlation of the negative results of the quantification by the absence of genetic profiles, as a result of DNA typing. Moreover, the validation of the Savant™ SPD131DDA SpeedVac™ Concentrator to get the maximum possible yield from DNA extracts was also investigated.     

Efficiency evaluation of the LT-DNA traces analysis modifications

Analysis of genetic profiles obtained from low template DNA samples (LT DNA) can be challenging because of increased probability of stochastic amplification artifacts occurrence. According to the recommendations of international genetic societies the quality of the LT-DNA traces results can be improved by applying low copy number (LCN) methods. Another strategy which allows to obtain better results of the analysis of LT-DNA traces is replicate the amplification of the same DNA sample and create consensus, composite (virtual pool profile) or real pool profile. The aim of the research was to analyze and compare the efficiency of modifications used in the testing of LT-DNA samples. Obtained results indicate that implementation of these methods in laboratory practice may lead to improvement in the quality of reported data from LT-DNA traces in genetic analyzes conducted to assist the justice system.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth.

Key points

• Bones and teeth often represent the only sources of DNA for identifying human remains.
• The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors.
• This study focuses on modifications to the previously available Qiagen-based protocols.
• The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols.
• The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

     

A multivariate statistical approach to for the evaluation of the biogeographical ancestry information from traditional STRs

The capability to achieve biogeographic ancestry (BGA) information from DNA profiles have been largely explored in forensic genetics because of its potential usefulness in providing investigative clues. For law enforcement and security purposes, when genetic data have been obtained from unknown evidence, but no reference samples are available and no hints come out from DNA databases, it would be extremely useful at least to infer the ethno-geographic origin of the stain donor by just examining traditional STRs DNA profiles.Current protocols for ethnic origin estimation using STRs profiles are usually based on Principal Component Analysis approaches and Bayesian methods. The present study provides an alternative approach that involves the use of target multivariate data analysis strategies for estimation of the BGA information from unknown biological traces. A powerful multivariate technique such as Partial Least Squares-Discriminant Analysis (PLS-DA) has been applied on NIST U.S. population datasets containing, for instance, the allele frequencies of African-American, Asian, Caucasian and Hispanic individuals. PLS-DA approach provided robust classifications, yielding high sensitivity and specificity models capable of discriminating the populations on ethnic basis. Finally, a real casework has been examined by extending the developed model to smaller and more geographically-restricted populations involving, for instance, Albanian, Italian and Montenegrian individuals.     

A brief history of the formation of DNA databases in forensic science within Europe

The introduction of DNA analysis to forensic science brought with it a number of choices for analysis, not all of which were compatible. As laboratories throughout Europe were eager to use the new technology different systems became routine in different laboratories and consequently, there was no basis for the exchange of results. A period of co-operation then started in which a nucleus of forensic scientists agreed on an uniform system. This collaboration spread to incorporate most of the established forensic science laboratories in Europe and continued through two major changes in the technology. At each step agreement was reached on which systems to use. From the beginning it was realised that DNA databases would provide the criminal justice systems with an efficient way of crime solving and consequently some local databases were created. It was not until the introduction of the amplification technology linked to the analysis of short tandem repeats that a sufficiently sensitive and robust system was available for the formation of efficient and effective DNA databases. Comprehensive legislation enacted in the UK in 1995 enabled forensic scientists to set up the first national DNA database which would hold both personal DNA profiles together with results obtained from crime scenes. Other countries quickly followed but in some the legislation has severely restricted the amount and type of data which can be retained and, therefore, effectiveness of the databases is limited. The widespread use of commercially produced multiplex kits has produced a situation in which nearly all European laboratories are using compatible systems and there is, therefore, the potential for the introduction of a pan-European DNA database. However, the exchange of results between countries is hampered by the various legislations which currently exist.     

Evidence in support of self-declaration as a sampling method for the formation of sub-population DNA databases

Well constructed sub-population databases are fundamental to the application of DNA-based forensic statistics. The size of such databases can affect the ability to examine adequately statistical or population genetic features, and the integrity of both the DNA profile and associated ethnicity information is also of importance. Use of short tandem repeat (STR) DNA profiling technology and the thoughtful construction of the governing legislation has seen large databases of DNA profiles collated for the four major sub-populations of New Zealand. Examination of the data illustrates the suitability of self-declaration as a means of categorizing samples on the basis of ethnicity.     

Application of the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System to the extraction of forensic casework samples

Robotic systems are commonly utilized for the extraction of database samples. However, the application of robotic extraction to forensic casework samples is a more daunting task. Such a system must be versatile enough to accommodate a wide range of samples that may contain greatly varying amounts of DNA, but it must also pose no more risk of contamination than the manual DNA extraction methods. This study demonstrates that the BioMek 2000 Laboratory Automation Workstation, used in combination with the DNA IQ System, is versatile enough to accommodate the wide range of samples typically encountered by a crime laboratory. The use of a silica coated paramagnetic resin, as with the DNA IQ System, facilitates the adaptation of an open well, hands off, robotic system to the extraction of casework samples since no filtration or centrifugation steps are needed. Moreover, the DNA remains tightly coupled to the silica coated paramagnetic resin for the entire process until the elution step. A short pre-extraction incubation step is necessary prior to loading samples onto the robot and it is at this step that most modifications are made to accommodate the different sample types and substrates commonly encountered with forensic evidentiary samples. Sexual assault (mixed stain) samples, cigarette butts, blood stains, buccal swabs, and various tissue samples were successfully extracted with the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System, with no evidence of contamination throughout the extensive validation studies reported here.