Results of the 2018 Rapid DNA Maturity Assessment.

Three commercially available integrated rapid DNA instruments were tested as a part of a rapid DNA maturity assessment in July of 2018. The assessment was conducted with sets of blinded single-source reference samples provided to participants for testing on the individual rapid platforms within their laboratories. The data were returned to the National Institute of Standards and Technology (NIST) for review and analysis. Both FBI-defined automated review (Rapid DNA Analysis) and manual review (Modified Rapid DNA Analysis) of the datasets were conducted to assess the success of genotyping the 20 Combined DNA Index System (CODIS) core STR loci and full profiles generated by the instruments. Genotype results from the multiple platforms, participating laboratories, and STR typing chemistries were combined into a single analysis. The Rapid DNA Analysis resulted in a success rate of 80% for full profiles (85% for the 20 CODIS core loci) with automated analysis. Modified Rapid DNA Analysis resulted in a success rate of 90% for both the CODIS 20 core loci and full profiles (all attempted loci per chemistry). An analysis of the peak height ratios demonstrated that 95% of all heterozygous alleles were above 59% heterozygote balance. For base-pair sizing precision, the precision was below the standard 0.5 bp deviation for both the ANDE 6C System and the RapidHIT 200.

Proposal for the International Society for Cell & Gene Therapy position statement on assays for the quality control and potency assessment of adoptive cellular immunotherapies.

Translation of cell and gene therapies from pre-clinical experiments to clinical trials and final drug licensing brings requires the development, verification and even validation of the assays essential for the definition of the drug product. The technical and scientific challenges in doing this are far greater than they seem at first and are compounded by a lack of approved standards for assays used to support (c)GMP manufacture. This paper highlights some of those challenges and proposes solutions based on the experience of our colleagues using similar assay platforms in regulated pathology laboratories.

Qualitative and quantitative assessment of Illumina's forensic STR and SNP kits on MiSeq FGx™.

Massively parallel sequencing (MPS) is a powerful tool transforming DNA analysis in multiple fields ranging from medicine, to environmental science, to evolutionary biology. In forensic applications, MPS offers the ability to significantly increase the discriminatory power of human identification as well as aid in mixture deconvolution. However, before the benefits of any new technology can be employed, a thorough evaluation of its quality, consistency, sensitivity, and specificity must be rigorously evaluated in order to gain a detailed understanding of the technique including sources of error, error rates, and other restrictions/limitations. This extensive study assessed the performance of Illumina's MiSeq FGx MPS system and ForenSeq™ kit in nine experimental runs including 314 reaction samples. In-depth data analysis evaluated the consequences of different assay conditions on test results. Variables included: sample numbers per run, targets per run, DNA input per sample, and replications. Results are presented as heat maps revealing patterns for each locus. Data analysis focused on read numbers (allele coverage), drop-outs, drop-ins, and sequence analysis. The study revealed that loci with high read numbers performed better and resulted in fewer drop-outs and well balanced heterozygous alleles. Several loci were prone to drop-outs which led to falsely typed homozygotes and therefore to genotype errors. Sequence analysis of allele drop-in typically revealed a single nucleotide change (deletion, insertion, or substitution). Analyses of sequences, no template controls, and spurious alleles suggest no contamination during library preparation, pooling, and sequencing, but indicate that sequencing or PCR errors may have occurred due to DNA polymerase infidelities. Finally, we found utilizing Illumina's FGx System at recommended conditions does not guarantee 100% outcomes for all samples tested, including the positive control, and required manual editing due to low read numbers and/or allele drop-in. These findings are important for progressing towards implementation of MPS in forensic DNA testing.

Production of high-fidelity electropherograms results in improved and consistent DNA interpretation: Standardizing the forensic validation process.

Samples containing low-copy numbers of DNA are routinely encountered in casework. The signal acquired from these sample types can be difficult to interpret as they do not always contain all of the genotypic information from each contributor, where the loss of genetic information is associated with sampling and detection effects. The present work focuses on developing a validation scheme to aid in mitigating the effects of the latter. We establish a scheme designed to simultaneously improve signal resolution and detection rates without costly large-scale experimental validation studies by applying a combined simulation and experimental based approach. Specifically, we parameterize an in silico DNA pipeline with experimental data acquired from the laboratory and use this to evaluate multifarious scenarios in a cost-effective manner. Metrics such as signal1copy-to-noise resolution, false positive and false negative signal detection rates are used to select tenable laboratory parameters that result in high-fidelity signal in the single-copy regime. We demonstrate that the metrics acquired from simulation are consistent with experimental data obtained from two capillary electrophoresis platforms and various injection parameters. Once good resolution is obtained, analytical thresholds can be determined using detection error tradeoff analysis, if necessary. Decreasing the limit of detection of the forensic process to one copy of DNA is a powerful mechanism by which to increase the information content on minor components of a mixture, which is particularly important for probabilistic system inference. If the forensic pipeline is engineered such that high-fidelity electropherogram signal is obtained, then the likelihood ratio (LR) of a true contributor increases and the probability that the LR of a randomly chosen person is greater than one decreases. This is, potentially, the first step towards standardization of the analytical pipeline across operational laboratories.

Results of an inter and intra laboratory exercise on the assessment of complex autosomal DNA profiles.

The interpretation of complex DNA profiles may differ between laboratories and reporting officers, which can lead to discrepancies in the final reports. In this study, we assessed the intra and inter laboratory variation in DNA mixture interpretation for three European ISO17025-accredited laboratories. To this aim, 26 reporting officers analyzed five sets of DNA profiles. Three main aspects were considered: 1) whether the mixed DNA profiles met the criteria for comparison to a reference profile, 2) the actual result of the comparison between references and DNA profiling data and 3) whether the weight of the DNA evidence could be assessed. Similarity in answers depended mostly on the complexity of the tasks. This study showed less variation within laboratories than between laboratories which could be the result of differences between internal laboratory guidelines and methods and tools available. Results show the profile types for which the three laboratories report differently, which informs indirectly on the complexity threshold the laboratories employ. Largest differences between laboratories were caused by the methods available to assess the weight of the DNA evidence. This exercise aids in training forensic scientists, refining laboratory guidelines and explaining differences between laboratories in court. Undertaking more collaborative exercises in future may stimulate dialog and consensus regarding interpretation. For training purposes, DNA profiles of the mixed stains and questioned references are made available.

A reliable and reproducible technique for DNA fingerprinting in biorepositories: a pilot study from BioBIM.

Standard operating procedures (SOPs) optimization for nucleic acid extraction from stored samples is of crucial importance in a biological repository, considering the large number of collected samples and their future downstream molecular and biological applications. However, the validity of molecular studies using stored specimens depends not only on the integrity of the biological samples, but also on the procedures that ensure the traceability of the same sample, certifying its uniqueness, and ensuring the identification of potential sample contaminations. With this aim, we have developed a rapid, reliable, low-cost, and simple DNA fingerprinting tool for a routine use in quality control of biorepositories samples. The method consists of a double ALU insertion/deletion genotyping panel suitable for uniqueness, identification of sample contaminations, and gender validation. Preliminary data suggest that this easy-to-use DNA fingerprinting protocol could routinely provide assurances of DNA identity and quality in a biorepository setting.

The optimization of a rapid pulsed-field gel electrophoresis protocol for the typing of Acinetobacter baumannii, Escherichia coli and Klebsiella spp.

Pulsed-field gel electrophoresis (PFGE) is the most common genotyping method used for the typing of a number of bacterial species. Generally, investigators use their own custom-developed protocol, but a standardized PFGE protocol would allow the comparison of typing results between laboratories and the tracing of strains around the country. In the present study, we optimized a PFGE protocol for subtyping of Acinetobacter baumannii, Escherichia coli and Klebsiella spp., which are commonly isolated from nosocomial infections in many hospitals. Reproducibility of our PFGE procedure was studied three times at 2- to 3-week intervals. Epidemiological concordance of the optimized PFGE procedure was tested on seven isolates of A. baumannii from a previous outbreak and seven A. baumannii isolates randomly selected among the clinical isolates. The optimized PFGE procedure was evaluated on a total of 174 clinical isolates including 62 A. baumannii, 50 E. coli, and 62 Klebsiella spp. The inter-laboratory reproducibility of the optimized protocol was tested at four laboratories. The optimized procedure is completed in 28 h after culturing. It is likely to be cost-effective, due to the reduction in the time, reagent volume and enzyme concentration needed. The procedure showed high concordance with epidemiological data. There were no non-typeable isolates among the tested bacteria. It is reproducible and versatile. This protocol can be used to identify outbreaks and monitor the spreading rate of nosocomial infections caused by the tested bacterial isolates. Furthermore, due to its high intra- and inter-laboratory reproducibility, the protocol has the potential to be useful for comparing PFGE fingerprinting profiles of the isolates from different settings.

Spanish public awareness regarding DNA profile databases in forensic genetics: what type of DNA profiles should be included?

The importance of non-codifying DNA polymorphism for the administration of justice is now well known. In Spain, however, this type of test has given rise to questions in recent years: (a) Should consent be obtained before biological samples are taken from an individual for DNA analysis? (b) Does society perceive these techniques and methods of analysis as being reliable? (c) There appears to be lack of knowledge concerning the basic norms that regulate databases containing private or personal information and the protection that information of this type must be given. This opinion survey and the subsequent analysis of the results in ethical terms may serve to reveal the criteria and the degree of information that society has with regard to DNA databases. In the study, 73.20% (SE 1.12%) of the population surveyed was in favour of specific legislation for computer files in which DNA analysis results for forensic purposes are stored.

Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii.

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains.

The feasibility of external blind DNA proficiency testing. I. Background and findings.

We describe the origins, purposes, and findings of a national study to determine whether a large-scale program of blind proficiency testing in U.S. DNA laboratories is feasible and/or practical. Proficiency testing in clinical laboratories is reviewed, particularly as mandated by the Clinical Laboratory Improvement Acts and its role in the regulation of those laboratories. Proficiency testing in forensic urine drug testing labs is also briefly reviewed. Studies involving comparisons between open and blind proficiency testing are discussed. The clinical laboratory proficiency testing and regulation landscape provides the background for the DNA Act of 1994, and the congressional mandate to investigate blind proficiency testing in forensic DNA laboratories. Four models of blind proficiency testing are defined and discussed, along with the advantages and disadvantages of each and estimates of the costs of a large-scale program. The purposes of proficiency testing in a quality-assurance context are likewise discussed and related to the models and the arguments generally proffered for and against blind vs. open proficiency testing.

The feasibility of external blind DNA proficiency testing. II. Experience with actual blind tests.

The background and goals of a national study to determine the feasibility of blind proficiency testing in U.S. forensic DNA laboratories are discussed. Part of the project involved designing and executing a series of fifteen blind proficiency tests. Execution included biological specimen donor recruitment and case evidence manufacturing. Simulated cases were submitted to DNA laboratories by law enforcement agencies and in some cases by other forensic-science laboratories. Replicate-manufactured evidence was submitted to reference laboratories to simulate the workings of a larger-scale program. Ten tests were straightforward, and essentially tested analytical ability. Five tests involved selecting on the basis of case facts appropriate bloodstains for typing from a bloodstain pattern. We describe in detail our experience in designing and conducting these blind proficiency test trials, and relate those experiences to the overall issue of blind proficiency testing as a quality-assurance tool in forensic DNA laboratories. In this feasibility test series, one blind test was detected by a laboratory, a second one was shown to the lab by law enforcement, and a third was never completed because of lapses in communication. Turnaround times were relatively fast in the independent/commercial labs and relatively slow in the larger public laboratories. Two cross-state case-to-case CODIS "hits" were "planted" among the first series of ten blind tests. One pair was detected. One member of the second pair went to a lab that was not CODIS-ready.

Quality assessment of Mycobacterium tuberculosis genotyping in a large laboratory network.

Quality assessment exercises were conducted to evaluate the reproducibility of IS6110 DNA fingerprinting performed by eight laboratories in the National Tuberculosis Genotyping and Surveillance Network. Three panels, each with 8 to 16 isolates, were typed at all laboratories, resulting in 280 images. When the pattern obtained by the majority for each isolate was used as the standard, exact matches were obtained for 73% of patterns; 90% and 97% of patterns matched within one- and two-band differences, respectively. A second approach involved retyping of randomly selected isolates at the Centers for Disease Control and Prevention. Retyping was done for 8-19 isolates per laboratory (76 total). Paired images matched exactly for 54% of isolates and within one and two band differences, 78% and 93%, respectively. We evaluated reasons for mismatching. We also evaluated the reproducibility of spoligotyping using a test panel of 13 isolates; a discrepancy of 1 in 91 results was noted.

Forensic DNA analysis and the United States Government.

All three branches of the United States Government are, directly or indirectly, promoting the use and judicial acceptance of forensic DNA analysis. In addition, the establishment of a US national DNA databank has been authorized. The US Congress has passed the 'DNA Identification Act of 1994', which provides, inter alia, funding to the states for developing and/or improving forensic laboratories capable of conducting DNA analysis, and also creates a framework for federal supervision of forensic DNA technology. Specifically, the Executive Branch, through the Department of Justice and particularly its Federal Bureau of Investigation, has been directed to develop standards and practices in order to speed the admissibility of forensic DNA analysis as scientifically acceptable evidence in US courts. Finally, the federal judiciary has been ordered by the US Supreme Court to abandon or modify the 70-year-old Frye standard, which the Federal courts previously used to determine whether scientific evidence is deemed admissible, a move that will directly impact the judicial acceptance of forensic DNA analysis in all federal courts and undoubtedly will affect the admissibility of DNA evidence in many American state courts.