Effects and considerations of multiplexing ForenSeq Kintelligence libraries with a negative control.

The negative template control or negative amplification control has been an essential component of the forensic DNA analysis workflow that helps monitor contamination. As such, the inclusion of a negative control in forensic DNA analysis has been a requirement for all laboratories audited under the FBI's Quality Assurance Standards. As massively parallel sequencing (MPS) becomes more conventional in forensic laboratories, considerations for the inclusion of a negative control in every sequencing run can be evaluated. Although the inclusion of a negative control in library preparation and the first sequencing run has a practical function, there is less utility for its inclusion in all subsequent sequencing runs for that library preparation. Although this is universal to all MPS assays, it is most relevant for an assay that has a low sample multiplexing capacity, such as the ForenSeq Kintelligence Kit (Qiagen/Verogen, Inc.). The ForenSeq Kintelligence Kit is an investigative genetic genealogy (IGG) sequencing-based assay that targets 10,230 forensically relevant single-nucleotide polymorphisms. The manufacturer recommends multiplexing 3 libraries per sequencing run, which includes controls. The purpose of this study was to investigate the effect of the inclusion of a negative control in every Kintelligence sequencing run. We observed that the library generated from a negative amplification control will take 7%-14% of the run output. The loss of sequencing space taken by a negative control decreased the available output for DNA-containing samples, leading in some cases to allele or locus dropout and accompanying higher numbers of sixth to seventh order unknown associations in GEDmatch PRO.

Discrepancy in responses to the surprise question between hemodialysis nurses and physicians, with focus on patient clinical characteristics: A comparative study.

INTRODUCTION: The surprise question (SQ) "Would I be surprised if this patient died within the next xx months" can be used by different professions to foresee the need of serious illness conversations in patients approaching end of life. However, little is known about the different perspectives of nurses and physicians in responses to the SQ and factors influencing their appraisals. The aim was to explore nurses' and physicians' responses to the SQ regarding patients on hemodialysis, and to investigate how these answers were associated with patient clinical characteristics. METHODS: This comparative cross-sectional study included 361 patients for whom 112 nurses and 15 physicians responded to the SQ regarding 6 and 12 months. Patient characteristics, performance status, and comorbidities were obtained. Cohen's kappa was used to analyze the interrater agreement between nurses and physicians in their responses to the SQ and multivariable logistic regression was applied to reveal the independent association to patient clinical characteristics. FINDINGS: Proportions of nurses and physicians responding to the SQ with "no, not surprised" was similar regarding 6 and 12 months. However, there was a substantial difference concerning which specific patient the nurses and physicians responded "no, not surprised", within 6 (κ = 0.366, p < 0.001, 95% CI = 0.288-0.474) and 12 months (κ = 0.379, p < 0.001, 95% CI = 0.281-0.477). There were also differences in the patient clinical characteristics associated with nurses' and physicians' responses to the SQ. DISCUSSION: Nurses and physicians have different perspectives in their appraisal when responding to the SQ for patients on hemodialysis. This may reinforce the need for communication and discussion between nurses and physicians to identify the need of serious illness conversations in patients approaching the end of life, in order to adapt hemodialysis care to patient preferences and needs.

Comparative study of Rapid DNA versus conventional methods on compromised bones.

Equivalent amounts of compromised bones were used to directly compare STR success of conventional and Rapid DNA methods. Conventional DNA extraction methods, including manual full demineralization and semi-automated PrepFiler BTA/ AutoMate Express (ThermoFisher Scientific), provided insights regarding the DNA quantity and extent of degradation of each compromised bone analyzed with ANDE 6C (ANDE Corp) and RapidHIT ID (ThermoFisher Scientific) Rapid systems. Full demineralization provided higher DNA yields than extraction with the AutoMate Express for quality control (QC) and environmentally challenged bones. The degradation indices ranged from ∼1.8 to 73. Both demineralization and AutoMate Express extracts benefited from additional clean-up with NucleoSpin XS devices, which usually resulted in more alleles being detected than without further clean-up. Complete "CODIS 20″ profiles could be obtained with bone QC1 with all methods. However, among the 14 compromised bones with low DNA content, complete CODIS 20 profiles were detected for 7, 4, and 0 bones analyzed with demineralization, AutoMate Express and ANDE methods, respectively. The RapidHIT ID was the least sensitive method, providing the fewest detectable alleles for the bones tested. Whereas extracted DNA of approximately 0.1 ng can yield complete GlobalFiler STR profiles, at least 30 ng was required for complete FlexPlex 27 profiles using the ANDE 6C Rapid DNA system. In addition to being less sensitive than conventional methods, the tested Rapid DNA approaches were less predictable when attempting to improve STR success and proved to be less reliable in genotyping accuracy.

Compound and Conditioned Likelihood Ratio Behavior within a Probabilistic Genotyping Context.

In cases where multiple questioned individuals are separately supported as contributors to a mixed DNA profile, guidance documents recommend performing a comparison to see if there is support for their joint contribution. Anecdotal observations suggest the summed log of the individual likelihood ratios (LR), termed the simple LR product, should be roughly equivalent to or less than the log(LR) for the joint likelihood ratio, termed the compound LR. To assist casework analysts in evaluating statistical weights applied to a case at hand, this study assessed how consistently compound LRs conform to an additive behavior when compared to the simple LR product counterparts. Two-, three-, and four-person DNA mixture data, of various mixture proportions and DNA inputs, were interpreted by STRmix® version 2.8 Probabilistic Genotyping Software. Relative magnitudes of LR increases were found to be dependent on both template level and mixture composition. The distribution of log(LR) differences between all compound/simple LR comparisons was ~-2.7 to ~28.3. This level of information gain was similar to that for compound LR comparisons, with and without interpretation conditioning (~-3.2 to ~27.7). In both scenarios, the probability density peaked at approximately 0.5, indicating the information gain from constrained genotype combinations has a comparable impact on the outcome of LR calculations whether the restriction is applied before or after interpretation.

Improving the Utilization of STRmix™ Variance Parameters as Semi-Quantitative Profile Modeling Metrics.

Distributions of the variance parameter values developed during the validation process. Comparisons of these prior distributions to the run-specific average are one measure used by analysts to assess the reliability of a STRmix deconvolution. This study examined the behavior of three different STRmix variance parameters under standard amplification and interpretation conditions, as well as under a variety of challenging conditions, with the goal of making comparisons to the prior distributions more practical and meaningful. Using information found in STRmix v2.8 Interpretation Reports, we plotted the log10 of each variance parameter against the log10 of the template amount of the highest-level contributor (Tc) for a large set of mixture data amplified under standard conditions. We observed nonlinear trends in these plots, which we regressed to fourth-order polynomials, and used the regression data to establish typical ranges for the variance parameters over the Tc range. We then compared the typical variance parameter ranges to log10(variance parameter) v log10(Tc) plots for mixtures amplified and interpreted under a variety of challenging conditions. We observed several distinct patterns to variance parameter shifts in the challenged data interpretations in comparison to the unchallenged data interpretations, as well as distinct shifts in the unchallenged variance parameters away from their prior gamma distribution modes over specific ranges of Tc. These findings suggest that employing empirically determined working ranges for variance parameters may be an improved means of detecting whether aberrations in the interpretation were meaningful enough to trigger greater scrutiny of the electropherogram and genotype interpretation.

A comparative study for the isolation of exogenous trace DNA from fingernails.

Often fingernails from a victim or suspect involved in a physical assault, such as murder or sexual assault, are submitted to crime laboratories for DNA testing of foreign/exogenous biological material; however, very few studies have been conducted comparing the effectiveness of different sampling methods on the removal of foreign/exogenous DNA while minimizing the fingernail endogenous DNA. In this study three different sampling methods (swabbing, PBS soak, and PrepFiler® lysis buffer soak) were compared in order to identify one that minimizes the amount of endogenous DNA removed and maximizes the amount of foreign/exogenous male DNA removed. The samples were processed using the Tecan HIDEVO150 robot in order to reduce analyst time and the DNA mixtures were interpreted using the probabilistic genotyping software STRmix™. For each sampling method the quantity of male DNA, the mixture proportions, the number of foreign/exogenous male alleles detected, the amount of DNA degradation, and the discrimination power via the likelihood ratio obtained for the foreign/exogenous male DNA donor were determined and compared. The PrepFiler® lysis buffer soak and swabbing sampling methods appear to be equally effective at removing foreign/exogenous DNA from fingernails; however, the lysis buffer soak sampling method extracts more female endogenous DNA from the fingernail and the female DNA is degraded. Marginally higher likelihood ratios were obtained for the swab samples versus the PrepFiler® lysis buffer soak samples; therefore, it was determined that the swabbing sampling method was the best sampling method for the recovery of foreign exogenous DNA from fingernails while minimizing the amount of endogenous DNA removed.

Constructing universal multiplex PCR systems for comparative genotyping.

Analysis of length polymorphisms at STR loci in the human genome has become a standard approach for comparative genotyping in many areas including disease research and diagnostics, parentage assessment, investigations of human diversity, and forensic science. The simultaneous analysis of multiple STR loci through multiplex PCR and multicolor fluorescence detection offers sample conservation, high throughput, and automated genetic analysis. Careful design and optimization of tetranucleotide STR multiplexes has led to reliable, standardized systems that powerfully differentiate and distinguish individual human DNA profiles. The development of these multiplex systems involved a rigorous experimental strategy that included careful selection of PCR primer sequences (for yield, specificity, and multiplex compatability), along with optimization of PCR component concentrations, thermal cycling parameters, and fluorescence detection conditions. This developmental approach rendered well-characterized DNA typing systems that are high performing (sensitive, specific, and balanced), optimized to universal parameters (same reaction conditions), resilient to fluctuations in reaction conditions, and simple to implement and use routinely.

TWGDAM validation of AmpFlSTR PCR amplification kits for forensic DNA casework.

Laboratory procedures used in short tandem repeat (STR) analysis were subjected to various scenarios that assessed reliability and identified potential limitations. These validation studies were designed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) and the DNA Advisory Board (DAB) (17,18). Various DNA samples were amplified by the polymerase chain reaction (PCR) using AmpFlSTR PCR Amplification Kits (i.e., AmpFlSTR Green I, Profiler, Profiler Plus, and COfiler kits), detected with ABI Prism instrumentation, and analyzed using GeneScan and Genotyper software. Data acquired in these studies reinforced an existing body of knowledge and expertise regarding application and interpretation of STR typing in the forensic science community. Consistent STR genotypes were detected in various body tissues and fluids. Inter-laboratory comparisons produced concordant genotype results. Quantitative interpretational aids for DNA mixtures were characterized. Ability of the typing systems to type potentially compromised samples reliably was evaluated. Nonprobative case evidentiary DNA was successfully amplified, genotyped, and interpreted. Potential limitations or cautionary factors in the interpretation of minimal fluorescence intensity were demonstrated. Differential amplification between loci was observed when PCR was inhibited; preferential amplification typically was not. Single AmpFlSTR locus amplification did not offer consistent benefit over AmpFlSTR multiplexing, even in cases of DNA degradation or PCR inhibition. During rigorous evaluation, AmpFlSTR PCR Amplification Kits reproducibly yielded sensitive and locus-specific results, as required in routine forensic analyses.