Modeling allelic analyte signals for aSTRs in NGS DNA profiles.

We describe an adaption of Bright et al.'s work modeling peak height variability in CE-DNA profiles to the modeling of allelic aSTR (autosomal short tandem repeats) read counts from NGS-DNA profiles, specifically for profiles generated from the ForenSeq™ DNA Signature Prep Kit, DNA Primer Mix B. Bright et al.'s model consists of three key components within the estimation of total allelic product-template, locus-specific amplification efficiencies, and degradation. In this work, we investigated the two mass parameters-template and locus-specific amplification efficiencies-and used MLE (maximum likelihood estimation) and MCMC (Markov chain Monte Carlo) methods to obtain point estimates to calculate the total allelic product. The expected read counts for alleles were then calculated after proportioning some of the expected stutter product from the total allelic product. Due to preferential amplicon selection introduced by the sample purification beads, degradation is difficult to model from the aSTR outputs alone. Improved modeling of the locus-specific amplification efficiencies may mask the effects of degradation. Whilst this model could be improved by introducing locus specific variances in addition to locus specific priors, our results demonstrate the suitability of adapting Bright et al.'s allele peak height model for NGS-DNA profiles. This model could be incorporated into continuous probabilistic interpretation approaches for mixed DNA profiles.

NucleoSpin(®) XS Columns for DNA Concentration and Clean-Up.

The phenol-chloroform (organic) extraction method continues to be a preferred method for extraction of DNA from forensic evidence samples that may contain low quantities of DNA and polymerase chain reaction (PCR) inhibitors. The aqueous extracts from the organic extraction of DNA require subsequent concentration and cleanup, which has traditionally been performed with microdialysis filter units, including the Centricon(®) and Microcon(®) centrifugal filter devices. Here, we describe the use of the NucleoSpin(®) XS silica columns as an alternative for the concentration and purification of the aqueous extracts from the organic extraction and for the removal of PCR inhibitors from existing DNA extracts.

Evaluation of the RapidHIT™ 200 and RapidHIT GlobalFiler(®) Express kit for fully automated STR genotyping.

The RapidHIT™ 200 Human Identification System and RapidHIT GlobalFiler(®) Express kit were evaluated and validated for use with single-source reference samples. It was of primary interest to evaluate the system for its efficacy as an expert system and to estimate a first pass success rate, as well as to identify the technical variables impacting that result. While results indicated that this instrument/kit combination can be used to accurately type single-source buccal samples, substantial variability in sensitivity and intra-color balance were observed, as were multiple artifacts, requiring extensive manual editing of the profiles. Artifacts included dye "blobs" and spectral overlap (pull-up) peaks that often originated from relatively low intensity allele peaks. Reduced intra-color balance, in combination with low sensitivity, occasionally resulted in instances of allelic dropout. Overall, 50% of the buccal samples analyzed in this study would have been successfully typed to give full GlobalFiler(®) profiles without the need for manual review and editing.

PCR inhibitor removal using the NucleoSpin® DNA Clean-Up XS kit.

Forensic evidence samples are collected from an unlimited variety of substrates, which may contain compounds known to inhibit the polymerase chain reaction (PCR). These PCR inhibitors are co-extracted with the DNA sample and can negatively affect the DNA typing results, which can range from partial to complete inhibition of the short tandem repeat (STR) PCR. One potential solution is to remove the PCR inhibitors from the extracts prior to the STR PCR with the NucleoSpin(®) DNA Clean-Up XS kit. The kit contains a NucleoSpin(®) XS silica column that has a special funnel design of thrust rings along with a very small silica membrane, which allows for sample elution in a small volume that is appropriate for use with current STR typing kits. The NucleoSpin(®) DNA Clean-Up XS kit was optimized for the best possible DNA recovery and then evaluated for its ability to remove eight commonly encountered PCR inhibitors including: bile salt, collagen, hematin, humic acid, indigo, melanin, tannic acid and urea. Each of these PCR inhibitors was effectively removed by the NucleoSpin(®) DNA Clean-Up XS kit as demonstrated by generating more complete STR profiles from the cleaned up inhibitor samples than from the raw inhibitor samples.

Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence.

We present a rapid alkaline lysis procedure for the extraction of DNA from sexual assault evidence that generates purified sperm fraction extracts that yield STR typing results similar to those obtained from the traditional organic/dithiothreitol differential extraction. Specifically, a sodium hydroxide based differential extraction method has been developed in a single-tube format and further optimized in a 96-well format. The method yields purified extracts from a small sample set (≈ 2-6 swabs) in approximately 2h and from a larger sample set (up to 96 swabs) in approximately 4h. While conventional differential extraction methods require vigorous sample manipulation to remove the spermatozoa from the substrate, the method described here exploits the propensity of sperm to adhere to a substrate and does not require any manipulation of the substrate after it is sampled. For swabs, sample handling is minimized by employing a process where the tip of the swab, including the shaft, is transferred to the appropriate vessel eliminating the need for potentially hazardous scalpels to separate the swab material from the shaft. The absence of multiple handling steps allows the process to be semi-automated, however the procedure as described here does not require use of a robotic system. This method may provide forensic laboratories a cost-effective tool for the eradication of backlogs of sexual assault evidence, and more timely service to their client agencies. In addition, we have demonstrated that a modification of the procedure can be used to retrieve residual sperm-cell DNA from previously extracted swabs.

The NucleoSpin® DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: an alternative to microdialysis filtration.

Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon(®) and Microcon(®) centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin(®) XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be used with standard organic extraction and dithiothreitol (DTT)-based differential extraction methods with no modifications to these methods prior to the concentration and clean-up step. Extracts from laboratory-prepared bloodstains, saliva and semen stains have been successfully amplified with both qPCR and STR assays. Finally, the total time to process a set of samples with the NucleoSpin(®) XS column is approximately 30 min vs. approximately 1.5h with the Centricon(®) YM-100 filter device.

A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA, human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: a diagnostic tool for STR typing.

A quadruplex real-time qPCR assay was developed to simultaneously assess total human DNA, human male DNA, DNA degradation and PCR inhibitors in forensic samples. Specifically, the assay utilizes a approximately 170-190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137 bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY), a 67 bp target sequence flanking the CSF1PO STR locus (nuCSF) to assess degradation (nuCSF:nuTH01 ratio) and a 77 bp synthetic DNA template used as an internal PCR control target sequence (IPC) for the assessment of PCR inhibition. Validation studies, performed on an ABI 7500 SDS instrument using TaqMan and TaqManMGB detection, indicate each of the targets in the quadruplex assay performs effectively and is informative even when challenged with DNase-degraded and hematin-inhibited samples. The nuTH01-nuSRY-nuCSF-IPC quadruplex qPCR assay is envisioned to assist in the choice of the most informative DNA typing system available, which may include standard autosomal STR typing when the results indicate the presence of non-degraded, single gender DNA or non-degraded, male:female mixtures at ratios expected to yield probative alleles; Y STR typing in samples containing a male component that is overwhelmed by the presence of an excess of female DNA; reduced amplicon size STR typing ("MiniSTRs") where the nuCSF:nuTH01 ratio indicates the sample is highly degraded; enhanced STR amplification with additional AmpliTaq Gold/BSA and/or sample clean-up when the presence of PCR inhibitors is suggested by a delayed IPC C(T) value or mitochondrial DNA typing in samples where little to no nuclear DNA is detected. The present study includes evaluations of species specificity, sensitivity, precision, reproducibility, male-female mixtures, population samples and applications to various casework-type samples as indicated by the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines.

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples.

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.