A tale of two PG systems: A comparison of the two most widely used continuous probabilistic genotyping systems in the United States.

The development of probabilistic genotyping (PG) systems to quantitatively analyze DNA mixture samples has been transformative in forensic science. TrueAllele® Casework (TA) and STRmix™ (STRmix) are the two most widely used PG systems in the United States. The two systems were challenged with 48 two-, three-, and four-person mock casework samples, for a total of 152 likelihood ratio (LR) comparisons. TA and STRmix converged on the same result (supportive, non-supportive, or inconclusive) for ~91% of contributor-specific comparisons. Where moderate or substantial differences in log(LR) values were observed, 9% affected the conclusion of the reference association to the mixture. The PG systems exhibited high correlations for estimated contributor-specific template quantities (~92%) and log(LR)s produced (>88%). When the log(LR)s for only low-template contributors (<100 pg) were compared, the R2 value dropped to ~68% and the difference became statistically significant. Of the 14 contributor comparisons where the conclusion differed, two were contradictory (supportive vs. non-supportive) and 12 were either inconclusive versus non-supportive or inconclusive versus supportive. The differing results were likely due to dissimilarities in the mixture input file as STRmix uses a lab-defined analytical threshold (AT) and TA models to 10 RFUs for each electropherogram. When 7 of the 14 mixtures were reanalyzed by STRmix using a 10 RFU AT, the log(LR)s for the low-template contributors became more similar to TAs. This study shows that while both systems may produce accurate and calibrated LRs, their results can deviate, especially for low-template, degraded contributors, and the deviation is generally predictable.

Use of hormone-specific antibody probes for differential labeling of contributor cell populations in trace DNA mixtures.

A significant proportion of casework analyzed by forensic science laboratories is often "touch" or trace forensic DNA evidence, which is deposited through physical contact and is comprised of sloughed epidermal cells. These samples can be challenging to analyze due to low DNA concentrations, frequent degradation, and the presence of cells from multiple individuals in the same sample. To address these challenges, we investigated a new approach for characterizing trace evidence prior to DNA profiling that labels epidermal cells with antibody probes targeting hormone molecules testosterone and dihydrotestosterone (DHT). The goal was to test whether cell populations derived from separate individuals showed different binding efficiencies to hormone probes and, thus, could be used to detect the presence of multiple cell populations. Additionally, we investigated whether antibody probes could be used to isolate contributor cell populations from an epidermal cell mixture and facilitate deconvolution of mixed DNA profiles recovered from touch/trace evidence. Results showed that cell populations from some individuals could differentiated in trace samples based on fluorescence histograms following probe labeling. However, certain pairs of contributors showed largely or completely overlapping histogram profiles and could not be resolved. Preliminary efforts to separate cell populations that could be differentiated with hormone probes with fluorescence-activated cell sorting (FACS) coupled to DNA profiling and probabilistic modeling indicated that it is possible to enrich contributor cell populations from touch/trace samples and produce more probative DNA profiles compared to the original mixture sample. The variability in labeling, differentiation, and physical separation of cell populations may be impacted by similarities in biochemical profiles across some contributors as well as imbalance of contributor DNA quantities in certain mixtures as is typical in casework involving touch/trace evidence. Ultimately, screening and separation of trace DNA samples with this approach may be presumptive and constrained by sample-specific parameters of the original mixture.

Simplification of complex DNA profiles using front end cell separation and probabilistic modeling.

Forensic samples comprised of cell populations from multiple contributors often yield DNA profiles that can be extremely challenging to interpret. This frequently results in decreased statistical strength of an individual's association to the mixture and the loss of probative data. The purpose of this study was to test a front-end cell separation workflow on complex mixtures containing as many as five contributors. Our approach involved selectively labelling certain cell populations in dried whole blood mixture samples with fluorescently labeled antibody probe targeting the HLA-A*02 allele, separating the mixture using Fluorescence Activated Cell Sorting (FACS) into two fractions that are enriched in A*02 positive and A*02 negative cells, and then generating DNA profiles for each fraction. We then tested whether antibody labelling and cell sorting effectively reduced the complexity of the original cell mixture by analyzing STR profiles quantitatively using the probabilistic modeling software, TrueAllele® Casework. Results showed that antibody labelling and FACS separation of target populations yielded simplified STR profiles that could be more easily interpreted using conventional procedures. Additionally, TrueAllele® analysis of STR profiles from sorted cell fractions increased statistical strength for the association of most of the original contributors interpreted from the original mixtures.

Analysis of cellular autofluorescence in touch samples by flow cytometry: implications for front end separation of trace mixture evidence.

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650-670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor's contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two-person touch mixture was separated into two fractions via fluorescence-activated cell sorting (FACS) using gating criteria based on intensity of 650-670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures. Graphical Abstract Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.

Establishing the Limits of TrueAllele® Casework: A Validation Study.

The limits of the expert system, TrueAllele® Casework (TA), were explored using challenging mock casework profiles that included 17 single-source and 18 two-, 15 three- and 7 four-person DNA mixtures. The sensitivity (ability to detect a minor contributor) of the TA analysis process was examined by challenging the system with mixture DNA samples that exhibited allelic and locus dropout and other stochastic effects. The specificity (ability to exclude nondonors) was rigorously tested by interrogating TA derived genotypes with 100 nondonor profiles. The accuracy with which TA estimated mixture weights of contributors to the two-person mixtures was examined. Finally, first-degree relatives of donors were used to assess the ability of the system to exclude close relatives. TA demonstrated great accuracy, sensitivity, and specificity. TA correctly assigned mixture weights and excluded nearly all first-degree relatives. This study demonstrates the analysis power of the TrueAllele® Casework system.

Integrated sample cleanup and microchip capillary array electrophoresis for high-performance forensic STR profiling.

Microfluidics has the potential to significantly improve the speed, throughput, and cost performance of electrophoretic short tandem repeat (STR) analysis by translating the process into a miniaturized and integrated format. Current STR analysis bypasses the post-PCR sample cleanup step in order to save time and cost, resulting in poor injection efficiency, bias against larger loci, and delicate injection timing controls. Here we describe the operation of an integrated high-throughput sample cleanup and capillary array electrophoresis microsystem that employs a streptavidin capture gel chemistry coupled to a simple direct-injection geometry for simultaneously analyzing 12 STR samples in less than 30 min with >10-fold improved sensitivity.

Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification.

A fully integrated microdevice and process for forensic short tandem repeat (STR) analysis has been developed that includes sequence-specific DNA template purification, polymerase chain reaction (PCR), post-PCR cleanup and inline injection, and capillary electrophoresis (CE). Fragmented genomic DNA is hybridized with biotin-labeled capture oligos and pumped through a fluidized bed of magnetically immobilized streptavidin-coated beads in microchannels where the target DNA is bound to the beads. The bead-DNA conjugates are then transferred into a 250 nL PCR reactor for autosomal STR amplification using one biotin and one fluorescence-labeled primer. The resulting biotin-labeled PCR products are electrophoretically injected through a streptavidin-modified capture gel where they are captured to form a concentrated and purified injection plug. The thermally released sample plug is injected into a 14 cm long CE column for fragment separation and detection. The DNA template capture efficiency provided by the on-chip sequence-specific template purification is determined to be 5.4% using K562 standard DNA. This system can produce full 9-plex STR profiles from 2.5 ng input standard DNA and obtain STR profiles from oral swabs in about 3 hours. This fully integrated microsystem with sample-in-answer-out capability is a significant advance in the development of rapid, sensitive, and reliable micro-total analysis systems for on-site human identification.

Integrated sample cleanup and capillary array electrophoresis microchip for forensic short tandem repeat analysis.

A twelve-lane capillary array electrophoresis (CAE) microsystem is developed that utilizes an efficient inline capture injection process together with the classical radial microfabricated capillary array electrophoresis (µCAE) format for high-sensitivity forensic short tandem repeat (STR) analysis. Biotin-labeled 9-plex STR amplicons are captured in a photopolymerized gel plug via the strong binding of streptavidin and biotin, followed by efficient washing and thermal release for CE separation. The analysis of 12 STR samples is completed in 30 min without any manual process intervention. A comparison between capture inline injection and conventional cross injection demonstrated at least 10-fold improvement in sensitivity. The limit-of-detection of the capture-CAE system was determined to be 35 haploid copies (17-18 diploid copies) of input DNA; this detection limit approaches the theoretical limits calculated using Poisson statistics and the spectral sensitivity of the instrument. To evaluate the capability of this microsystem for low-copy-number (LCN) analysis, three touch evidence samples recovered from unfired bullet cartridges in a pistol submerged in water for an hour were successfully analyzed, providing 53, 71, and 59% of the DNA profile. The high-throughput capture-CAE microsystem presented here provides a more robust and more sensitive platform for conventional as well as LCN and degraded DNA analysis.

Dual-domain microchip-based process for volume reduction solid phase extraction of nucleic acids from dilute, large volume biological samples.

A microfluidic device was developed to carry out integrated volume reduction and purification of nucleic acids from dilute, large volume biological samples commonly encountered in forensic genetic analysis. The dual-phase device seamlessly integrates two orthogonal solid-phase extraction (SPE) processes, a silica solid phase using chaotrope-driven binding and an ion exchange phase using totally aqueous chemistry (chitosan phase), providing the unique capability of removing polymerase chain reaction (PCR) inhibitors used in silica-based extractions (guanidine and isopropanol). Nucleic acids from a large volume sample are shown to undergo a substantial volume reduction on the silica phase, followed by a more stringent extraction on the chitosan phase. The key to interfacing the two steps is mixing of the eluted nucleic acids from the first phase with loading buffer which is facilitated by flow-mediated mixing over a herringbone mixing region in the device. The complete aqueous chemistry associated with the second purification step yields a highly concentrated PCR-ready eluate of nucleic acids devoid of PCR inhibitors that are reagent-based (isopropanol) and sample-based (indigo dye), both of which are shown to be successfully removed using the dual-phase device but not by the traditional microfluidic SPE (muSPE). The utility of the device for purifying DNA was demonstrated with dilute whole blood, dilute semen, a semen stain, and a blood sample inhibited with indigo dye, with the resultant DNA from all shown to be PCR amplifiable. The same samples purified using muSPE were not all PCR amplifiable due to a smaller concentration of the DNA and the lack of PCR-compatible aqueous chemistry in the extraction method. The utility of the device for the purification of RNA was also demonstrated, by the extraction of RNA from a dilute semen sample, with the resulting RNA amplified using reverse transcription (RT)-PCR. The vrSPE-SPE device reliably yields a volume reduction for DNA and RNA purification on the order of 50- and 14-fold, respectively, both compatible with downstream PCR analysis. In addition, purification of all samples consumed less reagents (2.6-fold) than traditional purification methods, with the added advantage of being a "closed system" that eliminates sample transfer steps, thereby reducing the possible entrance points for contaminants.

Implementation and validation of the Teleshake unit for DNA IQ robotic extraction and development of a large volume DNA IQ method.

Automated platforms used for forensic casework sample DNA extraction need to be versatile to accommodate a wide variety of sample types, thus protocols frequently need modification. In this study, DNA IQ methods previously developed for the Biomek 2000 Automation Workstation were adapted for the Teleshake Unit using normal volumes and all deepwell extraction, and a large volume DNA IQ method developed. DNA purification without detectable contamination of adjacent reagent blanks is reported in the extraction of tissue samples containing several micrograms of DNA. Sensitivity and contamination studies demonstrated similar performance with the manual organic extraction method for bloodstain dilution samples. Mock casework samples demonstrated the effectiveness of the Teleshake and Teleshake large volume methods. Because of the performance and increased versatility of the DNA IQ extraction with these modifications, the Teleshake Unit has been implemented in both normal and large volume automated DNA extractions at the Virginia Department of Forensic Science.

Development of STR profiles from firearms and fired cartridge cases.

Fired cartridge cases are a common type of evidence found at crime scenes. However, due to the high chamber temperatures and touch nature of this evidence, DNA testing is not commonly sought because it is believed DNA is only present in low levels, whether it is due to initial low levels of DNA and/or DNA degradation from the heat or inhibition of the PCR reaction. Moreover, very few laboratories report STR typing success with fired cases. This study focused on obtaining STR profiles from fired cartridge cases using the AmpFlSTR MiniFiler kit, which is designed to amplify DNA from low level, inhibited, and degraded samples. Comparisons to other STR amplification kits were also conducted. In attempt to simulate casework, random individuals loaded cartridges into a firearm. DNA was recovered from the fired cartridge cases using the double swab technique and extracted using an automated large volume DNA IQ method. Initially, testing focused on known shedders handling cartridges for 30s prior to firing. A significantly greater number of alleles was obtained following amplification with the MiniFiler kit versus the PowerPlex 16 BIO kit. No alleles were observed using the Identifiler kit. In an attempt to better simulate casework, a random selection of laboratory personnel handled shotshells for as long as needed to load and fire the weapon. In this mock sample study, the MiniFiler kit successfully amplified an average of 22% of expected alleles from DNA recovered from shotshell cases versus the PowerPlex 16 BIO kit where an average of 7% of alleles were observed. However, the total number of alleles obtained from the two kits was not significantly different. The quality of the DNA obtained from fired cases was studied with evidence of inhibition in at least 11% of shotshell case samples. After swabbing the head and the hull of three shotshell cases separately, a significantly greater number of alleles was obtained from the hull as opposed to the head of the fired shotshell case. In addition, after firing, various internal firearm surfaces were swabbed, including the chamber of barrel, ejection port, and breechface, in an attempt to obtain amplifiable DNA. DNA was obtained from the chamber of the barrel and was amplifiable using the MiniFiler kit, although mixtures were obtained with extensive drop-in and drop-out making this analysis unlikely to aid an investigation.

Integrated sample cleanup-capillary electrophoresis microchip for high-performance short tandem repeat genetic analysis.

An integrated PCR sample cleanup and preconcentration process is developed for forensic short tandem repeat (STR) analysis using a streptavidin-modified photopolymerized capture gel injector for microchip capillary electrophoresis (microCE). PCR samples generated with one biotinylated primer and one fluorescent primer provide the input to the streptavidin-based affinity capture-microCE device. Monoplex PCR samples processed by the device exhibited approximately 10- to 50-fold increased fluorescence intensities, and DNA profiles generated using 9-plex STR samples displayed approximately 14- to 19-fold higher signal intensities compared to those analyzed using traditional cross injection. Complete STR profiles were obtained with as few as 25 copies of DNA template using the capture-microCE device. Four DNA samples with various degrees of degradation were also tested. Samples analyzed using the capture-microCE device resulted in a significant increase of successful allele detection. The ability of our capture-microCE device and method to remove contaminating ions, to concentrate the sample injection plug, and to eliminate electrokinetic injection bias provides a powerful approach for integrating sample cleanup with DNA separation.

A forensic laboratory tests the Berkeley microfabricated capillary array electrophoresis device.

Miniaturization of capillary electrophoresis onto a microchip for forensic short tandem repeat analysis is the initial step in the process of producing a fully integrated and automated analysis system. A prototype of the Berkeley microfabricated capillary array electrophoresis device was installed at the Virginia Department of Forensic Science for testing. Instrument performance was verified by PowerPlex 16 System profiling of single source, sensitivity series, mixture, and casework samples. Mock sexual assault samples were successfully analyzed using the PowerPlex Y System. Resolution was assessed using the TH01, CSF1PO, TPOX, and Amelogenin loci and demonstrated to be comparable with commercial systems along with the instrument precision. Successful replacement of the Hjerten capillary coating method with a dynamic coating polymer was performed. The accurate and rapid typing of forensic samples demonstrates the successful technology transfer of this device into a practitioner laboratory and its potential for advancing high-throughput forensic typing.

Rapid determination of monozygous twinning with a microfabricated capillary array electrophoresis genetic-analysis device.

BACKGROUND: Microfabricated genetic-analysis devices have great potential for delivering complex clinical diagnostic technology to the point of care. As a demonstration of the potential of these devices, we used a microfabricated capillary array electrophoresis (microCAE) instrument to rapidly characterize the familial and genotypic relationship of twins who had been assigned fraternal (dizygous) status at birth. METHODS: We extracted the genomic DNA from buccal samples collected from the twin sons, the parents, another sibling, and an unrelated control individual. We then carried out multiplex PCR amplification of sequences at 16 short tandem repeat loci commonly used in forensic identity testing. We simultaneously separated the amplicons from all of the individuals on a microCAE device and fluorescently detected the amplicons with single-base resolution in <30 min. RESULTS: The genotypic analysis confirmed the identical status of the twins and revealed, in conjunction with the medical data, that their twin status arose from the rarer dichorionic, diamniotic process. CONCLUSIONS: The ability to rapidly analyze complex genetic samples with microCAE devices demonstrates that this approach can help meet the growing need for rapid genetics-based diagnostics.

Fluorescence energy transfer-labeled primers for high-performance forensic DNA profiling.

A fluorescence energy transfer (ET) dye-labeled STR typing system (ET 16-plex) is developed for the markers used in the commercial STR typing kit PowerPlex 16, and its performance assessed using a 96-lane microfabricated capillary array electrophoresis (muCAE) system. The ET 16-plex amplicons displayed 1.6-9-fold higher fluorescence intensities compared to those produced using the single-dye (SD)-labeled multiplex kits. The ET multiplex delivered full STR profiles from 62.5 pg of DNA; half the input required for the SD kits while maintaining a similar heterozygote allele balance. This increased sensitivity should improve typing of poor-quality DNA samples by making minor or imbalanced alleles more readily detectable at the low copy number (LCN) threshold. The ET 16-plex also generated complete profiles with only 28 PCR cycles; this capability should improve LCN typing by reducing the amplification time and drop-in allele incidence. To confirm the practical advantages of ET-labeled primers, six previously problematic casework samples were tested and only the ET 16-plex kit was able to capture additional allele data. The successful development and demonstration of ET primers for higher sensitivity STR typing offers a simple solution to improving current commercial multiplex typing capability. The superior spectral properties and universal compatibility with any primer sequence provided by ET cassettes will make future multiplex construction more facile and straightforward. The pairing of ET cassette technology with the muCAE system illustrates not only an enhanced STR typing platform, but a significant step toward a higher-efficiency forensic laboratory enabled by better chemistry and microfluidics.

Rapid and high-throughput forensic short tandem repeat typing using a 96-lane microfabricated capillary array electrophoresis microdevice.

A 96-channel microfabricated capillary array electrophoresis (muCAE) device was evaluated for forensic short tandem repeat (STR) typing using PowerPlex 16 and AmpFlSTR Profiler Plus multiplex PCR systems. The high-throughput muCAE system produced high-speed <30-min parallel sample separations with single-base resolution. Forty-eight previously analyzed single-source samples were accurately typed, as confirmed on an ABI Prism 310 and/or the Hitachi FMBIO II. Minor alleles in 3:1 mixture samples containing female and male DNA were reliably typed as well. The instrument produced full profiles from sample DNA down to 0.17 ng, a threshold similar to that found for the ABI 310. Seventeen nonprobative samples from various evidentiary biological stains were also correctly typed. The successful application of the muCAE device to actual forensic STR typing samples is a significant step toward the development of a completely integrated STR analysis microdevice.

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.

Validation and implementation of the PowerPlex 16 BIO System STR multiplex for forensic casework.

The PowerPlex 16 BIO multiplex short tandem repeat (STR) system contains the 13 CODIS loci (FGA, TPOX, D8S1179, vWA, D18S51, D21S11, TH01, D3S1358, CSF1PO, D16S539, D7S820, D13S317, and DS5S818), plus two pentanucleotide repeat loci (Penta D and Penta E) and the sex-identifying locus. Amelogenin. The PowerPlex 16 BIO System is optimized for use with the Hitachi FMBIO gel imaging systems. A consortium of seven independent laboratories collaborated to perform the studies defined by the FBI standards for performing a developmental validation, including the evaluation of sample concordance, percent stutter determination, nonprobative casework, precision, sensitivity, mixture determination, effect of substrates, the impact of environmental insults, and species specificity. All samples tested for concordance were consistent except for one sample from the Virginia Division of Forensic Science database that displayed discordance at D13S317, a locus whose primer sequence was altered. Stutter values were comparable to those of other STR multiplex systems, the precision was comparable to other multiplexes analyzed by gel electrophoresis, the DNA profiles were unchanged by the substrate upon which the blood samples were placed, and the nonprobative casework samples re-typed for the PowerPlex 16 BIO System were consistent with previous typing results. When greater than 0.125 ng of DNA was placed into the PowerPlex 16 BIO System amplification reaction, a full profile was generated by all laboratories. The mixture study results were comparable to those reported for other multiplex systems, the environmental study demonstrated a loss of larger molecular weight loci when samples were incubated at elevated temperatures for a prolonged period of time, and the only notable cross species hybridization was observed with primate DNA samples. This extensive validation work performed demonstrates that the PowerPlex 16 BIO System provides STR data of a quality comparable with other PowerPlex STR multiplex kits as well as other widely used STR multiplexes and is thus suitable for evidentiary casework analysis as well as database sample profiling.