DNA backlog reduction strategy: Law enforcement agency partnerships for a successful biological screening laboratory.

In 2009, the Palm Beach County Sheriff's Office Forensic Biology Unit developed an innovative DNA backlog strategy to construct and operate a centralized biological processing laboratory (BPL) within a law enforcement agency, the Boca Raton Police Services Department. The BPL became fully operational in 2012 and obtained accreditation in 2017. This coordinated, multi-agency agreement resulted in a streamlined process exemplifying several benefits such as communicating timely testing results, decreasing the case turnaround time, and decreasing the DNA case backlog. This paper provides a summary of the necessary considerations of location, construction, personnel, and services when constructing a BPL, as well as, provides a comparison of initial completion dates and ultimate completion dates over a three-year period from 2016 to 2018. Three LEAs submitted 613 cases to the BPL commensurate with jurisdictional population. Performance metrics such as types and number of criminal cases screened; the number of samples forwarded for PBSO DNA testing; the turnaround time to handle, screen, or analyze a forensic sample; evidentiary samples; and the number of profiles entered into the Combined DNA Index System (CODIS) database are reported. For example, prior to this DNA backlog reduction strategy, the FBU was taking an average of 153 days to handle, screen, or analyze a forensic sample from submission to final report and there was a backlog of 679 cases. From 2016 to 2018, the total average turnaround time for BPL decreased from 30.5 to 19.6 days, (35.8% decrease); and the FBU Request turnaround time decreased from 153 to 80 days (35% decrease). Monitoring laboratory metrics demonstrate the efficacy of the DNA backlog reduction strategy. There are several takeaway lessons from this experience, including (1) engaging legal counsel early to outline necessary legal procedures and the timeline; (2) bringing all stakeholders "to the table" early to discuss expectations, as well as legal and operational responsibilities; and (3) creating a realistic timeline as well as establishing a comprehensive memorandum of understanding by which all parties understand their roles and responsibilities. Understanding laboratory and non-laboratory policy issues is critical to implementation success and the efficacy of a BPL as a DNA backlog reduction strategy.

Combined DNA Index System (CODIS)-Based analysis of untested sexual assault evidence in Palm Beach County Florida.

In August 2015, the Palm Beach County Sheriff's Office (PBSO) initiated a CODIS-Based Sexual Assault Evidence Testing Initiative, hereto referred to as the Initiative, as a direct result of the ongoing national inquiry about the number of untested sexual assault cases having DNA evidence stored in law enforcement agency vaults. The Initiative was designed to research sexual assault related evidence including the estimated 1800 untested sexual assault kits stored in the custody of Palm Beach County Law Enforcement Agencies to determine if probative evidence was available, conduct DNA testing on the evidence, and enter all eligible DNA profiles into the CODIS database. Between December 2015 and July 2018 more than 5500 cases were researched and evaluated resulting in evidence from 1,558 cases spanning a 43-year period being tested at a cost of $1,032,496. Nearly 44% of all cases tested provided CODIS-eligible profiles and 36% of those eligible profiles generated a CODIS hit. The positive impact of this Initiative, while already realized by Initiative-related arrests, will continue to grow as additional CODIS hits are generated and investigated by PBSO law enforcement partners. Ultimately, the Initiative will provide information to criminal investigators and the courts including exonerating the innocent, bringing closure to victims, and helping convict the true perpetrators.

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.

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.

Characterization and validation studies of powerPlex 2.1, a nine-locus short tandem repeat (STR) multiplex system and penta D monoplex.

In order to increase the power of discrimination for human identification purposes, a nine-locus short tandem repeat (STR) multiplex, the GenePrint PowerPlex 2.1 system (PowerPlex 2.1) developed by Promega Corporation and a separate pentanucleotide-repeat locus, Penta D, were tested. This megaplex system includes the highly polymorphic loci FGA, TPOX, D8S1179, vWA, Penta E, D18S51, D21S11, TH01, and D3S1358 and may be used in combination with the eight-locus STR multiplex, the GenePrint PowerPlex 1.1 system (PowerPlex 1.1) that has been previously developed. Three of the loci, TPOX, TH01 and vWA, have been included in both systems for quality control purposes. As with PowerPlex 1.1, PowerPlex 2.1 is also based on a two-color detection of fluorescent-labeled DNA products amplified by polymerase chain reaction (PCR) and provides a valuable tool for accurate and rapid allele determination. The primer sequences used in the PowerPlex 2.1/Penta D system are also presented in this report. To meet the "Quality Assurance Standards for Forensic DNA Testing Laboratories" (FBI), we tested the efficiency and reproducibility of the PowerPlex 2.1/PentaD system by several validation studies that were conducted as a joint project among seven laboratories. Validation tests included concordance studies, sensitivity, and species specificity determination, as well as performance in forensic and environmentally impacted samples. The results produced from these tests demonstrated the consistency and reliability of the PowerPlex 2.1/Penta D system.

Assessing a novel room temperature DNA storage medium for forensic biological samples.

The ability to properly collect, analyze and preserve biological stains is important to preserving the integrity of forensic evidence. Stabilization of intact biological evidence in cells and the DNA extracts from them is particularly important since testing is generally not performed immediately following collection. Furthermore, retesting of stored DNA samples may be needed in casework for replicate testing, confirmation of results, and to accommodate future testing with new technologies. A novel room temperature DNA storage medium, SampleMatrix™ (SM; Biomatrica, Inc., San Diego, CA), was evaluated for stabilizing and protecting samples. Human genomic DNA samples at varying amounts (0.0625-200 ng) were stored dry in SM for 1 day to 1 year under varying conditions that included a typical ambient laboratory environment and also through successive freeze-thaw cycles (3 cycles). In addition, spiking of 1-4 × SM into samples prior to analysis was performed to determine any inhibitory effects of SM. Quantification of recovered DNA following storage was determined by quantitative PCR or by agarose gel electrophoresis, and evaluation of quantitative peak height results from multiplex short tandem repeat (STR) analyses were performed to assess the efficacy of SM for preserving DNA. Results indicate no substantial differences between the quality of samples stored frozen in liquid and those samples maintained dry at ambient temperatures protected in SM. For long-term storage and the storage of low concentration samples, SM provided a significant advantage over freezer storage through higher DNA recovery. No detectable inhibition of amplification was observed at the recommended SM concentration and complete profiles were obtained from genomic DNA samples even in the presence of higher than recommended concentrations of the SM storage medium. The ability to stabilize and protect DNA from degradation at ambient temperatures for extended time periods could have tremendous impact in simplifying and improving sample storage conditions and requirements. The current work focuses on forensics analysis; however this technology is applicable to all endeavors requiring storage of DNA.

Real-time forensic DNA analysis at a crime scene using a portable microchip analyzer.

An integrated lab-on-a-chip system has been developed and successfully utilized for real-time forensic short tandem repeat (STR) analysis. The microdevice comprises a 160-nL polymerase chain reaction reactor with an on-chip heater and a temperature sensor for thermal cycling, microvalves for fluidic manipulation, a co-injector for sizing standard injection, and a 7-cm-long separation channel for capillary electrophoretic analysis. A 9-plex autosomal STR typing system consisting of amelogenin and eight combined DNA index system (CODIS) core STR loci has been constructed and optimized for this real-time human identification study. Reproducible STR profiles of control DNA samples are obtained in 2h and 30min with

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.

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.

Improving efficiency of a small forensic DNA laboratory: validation of robotic assays and evaluation of microcapillary array device.

AIM: To present validation studies performed for the implementation of existing and new technologies to increase the efficiency in the forensic DNA Section of the Palm Beach County Sheriff's Office (PBSO) Crime Laboratory. METHODS: Using federally funded grants, internal support, and an external Process Mapping Team, the PBSO collaborated with forensic vendors, universities, and other forensic laboratories to enhance DNA testing procedures, including validation of the DNA IQ magnetic bead extraction system, robotic DNA extraction using the BioMek2000, the ABI7000 Sequence Detection System, and is currently evaluating a micro Capillary Array Electrophoresis device. RESULTS: The PBSO successfully validated and implemented both manual and automated Promega DNA IQ magnetic bead extractions system, which have increased DNA profile results from samples with low DNA template concentrations. The Beckman BioMek2000 DNA robotic workstation has been validated for blood, tissue, bone, hair, epithelial cells (touch evidence), and mixed stains such as semen. There has been a dramatic increase in the number of samples tested per case since implementation of the robotic extraction protocols. The validation of the ABI7000 real-time quantitative polymerase chain reaction (qPCR) technology and the single multiplex short tandem repeat (STR) PowerPlex16 BIO amplification system has provided both a time and a financial benefit. In addition, the qPCR system allows more accurate DNA concentration data and the PowerPlex 16 BIO multiplex generates DNA profiles data in half the time when compared to PowerPlex1.1 and PowerPlex2.1 STR systems. The PBSO's future efficiency requirements are being addressed through collaboration with the University of California at Berkeley and the Virginia Division of Forensic Science to validate microcapillary array electrophoresis instrumentation. Initial data demonstrated the electrophoresis of 96 samples in less than twenty minutes. CONCLUSION: The PBSO demonstrated, through the validation of more efficient extraction and quantification technology, an increase in the number of evidence samples tested using robotic/DNA IQ magnetic bead DNA extraction, a decrease in the number of negative samples amplified due to qPCR and implementation of a single multiplex amplification system. In addition, initial studies show the microcapillary array electrophoresis device (microCAE) evaluation results provide greater sensitivity and faster STR analysis output than current platforms.