Investigative Genetics

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    Massively parallel sequencing of 68 insertion/deletion markers identifies novel sequence variation for utility in human identity testing
    (2016-03-23) Song, Bing; Thompson, Lindsey; King, Jonathan; Budowle, Bruce; LaRue, Bobby; Wendt, Frank
    Short tandem repeat (STR) loci are traditionally used by the forensic science community for kinship, missing persons, and human identity testing. These markers hold considerable value due to their size, ability to be multiplexed, and highly polymorphic nature. However, they are unable to provide phenotypic and biogeographic ancestry estimates and are too large for use in analysis of DNA from highly compromised substrates such as explosives or human remains. Small bi-allelic polymorphisms, such as insertions/deletions (INDELs), have been of considerable interest within the forensic science community for their utility in filling such gaps. These markers range in size from 2-6 base pairs, making them ideal for highly compromised sample types. Additionally, the ease of multiplexing large INDEL panels allows for comparable discrimination power when compared to STRs. Capillary electrophoresis is a current mainstay in the forensic DNA workflow, generating fluorescent signals to detect alleles separated by size. This method is limited by number of dyes simultaneously utilized, number of loci capable of multiplexing, sample throughput, and required amplicon size. Massively parallel sequencing (MPS) provides a solution to these limitations by targeting many loci across the genomes of multiple samples simultaneously with relatively high sequence coverage. Herein, we describe the utility of MPS, using the Nextera™ Rapid Capture Custom Enrichment Kit (Illumina, Inc., San Diego, CA), to sequence 68 INDELs in four major US population groups on the Illumina MiSeq™. We also define a novel application of the STR Allele Identification Tool: Razor (STRait Razor) to analyze INDEL sequences and capture adjacent sequence variation in the form of single nucleotide polymorphisms (SNPs). This application has enabled the discovery of unique allelic variants, which increase the discrimination power and decrease the single-locus and combined random match probabilities of four well-characterized INDELs. These findings suggest that more valuable INDELs for human identification may exist elsewhere in the genome. As such, it is recommended that these four markers be included in future INDEL multiplex panels for human identification due to their enhanced individualization potential.
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    Effect of Detergent Selection on Quantity of DNA Extracted and on STR Profile Developed from Bone-Derived DNA
    (2016-03-23) Gaydosh Combs, Laura Ph.D.; Sun, Jie B.S.; Wendt, Frank B.S.; Warren, Joseph Ph.D.; Proctor, F. B.S.
    DNA analysis is often essential to make positive associations in cases of unidentified persons, missing persons, and mass fatality incidents. In cases such as these, human skeletal remains are frequently the only source of genetic material available, but bone-derived DNA characteristically provides lower quantities of DNA and lower quality short tandem repeat (STR) profiles than that of other sample types. Conventional DNA extraction methods were developed based on the biochemical composition of soft tissues or body fluids. DNA from these unmineralized sample types is more readily extracted and contains fewer inhibitors than DNA found in bone; furthermore, skeletal remains encountered in casework may be aged or subjected to environmental factors that reduce the quality of DNA obtained. These sample-specific issues support the development of specialized extraction techniques for bone in order to obtain the highest quantities of DNA and improved quality STR profiles. Prior work has demonstrated that increased DNA quantities are obtained from human skeletal remains when using Buffer ATL in conjunction with Collagenase Type II (CLSII) enzyme. It has also been determined that metals, particularly calcium, copurify with DNA when processing bone samples. These copurified metals have been shown to inhibit PCR amplification of STR markers. Building upon these findings, a protocol was designed to determine whether use of a detergent other than Buffer ATL would continue to improve upon current methods for DNA extraction from human bone. An unembalmed human cadaver diaphysis was obtained through the Willed Body Program of the University of North Texas Health Science Center. DNA was purified on the EZ1® Advanced XL System (Qiagen®, Hilden, Germany) after employing the modified digestion step. The DNA isolates were quantified using the Investigator® Quantiplex HYres Kit (Qiagen®), then STR markers were amplified using the Investigator® 24Plex QS Kit (Qiagen®) and fragment analysis performed with the 3500xL Genetic Analyzer (Thermo Fisher Scientific, Inc., Carlsbad, CA). STR profiles were assessed using GeneMapper® ID-X v1.4 (Thermo Fisher Scientific). All Real-Time PCR and electropherogram data were analyzed using Microsoft® Excel (Microsoft® Corp., Redmond, WA) and RStudio® (RStudio® Inc., Boston, MA). Though Buffer ATL yielded significantly higher quantities of DNA per milligram of bone, results indicate that using an increased strength anionic detergent, such as SDS or SLS, in conjunction with CLSII enzyme will improve the quality of STR profiles produced from human skeletal remains. Full profiles were recovered for all concentrations of SDS and SLS, while allelic drop out was observed for Buffer ATL and Triton™ X-100. Mean peak heights of profiles produced using all concentrations of SDS and SLS represented a quantitative improvement over both Buffer ATL and Triton™ X-100. Mean peak height ratios of profiles produced using all concentrations of SDS and SLS also represented a qualitative improvement compared to samples digested using CLSII enzyme with Buffer ATL or Triton™ X-100. Unlike SDS, there are no special considerations for storage or handling of SLS detergent solutions, making it an excellent choice for use in forensic laboratories. Use of SLS consistently produces sufficient quantities of DNA and full STR profiles at all concentrations tested, and as no significant differences were observed between concentrations of SLS, the lowest tested concentration of 1% should be employed in order to conserve resources.
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    A Novel Multiplex Assay for an Ancestry-Informative Marker (AIM) Panel of INDELs
    (2016-03-23) Sage, Kelly; LaRue, Bobby; King, Jonathan; Sturm, Sarah
    The current standard for forensic laboratories in criminal casework is to use Short Tandem Repeat (STR) markers to develop an evidentiary profile to compare with a reference profile. Commercially available STR amplification kits yield amplicons 100 to 500 base pairs (bp) in length. A common problem encountered by scientists is degraded DNA samples that are only 180-200 bps in length. These samples fail to amplify some loci and therefore produce an incomplete STR profile. STRs are used for identity testing because of their high discrimination power. However, there are cases where no STR match was obtained through a DNA database search and thus no investigative lead is obtained. The bioancestry of the donor of the sample could aid law enforcement in such cases. Another class of markers that could provide investigative value from degraded DNA samples is Ancestry-Informative Marker (AIM) Insertion/Deletions (INDELs). INDELs are polymorphisms that can be amplified from degraded samples due to their smaller amplicon size. AIMs have the ability provide bioancestry information. This project used a previously developed panel of AIM-INDEL markers to develop a multiplex PCR-based assay specifically for these identity-testing applications.
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    Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assualt Kit Swabs
    (2016-03-23) Warren, Joseph; Capt, Christina; Sun, Jie; Proctor, F. B.S.; Brignac, Francine J.
    Sexual assault kits comprise 40-50% of a typical Forensic Laboratory caseload. The traditional method to process these samples is time-consuming, and requires the use a dangerous chemical known as Phenol:Chloroform:Isoamyl Alcohol (PCIA). The purpose of this study is to assess the relative efficacy of the PCIA method when compared to three other currently available differential extraction methods. A single male volunteer and a single female volunteer donated semen and saliva, respectively. Aliquots of semen were serially diluted such that three decreasing concentrations of semen could be assessed alongside a consistent concentration of saliva. From these three different mixtures, swabs were made and allowed to dry in a 37 °C drying oven for two weeks, then at room temperature for an additional four weeks in order to simulate aged samples. Three days prior to DNA extraction and purification, another set of swabs were created to simulate fresh samples. The aged and unaged samples were tested in triplicate for each of the four extraction methods. The methods to be compared include two manual and two automated methods. The manual methods include the standard differential (SD) and the Lounsbury Method, which is a modified version of the SD. The two automated methods include the AutoMate ExpressTM DNA Extraction System (ThermoFisher Scientific, Carlsbad, CA), and a method employing the use of two of Qiagen’s DNA platforms: the QIAcube and the Qiagen EZ1® Advanced XL (Qiagen®, Hilden, Germany). Results indicate that as sperm sample concentration decreases, automated methods produce superior results both in DNA quantity obtained and in quality of STR profiles produced. Automated methods reduce hands-on time, facilitate higher through-put of samples, and reduce analyst contact with hazardous chemicals such as PCIA, making it an all around great choice for labs. All Real-Time PCR and electropherogram data were analyzed using Microsoft® Excel (Microsoft® Corp., Redmond, WA), and RStudio® (RStudio® Inc., Boston, MA).
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    Multiplex of INDELs for Human Identification Markers
    (2016-03-23) Sturm, Sarah; Thompson, Lindsey; Wiley, Rachel; King, Jonathan; LaRue, Bobby; Sage, Kelly
    Forensic laboratories commonly use short tandem repeat (STR) loci when comparing an evidentiary profile to that of a reference profile. In commercially available STR kits, the amplified products tend to range from 100- 500 base pairs (bp). For genomic DNA of degraded biological samples, the fragments are usually broken into product sizes of 180-200bps or less. Therefore, degraded biological samples may not produce a full STR profile. Another viable option has been proposed to enable successful typing of some degraded DNA samples. Insertion/ deletion (INDEL) polymorphisms are intergenic regions of the genome in which amplified products can be smaller in length than most STRs. Since forensic genotyping relies on comparison of an evidentiary sample DNA profile with that of a reference sample DNA profile, which usually come from suspects or victims, using highly discriminating markers is desirable. A multiplex panel of human identification (HID) INDEL markers that can individualize people would be beneficial. This project will test the hypothesis that INDELs, which can be used to identify individuals with high discriminatory power, can be developed as a multiplex PCR approach. In testing this hypothesis, primers were designed and multiplexed together to amplify specific INDELs that have been previously identified to be suitable for human identity testing purposes.
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    An evaluation of the RapidHIT® ID system for field forward applications
    (2016-03-23) Sage, Kelly; Budowle, Bruce; LaRue, Bobby; Wiley, Rachel
    The utilization of a new Rapid DNA (RDNA) platform to generate CODIS uploadable DNA profiles will serve to be instrumental in improving current DNA typing techniques and in reducing the backlog of forensic reference samples. The RapidHIT® ID (IntegenX; Pleasanton, CA) system is a second generation system in RDNA that has the potential to yield comparable DNA profiles to those achieved by traditional bench methods. The RapidHIT® ID platform is a self-contained, fully-automated, sample-to-profile system with a novel construction designed to reduce it’s footprint as well as the number of samples necessary to be run at a single time, making it condusive to both laboratory and field work application. The RapidHIT® ID system has the capacity to perform direct amplification, electrophoresis, and data analysis in approximately 90 minutes with nominal “hands-on” assistance required. Reliable DNA STR profiles have been generated from reference buccal swabs. The RapidHIT® ID platform was evaluated for concordance, reproducibility, and lack of contamination. Sensitivity and interpretation thresholds were established, and although the system was designed for reference buccal swabs, additional studies evaluating the effects of sample age, inhibitors, and sample collection methods were performed. This new instrumentation provided DNA STR profiles comparable to those obtained from traditionalDNA genotyping methodologies, in addition to complete or partial profiles from the sensitivity studies. Based on preliminary studies, the RapidHIT® ID system is a new RDNA platform that is robust and reliable for generating STR profiles from forensic reference samples.
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    Million Veteran Program (MVP)
    (2016-03-23) Rastogi, Padmashri; Reeves, Rustin; Bhat, Nikhil U.
    Purpose: The Million Veteran Program (MVP) is a national, voluntary research study conducted by the Department of Veterans Affairs Office of Research & Development. It is collaboration between the VA and veterans, whose goal is to illuminate potential links between genetic heterogeneity and disease. This is an important step in our scientific understanding about how genetic, as well as epigenetic makeup impinges upon disease characteristics and drug efficacy. Methods: Veterans who are treated in Veteran Affairs are eligible to participate. Those who provide consent are contacted by one of the researcher in the group. Veteran fills the survey related to their health and consent to give a blood sample. Central MVP biorepository saves the sample. Each sample is coded and so is their corresponding health information. Neither the person in the lab nor in the analysis knows the identity of the veteran. The key to the code is known to only a few personnel who are highly trained in research ethics thus safeguarding the privacy of the veterans. Results: Though the collection of data is ongoing, based on the analysis done so far, the correlation found between genetic and phenotypic pattern is helping to improve current treatment for certain cancers. Strong links that will very likely be found in this study, due to the large number of participating veterans (1 million), will be used to generate testable hypotheses for future study, such as if a particular gene polymorphism or epigenetic mark leads to a particular disease trait. This will enhance our understanding about how to better prevent and treat various diseases such as heart disease, diabetes, cancer, and post-traumatic stress disorder. Conclusions: Our site has contributed actively in the recruitment of veterans for this study by enrolling approximately 7000 veterans till now. With the help of research program, at our institution, we continue to work towards achieving our target. All over US, more than 450,000 patients have been enrolled in MVP. The research on the 400,000 samples has helped us discover a couple of useful drugs for cancer and schizophrenia. MVP aims to establish the largest of its kind database in the United States.