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Micromanipulation of single cells and fingerprints for forensic identification

Ostojic, Lana; O'Connor, Craig; Wurmbach, Elisa
Crime scene samples often include biological stains, handled items, or worn clothes and may contain cells from various donors. Applying routine sample collection methods by using a portion of a biological stain or swabbing the entire suspected touched area of the evidence followed by DNA extraction often leads to DNA mixtures. Some mixtures can be addressed with sophisticated interpretation protocols and probabilistic genotyping software resulting in DNA profiles of their contributors. However, many samples remain unresolved, providing no investigative information. Samples with many contributors are often the most challenging samples in forensic biology. Examples include gang rape situations or where the perpetrator's DNA is present in traces among the overwhelming amounts of the victim's DNA. If this is the only available evidence in a case, it is of paramount importance to generate usable information. An alternative approach, to address biological mixtures, could be the collection of individual cells directly from the evidence and testing them separately. This method could prevent cells from being inadvertently blended during the extraction process, thus resulting in DNA mixtures. In this study, multiple tools coupled with adhesive microcarriers to collect single cells were evaluated. These were tested on epithelial (buccal) and sperm cells, as well as on touched items. Single cells were successfully collected but fingerprints were swabbed in their entirety to account for the extracellular DNA of these samples and the poor DNA quality of shed skin flakes. Furthermore, micromanipulation devices, such as the P.A.L.M.® and the Axio Zoom.V16 operated manually or with a robotic arm aureka®, were compared for their effectiveness in collecting cells. The P.A.L.M.® was suitable for single cell isolation when smeared on membrane slides. Manual or robotic manipulations, by utilizing the Axio Zoom.V16, have wider applications as they can be used to isolate cells from various substrates such as glass or membrane slides, tapes, or directly from the evidence. Manipulations using the Axio Zoom.V16, either with the robotic arm aureka® or manually, generated similar outcomes which were significantly better than the outcomes by using the P.A.L.M.®. Robotic manipulations using the aureka® produced more consistent results, but operating the aureka® required training and often needed re-calibrations. This made the process of cell manipulations slower than when manually operated. Our preferred method was the manual manipulations as it was fast, cost effective, required little training, but relied on a steady hand of the technician.
PMID: 33260060
ISSN: 1878-0326
CID: 4694122

Analyzing degraded DNA and challenging samples using the ForenSeqâ„¢ DNA Signature Prep kit

Sharma, Vishakha; van der Plaat, Diana A; Liu, Yuexun; Wurmbach, Elisa
Typing short tandem repeats (STRs) is the basis for human identification in current forensic testing. The standard method uses capillary electrophoresis (CE) to separate amplicons by length and fluorescent labeling. In recent years new methods, including massively parallel sequencing (MPS), have been developed which increased the discriminative power of STRs through sequencing. MPS also offers the opportunity to test more genetic markers in a run than is possible with standard CE technology. Verogen's ForenSeq™ DNA Signature Prep kit includes over 150 genetic markers [STRs and single nucleotide polymorphisms (SNPs)]. Further, MPS separation depends on sequences rather than lengths; therefore, amplicons can be small or even of the same lengths. These improvements are advantageous when testing challenging forensic samples that could be severely degraded. This study tested the ForenSeq™ DNA Signature Prep kit in repeated experimental runs on series of degraded DNA samples, ranging from mild to severe degradation, as well as 24 mock case-type samples, derived from bones, blood cards, and teeth. Despite passing the quality metrics, positive controls (2800 M) showed drop-outs at some loci, mostly SNPs. Sequencing DNA samples repeatedly in two experimental runs as well as sequencing one pooled library in triplicate led to the assumption that spurious alleles of the Y-STRs in this study were not a result of sequencing artifacts but could be due to sequence structures (e.g. duplications, palindromes) of the Y-chromosome and/or might be accumulated during library preparation. Two sets of serially degraded DNA samples revealed that dropped-out loci were primarily loci with long amplicons as well as low read numbers (coverage), e.g. PentaE, DXS8378, and rs1736442. STRs started to drop out at degradation indices (DIs) > 4. However, severely degraded DNA (DI: 44) still resulted in 90% of the 20 CODIS loci, while only 35% were obtained using Promega's PowerPlex® Fusion kit, a current standard CE kit. Mock case-type samples confirmed these results. ForenSeq™ DNA Signature Prep kit demonstrated that it can be successfully used on degraded DNA samples. This study may be helpful for other laboratories assessing and validating MPS technologies.
PMID: 32381241
ISSN: 1876-4452
CID: 4430512

Evaluation of ForenSeqâ„¢ Signature Prep Kit B on predicting eye and hair coloration as well as biogeographical ancestry by using Universal Analysis Software (UAS) and available web-tools

Sharma, Vishakha; Jani, Krupa; Khosla, Pavan; Butler, Erin; Siegel, Donald; Wurmbach, Elisa
This study examined 266 individuals from various populations including African American, East Asian, South Asian, European, and mixed populations to evaluate the ForenSeqâ„¢ Signature Prep Kit Primer Mix B. Focus was placed on phenotypic and biogeographical ancestry predictions by Illumina's Universal Analysis Software (UAS). These outcomes were compared to those obtained through web-tools developed at the Erasmus Medical Center (EMC) and available from the Forensic Resource/Reference on Genetics-knowledge base (FROG-kb), as well as to eye color predictions by the 8-plex system. Due to drop-outs, predictions for eye and hair color by UAS failed for various samples in each run. By including reads below thresholds, predictions could be obtained for all samples through the web-tools. Eye and hair color predictions for African Americans, East Asians, and South Asians showed no errors. Difficulties however, were noted in intermediate (neither blue nor brown) eye color predictions. These were mitigated by the 8-plex system through exclusion of one eye color (e.g. "not brown"). Additionally, notable discrepancies were observed in hair color predictions, where some black/dark-brown haired individuals were predicted to have blond hair. Overall, ancestry predictions were more accurate by FROG-kb compared to UAS, which did not predict South Asian ancestry, particularly Indian individuals.
PMID: 30767247
ISSN: 1522-2683
CID: 3685302

Systematic assessment of the performance of illumina's MiSeq FGxâ„¢ forensic genomics system

Almalki, Nouran; Chow, Hoi Yan; Sharma, Vishakha; Hart, Katie; Siegel, Donald; Wurmbach, Elisa
This study assesses the performance of Illumina's MiSeq FGx System for forensic genomics by systematically analyzing single source samples, evaluating concordance, sensitivity and repeatability, as well as describing the quality of the reported outcomes. DNA from 16 individuals (9 males/7 females) in nine separate runs showed consistent STR profiles at DNA input ≥400 pg, and two full profiles were obtained with 50 pg DNA input. However, this study revealed that the outcome of a single sample does not merely depend on its DNA input but is also influenced by the total amount of DNA loaded onto the flow cell from all samples. Stutter and sequence or amplification errors can make the identification of true alleles difficult, particularly for heterozygous loci that show allele imbalance. Sequencing of 16 individuals' STRs revealed genetic variations at 14 loci at frequencies suggesting improvement of mixture deconvolution. The STR loci D1S1656 and DXS10103 were most susceptible to drop outs, and D22S1045 and DYS385a-b showed heterozygote imbalance.  Most stutters were typed at TH01 and DYS385a-b, while amplification or sequencing errors were observed mostly at D7S820 and D19S433. Overall, Illumina's MiSeq FGx System produced reliable and repeatable results.  aSTRs showed fewer drop outs than the Y- and X-STRs.
PMID: 27943350
ISSN: 1522-2683
CID: 3095252

Analysis of fingerprint samples, testing various conditions, for forensic DNA identification

Ostojic, Lana; Wurmbach, Elisa
Fingerprints can be of tremendous value for forensic biology, since they can be collected from a wide variety of evident types, such as handles of weapons, tools collected in criminal cases, and objects with no apparent staining. DNA obtained from fingerprints varies greatly in quality and quantity, which ultimately affects the quality of the resulting STR profiles. Additional difficulties can arise when fingerprint samples show mixed STR profiles due to the handling of multiple persons. After applying a tested protocol for sample collection (swabbing with 5% Triton X-100), DNA extraction (using an enzyme that works at elevated temperatures), and PCR amplification (AmpFlSTR® Identifiler® using 31cycles) extensive analysis was performed to better understand the challenges inherent to fingerprint samples, with the ultimate goal of developing valuable profiles (≥50% complete). The impact of time on deposited fingerprints was investigated, revealing that while the quality of profiles deteriorated, full STR profiles could still be obtained from samples after 40days of storage at room temperature. By comparing the STR profiles from fingerprints of the dominant versus the non-dominant hand, we found a slightly better quality from the non-dominant hand, which was not always significant. Substrates seem to have greater effects on fingerprints. Tests on glass, plastic, paper and metal (US Quarter dollar, made of Cu and Ni), common substrates in offices and homes, showed best results for glass, followed by plastic and paper, while almost no profiles were obtained from a Quarter dollar. Important for forensic casework, we also assessed three-person mixtures of touched fingerprint samples. Unlike routinely used approaches for sampling evidence, the surface of an object (bottle) was sectioned into six equal parts and separate samples were taken from each section. The samples were processed separately for DNA extraction and STR amplification. The results included a few single source profiles and distinguishable two person mixtures. On average, this approach led to two profiles ≥50% complete per touched object. Some STR profiles were obtained more than once thereby increasing the confidence.
PMID: 28063583
ISSN: 1355-0306
CID: 2943602

Qualitative and quantitative assessment of Illumina's forensic STR and SNP kits on MiSeq FGxâ„¢

Sharma, Vishakha; Chow, Hoi Yan; Siegel, Donald; Wurmbach, Elisa
Massively parallel sequencing (MPS) is a powerful tool transforming DNA analysis in multiple fields ranging from medicine, to environmental science, to evolutionary biology. In forensic applications, MPS offers the ability to significantly increase the discriminatory power of human identification as well as aid in mixture deconvolution. However, before the benefits of any new technology can be employed, a thorough evaluation of its quality, consistency, sensitivity, and specificity must be rigorously evaluated in order to gain a detailed understanding of the technique including sources of error, error rates, and other restrictions/limitations. This extensive study assessed the performance of Illumina's MiSeq FGx MPS system and ForenSeqâ„¢ kit in nine experimental runs including 314 reaction samples. In-depth data analysis evaluated the consequences of different assay conditions on test results. Variables included: sample numbers per run, targets per run, DNA input per sample, and replications. Results are presented as heat maps revealing patterns for each locus. Data analysis focused on read numbers (allele coverage), drop-outs, drop-ins, and sequence analysis. The study revealed that loci with high read numbers performed better and resulted in fewer drop-outs and well balanced heterozygous alleles. Several loci were prone to drop-outs which led to falsely typed homozygotes and therefore to genotype errors. Sequence analysis of allele drop-in typically revealed a single nucleotide change (deletion, insertion, or substitution). Analyses of sequences, no template controls, and spurious alleles suggest no contamination during library preparation, pooling, and sequencing, but indicate that sequencing or PCR errors may have occurred due to DNA polymerase infidelities. Finally, we found utilizing Illumina's FGx System at recommended conditions does not guarantee 100% outcomes for all samples tested, including the positive control, and required manual editing due to low read numbers and/or allele drop-in. These findings are important for progressing towards implementation of MPS in forensic DNA testing.
PMID: 29121662
ISSN: 1932-6203
CID: 3065002

Assay Development and Validation of an 8-SNP Multiplex Test to Predict Eye and Skin Coloration

Mushailov, Vladimir; Rodriguez, Stephanie A; Budimlija, Zoran M; Prinz, Mechthild; Wurmbach, Elisa
Identifying human remains is one of the many responsibilities of forensic scientists. An eye- and skin-color predictor translates genotypic information into phenotypic description. Eight single nucleotide polymorphisms (SNPs) are utilized for this predictor, five for eye, and six for skin coloration. Here, we describe the development and validation of an 8-SNP multiplex assay that consists of a multiplex PCR, followed by a multiplexed single-base primer extension reaction generating fluorescently labeled oligonucleotides of distinct length that are detected by multicolor capillary electrophoresis. Validation of this assay included tests for reproducibility, reliability, sensitivity, species specificity, its performance on degraded DNA, and on forensic samples. It can be concluded that the 8-SNP multiplex assay is robust and can be used on challenging samples, including bones, to reliably determine the genotypes to predict eye and skin color of individuals. This information can assist in the identification of human remains and missing persons.
PMID: 25782558
ISSN: 0022-1198
CID: 1506132

Deletion mapping in the Enhancer of split complex

Wurmbach, Elisa; Preiss, Anette
The Enhancer of split complex [E(spl)-C] comprises twelve genes of different classes. Seven genes encode proteins of with a basic-helix-loop-helix-orange (bHLH-O) domain that function as transcriptional repressors and serve as effectors of the Notch signalling pathway. They have been named E(spl)m8-, m7-, m5-, m3-, mbeta-, mgamma- and mdelta-HLH. Four genes, E(spl)m6-, m4-, m2- and malpha-BFM are intermingled and encode Notch repressor proteins of the Bearded-family (BFM). The complex is split by a single gene of unrelated function, encoding a Kazal-type protease inhibitor (Kaz-m1). All members within a family, bHLH-O or BFM, are very similar in structure and in function. In an attempt to generate specific mutants, we have mobilised P-element constructs residing next to E(spl)m7-HLH and E(spl)mgamma-HLH, respectively. The resulting deletions were mapped molecularly and by cytology. Two small deletions affected only E(spl)m7-HLH and E(spl)mdelta. The deficient flies were viable without apparent phenotype. Larger deletions, generated also by X-ray mutagenesis, uncover most of the E(spl)-C. The phenotypes of homozygous deficient embryos were analysed to characterize the respective loss of Notch signalling activity.
PMID: 25588303
ISSN: 0018-0661
CID: 1436352

Qualitative and quantitative assessment of single fingerprints in forensic DNA analysis

Ostojic, Lana; Klempner, Stacey A; Patel, Rosni A; Mitchell, Adele A; Axler-DiPerte, Grace L; Wurmbach, Elisa
Fingerprints and touched items are important sources of DNA for STR profiling, since this evidence can be recovered in a wide variety of criminal offenses. However, there are some fundamental difficulties in working with these samples, including variability in quantity and quality of extracted DNA. In this study, we collected and analyzed over 700 fingerprints. We compared a commercially available extraction protocol (Zygem) to two methods developed in our laboratory, a simple one-tube protocol and a high sensitivity protocol (HighSens) that includes additional steps to concentrate and purify the DNA. The amplification protocols that were tested were AmpFLSTR(R) Identifiler(R) using either 28 or 31 amplification cycles, and Identifiler(R) Plus using 32 amplification cycles. We found that the HighSens and Zygem extraction methods were significantly better in their DNA yields than the one-tube method. Identifiler(R) Plus increased the quality of the STR profiles for the one-tube extraction significantly. However, this effect could not be verified for the other extraction methods. Furthermore, microscopic analysis of single fingerprints revealed that some individuals tended to shed more material than others onto glass slides. However, a dense deposition of skin flakes did not strongly correlate with a high quality STR profile
PMID: 25098234
ISSN: 0173-0835
CID: 1105452

Improved eye- and skin-color prediction based on 8 SNPs

Hart, Katie L; Kimura, Shey L; Mushailov, Vladimir; Budimlija, Zoran M; Prinz, Mechthild; Wurmbach, Elisa
AIM: To improve the 7-plex system to predict eye and skin color by increasing precision and detailed phenotypic descriptions. METHODS: Analysis of an eighth single nucleotide polymorphism (SNP), rs12896399 (SLC24A4), showed a statistically significant association with human eye color (P=0.007) but a rather poor strength of agreement (kappa=0.063). This SNP was added to the 7-plex system (rs12913832 at HERC2, rs1545397 at OCA2, rs16891982 at SLC45A2, rs1426654 at SLC24A5, rs885479 at MC1R, rs6119471 at ASIP, and rs12203592 at IRF4). Further, the instruction guidelines on the interpretation of genotypes were changed to create a new 8-plex system. This was based on the analysis of an 803-sample training set of various populations. The newly developed 8-plex system can predict the eye colors brown, green, and blue, and skin colors light, not dark, and not light. It is superior to the 7-plex system with its additional ability to predict blue eye and light skin color. RESULTS: The 8-plex system was tested on an additional 212 samples, the test set. Analysis showed that the number of positive descriptions for eye colors as being brown, green, or blue increased significantly (P=6.98e-15, z-score: -7.786). The error rate for eye-color prediction was low, at approximately 5%, while the skin color prediction showed no error in the test set (1% in training set). CONCLUSIONS: We can conclude that the new 8-plex system for the prediction of eye and skin color substantially enhances its former version.
PMID: 23771755
ISSN: 0353-9504
CID: 508882