Cluster-Enhanced Nanopore Sensing of Ovarian Cancer Marker Peptides in Urine.

The development of novel methodologies that can detect biomarkers from cancer or other diseases is both a challenge and a need for clinical applications. This partly motivates efforts related to nanopore-based peptide sensing. Recent work has focused on the use of gold nanoparticles for selective detection of cysteine-containing peptides. Specifically, tiopronin-capped gold nanoparticles, trapped in the cis-side of a wild-type α-hemolysin nanopore, provide a suitable anchor for the attachment of cysteine-containing peptides. It was recently shown that the attachment of these peptides onto a nanoparticle yields unique current signatures that can be used to identify the peptide. In this article, we apply this technique to the detection of ovarian cancer marker peptides ranging in length from 8 to 23 amino acid residues. It is found that sequence variability complicates the detection of low-molecular-weight peptides (<10 amino acid residues), but higher-molecular-weight peptides yield complex, high-frequency current fluctuations. These fluctuations are characterized with chi-squared and autocorrelation analyses that yield significantly improved selectivity when compared to traditional open-pore analysis. We demonstrate that the technique is capable of detecting the only two cysteine-containing peptides from LRG-1, an emerging protein biomarker, that are uniquely present in the urine of ovarian cancer patients. We further demonstrate the detection of one of these LRG-1 peptides spiked into a sample of human female urine.

Droplet-based optical trapping for cell separation in mock forensic samples.

Optical tweezers have a wide range of uses for mechanical manipulation of objects in the microscopic range. This includes both living and static cells in a variety of biomedical and research applications. Single-focus optical tweezers, formed by focusing a laser beam through a high numerical aperture immersion objective, create a significant force, which enables controlled transport of a variety of different cell types and morphologies in three dimensions. Optical tweezers have been previously reported to capture and separate spermatozoa from a reconstituted simulated postcoital sample. We report herein the development of a simplified, more efficient cell transfer protocol that can separate and isolate both spermatozoa as well as leukocytes, with similar efficiencies as those previously reported. The new cell transfer method was used to separate sperm cells from a reconstituted mixture of spermatozoa and vaginal epithelial cells, with complete STR profiles developed from 50 cells with little evidence of contribution from the female contributor to the mixture. This modified protocol was then used to separate 21 samples of enriched leukocytes, with trapped cells ranging from 5 to 22 cells. Complete STR profiles were developed from as few as 10 leukocytes. Thus, with minimal sample preparation and a short trapping time, this method has the potential to provide an alternative to traditional differential extraction methods for separation of sperm:nonsperm mixtures while also providing versatility for separation of cells with differing morphologies.

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids.

Body fluid identification is an essential step in the forensic biology workflow that can assist DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the specificity that molecular techniques can achieve; therefore, molecular methods, including microRNA (miRNA) and microbial signature characterization, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase the discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance (blood and semen). This project synergized the benefits of microRNAs and microbial DNA to identify multiple body fluids using DNA extracts. A reverse transcription (RT)-qPCR duplex targeting miR-891a and let-7g was validated, and miR-891a differential expression was significantly different between blood and semen. The miRNA duplex was incorporated into a previously reported qPCR multiplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius to presumptively identify vaginal/menstrual secretions, feces, and saliva, respectively. The combined classification regression tree model resulted in the presumptive classification of five body fluids with 94.6% overall accuracy, now including blood and semen identification. These results provide proof of concept that microRNAs and microbial DNA can classify multiple body fluids simultaneously at the quantification step of the current forensic DNA workflow.

A quantifiler™ trio-based HRM screening assay for the accurate prediction of single source versus mixed biological samples.

At present, the forensic DNA workflow is not capable of providing information about the contributor status (single source vs. multiple contributors) of evidentiary samples prior to end-point analysis. This exacerbates the challenges inherent to mixtures and low-template DNA samples. If additional sample information could be provided earlier in the workflow, protocols could be implemented to mitigate these challenges. An integrated Quantiplex®- high resolution melt (HRM) assay was shown to be effective in distinguishing between single source and mixture DNA samples; however, integration of the HRM assay into a more commonly used chemistry would be beneficial to the practitioner community. Thus, the assay was redesigned as an integrated Quantifiler™ Trio-HRM assay, which included the identification of a new DNA-binding dye, an increased reaction volume, and the establishment of new data analysis and standard curve metrics for all targets. This redesigned assay produced quantification values and qualitative values that were comparable to those produced when the same samples were tested using the standard Quantifiler™ Trio chemistry and settings. Further, STR profiles generated with quantification values produced from the integrated Quantifiler™ Trio-HRM assay and standard Quantifiler™ Trio chemistry were complete and fully concordant. Most importantly, the integrated Quantifiler™ Trio-HRM assay was able to accurately predict whether a sample was single source or a mixture 79.2% of the time, demonstrating the potential of this approach. With the incorporation of an expanded training set for prediction modeling, and completion of critical developmental validation studies, this assay could prove useful to the forensic DNA practitioner community.

Evaluation and Verification of a microRNA Panel Using Quadratic Discriminant Analysis for the Classification of Human Body Fluids in DNA Extracts.

There is significant interest in the use of miRNA analysis for forensic body fluid identification. Demonstrated co-extraction and detection in DNA extracts could make the use of miRNAs a more streamlined molecular body fluid identification method than other RNA-based methods. We previously reported a reverse transcription-quantitative PCR (RT-qPCR) panel of eight miRNAs that classified venous and menstrual blood, feces, urine, saliva, semen, and vaginal secretions using a quadratic discriminant analysis (QDA) model with 93% accuracy in RNA extracts. Herein, miRNA expression in DNA extracts from 50 donors of each body fluid were tested using the model. Initially, a classification rate of 87% was obtained, which increased to 92% when three additional miRNAs were added. Body fluid identification was found to be reliable across population samples of mixed ages, ethnicities, and sex, with 72-98% of the unknown samples classifying correctly. The model was then tested against compromised samples and over biological cycles, where classification accuracy varied, depending on the body fluid. In conclusion, we demonstrated the ability to classify body fluids using miRNA expression from DNA extracts, eliminating the need for RNA extraction, greatly reducing evidentiary sample consumption and processing time in forensic laboratories, but acknowledge that compromised semen and saliva samples can fail to classify properly, and mixed sample classification remains untested and may have limitations.

A bacterial signature-based method for the identification of seven forensically relevant human body fluids.

Detection and identification of body fluids plays a crucial role in criminal investigation, as it provides information on the source of the DNA as well as corroborative evidence regarding the crime committed, scene, and/or association with persons of interest. Historically, forensic serological methods have been chemical, immunological, catalytic, spectroscopic, and/or microscopic in nature. However, most of these methods are presumptive, with few robust confirmatory exceptions. In recent years several new molecular methods (mRNA, miRNA, DNA methylation, etc.) have been proposed; although promising, these methods require high quality human DNA or RNA. Additional steps are required in RNA based methods. Additionally, RNA based methods cannot be used for old cases where only DNA extracts remain to sample from. In this study, a novel non-human DNA (microbiome) based method was developed for the identification of the majority of forensically relevant human biological samples. Eight hundred and twelve (n = 812) biological samples (semen, vaginal fluid, menstrual blood, saliva, feces, urine, and blood) were collected and preserved using methods commonly used in forensic laboratories for evidence collection. Variable region four (V4) of 16 S ribosomal DNA (16 S rDNA) was amplified using a dual-indexing strategy and then sequenced on the MiSeq FGx sequencing platform using the MiSeq Reagent Kit v2 (500 cycles) and following the manufacturer's protocol. Machine learning prediction models were used to assess the classification accuracy of the newly developed method. As there was no significant difference in bacterial communities between vaginal fluid, menstrual blood, and female urine, these were combined as female intimate samples. Except in urine, the bacterial structures associated with male and female body fluid samples were not significantly different from one another. The newly developed method accurately identified human body fluid samples with an overall accuracy of more than 88%. This newly developed bacterial signature-based method is fast (no additional steps are needed as the same DNA can be used for both body fluid identification and STR typing), efficient (consume less sample as a single test can identify all major body fluids), sensitive (needs only 5 pg of bacterial DNA), accurate, and can be easily added into a forensic high throughput sequencing (HTS) panel.

Developmental validation of a microRNA panel using quadratic discriminant analysis for the classification of seven forensically relevant body fluids.

Body fluid identification is an important step in the forensic DNA workflow, and more advanced methods, such as microRNA (miRNA) analysis, have been research topics within the community over the last few decades. We previously reported a reverse transcription-quantitative PCR (RT-qPCR) panel of eight miRNAs that could classify blood, menstrual secretions, feces, urine, saliva, semen, and vaginal secretions through analysis of differential gene expression. The purpose of this project was to evaluate this panel in a larger population size, develop a more statistically robust analysis method and perform a series of developmental validation studies. Each of the eight miRNA markers was analyzed in > 40 donors each of blood, menstrual secretions, feces, urine, saliva, semen, and vaginal secretions. A 10-fold cross-validated quadratic discriminant analysis (QDA) model yielded the highest classification accuracy of 93% after eliminating miR-26b and miR-1246 from the panel. Accuracy of body fluid predictions was between 84% and 100% when various population demographics and samples from the same donor over multiple time periods were evaluated, but the assay demonstrated limited scope and reduced accuracy when mixed body fluid samples were tested. Limit of detection was found to be less than 104 copies/µL across multiple commercially available RT-qPCR analysis methods. These data suggest that miR-200b, miR-320c, miR-10b, and miR-891a, when normalized to let-7 g and let-7i, can consistently and robustly classify blood, feces and urine, but additional work is important to improve classification of saliva, semen, and female intimate secretions before implementation in forensic casework.

Design and optimization of a 16S microbial qPCR multiplex for the presumptive identification of feces, saliva, vaginal and menstrual secretions.

Molecular methods for body fluid identification have been extensively researched in the forensic community over the last decade, mostly focusing on RNA-based methods. Microbial DNA analysis has long been used for forensic applications, such as postmortem interval estimations, but only recently has it been applied to body fluid identification. High-throughput sequencing of the 16S ribosomal RNA gene by previous research groups revealed that microbial signatures and abundances vary across human body fluids at the genus and/or species taxonomic level. Since quantitative PCR is still the current technique used in forensic DNA analysis, the purpose of this study was to design a qPCR multiplex targeting the 16S gene of Bacteroides uniformis, Streptococcus salivarius, and Lactobacillus crispatus that can distinguish between feces, saliva, and vaginal/menstrual secretions, respectively. Primers and probes were designed at the species level because these bacteria are highly abundant within their respective fluid. The validated 16S triplex was evaluated in DNA extracts from thirty donors of each body fluid. A classification regression tree model resulted in 96.5% classification accuracy of the population data, which demonstrates the ability of this 16S triplex to presumptively identify these fluids with high confidence at the quantification step of the forensic workflow using minimal input volume of DNA extracted from evidentiary samples.

Machine learning clustering and classification of human microbiome source body sites.

Distinct microbial signatures associated with specific human body sites can play a role in the identification of biological materials recovered from the crime scene, but at present, methods that have capability to predict origin of biological materials based on such signatures are limited. Metagenomic sequencing and machine learning (ML) offer a promising enhancement to current identification protocols. We use ML for forensic source body site identification using shotgun metagenomic sequenced data to verify the presence of microbiomic signatures capable of discriminating between source body sites and then show that accurate prediction is possible. The consistency between cluster membership and actual source body site (purity) exceeded 99% at the genus taxonomy using off-the-shelf ML clustering algorithms. Similar results were obtained at the family level. Accurate predictions were observed for genus, family, and order taxonomies, as well as with a core set of 51 genera. The accurate outcomes from our replicable process should encourage forensic scientists to seriously consider integrating ML predictors into their source body site identification protocols.

Comparison of DNA typing success in compromised blood and touch samples based on sampling swab composition.

Sample collection at the crime scene can introduce variations in DNA recovery based upon the substrate from which a sample is collected, the material of the collection device used, or the storage conditions after collection. There are many factors during this process that can degrade the sample during drying and storage, and before DNA extraction can be performed. The purpose of this study was to evaluate and compare the performance of standard cotton swab collection with the Bode BioSafe® swab, which includes both a desiccant at the swab head and proprietary compounds to prevent degradation of the sample during sample collection and preservation. Blood and touch DNA samples were collected from porous and nonporous substrates and stored at elevated temperatures to simulate accelerated time. DNA quantification and STR profile data were used to assess the performance of the swabs. BioSafe® swab collection resulted in similar DNA yields from blood samples and significantly higher DNA yields from touch samples when compared to collection with cotton swabs. BioSafe® swabs also resulted in higher DNA integrity during long-term storage, increased STR profile success and improved retention of low-level contributor alleles.

The effects of dithiothreitol (DTT) on fluorescent qPCR dyes.

DNA extractions of semen samples commonly utilize dithiothreitol (DTT) to reduce and disrupt disulfide bonds. Although traditional extraction techniques remove DTT before downstream analyses, the forensic DNA community has recently explored Y-screening, direct amplification, and direct cell lysis assays that omit purification but employ reducing agents to lyse spermatozoa. This study examined the impact of residual DTT on downstream processes involving fluorescent dyes. Quantification using Investigator® Quantiplex HYres revealed a significant increase in the male DNA yield (p = 0.00056) and a >150,000,000-fold increase in the male:human DNA ratio when DTT remained in extracts versus when it was filtered out using a traditional purification method. When DTT was present with Quantifiler™ Trio, the true mean DNA yield for the large autosomal target significantly increased (p = 0.038) and the average reported DNA yields increased 1.1-fold, >9.5-fold, and 1.3-fold for the small autosomal, large autosomal, and male targets, respectively. DTT-spiked DNA standards from both kits were impacted similarly to samples with residual DTT, demonstrating that observed effects were related to DTT and not the extraction method. This study corroborates other reports that DTT adversely affects multiple dyes (e.g., Cy5, Quasar 670, SYBR Green I, TMR, and Mustang Purple® ). Overall, DTT causes inaccurate quantities and, consequently, inaccurate calculated male:female ratios when used in conjunction with these kits. Thus, implementation of newer direct-to-PCR assays incorporating DTT should either be avoided or used only with carefully evaluated, compatible dyes.

microRNA Detection in Blood, Urine, Semen, and Saliva Stains After Compromising Treatments.

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification.

Molecular-based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT-qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA-specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.

The stability and persistence of blood and semen mRNA and miRNA targets for body fluid identification in environmentally challenged and laundered samples.

The identification of body fluids in evidentiary stains may provide investigators with probative information during an investigation. In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were performed to detect the presence of mRNA and miRNA in fresh and environmentally challenged samples. Blood, semen, and reference markers were chosen for both mRNA/miRNA testing. Samples of blood and semen were exposed to heat, humidity, and sunlight, and controlled conditions (room temperature, low humidity, and darkness) for 6 months. All mRNA targets were observed through six months under controlled conditions, but were undetected after 30 days in experimental conditions. However, miRNA targets persisted under all test conditions for the duration of the study. Additionally, cotton stained with blood or semen was laundered using a liquid detergent in various washing and drying conditions. An unstained cutting was evaluated for potential transfer. Both miRNA targets were observed in all stained samples regardless of the wash protocol used. Of the mRNA markers, HBB was detected in all bloodstained samples and PRM1 persisted in all but one semen stained sample. The unstained samples showed transfer of at least one body fluid specific miRNA marker in all samples and at least one body fluid specific mRNA in approximately half of the samples. These results support that RNA markers can be used for body fluid identification in challenging samples, and that miRNA markers may be more persistent than mRNA for blood and semen stains. However, some caution is warranted with laundered items due to possible transfer.

Optical tweezers as an effective tool for spermatozoa isolation from mixed forensic samples.

A single focus optical tweezer is formed when a laser beam is launched through a high numerical aperture immersion objective. This objective focuses the beam down to a diffraction-limited spot, which creates an optical trap where cells suspended in aqueous solutions can be held fixed. Spermatozoa, an often probative cell type in forensic investigations, can be captured inside this optical trap and dragged one by one across millimeter-length distances in order to create a cluster of cells which can be subsequently drawn up into a capillary for collection. Sperm cells are then ejected onto a sterile cover slip, counted, and transferred to a tube for DNA analysis workflow. The objective of this research was to optimize sperm cell collection for maximum DNA yield, and to determine the number of trapped sperm cells necessary to produce a full STR profile. A varying number of sperm cells from both a single-source semen sample and a mock sexual assault sample were isolated utilizing optical tweezers and processed using conventional STR analysis methods. Results demonstrated that approximately 50 trapped spermatozoa were required to obtain a consistently full DNA profile. A complete, single-source DNA profile was also achieved by isolating sperm cells via optical trapping from a mixture of sperm and vaginal epithelial cells. Based on these results, optical tweezers are a viable option for forensic applications such as separation of mixed populations of cells in forensic evidence.

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis.

A capillary electrophoresis method for identifying forensically relevant body fluids using miRNAs.

Body fluid identification (BFID) can provide crucial information during the course of an investigation. In recent years, microRNAs (miRNAs) have shown considerable body fluid specificity, are able to be co-extracted with DNA, and their small size (18-25 nucleotides) make them ideal for analyzing highly degraded forensic samples. In this study, we designed a preliminary 8-marker system for BFID including an endogenous reference gene (let-7g) to differentiate between venous blood (miR-451a and miR-142-3p), menstrual blood (miR-141-3p and miR-412-3p), semen (miR-891a and miR-10b), and saliva (miR-205) using a capillary electrophoresis approach. This panel uses a linear primer system in order to incorporate additional miRNA markers by forming a multiplex system. The miRNA system was able to distinguish between venous blood, menstrual blood, semen, and saliva using a rudimentary data interpretation strategy. All STR amplifications from co-extracted DNA yielded complete profiles from human identification purposes.

An Optimized Centrifugal Method for Separation of Semen from Superabsorbent Polymers for Forensic Analysis.

Connection of a perpetrator to a sexual assault is best performed through the confirmed presence of semen, thereby proving sexual contact. Evidentiary items can include sanitary napkins or diapers containing superabsorbent polymers (SAPs), complicating spermatozoa visualization and DNA analysis. In this report, we evaluated the impact of SAPS on the current forensic DNA workflow, developing an efficient centrifugal protocol for separating spermatozoa from SAP material. The optimized filtration method was compared to common practices of excising the top layer only, resulting in significantly higher sperm yields when a core sample of the substrate was taken. Direct isolation of the SAP-containing materials without filtering resulted in 20% sample failure; additionally, SAP material was observed in the final eluted DNA samples, causing physical interference. Thus, use of the described centrifugal-filtering method is a simple preliminary step that improves spermatozoa visualization and enables more consistent DNA yields, while also avoiding SAP interference.

High-throughput miRNA sequencing and identification of biomarkers for forensically relevant biological fluids.

microRNAs (miRNAs) are small noncoding RNAs that regulate cellular processes through modulation of proteins at the translational level. They tend to be highly stable as compared to other RNA species due to their small size and protection by protein and/or lipid matrices. Thus, it is likely that miRNAs, when fully evaluated, will make excellent candidates for body fluid identification. miRNA analysis of body fluids has been the subject of some recent interest in the forensic community. In this study, small RNAs were isolated from individual donations of eight forensically relevant biological fluids (blood, semen, vaginal fluid, menstrual blood, saliva, urine, feces, and perspiration) and subjected to next generation sequencing using the Illumina Hi-Seq platform. Sequencing reads were aligned and annotated against miRbase release 21, resulting in a list of miRNAs and their relative expression levels for each sample analyzed. Body fluids with high bacterial loads (vaginal fluid, saliva, and feces) yielded relatively low annotated miRNA counts, likely due to oversaturation of small RNAs from the endogenous bacteria. Both body fluid specific (miRs-200b, 1246, 320c, 10b-5p, 26b, and 891a) and potential normalization miRNAs (let-7g and i) were identified for further analysis as potential body fluid identification tools for each body fluid.

Infrared Laser Heating Applied to Nanopore Sensing for DNA Duplex Analysis.

Temperature studies coupled with resistive-pulse nanopore sensing enable the quantification of a variety of important thermodynamic properties at the single-molecule limit. Previous demonstrations of nanopore sensing with temperature control have utilized bulk chamber heating methodologies. This approach makes it difficult to rapidly change temperatures and enable optical access for other analytical techniques (i.e., single-molecule fluorescence). To address these issues, researchers have explored laser-based methodologies through either direct infrared (IR) absorption or plasmonic assisted heating. In this paper, we demonstrate the use of IR-based direct absorption heating with the DNA sensing capabilities of a biological nanopore. The IR heating enables rapid changes of the temperature in and around an α-hemolysin pore, and we use this to explore melting properties for short (≤50 bp) double-stranded DNA homopolymers. We also demonstrate that the IR heating enables one to measure the percentage of different-sized DNA molecules in a binary mixture.