DNA Barcoding and Metabarcoding Protocols for Species Identification.

The article presents the several steps to be performed on a plant, fungal, insect, or soil sample to obtain DNA sequences for DNA barcode analysis. The chapter begins with a description of sample preparation including procedures for cleaning and proceeds to DNA extraction with methods adapted for the specific type of sample. Next, DNA quantification is described so the proper amount is used for the amplification of the selected barcode regions. Information is provided for reaction mixes and amplification conditions for several referenced barcode primer pairs tuned for the individual sample of interest. This is followed by a description of procedures to access the success of amplification, cleanup, and quantification of the product ready for either Sanger sequencing or library preparation for massive parallel sequencing (MPS). Finally, procedures are provided for Sanger sequencing, library preparation, and MPS sequencing. The chapter provides several references of barcode regions for different sample types.

Guidelines for the Analysis of DNA Barcoding/Metabarcoding Sequencing Data and Interpretation of Publicly Available Databases.

This chapter describes procedures for the use of DNA sequence data to obtain and compare taxonomic identification using the public databases GenBank and Barcode of Life Data System (BOLD). The chapter begins by describing procedures used to prepare quality sequences for uploading into GenBank and BOLD. Next, steps used to query the DNA sequences against the public databases are described using GenBank BLAST and BOLD identification engines. Interpretation guidelines for the taxonomic identification assignments are presented. Finally, a procedure for evaluating the accuracy and reliability of sequences from GenBank and BOLD is provided.

Evaluation of Library Preparation Workflows and Applications to Different Sample Types Using the PowerSeq® 46GY System with Massively Parallel Sequencing.

This project evaluated the prototype PowerSeq® 46GY System using donor DNA and casework-type samples. The goal of this study was to determine whether modifications to the manufacturer's protocol could increase read coverage and improve sample results. Buccal and casework-type libraries were prepared using the TruSeq® DNA PCR-Free HT kit or the KAPA HyperPrep kit. Both kits were evaluated unmodified, and by substituting AMPure® XP beads for the beads of the most optimal kit. Two qPCR kits, the PowerSeq® Quant MS System and KAPA Library Quantification Kit, were also evaluated along with a KAPA size-adjustment workbook, which was compared as a third quantification method. Libraries were sequenced using the MiSeq® FGx and data were analyzed with STRait Razor. Results suggested that all three quantification methods overestimated library concentration, but the PowerSeq kit was most accurate. Samples prepared with the TruSeq library kit provided the highest coverage and the fewest instances of dropout and below-threshold alleles compared with the KAPA kit. Additionally, all bone and hair samples demonstrated full profile completeness, with bone samples yielding a higher average coverage than hair samples. Overall, our study demonstrated that the 46GY manufacturer's protocol produced the best quality results compared to alternative library preparation options.

Assessment of detection limits for dyed and mounted textile fibers using Raman spectroscopy.

Trace evidence in the form of textile fibers can be used to link objects and places during an investigation. Raman spectroscopy is a well-established technique that has been used for the examination of various pigments, paints, inks, and dyes. The objective of this study was to determine the capability of Raman spectroscopy to detect several different dye classes and colors on a variety of textile fibers. To test this, four categories of dyes, reactive, disperse, acid, and direct were examined with Raman microscopy while applied to one of five fiber types (cotton, polyester, nylon, wool, and rayon). Each dye category was tested using four colors, black, blue, red, and yellow, while at four concentrations of dye (w/w), 4% (black only), 1%, 0.5%, and 0.05% (blue, red, and yellow). Finally, each dye, fiber, color, and dye concentration combination were examined with Raman using one of two laser excitation sources (532 nm and 780 nm) while mounted in one of two mounting media, Permount™ and Entellan® new, as well as unmounted. Raman spectroscopy could detect some dyes at low concentrations (0.5% and 0.05%) even when mounted in mounting media and covered with a glass coverslip. Excitation source, dye category, dye concentration, fiber type, and mounting method all influence the ability to detect any given dye. These results support the continued study of Raman as a tool for the examination of fiber dyes as it has shown the potential to be effective even under constraints experienced by forensic examiners.

Rapid, presumptive identification of seed-based toxins using direct analysis in real time mass spectrometry (DART-MS) and its variants.

Detection of seed-based toxins is a need for forensic chemists when suspected poisonings occur. The evidence that is found is often physically unidentifiable, as the seeds are mashed to extract the toxin. This work investigates potential strategies for rapid detection of seed-based toxins and seed mashes containing these toxins using chemical signatures obtained by direct analysis in real time mass spectrometry (DART-MS). Seven toxins (digoxin, digitoxin, hypaconitine, hyoscyamine, lanatoside, oleandrin, and scopolamine) and six seeds containing these toxins were studied. While detection of four of the toxins was readily attainable, detection of digoxin, digitoxin, and lanatoside was hindered by the inability to thermally desorb these larger compounds under normal operating conditions. The use of DART-MS variants capable of higher desorption temperatures (thermal desorption (TD)-DART-MS and infrared thermal desorption (IRTD)-DART-MS) enabled detection of these compounds. Detection of toxins from direct analysis of seed mashes and methanolic seed mash extracts was found to be compound and technique dependent. Principal component analysis (PCA) of generated mass spectra enabled differentiation of seed species, even in cases where the toxins were undetectable.

Comparison of polymerases used for amplification of mitochondrial DNA from challenging hairs and hairs of various treatments.

Forensic DNA analysis of hair evidence typically involves the amplification and sequencing of the control region (CR) of the mitochondrial genome (mtgenome). In compromised hair samples, such as shed hairs, the number of mtgenome copies could be low; thus, it is imperative that the polymerase used in PCR is efficient to ensure maximum amplification. Considering this, the first phase of this study compared the yields obtained from 12 polymerases (sourced from a range of commercial companies) when amplifying the CR, hypervariable (HV) region II (HV2), and hypervariable subregion II-B (HV2B). This initial assessment was performed using mitochondrial DNA (mtDNA) extracted from 2 cm of hair adjacent to the root from three donors of different self-reported ancestries and hair color/texture. PrimeSTAR HS and KAPA HiFi HotStart consistently generated significantly higher amplicon yields (p < 0.05, ~5-fold increase) for most regions than AmpliTaq Gold DNA polymerase (the polymerase validated for use in most forensic laboratories). The second phase of this project was focused on assessing the broad utility of these top two performing polymerases for amplifying two regions of the mtgenome (CR and HV2B) from hair samples representing diverse self-reported ancestral origins (European, Latin American, African American, Asian, and Native American), characteristics/treatments (bleached, dyed, and chemically straightened), and anatomical origins (e.g., head and pubic region) (n = 41). These regions were chosen as they are the most challenging to amplify and sequence in compromised hair samples due to length (i.e., the CR is ~1.2 kb) and repeat structure (i.e., the polycytosine stretch within HV2B). The results indicated that regardless of sample type, PrimeSTAR HS and KAPA HiFi HotStart polymerases outperformed (p < 0.05) AmpliTaq Gold DNA polymerase (averaging 11- and 8-fold increased yields, respectively). The results from this study highlight that enhanced commercially available polymerases appear to significantly improve the amplification of mtDNA from challenging hair samples.

Performance Comparison of Massively Parallel Sequencing (MPS) Instruments Using Single-Nucleotide Polymorphism (SNP) Panels for Ancestry.

Thermo Fisher Scientific released the Precision ID Ancestry Panel, a 165-single-nucleotide polymorphism (SNP) panel for ancestry prediction that was initially compatible with the manufacturer's massively parallel sequencer, the Ion Torrent Personal Genome Machine (PGM). The semiautomated workflow using the panel with the PGM involved several time-consuming manual steps across three instruments, including making templating solutions and loading sequencing chips. In 2014, the manufacturer released the Ion Chef robot, followed by the Ion S5 massively parallel sequencer in late 2015. The robot performs the templating with reagent cartridges and loads the chips, thus creating a fully automated workflow across two instruments. The objective of the work reported here is to compare the performance of two massively parallel sequencing systems and ascertain if the change in the workflow produces different ancestry predictions. For performance comparison of the two systems, forensic-type samples (n = 16) were used to make libraries. Libraries were templated either with the Ion OneTouch 2 system (for the PGM) or on the Ion Chef robot (for the S5). Sequencing results indicated that the ion sphere particle performance metrics were similar for the two systems. The total coverages per SNP and SNP quality were both higher for the S5 system. Ancestry predictions were concordant for the mock forensic-type samples sequenced on both massively parallel sequencing systems. The results indicated that automating the workflow with the Ion Chef system reduced the labor involved and increased the sequencing quality.

Assessment of BOLD and GenBank - Their accuracy and reliability for the identification of biological materials.

Taxonomic identification of biological materials can be achieved through DNA barcoding, where an unknown "barcode" sequence is compared to a reference database. In many disciplines, obtaining accurate taxonomic identifications can be imperative (e.g., evolutionary biology, food regulatory compliance, forensics). The Barcode of Life DataSystems (BOLD) and GenBank are the main public repositories of DNA barcode sequences. In this study, an assessment of the accuracy and reliability of sequences in these databases was performed. To achieve this, 1) curated reference materials for plants, macro-fungi and insects were obtained from national collections, 2) relevant barcode sequences (rbcL, matK, trnH-psbA, ITS and COI) from these reference samples were generated and used for searching against both databases, and 3) optimal search parameters were determined that ensure the best match to the known species in either database. While GenBank outperformed BOLD for species-level identification of insect taxa (53% and 35%, respectively), both databases performed comparably for plants and macro-fungi (~81% and ~57%, respectively). Results illustrated that using a multi-locus barcode approach increased identification success. This study outlines the utility of the BLAST search tool in GenBank and the BOLD identification engine for taxonomic identifications and identifies some precautions needed when using public sequence repositories in applied scientific disciplines.

The Gliocentric Brain.

The Neuron Doctrine, the cornerstone of research on normal and abnormal brain functions for over a century, has failed to discern the basis of complex cognitive functions. The location and mechanisms of memory storage and recall, consciousness, and learning, remain enigmatic. The purpose of this article is to critically review the Neuron Doctrine in light of empirical data over the past three decades. Similarly, the central role of the synapse and associated neural networks, as well as ancillary hypotheses, such as gamma synchrony and cortical minicolumns, are critically examined. It is concluded that each is fundamentally flawed and that, over the past three decades, the study of non-neuronal cells, particularly astrocytes, has shown that virtually all functions ascribed to neurons are largely the result of direct or indirect actions of glia continuously interacting with neurons and neural networks. Recognition of non-neural cells in higher brain functions is extremely important. The strict adherence of purely neurocentric ideas, deeply ingrained in the great majority of neuroscientists, remains a detriment to understanding normal and abnormal brain functions. By broadening brain information processing beyond neurons, progress in understanding higher level brain functions, as well as neurodegenerative and neurodevelopmental disorders, will progress beyond the impasse that has been evident for decades.

Effects of Management Treatments on Regeneration of a Geographically Disjunct, Relictual Hybrid Aspen (Populus xsmithii) Population in the Central Great Plains, USA.

Populus xsmithii is an uncommon hybrid of quaking aspen (Populus tremuloides) and bigtooth aspen (Populus grandidentata). Like its parents, Populus xsmithii is an early successional member of boreal forest communities, dependent on disturbance events that clear areas of competitive stems and spur an increase in clonal suckering. In recent years, aspen dieback has been noted across much of the western United States, a condition characterized by mortality of older stems and a lack of recruitment of suckers to maturity. In the Niobrara River Valley of Cherry County, Nebraska, USA, a disjunct population of Pleistocene relict Populus xsmithii has been targeted for management via clearing of competitive conifer species and establishment of fenced refugia to protect suckers from herbivory. The stands currently contain abundant suckers, which occur in three types of sites: the fenced refugia created by managers, the open habitat cleared of other species in the stands, and the woodpiles left by said clearing. This study assessed the growth and vigor of these aspen suckers over a nine-month period (summer 2013-spring 2014) and compared the effects of different site treatments. We found that aspen suckers in the open areas were significantly shorter, had smaller basal diameter, and had higher damage scores than those in the two protected site types (fenced and woodpile). Because this population is on the margin of the distribution for aspen, evaluating the effectiveness of management techniques will provide valuable information for those who seek to ensure the survival of this aspen population and others.

An automated independent workflow for the analysis of massively parallel sequence data from forensic SNP assays.

Illumina and Thermo Fisher Scientific have developed assays that permit the sequencing of forensically relevant single nucleotide polymorphisms (SNPs), along with software to determine the associated genotypes. Currently there is no method to either independently confirm the genotypes determined using the manufacturer's software, or to compare genotypes and quality metrics among samples processed using both platforms. This paper outlines an automated workflow developed in CLC Genomics Workbench that permits accurate, fast and independent analysis of SNP sequence data from either platform. To facilitate the straightforward comparison of genotypes generated from both the manufacturer's software and the independent CLC analysis, a Python script was written. Data for a total of 323 forensically relevant ancestry, identity and phenotypic SNPs can be analyzed, and the resulting genotypes, coverage, quality flags and major allele frequencies are easily compared across samples and platforms.

A protocol for obtaining DNA barcodes from plant and insect fragments isolated from forensic-type soils.

Soil is often collected from a suspect's tire, vehicle, or shoes during a criminal investigation and subsequently submitted to a forensic laboratory for analysis. Plant and insect material recovered in such samples is rarely analyzed, as morphological identification is difficult. In this study, DNA barcoding was used for taxonomic identifications by targeting the gene regions known to permit discrimination in plants [maturase K (matK) and ribulose 1,5-biphosphate carboxylase (rbcL)] and insects [cytochrome oxidase subunit I (COI)]. A DNA barcode protocol suitable for processing forensic-type biological fragments was developed and its utility broadly tested with forensic-type fragments (e.g., seeds, leaves, bark, head, legs; n, 213) isolated from soils collected within Virginia, USA (n, 11). Difficulties with PCR inhibitors in plant extracts and obtaining clean Sanger sequence data from insect amplicons were encountered during protocol development; however, the final protocol produced sequences specific to the expected locus and taxa. The overall quantity and quality of DNA extracted from the 213 forensic-type biological fragments was low (< 15 ng/µL). For plant fragments, only the rbcL sequence data was deemed reliable; thus, taxonomic identifications were limited to the family level. The majority of insect sequences matched COI in both GenBank and Barcode of Life DataSystems; however, they were identified as an undescribed environmental contaminant. Although limited taxonomic information was gleaned from the forensic-type fragments processed in this study, the new protocol shows promise for obtaining reliable and specific identifications through DNA barcoding, which could ultimately enhance the information gleaned from soil examinations.

Isolation of Mitochondrial DNA from Single, Short Hairs without Roots Using Pressure Cycling Technology.

Hairs are commonly submitted as evidence to forensic laboratories, but standard nuclear DNA analysis is not always possible. Mitochondria (mt) provide another source of genetic material; however, manual isolation is laborious. In a proof-of-concept study, we assessed pressure cycling technology (PCT; an automated approach that subjects samples to varying cycles of high and low pressure) for extracting mtDNA from single, short hairs without roots. Using three microscopically similar donors, we determined the ideal PCT conditions and compared those yields to those obtained using the traditional manual micro-tissue grinder method. Higher yields were recovered from grinder extracts, but yields from PCT extracts exceeded the requirements for forensic analysis, with the DNA quality confirmed through sequencing. Automated extraction of mtDNA from hairs without roots using PCT could be useful for forensic laboratories processing numerous samples.

Effect of Sterilants on Amplification and Detection of Target DNA from Bacillus cereus Spores.

To conceal criminal activity of a bioterrorist or agroterrorist, the site of pathogen generation is often treated with sterilants to kill the organisms and remove evidence. As dead organisms cannot be analyzed by culture, this study examined whether DNA from sterilant-treated Bacillus cereus spores was viable for amplification. The spores were exposed to five common sterilants: bleach, Sterilox®, oxidizer foam (L-Gel), a peroxyacid (Actril®), and formaldehyde vapor. The spores were inoculated on typical surfaces found in offices and laboratories to test for environmental effects. It was found that the surface influenced the efficiency of recovery of the organisms. The DNA isolated from the recovered spores was successfully detected using RT-qPCR for all treatments except for formaldehyde, by amplifying the phosphatidylinositol phospholipase C and sphingomyelinase genes. The results demonstrated that evidence from sites treated with sterilants can still provide information on the uncultured organism, using DNA amplification.

Evaluation of the Precision ID Identity Panel for the Ion Torrent™ PGM™ sequencer.

In cases where only a partial or incomplete STR profile is obtained from a sample, information contained in single nucleotide polymorphisms (SNPs) can prove informative for human identification. Thermo Fisher Scientific, which developed the high throughput Ion Torrent™ PGM™ sequencer, released the Precision ID Identity Panel, a multiplex SNP panel for human identity. We evaluated the reproducibility and sensitivity of this multiplex, which contains primers for the amplification of 90 autosomal SNPs and 34 Y-clade SNPs. The manufacturer's protocol was tested using five commercially available pure native DNAs and six forensic type samples at a range of DNA input amounts (0.2-1.0ng; n, 90). In addition to analyzing the data using the manufacturer's software, HID SNP Genotyper (v4.3.1), we also used CLC Genomics Workbench (Qiagen). Although library yields and templating of ion sphere particles (ISPs) were low, downstream sequencing was still successful. Across all samples, only 1.5% of all possible quality control (QC) flags were raised by both the plugin QC filter and CLC; 85% of those flags were raised as the SNP had a major allele frequency outside the thresholds specified by the manufacturer. For the remaining SNPs, coverage of >1500 X and >780 X was obtained for autosomal and Y-clade SNPs respectively, and 100% congruence among genotype calls from both analysis programs was observed. Our results demonstrate that it is possible to obtain reliable and reproducible genotypes using the Precision ID Identity Panel, when using low quantities (≥0.2ng) of either pure native DNA or forensic type DNA samples.

Anxious behavior induces elevated hippocampal Cb2 receptor gene expression.

Anxiety is differentially expressed across a continuum of stressful/fearful intensity, influenced by endocannabinoid systems and receptors. The hippocampus plays important roles in the regulation of affective behavior, emotion, and anxiety, as well as memory. Location of Cb1/Cb2 receptor action could be important in determining emotional valence, because while the dorsal hippocampus is involved in spatial memory and cognition, the ventral hippocampus has projections to the PFC, BNST, amygdala, and HPA axis, and is important for emotional responses to stress. During repeated social defeat in a Stress-Alternatives Model arena (SAM; an oval open field with escape portals only large enough for smaller mice), smaller C57BL6/N mice are subject to fear conditioning (tone=CS), and attacked by novel larger aggressive CD1 mice (US) over four daily (5min) trials. Each SAM trial presents an opportunity for escape or submission, with stable behavioral responses established by the second day of interaction. Additional groups had access to a running wheel. Social aggression plus fear conditioning stimulates enhanced Cb2 receptor gene expression in the dorsal CA1, dorsal and ventral dentate gyrus subregions in animals displaying a submissive behavioral phenotype. Escape behavior is associated with reduced Cb2 expression in the dorsal CA1 region, with freezing and escape latency correlated with mRNA levels. Escaping and submitting animals with access to running wheels had increased Cb2 mRNA in dorsal DG/CA1. These results suggest that the Cb2 receptor system is rapidly induced during anxiogenic social interactions plus fear conditioning or exercise; with responses potentially adaptive for coping mechanisms.

Intensity of anxiety is modified via complex integrative stress circuitries.

Escalation of anxious behavior while environmentally and socially relevant contextual events amplify the intensity of emotional response produces a testable gradient of anxiety shaped by integrative circuitries. Apprehension of the Stress-Alternatives Model apparatus (SAM) oval open field (OF) is measured by the active latency to escape, and is delayed by unfamiliarity with the passageway. Familiar OF escape is the least anxious behavior along the continuum, which can be reduced by anxiolytics such as icv neuropeptide S (NPS). Social aggression increases anxiousness in the SAM, reducing the number of mice willing to escape by 50%. The apprehension accompanying escape during social aggression is diminished by anxiolytics, such as exercise and corticotropin releasing-factor receptor 1 (CRF1) antagonism, but exacerbated by anxiogenic treatment, like antagonism of α2-adrenoreceptors. What is more, the anxiolytic CRF1 and anxiogenic α2-adrenoreceptor antagonists also modify behavioral phenotypes, with CRF1 antagonism allowing escape by previously submissive animals, and α2-adrenoreceptor antagonism hindering escape in mice that previously engaged in it. Gene expression of NPS and brain-derived neurotrophic factor (BDNF) in the central amygdala (CeA), as well as corticosterone secretion, increased concomitantly with the escalating anxious content of the mouse-specific anxiety continuum. The general trend of CeA NPS and BDNF expression suggested that NPS production was promoted by increasing anxiousness, and that BDNF synthesis was associated with learning about ever-more anxious conditions. The intensity gradient for anxious behavior resulting from varying contextual conditions may yield an improved conceptualization of the complexity of mechanisms producing the natural continuum of human anxious conditions, and potential therapies that arise therefrom.

52 additional reference population samples for the 55 AISNP panel.

Ancestry inference for a person using a panel of SNPs depends on the variation of frequencies of those SNPs around the world and the amount of reference data available for calculation/comparison. The Kidd Lab panel of 55 AISNPs has been incorporated in commercial kits by both Life Technologies and Illumina for massively parallel sequencing. Therefore, a larger set of reference populations will be useful for researchers using those kits. We have added reference population allele frequencies for 52 population samples to the 73 previously entered so that there are now allele frequencies publicly available in ALFRED and FROG-kb for a total of 125 population samples.

Nuance and behavioral cogency: How the Visible Burrow System inspired the Stress-Alternatives Model and conceptualization of the continuum of anxiety.

By creating the Visible Burrow System (VBS) Bob Blanchard found a way to study the interaction of genetics, physiology, environment, and adaptive significance in a model with broad validity. The VBS changed the way we think about anxiety and affective disorders by allowing the mechanisms which control them to be observed in a dynamic setting. Critically, Blanchard used the VBS and other models to show how behavioral systems like defense are dependent upon context and behavioral elements unique to the individual. Inspired by the VBS, we developed a Stress Alternatives Model (SAM) to further explore the multifaceted dynamics of the stress response with a dichotomous choice condition. Like the VBS, the SAM is a naturalistic model built upon risk assessment and defensive behavior, but with a choice of response: escape or submission to a large conspecific aggressor. The anxiety of novelty during the first escape must be weighed against fear of the aggressor, and a decision must be made. Both outcomes are adaptively significant, evidenced by a 50/50 split in outcome across several study systems. By manipulating the variables of the SAM, we show that a gradient of anxiety exists that spans the contextual settings of escaping an open field, escaping from aggression, and submitting to aggression. These findings correspond with increasing levels of corticosterone and increasing levels of NPS and BDNF in the central amygdala as the context changes.Whereas some anxiolytics were able to reduce the latency to escape for some animals, only with the potent anxiolytic drug antalarmin (CRF1R-blocker) and the anxiogenic drug yohimbine (α2 antagonist) were we able to reverse the outcome for a substantial proportion of individuals. Our findings promote a novel method for modeling anxiety, offering a distinction between low-and-high levels, and accounting for individual variability. The translational value of the VBS is immeasurable, and it guided us and many other researchers to seek potential clinical solutions through a deeper understanding of regional neurochemistry and gene expression in concert with an ecological behavioral model.

Forensic differentiation of Bacillus cereus spores grown using different culture media using Raman spectroscopy.

Some microorganisms have been shown to retain a chemical signature indicative of the medium used for culturing. However, the repeatability of medium-specific chemical signatures has not been demonstrated from samples of microorganisms produced in the same batch or in different batches by the same sporulation protocol. Here, the variation in Raman spectra of bacterial endospores repeatedly prepared by the same procedure is compared to the variation between Raman spectra of spores prepared using different media. Bacillus cereus T strain (BcT) samples were correctly classified according to the medium used to induce sporulation for 100 % of spores grown in a controlled manner by the same scientist using Raman spectroscopy and multivariate data analysis. The proof-of-concept results from BcT spores produced in 12 different sporulation media showed correct classification by medium for 98 % of samples (with 100 % classification accuracy for all but one sporulation medium in this data set). Spectral differences were discerned between spores that had been freshly prepared or freeze-dried and spores that had been frozen; however, the differences did not impact the classification of the sporulation medium. Latent variables reduced the classification accuracy of BcT sporulated in G medium by different scientists using different media lots and stored for different periods of time and requires further study.