Forensic mitochondrial DNA analysis is a vital investigative tool for wildlife casework, but numts continue to complicate DNA analysis. A major goal of wildlife forensic DNA analysis is to identify non-human biological evidence to its taxonomic source. Species identification is accomplished by sequencing genetic markers on the mitochondrial genome and comparing evidentiary sequence data to published reference sequences. Due to the high level of sequence similarity between mitochondrial genes and numts, current sequencing methods result in co-amplification of the target gene marker and non-target numt. Co-amplification of target and non-target loci results in ambiguous nucleotide calls in sequence data. Reducing the influence of numts during sequence analysis will provide a technique that will maximize accuracy and minimize error in taxonomic identifications. To overcome the analytical burden of numts, we studied the enzymatic removal of numts using exonuclease V. To evaluate the feasibility of exonuclease V as a numt removal method, total Panthera tigris DNA was extracted from blood and liver and divided into three treatment groups: untreated, 1stdigest, and 2nddigest. For the untreated sample, 7.6% of the 620bp sequence data was classified as ambiguous. Following treatment, all samples demonstrated a reduction in ambiguous calls: liver-1st digest(48 hrs): 6.6%, liver-2nddigest(48 hrs + 16 hrs): 1.8%, and blood-1stdigest(48 hrs): 0%. Based on this preliminary study, exonuclease V treatment effectively removed numts before sequencing analysis. While exonuclease V treatment has demonstrated potential, additional studies are required to optimize the reaction and fully validate the methodology.

Forensic mitochondrial DNA (mtDNA) analysis is a vital investigative tool for both human and wildlife casework, but nuclear-located mitochondrial pseudogenes (numts) continue to complicate DNA sequencing analysis. The primary goal of wildlife forensic DNA analysis is to identify non-human biological evidence to its taxonomic source. For animal-based evidence, this is often accomplished by sequencing genetic markers located within the mitochondrial genome and comparing the sequence data from the evidentiary sample to that of the database reference sequence. Due to the high level of sequence similarity between mitochondrial genes and numts, current sequencing methods result in the co-amplification of both the desired and undesired genetic locations. Sequencing data from the co-amplification of both loci result in ambiguous nucleotide base calls. Consequently, downstream taxonomic identification analysis is inaccurate and unsuitable for legal purposes. To overcome the analytical burden of numts, we have developed an enzymatic method for removing nuclear pseudogenes. Exonuclease V is a DNA-specific exonuclease and single stranded DNA (ssDNA)endonuclease that cleaves linear duplex DNA while leaving circular DNA intact. To evaluate the effectiveness of our numt purification protocol, total Panthera tigris DNA was extracted from whole blood and liver tissue samples and divided into three treatment groups: untreated, 1st digest, and 2nd digest. Following treatment, 1958bp of the mitochondrial genome (~100bp of ND6 and all CYTB) was sequenced and analyzed for each sample and treatment combination. For the untreated sample, 8.1% of the 1958bp amplicon was classified as ambiguous. Following exonuclease V treatment, all samples demonstrated a reduction in ambiguous calls: 1st Digest Liver - 6.6%, 2nd Digest Liver - 1%, and 1st Digest Blood - 0%. The method presented here is an effective and robust process for the enzymatic removal of numts from total DNA extracts prior to sequencing analysis.

Because Kemp’s ridley sea turtles ( Lepidochelys kempii ) are critically endangered and closely related to the vulnerable olive ridleys ( L. olivacea ), it is essential for forensic investigations and conservation efforts to distinguish these species when only skeletal elements remain. DNA extraction and analysis by DNA sequencing of genetic markers is the only method to determine species identity reliably, yet these methods are significantly compromised when DNA becomes degraded. To evaluate the role that time and environment play in obtaining high-quality DNA sequencing data, we placed skeletal elements of a terrestrial mammal ( Bos taurus) and L. kempii in a supratidal and subtidal environment for 3 years. Bi-annual sampling revealed that after 3 years, mitochondrial DNA (mtDNA) consistently identified each species from each environment. Our results show that mtDNA recovery from bone and identification for Kemp’s ridley sea turtles was possible up to 3 years in both environments. All sequencing data obtained was accurate and robust, but DNA sequencing results were not consistent after 664-days of exposure. Our findings led us to conclude that if sufficient DNA is extracted from bone samples, then high-quality sequence data can be obtained, and the resulting sequence data accurately reflects the reference sequence for the given gene marker. This study provides evidence that DNA can be extracted and analyzed from challenging biological substrates, like bone, when these substrates are exposed to seasonally dynamic maritime environmental conditions for up to 3-years.

Wildlife crime continues unabated contributing to the extinction or near extinction of many plant and animal species. Species identification is a key tool in the enforcement of national legislation. If no morphology exists, comparison of DNA sequences generated from a mitochondrial gene are compared to those on a reference database, commonly GenBank. Sequences up-loaded to GenBank are unregulated and can lead to uncertainty with the adequacy of this DNA sequence repository for identification in a forensic context. We propose the establishment of ForCyt as a fully-regulated database of species that are commonly encountered in forensic investigations. The establishment of ForCyt will allow confidence in future species identification; something that is an absolute requirement to ensure high quality forensic science.

White-nose syndrome is an often lethal fungal infection of bats that is caused by the fungus Pseudogymnoascus destructans, formerly Geomyces destructans. The fungal spores can persist for extended periods of time in the soil and on surfaces in caves where it might be found even after the bats depart. In 2010, a single bat, Myotis velifer, from a western Oklahoma gypsum cave, was initially diagnosed by the U.S. Geological Survey National Wildlife Health Center, as "suspect white-nose syndrome.'' Based on this, we decided to examine soil samples from various bat caves across Oklahoma for the presence of P. destructans. We used Real-time Polymerase Chain Reaction to analyze 83 soil samples from 17 caves in Oklahoma. None of the soil samples were found to contain genetic material from P. destructans. We postulate that P. destructans has not yet reached Oklahoma because of the negative Real-time Polymerase Chain Reaction results, because a National Wildlife Health Center reexamination of the original bat suggested the bat was negative for P. destructans, and subsequent analyses of bat nose and wing swabs, bat tissues, and physical examination of hibernating bats have all been negative for white-nose syndrome.

Using quantitative PCR analysis and DNA sequencing, we provide evidence for the presence of rat lungworm (Angiostrongylus cantonensis) in Oklahoma, USA, and identified a potentially novel rat host (Sigmodon hispidus). Our results indicate a geographic range expansion for this medically and ecologically relevant parasite in North America.

RNA sequencing has emerged as the premier approach to study bacterial transcriptomes. While the earliest published studies analyzed the data qualitatively, the data are readily digitized and lend themselves to quantitative analysis. High-resolution RNA sequence (RNA-seq) data allows transcriptional features (promoters, terminators, operons, among others) to be pinpointed on any bacterial transcriptome. Once the transcriptome is mapped, the activity of transcriptional features can be quantified. Here we highlight how quantitative transcriptome analysis can reveal biological insights and briefly discuss some of the challenges to be faced by the field of bacterial transcriptomics in the near future.

We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.

A continual challenge in the field of forensic DNA analysis is the amplification and interpretation of degraded and low-copy number (LCN) DNA obtained from amounts of limited biological evidence. It has been well established that DNA profiles obtained from the amplification of low quality, degraded, and/or LCN DNA samples are often of limited value due to the frequent occurrence of preferential amplification during polymerase chain reaction (PCR). The by-products of preferential PCR amplification are often observed as inter- and intra-locus peak imbalance, allelic dropout, and/or locus dropout. These are all artifacts that are identified during the interpretation phase of analysis rather than by improving the quality of the DNA present. While it is theoretically possible to obtain a complete DNA profile from a single cell, in reality, profiles obtained from suboptimal amounts of DNA are difficult to interpret and frequently inconsistent when replicated. Inspired by advances in next-generation sequencing techniques, we propose a methodology for simultaneously normalizing the abundance of PCR products across all short tandem repeat (STR) loci using the DNA exonuclease, duplex-specific nuclease (DSN). DSN is an enzyme isolated from the hepatopancreas of Red King (Kamchatka) crab that possesses a strong affinity for digesting double stranded DNA (dsDNA) and has limited activity toward single stranded DNA (ssDNA). Degraded DNA known to display peak imbalance and allele dropout was amplified using AmpFlSTR® Identifiler® Plus for 28 cycles. Following amplification, samples were denatured at 99.9°C for 5min and incubated with one unit of DSN at 62°C in a 28μl volume for 1min. Nuclease activity was terminated through the addition of equal volume of 10mM EDTA and 95°C incubation for 2min. Following DSN treatment, 21 of 30 alleles within the known profile exhibited some improvement in peak height balance. The findings obtained support the potential use of DSN treatment as a method for normalizing STR profiles and improving the quality of data from degraded and low quantity DNA samples.