Microscale microbial distributions are patchy, with abundance hotspots and coldspots that provide... more Microscale microbial distributions are patchy, with abundance hotspots and coldspots that provide important microenvironments for microbial interactions. However, studies are often restricted to abundance estimates alone. At the riverbed of the Murray River, we used taxonomy to complement quantitative analysis to show that abundance hotspots, coldspots and background levels are taxonomically distinct at all taxonomic levels. Abundance hotspots varied 115- and 5.9-fold above background over 0.9 cm for viruses and bacteria, respectively. For bacteria, hotspots represent increases in particular taxa rather than in all bacteria. Genera with increased abundances—Pseudomonas, Parasporobacterium, Lachnospiraceae incertae sedis and Bacteroides—were indicative of human and animal inputs, and represented up to 14.7% of the community. Distinct dominant genera led to high taxonomic dissimilarity among hotspots. Genera exclusivity was still higher in the background and coldspots, with 54 and 48 exclusive genera compared to 7 and 4 genera in hotspots, suggesting hotspots from persistent genera, rather than introduced genera. Hotspots were more similar to coldspots than background, suggesting coldspots may represent dying hotspots. Sample category predicted taxonomic similarity better than proximity, further indicating these heterogeneities are distinct from the background at the sub-centimetre scale. Hotspots and coldspots represent distinct spatial taxonomic distributions, rather than changes of the overall community. This suggests 0.3 ml volumes are cohesive long enough for particular operational taxonomic units (OTUs) to increase and create taxonomically distinct microscale communities within the Kolmogorov and Batchelor scales.
We report on the effects of six dyes used in the detection of DNA on the process of DNA extractio... more We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profil...
Although endangered species are afforded protection under national and international laws, trade ... more Although endangered species are afforded protection under national and international laws, trade in products containing endangered species is still one of the most lucrative criminal enterprises in the world. Forensic science is employed to determine if endangered species are present in commercial products but runs into problems due to the extremely low levels of DNA and degraded nature of the
ABSTRACT Despite renewed public interest, education, research and legislation, Traditional East A... more ABSTRACT Despite renewed public interest, education, research and legislation, Traditional East Asian Medication (TEAM) continues to incorporate animal parts from endangered species and can be obtained openly in many countries. Products sold as having medicinal properties include plasters, pills, ointments, tonic wine and others. Due to the low population levels of some endangered species other more common species may be substituted or very small amounts may be used. This results in low levels of DNA which can be difficult to identify. Most species identification tests rely on sequence comparison with a known reference sample. Most TEAM contains mixtures of several different species making this technique unsuitable. We describe a novel single step assay to rapidly and simultaneously identify rhino, tiger, bear, leopard, pangolin, musk deer and several non-endangered mammals often substituted in TEAM. The test targets the mitochondrial genome to amplify species-specific fragments that can be separated easily using a genetic analyzer. Each fragment is of a different size so that none can be confused. The specificity of each primer pair allows for species identification to be made even if a mixture of several species is present. Further, if more than one of the target species is present then all the species will be amplified and identified simultaneously. The test is sensitive to very low levels of DNA equating to several hundred mitochondrial copies (a fraction of single cell).
Current histological investigation of vaginal swabs after alleged sexual assault includes the sco... more Current histological investigation of vaginal swabs after alleged sexual assault includes the scoring of spermatozoa (0, + to ++++) and the recording of visible tails. It is a method that is universally employed. Despite this method being used for 40 years, there has never been a study investigating its suitability for forensic science. Here, we investigate the reproducibility and subjectivity of sperm scoring among different investigators. Dilutions of seminal fluid were randomly distributed onto 20 slides, stained with haematoxylin/eosin and assessed by 37 investigators, over 2 years. Slides were assessed for levels of spermatozoa and the presence of tails. Each slide was scored by a minimum of 25 investigators. On no slide was there a consensus between all scores. Standard deviation remained below 1, but relative standard deviation (RSD) ranged from 6 to 105% in a positive correlation as the average score decreased. Spermatozoa were not observed 56 times (9.6%) and 27 investigators (73%) did not observe spermatozoa on at least one slide. Spermatozoa with tails were observed on every slide by at least 10 examiners, but as the average score of the slide decreased, so did the observation of tails. The current sperm scoring method is highly subjective with a particularly high %RSD in slides with low overall sperm counts. Moreover, the recording of tails does not add value to the current technique of sperm scoring. Further research might improve the objectivity of sperm scoring and the reliability of recording of tails.
ABSTRACT Non-invasive genetic sampling using scats has a well established role in conservation bi... more ABSTRACT Non-invasive genetic sampling using scats has a well established role in conservation biology, but has rarely been applied to reptiles. Using scats from captive and wild Egernia stokesii (Squamata, Scincidae) we evaluated two storage and six DNA-extraction methods and the reliability of subsequent genotype and sequence data. Accurate genotype and sequence data were obtained from frozen and dried captive lizard scat DNA extracted using a QIAamp® DNA Stool Mini Kit and a modified Gentra® Puregene® method, but success rates were reduced for wild lizard scats. Wild E. stokesii eat more plants than their captive counterparts, possibly resulting in scat DNA extracts containing plant compounds that inhibit PCR-amplifications. Notably, reliable genotypes and sequences were obtained from wild E. stokesii scat DNA extracted using a Qiagen DNeasy® Plant Mini Kit, a method designed to remove plant inhibitory compounds. Results highlight the opportunity for using scat-derived DNA in lizard studies, particularly for species that deposit scats in piles.
The trade in samples of tiger (Panthera tigris), or parts derived from tiger, is controlled throu... more The trade in samples of tiger (Panthera tigris), or parts derived from tiger, is controlled through the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), which lists all subspecies as protected at the highest level. Tiger has been used as a component in traditional medicines for centuries, often as powder thus making its presence difficult
Species identification using mitochondrial DNA (mtDNA) loci is a standard method for mammalian sp... more Species identification using mitochondrial DNA (mtDNA) loci is a standard method for mammalian species testing. Less is understood about the conservation and variability in the avian mitochondrial genome, yet many exotic bird species are threatened with extinction and are traded illegally. In this study 80 different avian species were chosen from 22 different Orders and their gene sequences for the
ABSTRACT Species identification is a tool used extensively in forensic science; particularly in t... more ABSTRACT Species identification is a tool used extensively in forensic science; particularly in the investigation of wildlife crime. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b), and identification is generally carried out through the use of DNA sequencing. However, there is currently no standard method to quantify the data from sequence comparisons for presentation in reports and to courts as there have been no detailed studies of the expected levels of inter- and intraspecific variation. For the first time this study provides a detailed comparison of the effectiveness of these two loci. Interspecific and intraspecific variation are assessed and statistical confidence is applied to sequence comparisons. Comparison of 217 different mammalian species revealed that cyt b more accurately reconstructed their phylogeny and known relationships, and gave better resolution when separating species based on sequence data. Intraspecific variation was assessed using three model species and showed variation ranging from 0 to 1.16% (Kimura 2-parameter p-distance (K2P) × 100%), indicating that some level of variation should be expected. Interspecific variation was greater in cyt b than in COI. Using a K2P (×100) threshold of 1.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI. This study allows, for the first time, application of statistical confidence to sequences comparisons for species identification.
Microscale microbial distributions are patchy, with abundance hotspots and coldspots that provide... more Microscale microbial distributions are patchy, with abundance hotspots and coldspots that provide important microenvironments for microbial interactions. However, studies are often restricted to abundance estimates alone. At the riverbed of the Murray River, we used taxonomy to complement quantitative analysis to show that abundance hotspots, coldspots and background levels are taxonomically distinct at all taxonomic levels. Abundance hotspots varied 115- and 5.9-fold above background over 0.9 cm for viruses and bacteria, respectively. For bacteria, hotspots represent increases in particular taxa rather than in all bacteria. Genera with increased abundances—Pseudomonas, Parasporobacterium, Lachnospiraceae incertae sedis and Bacteroides—were indicative of human and animal inputs, and represented up to 14.7% of the community. Distinct dominant genera led to high taxonomic dissimilarity among hotspots. Genera exclusivity was still higher in the background and coldspots, with 54 and 48 exclusive genera compared to 7 and 4 genera in hotspots, suggesting hotspots from persistent genera, rather than introduced genera. Hotspots were more similar to coldspots than background, suggesting coldspots may represent dying hotspots. Sample category predicted taxonomic similarity better than proximity, further indicating these heterogeneities are distinct from the background at the sub-centimetre scale. Hotspots and coldspots represent distinct spatial taxonomic distributions, rather than changes of the overall community. This suggests 0.3 ml volumes are cohesive long enough for particular operational taxonomic units (OTUs) to increase and create taxonomically distinct microscale communities within the Kolmogorov and Batchelor scales.
We report on the effects of six dyes used in the detection of DNA on the process of DNA extractio... more We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profil...
Although endangered species are afforded protection under national and international laws, trade ... more Although endangered species are afforded protection under national and international laws, trade in products containing endangered species is still one of the most lucrative criminal enterprises in the world. Forensic science is employed to determine if endangered species are present in commercial products but runs into problems due to the extremely low levels of DNA and degraded nature of the
ABSTRACT Despite renewed public interest, education, research and legislation, Traditional East A... more ABSTRACT Despite renewed public interest, education, research and legislation, Traditional East Asian Medication (TEAM) continues to incorporate animal parts from endangered species and can be obtained openly in many countries. Products sold as having medicinal properties include plasters, pills, ointments, tonic wine and others. Due to the low population levels of some endangered species other more common species may be substituted or very small amounts may be used. This results in low levels of DNA which can be difficult to identify. Most species identification tests rely on sequence comparison with a known reference sample. Most TEAM contains mixtures of several different species making this technique unsuitable. We describe a novel single step assay to rapidly and simultaneously identify rhino, tiger, bear, leopard, pangolin, musk deer and several non-endangered mammals often substituted in TEAM. The test targets the mitochondrial genome to amplify species-specific fragments that can be separated easily using a genetic analyzer. Each fragment is of a different size so that none can be confused. The specificity of each primer pair allows for species identification to be made even if a mixture of several species is present. Further, if more than one of the target species is present then all the species will be amplified and identified simultaneously. The test is sensitive to very low levels of DNA equating to several hundred mitochondrial copies (a fraction of single cell).
Current histological investigation of vaginal swabs after alleged sexual assault includes the sco... more Current histological investigation of vaginal swabs after alleged sexual assault includes the scoring of spermatozoa (0, + to ++++) and the recording of visible tails. It is a method that is universally employed. Despite this method being used for 40 years, there has never been a study investigating its suitability for forensic science. Here, we investigate the reproducibility and subjectivity of sperm scoring among different investigators. Dilutions of seminal fluid were randomly distributed onto 20 slides, stained with haematoxylin/eosin and assessed by 37 investigators, over 2 years. Slides were assessed for levels of spermatozoa and the presence of tails. Each slide was scored by a minimum of 25 investigators. On no slide was there a consensus between all scores. Standard deviation remained below 1, but relative standard deviation (RSD) ranged from 6 to 105% in a positive correlation as the average score decreased. Spermatozoa were not observed 56 times (9.6%) and 27 investigators (73%) did not observe spermatozoa on at least one slide. Spermatozoa with tails were observed on every slide by at least 10 examiners, but as the average score of the slide decreased, so did the observation of tails. The current sperm scoring method is highly subjective with a particularly high %RSD in slides with low overall sperm counts. Moreover, the recording of tails does not add value to the current technique of sperm scoring. Further research might improve the objectivity of sperm scoring and the reliability of recording of tails.
ABSTRACT Non-invasive genetic sampling using scats has a well established role in conservation bi... more ABSTRACT Non-invasive genetic sampling using scats has a well established role in conservation biology, but has rarely been applied to reptiles. Using scats from captive and wild Egernia stokesii (Squamata, Scincidae) we evaluated two storage and six DNA-extraction methods and the reliability of subsequent genotype and sequence data. Accurate genotype and sequence data were obtained from frozen and dried captive lizard scat DNA extracted using a QIAamp® DNA Stool Mini Kit and a modified Gentra® Puregene® method, but success rates were reduced for wild lizard scats. Wild E. stokesii eat more plants than their captive counterparts, possibly resulting in scat DNA extracts containing plant compounds that inhibit PCR-amplifications. Notably, reliable genotypes and sequences were obtained from wild E. stokesii scat DNA extracted using a Qiagen DNeasy® Plant Mini Kit, a method designed to remove plant inhibitory compounds. Results highlight the opportunity for using scat-derived DNA in lizard studies, particularly for species that deposit scats in piles.
The trade in samples of tiger (Panthera tigris), or parts derived from tiger, is controlled throu... more The trade in samples of tiger (Panthera tigris), or parts derived from tiger, is controlled through the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), which lists all subspecies as protected at the highest level. Tiger has been used as a component in traditional medicines for centuries, often as powder thus making its presence difficult
Species identification using mitochondrial DNA (mtDNA) loci is a standard method for mammalian sp... more Species identification using mitochondrial DNA (mtDNA) loci is a standard method for mammalian species testing. Less is understood about the conservation and variability in the avian mitochondrial genome, yet many exotic bird species are threatened with extinction and are traded illegally. In this study 80 different avian species were chosen from 22 different Orders and their gene sequences for the
ABSTRACT Species identification is a tool used extensively in forensic science; particularly in t... more ABSTRACT Species identification is a tool used extensively in forensic science; particularly in the investigation of wildlife crime. The two most commonly used genetic loci in species identification are the cytochrome oxidase I gene (COI) and the cytochrome b gene (cyt b), and identification is generally carried out through the use of DNA sequencing. However, there is currently no standard method to quantify the data from sequence comparisons for presentation in reports and to courts as there have been no detailed studies of the expected levels of inter- and intraspecific variation. For the first time this study provides a detailed comparison of the effectiveness of these two loci. Interspecific and intraspecific variation are assessed and statistical confidence is applied to sequence comparisons. Comparison of 217 different mammalian species revealed that cyt b more accurately reconstructed their phylogeny and known relationships, and gave better resolution when separating species based on sequence data. Intraspecific variation was assessed using three model species and showed variation ranging from 0 to 1.16% (Kimura 2-parameter p-distance (K2P) × 100%), indicating that some level of variation should be expected. Interspecific variation was greater in cyt b than in COI. Using a K2P (×100) threshold of 1.5, cyt b gives a better resolution for separating species with a lower false positive rate and higher positive predictive value than those of COI. This study allows, for the first time, application of statistical confidence to sequences comparisons for species identification.
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