Browsing by Subject "Sequence Analysis, DNA"
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Item Effects of temperature on bacterial microbiome composition in Ixodes scapularis ticks(John Wiley & Sons, Inc., 2018-09-21) Thapa, Santosh; Zhang, Yan; Allen, Michael S.Ixodes scapularis, the blacklegged deer tick, is the principal vector of Lyme disease in North America. Environmental factors are known to influence regional and seasonal incidence of Lyme disease and possibly the endemicity of the disease to the northeastern and upper mid-western regions of the United States. With a goal to understand the impact of environmental temperature on microbial communities within the tick, we investigated the bacterial microbiome of colony-reared I. scapularis ticks statically incubated at different temperatures (4, 20, 30, and 37°C) at a constant humidity in a controlled laboratory setting by comparison of sequenced amplicons of the bacterial 16S V4 rRNA gene to that of the untreated baseline controls. The microbiomes of colony-reared I. scapularis males were distinct than that of females, which were entirely dominated by Rickettsia. In silico removal of Rickettsia sequences from female data revealed the underlying bacterial community, which is consistent in complexity with those seen among male ticks. The bacterial community composition of these ticks changes upon incubation at 30°C for a week and 37°C for more than 5 days. Moreover, the male ticks incubated at 30 and 37°C exhibited significantly different bacterial diversity compared to the initial baseline microbiome, and the change in bacterial diversity was dependent upon duration of exposure. Rickettsia-free data revealed a significantly different bacterial diversity in female ticks incubated at 37°C compared to that of 4 and 20°C treatments. These results provide experimental evidence that environmental temperature can impact the tick bacterial microbiome in a laboratory setting.Item Examining the skin and workplace microbiome following the return to the University of North Texas Health Science Center after self-isolation(2021-08) Khichi, Ophelia J.; Allen, Michael S.; Zhang, Yan; Zascavage, Roxanne R.; Rosales, ArmandoThe human skin microbiome contains trillions of microbiomes that colonize the human body. It is unknown how social distancing and an increase in handwashing due to the COVID-19 pandemic affect the bacterial communities that reside on the human hands & fomites. In this research, bacterial swabs from individual's dominant hand, forearm of their dominant hand, and a fomite from the workplace environment were taken, and the resident microflora, i.e., the skin microbiome, was investigated. The DNA from the samples were extracted and amplified by PCR. Samples were then pooled for sequencing of the V4 region of the 16S ribosomal RNA gene using Illumina's MiSeq platform and subsequently analyzed for community composition. The results were compared against each other to determine how an individual and a fomite's microbiome shifted following their return to work. Furthermore, the results were used to determine if individual's microbiome became more similar to each other as they returned to work in the same building.Item Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU(PLOS, 2017-05-17) Durrer, Katherine E.; Allen, Michael S.; Hunt von Herbing, IonePhenylketonuria (PKU) is a genetic disease characterized by the inability to convert dietary phenylalanine to tyrosine by phenylalanine hydroxylase. Given the importance of gut microbes in digestion, a genetically engineered microbe could potentially degrade some ingested phenylalanine from the diet prior to absorption. To test this, a phenylalanine lyase gene from Anabaena variabilis (AvPAL) was codon-optimized and cloned into a shuttle vector for expression in Lactobacillus reuteri 100-23C (pHENOMMenal). Functional expression of AvPAL was determined in vitro, and subsequently tested in vivo in homozygous PAHenu2 (PKU model) mice. Initial trials of two PAHenu2 homozygous (PKU) mice defined conditions for freeze-drying and delivery of bacteria. Animals showed reduced blood phe within three to four days of treatment with pHENOMMenal probiotic, and blood phe concentrations remained significantly reduced (P < 0.0005) compared to untreated controls during the course of experiments. Although pHENOMMenal probiotic could be cultured from fecal samples at four months post treatment, it could no longer be cultivated from feces at eight months post treatment, indicating eventual loss of the microbe from the gut. Preliminary screens during experimentation found no immune response to AvPAL. Collectively these studies provide data for the use of a genetically engineered probiotic as a potential treatment for PKU.Item USAT: a bioinformatic toolkit to facilitate interpretation and comparative visualization of tandem repeat sequences(BioMed Central Ltd., 2022-11-20) Wang, Xuewen; Budowle, Bruce; Ge, JianyeBACKGROUND: Tandem repeats (TR), highly variable genomic variants, are widely used in individual identification, disease diagnostics, and evolutionary studies. The recent advances in sequencing technologies and bioinformatic tools facilitate calling TR haplotypes genome widely. Both length-based and sequence-based TR alleles are used in different applications. However, sequence-based TR alleles could provide the highest precision in characterizing TR haplotypes. The need to identify the differences at the single nucleotide level between or among TR haplotypes with an easy-use bioinformatic tool is essential. RESULTS: In this study, we developed a Universal STR Allele Toolkit (USAT) for TR haplotype analysis, which takes TR haplotype output from existing tools to perform allele size conversion, sequence comparison of haplotypes, figure plotting, comparison for allele distribution, and interactive visualization. An exemplary application of USAT for analysis of the CODIS core STR loci for DNA forensics with benchmarking human individuals demonstrated the capabilities of USAT. USAT has user-friendly graphic interfaces and runs fast in major computing operating systems with parallel computing enabled. CONCLUSION: USAT is a user-friendly bioinformatics software for interpretation, visualization, and comparisons of TRs.Item Validation of the ParaDNA[R] Screening System with touch DNA using mock evidence samples(2018-05) West, Whitney R.; Allen, Michael S.; Warren, Joseph E.; Staub, Rick W.; Schreihofer, Ann M.Short tandem repeat, or STR, analysis is expensive and often creates a waiting game for law enforcement agencies to receive these results due to high demand and the current backlog at forensic laboratories. ParaDNA[R] by LGC utilizes HyBeacon Probe technology to rapidly analyze DNA and provide a percentage value of the amount of DNA present, as well as 2-5 loci, depending on whether the Screening System or Intelligence System is being used. Currently, this technology is novel and is intended for sample screening and prioritizing purposes for the Plano Police Department. This validation study has shown that the ParaDNA[R] Screening System can obtain genetic data from touch DNA and trace DNA samples by using both direct and indirect sampling methods. The results from this validation have further indicated that if the Screening System provides a score higher than 60%, these samples should be prioritized and sent to forensic laboratories for full STR analysis.