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11th Annual Sequencing, Finishing, and Analysis in the Future Meeting IMPACTS OF FEEDING IN A RESISTANT TREE ON THE ASIAN LONGHORNED BEETLE AND ITS GUT MICROBIAL COMMUNITY Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.01) Erin Scully USDA‐ARS Center for Grain and Animal Health Research The Asian longhorned beetle (ALB; Anoplophora glabripennis) is an invasive, wood‐boring pest capable of thriving in the heartwood of over 47 tree species worldwide where it faces a number of nutritional challenges, including digestion of lignocellulose and hemicellulose and acquisition of nitrogen, essential amino acids and nutrients that are present in low abundances in woody tissue. Through transcriptome sequencing, we have previously demonstrated that this insect possesses a rich repertoire of metabolic machinery that enables it to degrade major hardwood polysaccharides, such as cellulose, xylan, and pectin; detoxify host plant defensive compounds; recycle essential nutrients; and efficiently acquire protein and nitrogen from woody tissue or microbes that inhabit the gut. Furthermore, through metagenome and metatranscriptome sequencing efforts, we have also demonstrated that the taxonomically diverse gut microbiota encode diverse suites of genes that complement and augment ALB’s endogenous physiological capacities, including the abilities to convert xylose sugars into compounds that can be directly utilized by ALB for the synthesis of fatty acids and amino acids, fix atmospheric nitrogen and recycle nitrogenous waste products, and synthesize several essential amino acids and nutrients that are present in low abundances in woody tissue. Further, it also encodes genes with the capacity to degrade large aromatic compounds and may collaborate with ALB to facilitate digestion of the lignin biopolymer. Thus, the metabolic potential of the gut community encodes an extensive suite of enzymes, which has been hypothesized to contribute to ALB’s broad host range. Despite its broad host range, there are several tree species which display considerable resistance to ALB and other wood‐boring pests, but the mechanisms underlying this resistance have not yet been characterized. In this study, we investigate the impacts of feeding in a resistant poplar tree on ALB and its gut microbiota, revealing that feeding in this resistant host causes substantial disruptions to the gut bacterial and fungal communities, interferes with the expression of beetle genes with predicted roles in detoxification and interactions with gut microbes, and reduces the abundances of proteins with key roles in digestion. 67
11th Annual Sequencing, Finishing, and Analysis in the Future Meeting TOWARDS DEVELOPMENT OF A STANDARD INPUT FOR DETECTION OF MICROBES BY NEXT GENERATION SEQUENCING Wednesday, 1st June 20:00 La Fonda NM Room (1st floor) Poster (PS‐1b.02) Rachel Spurbeck, Richard Chou, Nick Fackler, Jazmine Quinn Battelle Memorial Institute The utilization of Next Generation Sequencing (NGS) for bacterial pathogen detection is a powerful technique for biosurveillance purposes. However, the field suffers from a lack of standards for validation of NGS pipelines necessary to determine the limit of detection. Currently, there are several different methods for DNA extraction, library preparation, sequencing, and bioinformatics from which a researcher can pick and choose, without a means for determining which is best for their application. Presented here is the initial development of a standard for microbial detection by NGS. This standard is a quantified mixture of bacterial cells, which when sequenced using our chosen method, produce an expected number of reads mapping to the genomes of the bacteria present in the standard mix. To develop this standard, pure cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were quantified by spectrophotometry (A600) and spread plates. Pure cultures were then diluted in a titration series from 1010 CFU to 102 CFU, DNA was extracted, and PCR‐free libraries were prepared. Prior to quantification and sequencing on the Illumina MiSeq, the libraries were pooled and concentrated. Similarly, two organism mixtures (P. aeruginosa + E. coli, P. aeruginosa + S. aureus, E. coli + P. aeruginosa, and S. aureus + P. aeruginosa) were prepared from the quantified pure cultures with one organism being held constant at 108 CFU and the other organism in a titration series from 108 to 102 CFU. DNA was then extracted from the mixtures, PCR‐free libraries prepared, and sequenced. The number of reads that mapped to each genome in the pure culture library titrations or in the mixed samples were quantified. For each of the bacteria in pure culture, read counts plateaued near 105 reads between 106 and 102 CFU. Thus, for individual or low DNA samples, each bacteria was detectable, but not quantifiable below 106 CFU. For TSB negative culture control, read counts were under 100 reads mapping to any of the queried genomes. For mixed cultures, where there was more background DNA to enable efficient reactions during library preparation, the plateau effect below 106 was not observed, consistent with the hypothesis that the presence of a threshold of DNA is necessary for efficient library preparation. With a background present, the coefficient of determination (R2) is 0.9667 with a P‐value of 0.000421 for a titration of E. coli in a P. aeruginosa background, demonstrating that there is a correlation between the number of bacteria in a sample and the number of reads mapping to that organism. Future work is necessary to develop the standard further, by including more organisms, and testing the mix in different backgrounds. Using a known mix of organisms in a known quantity will enable true comparison and evaluation of bacterial detection systems and workflows, which is necessary for the general acceptance of pathogen detection results. 68
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Reliable solutions for focused NGS
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