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11th Annual Sequencing, Finishing, and Analysis in the Future Meeting DETECTION AND GENETIC CHARACTERIZATION OF BURKHOLDERIA PSEUDOMALLEI AND CLOSELY RELATED SPECIES DIRECTLY FROM SOIL USING A CUSTOM AMPLICON SEQUENCING ASSAY. Wednesday, 1st June 18:30 La Fonda NM Room (1st floor) Poster (PS‐1a.17) James Schupp 1 , Jason Sahl 2 , Rebecca Colman 1 , Jordan Buchaggen 1 , Josie Delisle 3 , John Gillece 1 , Adam Vazquez 2 , Joseph Gennarelli 2 , Carina Hall 2 , Joseph Busch 2 , Vanessa Theobald 3 , Glenda Harrington 3 , Mirjam Kaestli 3 , Mark Mayo 3 , Bart Currie 3 , David Engelthaler 1 , Paul Keim 1,2 , David Wagner 2 1 TGen, 2 Northern Arizona University, 3 Menzies School of Health Research Rapid detection and characterization of clinical and forensic materials suspected of containing Burkholderia pseudomallei, a public health and potential bioterrorism agent endemic to Southeast Asia and Northern Australia, would be of enormous benefit to epidemiological and forensic investigations. Current methodologies, such as real time PCR, allow rapid detection but only limited characterization. High Throughput Sequencing (HTS) of multiple informative genetic loci can provide efficient, rapid detection and differentiation from near neighbor species, as well as fine scale genetic characterization. We have developed a 67 locus amplicon sequencing system that results in 1) detection of B. pseudomallei; 2) differentiation from B. mallei and near neighbor species; 3) potential detection of strain mixtures; 4) differentiation within B. pseudomallei; and 5) virulence gene characterization (10 vir genes), within 24‐48 hours. The system couples highly multiplexed amplification reactions with a universal amplicon indexing system, resulting in efficient multilocus amplicon sequencing from potentially hundreds of samples in a single Illumina MiSeq sequencing run. We have detected B. pseudomallei in soils from Northern Australia, down to near single genome copy, as well as detection of near neighbor species, such as B. ubonensis. In analyzing soils from AZ, which show a very different bacterial community, no detection of B. pseudomallei and low level detection of other near neighbor species. We demonstrate detection of Burkholderia species mixtures as well as B. pseudomallei strain mixtures, utilizing variation within the targeted species specific, MLST and virulence loci. In samples containing a single predominant strain of B. pseudomallei, we also demonstrate strain level phylogenetic classification using the Whole Genome Focused Array SNP Typing (WG‐FAST) pipeline directly from the soil sample. 51
11th Annual Sequencing, Finishing, and Analysis in the Future Meeting INCREASED RATES OF SPONTANEOUS DUPLICATIONS AND DELETIONS UNDER HEAVY METAL EXPOSURE IN DAPHNIA MUTATION ACCUMULATION LINES Wednesday, 1st June 18:30 La Fonda NM Room (1st floor) Poster (PS‐1a.18) Frédéric Chain, Jullien Flynn, James Bull 1 , Melania Cristescu McGill University Heavy metals are pervasive environmental pollutants and toxic contaminants that are of major con‐ cern for ecosystem and human health. Metals can induce oxidative and DNA damage, promoting mutations through DNA repair and replication. Errors occurring during these processes are particularly important mechanisms generating copy number variations (CNVs) deletions, duplications and insertions but little is known about the long‐term effects of metals on genome‐wide mutations such as CNVs. Genomics approaches allow for nucleotide‐resolution detection of CNVs, although these methods are limited by the quality and architecture of the reference genome. We analyzed CNVs among 42 whole genomes of clonal Daphnia mutation accumulation lines, all seeded by the same progenitor ancestor, but maintained in conditions either with or without metals at ecologically relevant concentrations. After propagating lines for an average of 100 generations, we found higher basal rates of both gene deletions and gene duplications in lines exposed to a mixture of nickel and copper compared to controls. However, our CNV rate estimates are limited to unique genomic regions to reduce the bias from reads mapping to multiple locations, which can be due to the mis‐assembly of divergent alleles in diploid genomes. Our results show that metal exposure can increase the incidence of large‐scale heritable mutations, but that complex genomes and assemblies limit the extent to which we can perform truly “genome‐wide” analyses. 52
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Reliable solutions for focused NGS
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