Sequencing

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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting WHOLE GENOME APPROACH TO MICROBIAL FORENSICS OF PLANT PATHOGENS Wednesday, 1st June 18:30 La Fonda Mezzanine (2nd Floor) Poster (PS‐2a.09) Jacqueline Fletcher 1 , Trenna Blagden 1 , Ulrich Melcher 1 , Brittany Campos 2 , David Lin 3 , Kumar Hari 3 , Richard Winegar 2 1 National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Oklahoma State University, 2 MRIGlobal, 3 cBio, Inc A number of bacteria, viruses, and fungi pose serious health concerns to humans, threaten the U.S. economy, food and energy supplies, and/or the environment. The potential for pathogen use as biological weapons has been demonstrated, and the U.S. needs strong capabilities to respond to incidents involving such activity. Use of next‐generation sequencing (NGS) for applications in forensic investigation enhances microbial detection, strain matching and discrimination capabilities. The technology also allows for detection of all microbes in complex samples, detection of previously unknown organisms, and identification of specific signatures or genes. A multidisciplinary and multi‐institutional team, led by MRIGlobal.,Inc. in cooperation with the Department of Homeland Security, assessed the genome sequencing status and then performed whole genome sequencing and assembly for a collection of high threat human, animal and plant pathogens, including U.S. select agents, Genome sequence assessment surveys and surveillance plans were completed for the plant pathogenic bacteria Ralstonia solanacearum, Rathayibacter toxicus, Xylella fastidiosa, and Xan‐ thomonas oryzae, fungi Coniothyrium glycines, Puccinia graminis, Puccinia striiformis, Puccinia triticina, and Sybchytrium endobioticum, and oomycetes Peronosclerospora philippinensis and Sclerophthora rayssiae. Information and data gaps in existing genetic databases were used to set priorities for project collections and genome sequencing efforts. Nucleic acid purified from the plant pathogens Ralstonia solanacearum (four strains), Rathayibacter toxicus (two strains), Xylella fastidiosa subsp. pauca (six strains), Xanthomonas oryzae (pv. USA) and its near neighbor X. campestris pv. leersiae, and Coniothyrium glycines were obtained from expert collaborators. Illumina sequence libraries were prepared using Nextera XT. Whole genome 2×300 paired‐end sequencing was performed using the Illumina MiSeq instrument. Reads were filtered and trimmed using Trimmomatic. The iMetAMOS pipeline was used to optimize de novo assembly and perform quality checks. Elements of the pipeline include FastQC, SPAdes, IDBA, KmerGenie, and QUAST. Resulting assemblies were polished using Pilon; with Samtools and BLAST used to remove low coverage and contaminating contigs. Final assemblies were annotated through the NCBI Prokaryotic Genome Automatic Annotation Pipeline. An important outcome of this work was the creation of a NCBI‐hosted genomic database for selected microbial biothreat plant pathogens of priority interest to the United States. The project generated provided genomic information necessary to assist forensic investigators in genotyping, phylogenetic analysis, and sample matching of evidentiary materials. 65
11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting DISPLAYING METAGENOME SEQUENCING METADATA USING AN R SHINY APPLICATION Wednesday, 1st June 18:30 La Fonda Mezzanine (2nd Floor) Poster (PS‐2a.10) Chris Stubben, Patrick Chain Los Alamos National Laboratory Over 200,000 metagenome sequencing runs have been deposited in the Short Read Archive since 2015 and in March 2016 over 35 runs are submitted every hour. The FASTQ files from each run are associated with a large amount of metadata describing experiments, samples and studies. We created an R Shiny app that connects to the REST API at the European Nucleotide Archive and retrieves metadata from studies, samples and experiments and allows users to filter, map and summarize metagenome metadata. Nearly two‐thirds of metagenome samples since 2015 include locations and half of the remaining samples have place names that are geocoded using the Google maps API. The mean time between collection date and public release is 3.2 years and this delay has only declined slightly over the past 4 years. The app updates daily and displays studies and maps samples released over the last two weeks by default. 66
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Reliable solutions for focused NGS
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