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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting SEQUENCING STRATEGY FOR THE WHOLE GENOME OF SHEEPPOX VIRUS Wednesday, 1st June 18:30 La Fonda NM Room (1st floor) Poster (PS‐1a.13) Vitaliy Strochkov, Yerbol Burashev, Nurlan Sandybayev Sandybayev Research Institute for Biological Safety Problems, MES Republic of Kazakhstan Sheeppox virus (SPPV) is a widespread and economically significant pathogen considered endemic in central Asia and member of the genus Capripoxvirus. Capripoxvirus genomic DNA are doublestranded with lengths of around 150 kbp, with SPPV, goatpox virus (GTPV), and lumpy skin disease virus species generally sharing at least 96% nucleotide identity. Strains of SPPV and GTPV share at least 147 genes. A previous strategy of shotgun sequencing of poxviruses is incomplete cleavage of genomic DNA with restriction endonucleases, e.g. Tsp509I. Fragments ranging in size from 1 to 2.5 kbp were randomly cloned into a plasmid and sequenced. By using this strategy, complete nucleotide sequences for several strains of sheep pox and goat poxvirus was defined by our group (JVI, 2002, 76(12):6054‐ 6061). We have adopted directed sequencing methods to rapidly obtain complete genome sequences from capripoxviruses. PCR arrays yielding overlapping PCR amplicons are being used to generate genomically tiled Sanger sequence data. Initial primer candidates were generated against the A strain genomic sequence, and targeting tiled amplicons of 1200 bp and overlapping by 500 bp. Specific primer selection was conducted to match annealing temperatures and to best match conserved regions relative to other capripoxviruses to reduce PCR failures resulting from sequence diversity. Primers were manufactured in 96‐well plates and arranged to group primer sets with similar annealing temperatures. This enables generation of specific sequencing templates with redundancy at all sites using amplification. DNA sequencing is performed by dideoxy sequencing method with use of termination dideoxy nucleotide (Sanger’s method) on an automatic 16‐capillary Genetic Analyzer 3130xl, Applied Biosyste As a polymer for capillaries POP‐7 is used. Production of termination DNA outputs is carried out by the method of cyclic sequencing. These long read (up to approximately 1kbp) products are of particular use in sequence assembly, which is conducted de novo and by mapping against genomic reference genomes using both commercial and open source software. With the use of direct sequencing of PCR products, we obtained nucleotide sequence of complete genomes for three sheeppox virus strains from different geographic regions using PCR and Sanger sequencing technologies currently in place at RIBSP. The work was performed under a study for Comparative genomics especially dangerous poxviruses (orthopox and capripox) in Kazakhstan. 47
11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting USING NOVEL METHODS TO ASSEMBLY PLANT MITOCHONDRIAL GENOMES: AN EXAMPLE FROM THE MIMOSOID LEGUME GENUS LEUCAENA Wednesday, 1st June 18:30 La Fonda NM Room (1st floor) Poster (PS‐1a.14) Sealtiel Ortega Rodriguez, Donovan Bailey New Mexico State University Plant mitochondria host structurally complex low gene content genomes known for high rates intracellular recombination, sequence loss, and sequence acquisition. As a result of these complexities very few such genomes have been published. This is even true for the ecologically and economically important legumes, where mitochondrial genomes are available for just fives species of closely related papilionoids. To diversify the representation of available legume mitochondrial genomes, we generated a draft sequence assembly for a member of the subfamily Mimosoideae, Leucaena trichandra. Mimosoid legumes are best represented throughout the tropics, where they are often critical species in natural ecosystems while also providing nearby human populations with a variety of important resources. Using 60X PacBio sequence data with 14.5kb average read length we employed a novel iterative reference seeded approach to de novo assemble the L. trichandra mitochondrial genome data from among a complex mix of nuclear and cpDNA reads. We discuss what we have learned about this iterative assembly approach along with the preliminary mitofy annotations of the draft genome. 48
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Reliable solutions for focused NGS
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