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11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting BAC SUDOKU SEQUENCING STRATEGY FOR IN SILICO SCREENING OF LARGE-INSERT SOIL METAGENOMIC LIBRARIES Wednesday, 1st June 18:30 La Fonda NM Room (1st floor) Poster (PS‐1a.19) Scott Monsma 1 , Jinglie Zhou 2 , Alinne Pereira 2 , Blaine Pfeifer 3 , Timothy Bugni 4 , Scott R. Santos 2 , Megan Niebauer 1 , Erin Ferguson 1 , Ronald Godiska 1 , Chengcang Wu 5 , David Mead 1 , Mark R. Liles 2 1 Lucigen Corporation, 2 Auburn University, 3 State University of New York at Buffalo, 4 University of Wisconsin‐Madison, 5 Intact Genomics Inc Soil microorganisms express diverse bioactive natural products; however, the majority of soil microbes are recalcitrant to cultivation. We are using a metagenomic approach to bypass cultivation and directly capture the DNA from diverse microbial genomes in natural environments such as soils. A metagenomic library from an agricultural soil (Cullars Rotation, Auburn, AL) was constructed in a broad host‐range BAC vector that contained 19,200 clones with an average insert size of 110kb. Screening was accomplished using strategy termed BAC Sudoku sequencing, wherein a pooling strategy is used to multiplex the results, while still providing the ID of individual clones. BAC clones were sequenced in pools (row, column, and plate) using indexed primers and paired end reads on an Illumina HiSeq. Contigs were assembled for each pool and screened for secondary metabolite gene clusters using antiSMASH, resulting in identification of >1000 novel PKS/NRPS pathway‐containing clones. The cloned pathways are very divergent from known pathways, with the GC content varying from 41 to 76% and the amino acid identity of the KS domains ranging from 32 to 83% to the best matching BLAST hit. Expression of these PKS pathway‐containing clones in E. coli strain BTRA (engineered for polyketide precursor expression) has resulted in multiple clones with evidence for heterologous expression of a cloned PKS pathway. These results indicate a high degree of unique sequence space has been recovered from large‐insert metagenomic clones and a subset of these clones are capable of being heterologously expressed to produce secondary metabolites, thereby expanding our available resources for natural product discovery. 53
11th Annual <strong>Sequencing</strong>, Finishing, and Analysis in the Future Meeting ROLE OF PIRNAS IN THE ABSENCE OF ACTIVE TRANSPOSABLE ELEMENTS Wednesday, 1st June 18:30 La Fonda NM Room (1st floor) Poster (PS‐1a.20) Michael Vandewege 1 , Roy Platt 2 , David Ray 2 , Federico Hoffmann 1 1 Mississippi State University, 2 Texas Tech University Transposable elements (TEs) have the ability to mobilize throughout a genome and make up sizable proportions of mammalian genomes. A class of small RNAs, PIWI interacting RNAs (piRNAs), are part of a cellular pathway that protects genomes against the expression and proliferation of TEs. Posttranscriptional regulation of TE expression involves the guidance of PIWI proteins with endonuclease activity to TE transcripts via piRNAs that share sequence complementarity with TEs. In most mammals Long INterspersed Elements (LINEs) and Short INterspersed Elements (SINEs), are the dominant mobilizing TEs. However, in a rare instance, LINE and SINE elements are no longer mobilizing in the 13 lined ground squirrel (Ictidomys tridecemlineatus). This extinction occurred approximately 5 million years ago, and it is likely that piRNAs and PIWIs are no longer required to protect the genome against the expression of these elements. To determine the role of piRNAs in the absence of TEs, we sequenced and analyzed the piRNA repertoire of the ground squirrel at different life stages between birth and adulthood. The life stages coincide with the development of testis and expression of discrete classes of piRNAs. For direct comparisons, we also sequenced the piRNA repertoire of a juvenile and adult rabbit with active LINE and SINE elements. Among the rabbit piRNAs, there was a clear enrichment of LINE‐like sequences. By contrast, there was no TE enrichment among any of the squirrel piRNA repertoires. We found evidence that piRNAs were derived from different locations of the genome among the squirrel juvenile and adult life stages, suggesting the presence different piRNA biogenesis pathways during testis development. There was evidence of residual LINE expression and piRNA directed cleavage of LINE elements in the squirrel, although it is unclear whether or not LINE mRNA is haphazardly derived from immobile LINE insertions. It is apparent that piRNAs are still produced through typical pathways in ground squirrel testis, although it is not apparent they are actively defending the genome. It is possible that piRNAs are continuously produced and used in a preventive manner. 54
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Sequencing

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