October 25 2018

5:00 pm 1100 TLSB

EcoEvoPub Series

Graduate Student Presentations


Department of Ecology and Evolutionary Biology, UCLA

Anacapa Toolkit: an environmental DNA toolkit for processing multilocus metabarcode datasets

1. Environmental DNA (eDNA) metabarcoding holds promise as a rapid, affordable, noninvasive approach to monitor species and communities. Longstanding needs of the eDNA community are flexible informatics tools, comprehensive and customizable reference databases, flexibility across NGS platforms, fast multilocus metabarcode processing, and accurate taxonomic assignment. As bioinformatics tools continue to improve, addressing each of these demands within a single bioinformatics pipeline is becoming a reality.
2. Here we developed an open access flexible metabarcode sequence toolkit, Anacapa, that addresses the above goals to allow users to build comprehensive reference databases and process raw multilocus metabarcode sequence data to generate accurate community tables. Our database builder, Creating Reference libraries Using eXisting tools (CRUX), generates comprehensive reference databases for specific user defined metabarcode loci. The Anacapa toolkit sorts and process multiple metabarcode loci and is designed to process both unmerged and unpaired reads. The tool kit uses DADA2 to denoise, dereplicate, remove chimeras, and detect amplicon sequence variants (ASVs). Anacapa toolkit then utilizes Bowtie2 to match ASVs to the CRUX database. Taxonomy is then assigned to ASVs with confidence scores using Bayesian Least Common Ancestor (BLCA) method. The toolkit also includes an R package, ranacapa, for automated results exploration through standard biodiversity statistical analysis.
3. We demonstrate the performance of the Anacapa Toolkit to generate comprehensive user defined reference databases relative to published databases and compare taxonomic assignment of the Anacapa Bowtie 2 BLCA classifier relative to the progenitory BLAST BLCA classifier (Gao et al. 2017) using standardized cross validation methods. We further demonstrate the Anacapa tool kit using empirical eDNA from terrestrial and marine samples collected in southern California through the CaleDNA project.
4. The ability of Anacapa toolkit to generate metabarcode specific databases, process multilocus data, retain all read types, and expand non traditional eDNA targets broadens the exploration of eDNA from soils, sediments, and water to assist in biodiversity management. Anacapa software and source code are open and additionally available in a virtual container to ease installation.

Department of Ecology and Evolutionary Biology, UCLA

Application of CRISPR-Cas9 Edited Cells to Evaluate Gene Function in the North American Gray Wolf (Canis lupus)

With the advent of next generation sequencing technologies, the identification of genes under selection has become a central focus of evolutionary studies. Yet, such studies are often speculative and without independent support. In this study, we utilize the resource of CRISPR-Cas9 edited North American gray wolf (Canis lupus) keratinocytes to functionally test the differential antibacterial properties of canine beta-defensin 3 (CBD103) genotypes. CBD103 is a particularly interesting and suitable gene to consider given: 1) a mutation in CBD103 has undergone a selective sweep in gray wolves, leading to an easily observable phenotype (coat color) in wild populations; 2) the mutation has been well-described and can be manipulated in a laboratory setting; and 3) the functional role that CBD103 plays in innate immune response can be tested using in vitro methods. The cell lines used in this study were cultured from wild type keratinocytes and immortalized. After immortalization, CRISPR-Cas9 was used to insert the naturally observed 3bp deletion and generate heterozygous and homozygous mutant lines. We report preliminary results of a bacterial killing assay where lysate from heterozygous and homozygous mutant cells diminish the growth of Staphylococcus aureus relative to lysate from wild type cells. This framework of developing CRISPR-Cas9 cells cultured from a non-model system can be more broadly applied. Other candidate genes under selection in a wide variety of systems beyond wolves can be tested with challenges such as pathogens, varied temperatures, or altered cell conditions to test functional hypotheses to more definitively build a case for genotype-phenotype relationships.




































































































































































































































































































































































































































































































































































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