Date of Award
Open Access Thesis
Master of Science (MS)
David M. Hayes
Sherry L. Harrel
DNA is found to be free and ubiquitous in the environment where it is no longer associated with the source organism, and is also known as environmental-DNA (eDNA). Methods optimized for specific environments may be able supplement insight to local taxa richness. With the advent of high throughput sequencing and the proliferation of sequence data in public repositories, insights to the biodiversity of communities at the molecular level have been possible. Thus, this study compared commonly used DNA capture (water precipitation and filtration) and extraction (MoBio's PowerWater, Qiagen's DNeasy Blood and Tissue Kit, and a CTAB protocol) methods in their ability to isolate eDNA for the purpose of metabarcoding a section of the ribosomal small subunit 18 S (18s) and the cytochrome oxidase I (COI) gene regions. The 18s sequence data is non-reportable due to lack of sequence quality, and MoBio's PowerWater did not yield DNA suitable concentrations. CTAB and DNeasy extractions yielded successful PCR reactions and high-throughput sequencing (HTS). When combined with their respective replicates, CTAB and DNeasy were determined to have genus richness ( -diversity) of 25 and 24, respectively of benthic macroinvertebrates with 20 taxonomic determinations being shared between the two methods. After conducting Jaccard's dissimilarity index and constructing ordination plots using non-metric multidimensional scaling (NMDS), this study was not able to reveal differences in the amount of taxa richness between CTAB and DNeasy, which implied extraction methods may not be a limiting factor in detected taxa richness.
Copyright 2017 Michael Triston Mullins
Mullins, Michael Triston, "Sample Collection and DNA Extraction Methods for Environmental DNA Metabarcoding in Headwater Streams" (2017). Online Theses and Dissertations. 544.