Abstract
BackgroundThe complex cell wall structure of algae often precludes efficient extraction of their genetic material. The purpose of this study was to design a next-generation sequencing-suitable DNA isolation method for unicellular, achlorophyllous, yeast-like microalgae of the genus Prototheca, the only known plant pathogens of both humans and animals. The effectiveness of the newly proposed scheme was compared with five other, previously described methods, commonly used for DNA isolation from plants and/or yeasts, available either as laboratory-developed, in-house assays, based on liquid nitrogen grinding or different enzymatic digestion, or as commercially manufactured kits.ResultsAll five, previously described, isolation assays yielded DNA concentrations lower than those obtained with the new method, averaging 16.15 ± 25.39 vs 74.2 ± 0.56 ng/µL, respectively. The new method was also superior in terms of DNA purity, as measured by A260/A280 (−0.41 ± 4.26 vs 2.02 ± 0.03), and A260/A230 (1.20 ± 1.12 vs 1.97 ± 0.07) ratios. Only the liquid nitrogen-based method yielded DNA of comparable quantity (60.96 ± 0.16 ng/µL) and quality (A260/A280 = 2.08 ± 0.02; A260/A230 = 2.23 ± 0.26). Still, the new method showed higher integrity, which was best illustrated upon electrophoretic analysis. Genomic DNA of Prototheca wickerhamii POL-1 strain isolated with the protocol herein proposed was successfully sequenced on the Illumina MiSeq platform.ConclusionsA new method for DNA isolation from Prototheca algae is described. The method, whose protocol involves glass beads pulverization and cesium chloride (CsCl) density gradient centrifugation, was demonstrated superior over the other common assays in terms of DNA quantity and quality. The method is also the first to offer the possibility of preparation of DNA template suitable for whole genome sequencing of Prototheca spp.
Highlights
The complex cell wall structure of algae often precludes efficient extraction of their genetic material
The low A260:280 ratios may either be due to heavy protein contamination or residual phenol associated with the Quality assessment of extracted DNA To assess the quality and purity of nuclear DNA extracted with a new, optimized protocol, and to evaluate its usefulness for genome sequencing, the whole-genome sequencing (WGS) was performed with the Illumina MiSeq platform (Illumina, San Diego, USA)
This was done for two genomic DNA samples obtained by the N method with and without cesium chloride (CsCl) ultracentrifugation to ensure that this step is crucial for separation of nuclear from organellar DNA
Summary
The complex cell wall structure of algae often precludes efficient extraction of their genetic material. The purpose of this study was to design a next-generation sequencing-suitable DNA isolation method for unicellular, achlorophyllous, yeast-like microalgae of the genus Prototheca, the only known plant pathogens of both humans and animals. The genus Prototheca (Trebouxiophyceae) accommodates unicellular, achlorophyllous, yeast-like microalgae, ubiquitously distributed in the environment. Normally saprophytic, these organisms may, under certain conditions, give rise to infections in both humans and animals. One of the major gaps has been a paucity of understanding of the pathobiology and mechanisms underlying the protothecal disease. For this to be revealed, a considerable amount of genetic data is required. No such reports have been published, a possible reason for this being the lack of a rapid and efficient method for high-quality genomic DNA extraction from Prototheca spp
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