An optimized method for high quality DNA extraction from microalga Prototheca wickerhamii for genome sequencing

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.

A survey on the incidence of Prototheca mastitis in dairy herds in Lublin province, Poland

“…Phenotype-based identification was then confirmed with molecular methods. For these to be applied, DNA extraction was performed, essentially as reported elsewhere (Jagielski et al, 2017b). Species-and genotype-level identification was carried out using genotype-specific PCR assays, as described by Roesler et al (2006) and with a newly described PCR-restriction enzyme analysis (PCR-REA) assay of partial cytB gene, as proposed by Jagielski et al (2018).…”

Identification and characterization of yeasts from bovine rumen for potential use as probiotics

“…There are no reports of isolation of these microalgae in the rumen. However, members of this genera are ubiquitously distributed in the environment (Jagielski et al ).…”

Prevalence of Prototheca spp. on dairy farms in Poland – a cross‐country study

“…This included evaluation of colony and cellular morphology, and carbohydrate assimilation profiling, with the API 20C AUX system (Biomerieux ® , Marcy‐l'Étoile, France). Phenotype‐based identification was confirmed by molecular methods, performed on total genomic DNA whose extraction procedure was detailed elsewhere (Jagielski et al ., ). Species‐ (genotype‐)level identification was carried out using genotype‐specific PCR assays, as described by Roesler et al .…”

“…Etoile, France). Phenotype-based identification was confirmed by molecular methods, performed on total genomic DNA whose extraction procedure was detailed elsewhere (Jagielski et al, 2017). Species-(genotype-) level identification was carried out using genotype-specific PCR assays, as described by Roesler et al (2006) and with a PCR restriction-enzyme analysis (PCR-REA) assay of partial cytB gene, as proposed by Jagielski et al (2018).…”