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Culture purification and DNA extraction procedures suitable for next-generation sequencing of euglenids

Author:
Gumińska, Natalia, Płecha, Magdalena, Walkiewicz, Halszka, Hałakuc, Paweł, Zakryś, Bożena, Milanowski, Rafał
Source:
Journal of applied phycology 2018 v.30 no.6 pp. 3541-3549
ISSN:
0921-8971
Subject:
DNA, Euglena, absorbance, cetyltrimethylammonium bromide, fluorometry, gel electrophoresis, high-throughput nucleotide sequencing, molecular weight, washing
Abstract:
In the present study, five different DNA extraction procedures were examined to determine their effectiveness for extracting DNA suitable for NGS applications. This included two silica-membrane spin column kits, phenol:chloroform, and two CTAB-based methods. Spectrophotometric and fluorimetric measurements as well as standard gel electrophoresis were used as criteria for evaluating the quantity and quality of the isolated DNA prior to the sequencing. Herein, the method of establishing and maintaining axenic Euglena cultures is also presented. The modified CTAB-based method proved to be highly efficient. In terms of DNA quantity and purity (according to the absorbance ratios), the chosen method resulted in DNA of high molecular weight and quality, which fulfills the library construction requirements. Genomic DNA of Euglena hiemalis (CCAP 1224/35) and E. longa (CCAP 1204-17a) isolated using the suggested protocol had been successfully sequenced on the Illumina HiSeq platform. A modified, rapid CTAB-based method of total DNA isolation from Euglena has been described. In terms of the DNA quantity and quality, the protocol devised involving the washing step with DMSO:acetonitrile proved superior to the commonly used, commercially manufactured kits and isolation with phenol:chloroform. The method is also less labor-intensive and time-consuming than the traditional CTAB-based protocol.
Agid:
6273324