Optimisation of DNA extraction from the crustacean Daphnia.
Optimisation of DNA extraction from the crustacean Daphnia.
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作者：
Athanasio CG;Chipman JK;Viant MR;Mirbahai L
水蚤是表型可塑性、适应性和微进化机制研究的重要模式生物，对基因组学资源的需求日益增加。任何基因组学分析的关键步骤，如高通量测序，都是获得足够高质量的DNA。尽管存在从一些物种中提取基因组DNA的商业试剂盒，但从水蚤和其他几丁质物种中制备高质量的DNA可能具有挑战性。在这里，我们优化了针对不同下游分析定制的组织均质化、DNA提取和定量方法（例如LC-MS/MS、Hiseq、配偶对测序或Nanopore）。我们证明，如果大水蚤作为整个动物（包括甲壳）均质，基于吸光度的DNA定量方法明显高估了DNA的数量，导致实验使用的起始材料不足，例如测序文库的制备。这是由于水蚤甲壳中几丁质在260 nm处具有较高的折射率。因此，除非通过隔夜蛋白酶消化去除甲壳，否则提取的DNA应采用基于荧光的方法进行定量。然而，隔夜蛋白酶消化会导致DNA部分断裂，因此制备的DNA不适合下游需要高分子量DNA的方法，如PacBio，配偶对测序和Nanopore。综上所述，我们发现MasterPure DNA纯化试剂盒，结合冷冻组织研磨，是提取高分子量DNA的最佳方法，只要提取的DNA采用荧光定量方法。该方法产生高收率和高分子量的DNA(3.10±0.63 ng/µg干质量，片段>60 kb)，不含有机污染物（苯酚，氯仿），适用于大量下游分析。
Daphnia are key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA from Daphnia spp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that if Daphnia magna are homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin in Daphnia’s carapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses.