Optimisation of DNA extraction from the crustaceanDaphnia

Abstract

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.

Home-Made Cost Effective Preservation Buffer Is a Better Alternative to Commercial Preservation Methods for Microbiome Research

“…Because NAP and RNA later ® have similar densities to those of the bacterial cells that were released from the swab into solution, it is difficult to re-pellet the cells via centrifugation. Furthermore, RNA later ® can interfere with the DNA extraction process (Athanasio et al, 2016 ). To remove NAP and RNA later ® from our samples, we diluted them by adding equal volumes of ice-cold phosphate-buffered saline (PBS) before centrifugation at 6000 g for 15 min as suggested in the manual (Life Technologies, 2011 ) and discarded the supernatant.…”

A comprehensive epigenomic analysis of phenotypically distinguishable, genetically identical female and male Daphnia pulex

“…Cultures of Daphnia pulex Eloise Butler strain (genotypes EB31 and EB45, originally sampled from Eloise Butler pond in Minnesota, [59] were maintained in standard COMBO as previously described [30,60,61]. To induce male Daphnia, sexually mature individual female Daphnia were treated with the crustacean reproductive hormone, methyl (2E, 6E)-farnesoate (MF) at a final concentration of 400 nM.…”

“…Genomic DNA and RNA were extracted from a pool of samples with a mixture of different ages (3, 8 and 15 days old) using MasterPure DNA purification kit (Epicentre, USA) and RNeasy Micro Kit (Qiagen Ltd., UK), respectively as described by Athanasio et al 2016 and 2018 [61,62]. DNA for the whole genome bisulfite sequencing (WGBS) was extracted from both genotypes (EB31 & EB45), from 3 female and 3 male Daphnia pools from each genotype.…”

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

“…The ideal measures for pure DNA are shown in Figure 2, yet in some cases, they are exceeded. These measures can be influenced by many aspects, such as invertebrate chitin (Athanasio et al 2016) and RNA. Spectrophotometry of extracted DNA can be affected by the presence of co-extracted RNA by inflating A 260 values, therefore the ratios used for purity evaluations are skewed upwards.…”