Optimisation of DNA extraction from the crustacean Daphnia

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.

Keywords: Epigenetics; Genomics; High throughput sequencing; Omics.

Figure 1. Average DNA concentration measured with NanoDrop 8000 and SYBR Green I.

Error bars indicate standard error of the mean. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001; No statistically significant differences were observed for CTAB probably due to greater data variability.

Figure 2. Quality assessment of DNA samples extracted with MasterPure DNA purification kit and Agencourt DNAdvance.

(A) 1% agarose gel for MasterPure samples. (B) 1% agarose gel for DNAdvance samples; same amount of DNA was loaded onto each lane. Lanes 1–3 present fragmented DNA spread along the lane, while lanes 4–9 present majority of the DNA in a distinct band above the 10 kb marker position. (C) TapeStation results for MasterPure. (D) TapeStation results for DNAdvance. Dark grey—Homogenisation with plastic pestle; Light grey—homogenisation ceramic beating beads. (E) DNA fragments distribution for MasterPure samples. (F) DNA fragments distribution for DNAdvance samples. Error bars in (E) and (F) indicate standard error of the mean.

Figure 3. Example of downstream analyses of DNA samples.

(A) Chromatogram of hydrolysed Daphnia DNA sample extracted with CTAB. (B) Chromatogram of hydrolysed Daphnia DNA sample extracted with MasterPure DNA purification kit. Shadowed area indicates 5-methylcytosine position. (C) Artificially methylated DNA generated from DNA extracted from Daphnia with MasterPure DNA purification kit. Sample was methylated using SssI methyltransferase (NEB, USA), treated with sodium bisulfite to preserve DNA methylation patterns, amplified using PCR and sequenced on the ABI 3730. (D) Unmethylated DNA generated from Daphnia with whole genome amplification (Sigma Aldrich, Dorset, UK) using MasterPure DNA purification kit. Shadowed area indicates cytosine-guanine position. (E) DNA fragment size selection (3 kb–9 kb) for mate pair library construction. (F) Quality scores for mate pair sequencing data.

Figure 4. Summary of conclusion regarding the best methods of tissue homogenisation and DNA extraction.

^∗ CTAB is not suitable for samples that will be used for mass spectrometry. ^∗∗ Samples need to be quantified with fluorescence-based method.