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Optimisation of DNA extraction from the crustacean Daphnia
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Gonçalves Athanasio, Camila, Chipman, James K., Viant, Mark R. and Mirbahai, Leda (2016) Optimisation of DNA extraction from the crustacean Daphnia. PeerJ, 4 . e2004. doi:10.7717/peerj.2004
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WRAP-optimisation-DNA-extraction-crustacean-daphnia-Mirbahai-2016.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (977Kb) | Preview |
Official URL: http://dx.doi.org/10.7717/peerj.2004
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.
| Item Type: | Journal Article | |||||||||
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| Subjects: | Q Science > QL Zoology | |||||||||
| Divisions: | Faculty of Medicine > Warwick Medical School > Biomedical Sciences > Cell & Developmental Biology Faculty of Medicine > Warwick Medical School > Biomedical Sciences Faculty of Medicine > Warwick Medical School |
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| Library of Congress Subject Headings (LCSH): | Daphnia -- Functional genomics | |||||||||
| Journal or Publication Title: | PeerJ | |||||||||
| Publisher: | PeerJ, Ltd. | |||||||||
| ISSN: | 2167-8359 | |||||||||
| Official Date: | 10 May 2016 | |||||||||
| Dates: |
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| Date of first compliant deposit: | 4 February 2019 | |||||||||
| Volume: | 4 | |||||||||
| Article Number: | e2004 | |||||||||
| DOI: | 10.7717/peerj.2004 | |||||||||
| Status: | Peer Reviewed | |||||||||
| Publication Status: | Published | |||||||||
| Access rights to Published version: | Open Access | |||||||||
| RIOXX Funder/Project Grant: |
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