Developing methods for on-site DNA sequencing

Student thesis: Master thesis (including HD thesis)

  • Peter Rendbæk
4. term, Biotechnology, Master (Master Programme)
The efficiency of wastewater treatment plants is largely determined by their microbial composition. Therefore, identification of the microbial community is an important part of running a particular wastewater treatment plant and understanding how it functions. Currently, this is done in highly specialized laboratories, but this limits the method to being only able to look back at changes, and it is not being used to guide operational decisions. However, ongoing advancement of sequencing technology (Oxford Nanopore MinION) and automated sample preparation makes it theoretically possible to move sequencing out of the laboratory. However, to make this a reality, there is a need for a fast, cheap, reliable and highly mobile DNA extraction that works on par with state-of-the-art extraction methods.

In this thesis, an easy to use, fast and highly mobile DNA extraction method is developed. The method is based on a power tool with a 3D printed adapter for bead-beating based lysis of cells, and DNA is isolated using solid phase reversible immobilization beads. The method was compared to the state-of-the-art and recommended DNA extraction method for the field of activated sludge: the MiDAS field guide. The comparison of the methods was made on several levels including the amount of extracted DNA, purity and fragmentation. Furthermore, 16S rRNA amplicon sequencing was used to evaluate any potential extraction bias in the observed microbial community.

It was shown that the proposed DNA extraction method did not introduce a bias in microbial community composition and performed just as good on yield and purity. Correspondingly, it cut the total time for DNA extraction down to roughly 10 minutes compared to the 1-hour standard protocol.

However, further optimization is needed to make sure the method fulfills the high purity and long DNA fragment length requirement for the MinION. Overall, the developed approach provides a foundation for moving the DNA extraction and sequencing out of the laboratory and into the field.
LanguageEnglish
Publication date5 Jun 2017
Number of pages76
ID: 259181974