Developing methods for on-site DNA sequencing

Abstract

Effektiviteten af spildevandsrensningsanlæg er i høj grad bestemt af deres mikrobielle sammensætning. Derfor er identifikation af det mikrobielle samfund en vigtig del af at køre et bestemt spildevandsrensningsanlæg og forstå, hvordan det fungerer. I øjeblikket gøres dette i højt specialiserede laboratorier, men det begrænser metoden til at se tilbage ved ændringer og bruges ikke til at styre operationelle beslutninger. Imidlertid gøres der løbende fremskridt med sekventeringsteknologi (Oxford Nanopore Minion) og automatiseret prøveforberedelse gør det teoretisk muligt at flytte sekventering ud af laboratoriet. Men for at gøre dette til en realitet er der et behov for en hurtig, billig, pålidelig og meget mobil DNA-ekstraktion, der fungerer i lighed med State-of-the-art ekstraktionsmetoder. I denne afhandling er der udviklet en brugervenlig, hurtig og meget mobil DNA-ekstraktionsmetode. Metoden er baseret på et kraftværktøj med en 3D-trykt adapter til bead beating baseret lysering af celler, og DNA isoleres ved anvendelse af fastfase-reversible immobiliseringsbeads . Metoden blev sammenlignet med den nyeste og anbefalede DNA-ekstraktionsmetode for feltet af aktiveret slam-MiDAS-feltguide. Sammenligningen af fremgangsmåderne blev lavet på flere niveauer, inklusive mængden af ekstraheret DNA, renhed og fragmentering. Desuden blev 16S rRNA amplicon-sekventering brugt til at evaluere enhver potentiel ekstraktionsforstyrrelse i det observerede mikrobielle samfund. Det blev vist, at den foreslåede DNA-ekstraktionsmetode ikke introducerede en bias i mikrobielle samfunds sammensætning og præsterede lige så godt på udbytte og renhed. Tilsvarende reduceres den samlede tid til DNA-extraction ned til ca. 10 minutter sammenlignet med 1-timers standard protokollen. Der er dog behov for yderligere optimering for at sikre, at metoden opfylder kravet om høj renhed og lang DNA-fragmentlængde for MinION. Samlet set giver den udviklede tilgang et fundament for at flytte DNA-ekstraktionen og sekventeringen ud af laboratoriet og ind i feltet.

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.

A master thesis from Aalborg University

Developing methods for on-site DNA sequencing

[Udvikling af metoder til on-site DNA sekventeringdvilking]