Developing methods for on-site DNA sequencing

Udvikling af metoder til on-site DNA sekventeringdvilking

Rendbæk, Peter

4. term

Biotechnology, Master

2017

2017-06-05

76

Rensningsanlægs effektivitet afhænger i høj grad af deres mikrobielle sammensætning. At kende de tilstedeværende mikroorganismer er derfor vigtigt for driften og for at forstå, hvordan anlægget fungerer. I dag sker analysen primært i specialiserede laboratorier, hvilket betyder, at resultaterne mest bruges til at se tilbage på ændringer og sjældent til at styre daglige beslutninger. Bærbare sekventeringsværktøjer som Oxford Nanopore MinION og automatiseret prøveforberedelse gør det teoretisk muligt at flytte sekventering ud af laboratoriet. For at realisere dette kræves en hurtig, billig, pålidelig og meget mobil DNA-ekstraktionsmetode, der kan matche de bedste standarder. Dette speciale udvikler en brugervenlig, hurtig og feltklar DNA-ekstraktionsmetode. Metoden bruger et elværktøj med en 3D-printet adapter til bead-beating (kraftig omrystning med små kugler for at bryde celler), og DNA isoleres med solid phase reversible immobilization (SPRI)-perler, hvor magnetiske perler midlertidigt binder DNA, så det kan vaskes og frigives. Metoden blev sammenlignet med den anbefalede state-of-the-art for aktivt slam (MiDAS feltguide) med fokus på mængden af udtrukket DNA, renhed og fragmentering. Derudover blev 16S rRNA amplicon-sekventering (markergen-sekventering til at profilere mikrobielle fællesskaber) brugt til at teste, om ekstraktionen skævvrider den observerede mikrobesammensætning. Resultaterne viste, at den foreslåede metode ikke introducerede bias i den observerede mikrobielle sammensætning og matchede standarden i udbytte og renhed. Samtidig reducerede den den samlede tid til DNA-ekstraktion til omkring 10 minutter sammenlignet med cirka 1 time for standardprotokollen. Der er dog behov for yderligere optimering for at opfylde MinIONs krav om høj renhed og lange DNA-fragmenter. Samlet set lægger tilgangen et fundament for at flytte DNA-ekstraktion og sekventering fra laboratoriet og ud i felten.

The performance of wastewater treatment plants relies heavily on their microbial makeup. Knowing which microorganisms are present is important for day-to-day operation and for understanding how a plant works. Today, this analysis is mostly done in specialized laboratories, so results tend to be retrospective and rarely guide real-time decisions. Portable sequencing tools like the Oxford Nanopore MinION and automated sample preparation make it theoretically possible to move sequencing out of the lab. To do so, we need a fast, inexpensive, reliable, and highly mobile DNA extraction method that performs on par with current best practices. This thesis develops a simple, rapid, field-ready DNA extraction method. It uses a common power tool with a 3D-printed adapter for bead-beating (vigorous shaking with small beads to break open cells), and isolates DNA with solid phase reversible immobilization (SPRI) beads, where magnetic beads temporarily bind DNA for washing and release. The method was compared to the recommended state-of-the-art for activated sludge (the MiDAS field guide), assessing DNA yield, purity, and fragmentation. To check for extraction bias, 16S rRNA amplicon sequencing (a marker-gene approach to profile microbial communities) was used to see whether the observed community composition was skewed. The results showed that the proposed method did not introduce bias in the observed microbial community and matched the standard in yield and purity. It also cut total DNA extraction time to roughly 10 minutes compared to about 1 hour for the standard protocol. However, further optimization is needed to meet the MinION’s requirements for high purity and long DNA fragments. Overall, this approach lays the groundwork for moving DNA extraction and sequencing from the lab into the field.

[This abstract was generated with the help of AI]

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