The Scientific component of the expedition
We wanted our project to have solid scientific foundations. To achieve this, we read many scientific publications and compiled a broad collection of protocols about the experiments we were going to do. We also asked for advice from researchers and other PhD students in the university. We already had good experience in molecular biology, microbiology and DNA sequencing, however, doing these techniques outside the lab was an additional challenge.
Our scientific interest was, in fancy words, to explore biodiversity in different lochs using an on-site metagenomics approach and do gene-mining to characterise genes with a potential industrial application. In other words, we wanted to know which microorganisms live in Loch Ness (constantly between 4-6˚C. degrees) and compare with microorganisms living in other lochs. We also wanted to understand how these microorganisms survive in constant cold conditions.
The process to identify the microorganisms involve five steps.
1) Sampling and processing. First, water and sediment was collected from the lochs. We registered the coordinates where the samples were collected and kept a record of the water temperature at each location. The processing involved filtering the water samples to concentre the microorganisms. The sediment was weighted and processed using a kit for DNA extraction from environmental samples (Power Soil from Qiagen). This enabled us to isolate a large number of microbes in the sample and keep all its content in a water solution.
2) DNA extraction. After lysing all the microbes, we centrifuge the samples to remove all the soil and microbial debris and recover the liquid phase where DNA is located. We then used a spin column, which contains a resin to which DNA binds. We passed the liquid phase through the column to isolate the DNA. After washing the column several times we eluted the DNA in a fresh tube and measured its quality and concentration.
3) Library preparation. From the previous step, we had a tube that contained a mix of all the DNA from the microbes that were present in the sediment sample. To read the samples, we fragmented the DNA into large chunks (80 Kbp) and labelled the DNA ends with adaptors required for the sequencing process.
4) DNA sequencing. To read the DNA sequences, we used the MinION from Oxford Nanopore. This machine has an array of very small pores that can measure electric charges in a very precise way. The adaptors added on the DNA ends directed the DNA fragments to the pores and the DNA strand passed through the pore. The DNA is composed of four nitrogenous bases, represented as ‘A’, ‘T’, ‘C’ and ‘G’. Each of these bases will produce a different electric signal. As the DNA slides through the pore, the machine measures the electric charge, giving a specific signal for each base, which is subsequently translated into a sequence of ATGC.
5) Data analysis. The first thing we did was to check the quality of the sequences obtained and discarded those sequences (reads) which failed the quality control. We then had two options, identify those pieces of DNA which belong to the same organism to rebuild their genome or look for specific signatures in the fragments (long DNA fragments) which are specific for each microbe. Afterwards, we compared the sequences we obtained with different DNA databases and evaluated the similarity of our sequences with those sequences previously characterised by other researchers.