“First of all, I would like to say thank you for providing me an opportunity to be awarded an international mobility scholarship for my summer course program in the University of Tartu, Estonia. This opportunity allows me to broaden my horizon not only in terms of academic mission but also in soft skill development. “
Mayang Christy Perdana
PhD student, Department of Applied Ecology, FŽP, CZU
The day 1 program started by gathering all the summer course participants in the building Vana Anatoomikum, University of Tartu, to deliver an overview of the course. After that, all the participants were separated into several groups based on their enrolled sub course. The day 1 program in the metabarcoding sub course group was initiated by lecture from Prof. Leho Tedersoo. This lecture provided a general understanding of DNA barcoding and metabarcoding. DNA barcoding is the identification of specimens based on the specific DNA sequence while metabarcoding identification of multiple species from a single bulk sample and meta itself means “beyond”. This course emphasized that DNA is divided into two categories namely extracellular and intracellular DNA. Extracellular DNA represents fine particles and free-floating or sediment, while intracellular DNA is DNA derived from living organisms. Metabarcoding is commonly characterized by using next-generation sequencing. An outline of metabarcoding approach consists of:
- Sampling
- DNA Extraction
- DNA amplification
- Sequencing
- Sequence analyses
- Identification of taxa
Solid samples such as soil, the short-term storage can be done in freezing condition -80°C called the golden standard (maximum -20°C). It was suggested to avoid Freeze-thaw-freeze sample as it may change the DNA composition. A larger sample is not required for bacteria. Short-term storage can be done at 4°C in 14 days. The greater organism is, the more sample is needed.
The second lecture was delivered by Dr. Sten Anslan which was going deeper not only into introduction of metabarcoding but also into the basics of DNA extraction, PCR, and primer selection. The primer selection should be adjusted by the organism target. For bacteria, 16srRNA is used. For eukaryotes and fungi, regions of 18srRNA and ITS are suitable, respectively. It is recommended to use the same method for DNA extraction to decrease the variability in our the studies.
The day 2 was allocated for practical lab session in the lab namely the activities of DNA extraction, DNA quality control, and PCR preparation. The DNA extraction was conducted from the soil sample using the kit of NucleoSpin™Soil. While the DNA quality control was using nanodrop, Qubit, and gel electrophoresis. The gel electrophoresis was conducted before putting into PCR machine. Normally, it is conducted to visualize the PCR result.
For the day 3 to day 5, the activity was mostly conducted for the data analyses. The data were derived as a result of DNA sequencing from the company (if the DNA sequencing process is sent to the company). During the session, we used the application software called “pipecraft” which can be operated using “Docker” desktop. This software can be used before the continued analyses using R programming or other software such as “Vegan”. From the “pipecraft”, the data resulted clusters of species or genus depending on the microorganisms target of our interest, for example, fungi, which refers to ITS database, or bacteria which refers on silva database. We were also given a task to earn 4 ECTS credits. The task was data processing using “pipecraft” to analyze data sequencing for fungi community from ITS database.
