Introduction of next-generation sequencing in the industry to improve molecular microbiology analysis
Kukkurainen, Veera (2020)
Kukkurainen, Veera
2020
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2020-11-02
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202010197384
https://urn.fi/URN:NBN:fi:tuni-202010197384
Tiivistelmä
Microbiology plays an important role in several areas, such as human health and biotechnological and industrial processes. In the forest industry, microbiology has an impact on both product purity and occupational safety. The next- generation sequencing method is an analysis that allows a new way to study microbiological phenomena. As a method, sequencing adds value to existing microbiological analyzes both in the forest industry and in other fields of research. In microbiological taxonomic analysis, amplicon sequencing is largely based on universal genes, such as the 16s rRNA of bacteria and the 18s rRNA of the fungal gene. In amplicon sequencing, a primer pair is designed for the desired target gene that amplifies the gene of interest prior to sequencing. In addition, for successful sequencing, the samples must be modified into a sequencing library. In this work, a method based on target gene amplification and two-step PCR was optimized. This method was designed to be used for the analysis of samples from Stora Enso's industrial environment. The class of organisms to be studied in the work were fungi. An appropriate primer pair was first selected for the preparation of the sequencing library, after which the method used to prepare the sequencing library was optimized. Optimization of the method was followed by sequencing and final analysis of 24 samples. The results show in the optimization of the method that the initial DNA content of the samples is of great importance in the preparation of the sequencing library. In addition, the 18s rRNA primer pair has a higher amplification efficiency than other primer pairs tested. From the sequencing results, it is found that the primer pair targeted to the 18s rRNA gene also amplifies non-desired fungal sequences. This nonspecific amplification may be due to primer pair selection or the 18s rRNA gene itself. Sequencing results indicate that sample types may have effects on fungal diversity. The results of the work help to better understand the use of sequencing methods as an analysis tool in industrial microbiology. In addition, the results clarify the diversity of fungi in the industrial environment and help to select potential target genes for the analysis of fungal gene material.