| Popis |
The presence of microbial communities in human clinical samples can be studied by DNA sequencing. Unlike bacterial microbiome (bacteriome), which has become a common topic in the literature, fungal microbiome (mycobiome) is relatively rarely studied. Highthroughput sequencing experiments require a mock community as a positive control. Including such control allows us to evaluate the extent to which the sequencing result is biased away from the correct fungal composition of the samples. However, studies using fungal mock communities are not available, although some studies use mock communities in the form of fruiting bodies or spores collected from sporocarps. This work aims to design a fungal internal standard that can be spiked into samples with human DNA while showing no similarities to fungi that might be present in such samples and to suggest a suitable dilution of that internal standard. The sequence of specific fungus was chosen. In the NCBI database, an ITS2 region, into which our chosen primers fit, was defined; the defined single-stranded sequence was commercially obtained. The synthetized ssDNA internal standard sample was cloned to produce dsDNA and the sequence of this DNA was verified by whole genome sequencing. Classic ITS PCR reactions with 1 µl of the internal standard were performed at several dilutions – 1,000×, 100,000×, 200,000×, 500,000×, 800,000× and 1,000,000× to determine the appropriate concentration of the added internal standard to the clinical samples; the quality and quantity control of PCR products was evaluated using electrophoresis and fluorometry. The sequencing of dsDNA confirms successful cloning. The results from gel electrophoresis show robust bands in dilution 1,000×, 100,000×, 200,000×, and 500,000×. However, the bands of 800,000× and 1,000,000× dilution seem appropriate for spiking the clinical samples for sequencing. Nevertheless, since the cloned DNA degraded over time, for future analyses it is necessary to first verify the concentration of the stock DNA by fluorometric measurement and visualize PCR products on a gel electrophoresis. A synthetic fungal internal standard was created that does not interfere with real clinical samples. The dilution 800,000× and 1,000,000× of the internal standard was chosen as appropriate dilution for spiking clinical samples for sequencing. However, the suitability of the selected internal standard dilution must be confirmed by sequencing analysis. Results from this analysis will be further used in the work with clinical samples.
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