Priprava modelnih mikrobnih združb za raziskave diverzitete z visokozmogljivim sekvenciranjem

Zaman, Jure (author), Turk, Martina (mentor)

Za preučevanje mikrobnih združb v veliki meri uporabljamo visokozmogljiva sekvenciranja z analizo pridobljenih nukleotidnih zaporedij. Najpogosteje uporabljana metoda za take analize je sekvenciranje pomnožkov (angl. Amplicon Sequencing). Ta omogoča sekvenciranje delov specifičnih genov, ki služijo kot taksonomski označevalci, to so tako imenovane črtne kode DNA (angl. DNA Barcode). Poglavitni taksonomski označevalci, ki jih pri uporabljamo, so geni za rRNA (16S rDNA za prokarionte in 18S rDNA ter notranji prepisani vmesnik (ITS) za evkarionte). Metoda je sestavljena iz več zaporednih korakov: izolacije DNA, pomnoževanja delov specifičnih genov z verižno reakcijo s polimerazo (PCR), sekvenciranja le-teh in bioinformatske obdelave podatkov. Vsak korak v tem procesu lahko povzroči pristranskost, kar vodi do napačnih zaključkov. Da bi se temu izognili, je potreben dober referenčni standard. Modelna mikrobna združba (MC) je standard z znano vrstno in številčno sestavo mikrobov. Pripravili smo modelno združbo, sestavljeno iz trinajstih vrst gliv in sicer v dveh oblikah, iz samih celic in iz izolirane DNA. Celično MC smo pripravili v dveh kombinacijah, kot mešanico, kjer je bila vsaka vrsta zastopana v enakem številu celic (enakomerna MC), ter kot mešanico, kjer so bile vrste zastopane v različnem številu (vrednosti desetiškega logaritma; logaritemska MC). Testno združbo na osnovi DNA smo pripravili iz DNA čistih kultur, kjer smo iz definiranega števila celic ali spor izolirali DNA in jih nato združili. Glive smo najprej gojili, določili število celic, izbrano število celic mehansko lizirali in izolirali DNA. Uspešnost izolacije DNA smo preverili z gelsko elektroforezo in izmerili njeno koncentracijo. V celični MC in MC iz DNA smo nato s PCR pomnožili regijo ITS2, ki je taksonomski označevalec za taksonomsko identifikacijo gliv. Pomnoženo DNA smo nato sekvencirali. Dobili smo grafe relativne zastopanosti variant zaporedja pomnožkov posameznega seva znotraj MC. Prisotnosti nekaterih sevov v končnih rezultatih nismo zaznali, številni pa so se pojavili samo v sledeh. Uspešno smo pripravili MC gliv. Vseh taksonov nismo zaznali v vseh MC, prav tako vrste niso bile zastopane v pričakovanih razmerjih. Kljub temu lahko pripravljene MC uporabimo kot referenčni standard v raziskavah mikrobne diverzitete s HTS.

• Language: Slovenian
• Keywords: glivna modelna združba, sekvenciranje amplikonov, ITS, standard
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [J. Zaman]
• Year: 2024
• PID: 20.500.12556/RUL-155553
• UDC: 579.222:577(043.2)
• COBISS.SI-ID: 193128707
• Publication date in RUL: 06.04.2024

Secondary language

• Language: English
• Title: Preparation of model microbial communities for use in high-throughput sequencing microbial diversity research

Abstract

High-throughput sequencing with analysis of the resulting nucleotide sequences is widely used to study microbial communities. The most commonly used method for microbial community analysis is amplicon sequencing, which allows the sequencing of parts of specific genes that serve as taxonomic markers, so-called DNA barcodes. The main taxonomic markers used in amplicon sequencing are rRNA genes (16S rDNA for prokaryotes and 18S rDNA and internal transcribed spacers (ITS) for eukaryotes). The method consists of several consecutive steps: DNA isolation, polymerase chain reaction (PCR) amplification of parts of specific genes, their sequencing and bioinformatic data processing. Each step in this process can introduce bias, leading to erroneous conclusions, thus a good reference standard is needed. A model microbial community (MC) is a standard with a known species and abundance composition of microbes. A model fungal community consisting of 13 species was prepared in two varieties, from the cells themselves and from isolated DNA. Cell MC was prepared in two further subvarieties, as a mixture where each species was represented with the same number of cells (uniform MC) and as a mixture where the species were represented with different numbers (decimal logarithm values; logarithmic MC). A DNA-based MC was constructed from DNA of purified cultures in which DNA was isolated from a defined number of cells or spores and then pooled. Fungi were cultured, the number of cells determined, a selected number of cells mechanically lysed and DNA isolated. The success of the DNA isolation was examined by electrophoresis and DNA concentration was measured. The ITS2 region, a marker gene for taxonomic identification of fungi, was then amplified by PCR from the cell model fungal community (MFC) and the DNA MFC. The amplified DNA was sequenced, and the data processed. We obtained graphs of the relative abundance of the ASV of each strain within the MC. The presence of some strains was not detected in the final results, and many appeared only in traces. We have successfully prepared MFCs. Not all taxa were detected in all MCs, and species were not represented in the expected ratios. Nevertheless, the prepared MCs can be used as a reference standard in microbial diversity studies with HTS.

• Keywords: fungal mock community, amplicon sequencing, ITS, standard