The design of internal standard for mycobiome analysis of
clinical samples

a 2023

The design of internal standard for mycobiome analysis of clinical samples

FARNÍKOVÁ, Simona, Kateřina VYKLICKÁ, Petra BRENEROVÁ a Petra BOŘILOVÁ LINHARTOVÁ

Základní údaje

Originální název

The design of internal standard for mycobiome analysis of clinical samples

Autoři

FARNÍKOVÁ, Simona (203 Česká republika), Kateřina VYKLICKÁ (203 Česká republika), Petra BRENEROVÁ (203 Česká republika) a Petra BOŘILOVÁ LINHARTOVÁ (203 Česká republika, garant, domácí)

Vydání

Setkání biochemiků a molekulárních biologů, 2023

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Kód RIV

RIV/00216224:14310/23:00133554

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova anglicky

Microbiome; mycobiome; internal standard; fungi

Změněno: 24. 5. 2024 10:02, doc. RNDr. Petra Bořilová Linhartová, Ph.D., MBA

Anotace

V originále

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.

Návaznosti

LM2023069, projekt VaV

Název: Výzkumná infrastruktura RECETOX

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Výzkumná infrastruktura RECETOX

Citovat

FARNÍKOVÁ, Simona, Kateřina VYKLICKÁ, Petra BRENEROVÁ a Petra BOŘILOVÁ LINHARTOVÁ. The design of internal standard for mycobiome analysis of clinical samples. In Setkání biochemiků a molekulárních biologů. 2023.

@proceedings{2374664,
   author = {Farníková, Simona and Vyklická, Kateřina and Brenerová, Petra and Bořilová Linhartová, Petra},
   booktitle = {Setkání biochemiků a molekulárních biologů},
   keywords = {Microbiome; mycobiome; internal standard; fungi},
   language = {eng},
   title = {The design of internal standard for mycobiome analysis of clinical samples},
   year = {2023}
}

TY  - CONF
ID  - 2374664
AU  - Farníková, Simona - Vyklická, Kateřina - Brenerová, Petra - Bořilová Linhartová, Petra
PY  - 2023
TI  - The design of internal standard for mycobiome analysis of clinical samples
KW  - Microbiome
KW  - mycobiome
KW  - internal standard
KW  - fungi
N2  - 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.
ER  -

FARNÍKOVÁ, Simona, Kateřina VYKLICKÁ, Petra BRENEROVÁ a Petra BOŘILOVÁ LINHARTOVÁ. The design of internal standard for mycobiome analysis of clinical samples. In \textit{Setkání biochemiků a molekulárních biologů}. 2023.

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