Fecal Microbiome Analysis by Illumina Sequencing

Total DNA was isolated from frozen stool samples using MagnaPure LC DNA Isolation Kit III (Bacteria, Fungi) (Roche, Mannheim, Germany) according to manufacturer’s instructions including mechanic and enzymatic lysis [13 (link)]. Hypervariable regions V1-2 were amplified in a target specific PCR using the primers 27F and R357 (27F-AGAGTTTGATCCTGGCTCAG; R357-CTGCTGCCTYCCGTA) and sequenced with the Illumina MiSeq technique (Illumina, Eindhoven, The Netherlands) [13 (link)]. Sequencing was done in cooperation with the Core Facility for Molecular Biology at the Center for Medical Research in Graz.

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Stadlbauer V., Horvath A., Komarova I., Schmerboeck B., Feldbacher N., Wurm S., Klymiuk I., Durdevic M., Rainer F., Blesl A., Stryeck S., Madl T., Stiegler P, & Leber B. (2019). A single alcohol binge impacts on neutrophil function without changes in gut barrier function and gut microbiome composition in healthy volunteers. PLoS ONE, 14(2), e0211703.

Publication 2019

MiSeq technique

Bacteria Enzymatic Frozen Fungi Isolation Mechanic Primers Stool

Corresponding Organization : BioTechMed-Graz

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Variable analysis

independent variables

Mechanic and enzymatic lysis of frozen stool samples

dependent variables

Composition of bacterial and fungal communities in stool samples

control variables

Use of MagnaPure LC DNA Isolation Kit III (Bacteria, Fungi) according to manufacturer's instructions
Amplification of hypervariable regions V1-2 using primers 27F and R357
Sequencing with the Illumina MiSeq technique

controls

No positive or negative controls were explicitly mentioned in the protocol.

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