Zoltán Kiss1, Nóra Makra2, Bálint Tél1, Kriszta Katinka Boros1, Dóra Szabó2, Gábor Veres1
1: 1st Department of Pediatrics, Semmelweis University, Budapest, Hungary
2: Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary
Introduction: Inflammatory bowel disease (IBD) is an idiopathic gastrointestinal disease caused by a dysregulated immune response to host intestinal microflora. The changes in the gut microbiome in pediatric patients receiving biological therapy is not characterized.
Aims: We aimed to establish a stool sample bank for investigating the alterations of the gut microbiome under biological therapies in pediatric patients with IBD. In the first phase of the study we investigated the feasibility of preparation of stool samples for DNA extraction and library generation for next generation sequencing measurements.
Methods: The stool samples were collected in an inpatient setting, aliquoted in native form and stored frozen on -80 C. DNA were extracted using QIAamp Fast DNA Stool Mini Kit. 180-220 mg of sample were used in the extraction. Yielded DNA concentration was measured on a Qubit 2.0 Fluorometer with Qubit dsDNA HS Assay Kit. Library generation for the 16s gene sequencing was made with Q5 Hot Start High-Fidelity 2X Master Mix and Nextera XT Index Kit. Quality validation was made on Agilent 2100 Bioanalyzer.
Results: We were able to establish a stool sample bank of pediatric patients in the following groups: control patients, newly diagnosed IBD, IBD with remission with and without biological therapy. The manufacturer advised protocol of DNA extraction were modified for optimization, as the DNA yield was unsatisfactory for library preparation. Optimization were able to achieve significantly higher DNA yield.
Conclusion: We were able to optimize the sample preparation method for the collected stool samples of different consistency, which made the preparation of libraries for 16s RNS sequencing feasible in our experimental setting. In the next step the libraries will undergo 16s RNS sequencing in bulk.
Grants: Supported by ÚNKP-17- 3-III New National Excellence Program of the Ministry
of Human Capacities.
PhD school: Doctoral School of Clinical Medicine
Program: Prevention of Chronic Diseases in Childhood
Supervisor: Gábor Veres
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