engleski

Methodology challenges in studying human gut microbiota

The human gut contains dense and diverse microbial communities which have deep influence on human health. In order to understand complex features of these communities, it is necessary to obtain high quality microbial nucleic acids out of human faecal samples. Methodologies for collection and storage of human faecal samples, as well as DNA extraction, finally determine the quality of isolated DNA. These methodologies and procedures are crucial steps in obtaining representative models of bacterial community structure after DNA sequencing. High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. In this study we compared two different approaches for faeces collection and storage, three commercially available DNA extraction kits (MP Biomedicals, Qiagen and MoBio) and Ion Torrent Personal Genome Machine (PGM) sequencing platform for bacterial community profiling by 16S rRNA amplicon sequencing. Faeces from 4 healthy human donors were collected fresh and in OMNIgene.GUT system, and then stored for 14 days at -20°C and at room temperature, respectively. OMNIgene.GUT was proven as convenient and reliable faecal collection, transport and storage system for human faecal microbiota analyses. Although OMNIgene.GUT faeces collection system slightly decreased the richness of detected bacteria, the change was not statistically significant. DNA yield and quality varied between DNA extraction kits in the order MP>QIAGEN>MoBio. The results of bacterial community profiling performed on the human-derived microbiological specimens were generally in good agreement for all extraction kits. Donor-specific bacterial diversity was maintained irrespective of the collection, storage or extraction.

human gut, human faecal samples, collection and storage, bacterial community structure, microbial diversity

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