engleski

Studying human gut microbiota methodology challenges

The microbiota are all the microorganisms that live in a specific ecosystem. In humans, specific clusters of microbiota are found on the skin, gastrointestinal tract, mouth, vagina, lungs and other organs in touch with the environment. The field of microbiota research experienced a renaissance with the introduction of next generation sequencing (NGS) technologies. The acquired information provided deeper insights into microbiota composition and function. The gastrointestinal tract harbours the largest microbial ecosystem in the human body and the analyses of faeces provided new knowledge on the importance of GI microorganisms for the human health. However, the detection of the faecal bacterial community composition can be affected by various factors including experimental design and procedures including: different protocols for collection and storage of faecal samples, bacterial DNA extraction, NGS platforms used for sequencing and data analysis pipeline. In our research, we investigated available commercial tools and technologies and compared their performance during analyses of human faeces from healthy donors. OMNIgene.GUT proved as a reliable and convenient system for collection and storage of faecal samples although favouring Sutterella genus. Among the DNA isolation kits, MP provided superior DNA yield and quality, MO BIO depleted Gram positive organisms while using QIAGEN with OMNIgene.GUT resulted in greatest variability compared to other two kits. MiSeq and IT platforms in their supplier recommended setups provided comparable reproducibility of donor faecal microbiota. The differences included higher diversity observed with MiSeq and increased capacity of MiSeq to detect Akkermansia muciniphila, [Odoribacteraceae], Erysipelotrichaceae and Ruminococcaceae (primarily Faecalibacterium prausnitzii). The results of our study could assist the investigators using NGS technologies to make informed decisions on appropriate tools for their experimental pipelines.

human gut microbiota, methodology

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