Methodology challenges in studying human gut microbiota – effects of collection, storage, DNA extraction and next generation sequencing technologies

The information on microbiota composition in the human gastrointestinal tract predominantly originates from the analyses of human feces by application of next-generation sequencing (NGS). However, the detected composition of the fecal bacterial community can be affected by various factors including experimental design and procedures. This study evaluated the performance of different protocols for the collection and storage of faecal samples (native and OMNIgene.GUT system) and bacterial DNA extraction (MP Biomedicals, QIAGEN, and MO BIO kits), using two NGS platforms for 16S rRNA gene sequencing (Illumina MiSeq and Ion Torrent PGM). OMNIgene.GUT proved as a reliable and convenient system for collection and storage of fecal samples although favoring Sutterella genus. 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 fecal microbiota. The differences included higher diversity observed with MiSeq and increased the 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