Abstract
The information on microbiota composition in the human gastrointestinal tract predominantly originates from the analyses of human faeces by application of next generation sequencing (NGS). However, the detected composition of the faecal bacterial community can be affected by various factors including experimental design and procedures. This study evaluated the performance of different protocols for 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 (Ilumina MiSeq and Ion Torrent PGM). OMNIgene.GUT proved as a reliable and convenient system for collection and storage of faecal samples although favouring 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 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.
Highlights
The scientific interest and mounting knowledge on human gut microbiota have led to many important findings associating the composition of bacterial taxa in the human gastrointestinal tract with disorders from the neurologic, psychiatric, respiratory, cardiovascular, gastrointestinal, hepatic, autoimmune, metabolic and oncologic spectra[1,2]
This study evaluated the performance of different protocols for collection and storage of faeces as well as bacterial DNA extraction using the manufacturer recommended methodology for 16S rRNA gene sequencing on two commercially available platforms (Ilumina MiSeq and Ion Torrent (IT) Personal Genome Machine (PGM))
The yield and quality of DNA differed between kits, with the average yield and the estimated purity of DNA, both of which varied between kits in the MP > QIA > MO BIO order (Fig. 2)
Summary
The scientific interest and mounting knowledge on human gut microbiota have led to many important findings associating the composition of bacterial taxa in the human gastrointestinal tract with disorders from the neurologic, psychiatric, respiratory, cardiovascular, gastrointestinal, hepatic, autoimmune, metabolic and oncologic spectra[1,2]. The main source of the information regarding human gut microbiota originated from the analyses of the faeces The importance of this available metabolic waste comes from its microbial content found to hold the potential in diagnosis, disease prediction and therapeutic intervention. Illumina and Thermo platforms with their MiSeq and Ion Torrent (IT) Personal Genome Machine (PGM) benchtop sequencers are increasingly being used for 16S rRNA-based analyses of diverse bacterial populations[7,9,19,21,22] Both Illumina and IT platforms sequence DNA by detecting the nucleotide addition during DNA synthesis, the two platforms function on different principles, which could affect their performance and result in variation of the obtained data[7,9,24,25,26,27]. This study evaluated the performance of different protocols for collection and storage of faeces (native versus OMNIgene.GUT system) as well as bacterial DNA extraction (with MP Biomedicals, Qiagen and MO BIO commercial kits) using the manufacturer recommended methodology for 16S rRNA gene sequencing on two commercially available platforms (Ilumina MiSeq and IT PGM)
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