engleski

Methodology challenges in studying human gut microbiota – comparison of illumina and Ion Torrent NGS platforms

Microbial communities of human gut have deep impact on health. To understand microbiota complexity, representative models of bacterial community structure after DNA sequencing are needed. The quality of final output is influenced by the methodologies along the pipeline – sample collection, storage, DNA extraction and sequencing approach. The aim of this study was to compare results obtained by two NGS platforms – illumina and Ion Torrent. Faecal bacterial communities from four healthy donors were profiled by 16S rRNA amplicon sequencing using two NGS platforms (illumina MiSeq, regions V3-V4 and Ion Torrent PGM, regions V2-V9) and manufacturer recommended reagents and protocols. Two different approaches for faeces collection (fresh and in OMNIgene.GUT) and three commercially available DNA extraction kits (MP Biomedicals, Qiagen and MoBio) were employed. Analyses were performed using QIIME protocol [2]: OTU assignment procedure for de-multiplexed and quality-trimmed raw reads was performed against the Greengenes reference database version 13_8 (May 2013) [3]. The analysis of sequencing results obtained by two different NGS platforms, using platform-specific experimental approach pipeline, revealed that differences can be observed and are pertinent to the appropriate technology for specific scientific questions. Irrespective of the differences observed between platforms, donor-specificities are in general maintained.

human faecal samples, microbiota, illumina MiSeq, Ion Torrent PGM, 16S rRNA gene sequencing

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