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

Panek, Marina and Čipčić Paljetak, Hana and Barešić, Anja and Perić, Mihaela and Matijašić, Mario and Lojkić, Ivana and Vranešić Bender, Darija and Krznarić, Željko and Verbanac, Donatella (2018) Methodology challenges in studying human gut microbiota – effects of collection, storage, DNA extraction and next generation sequencing technologies. Scientific Reports, 8 (1). p. 5143. ISSN 2045-2322

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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.

Item Type: Article
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MeSH: Adult ; DNA, Bacterial/genetics ; DNA, Bacterial/isolation & purification ; Feces/microbiology ; Female ; Gastrointestinal Microbiome ; Gram-Positive Bacteria/chemistry ; Gram-Positive Bacteria/classification ; Gram-Positive Bacteria/genetics ; Gram-Positive Bacteria/isolation & purification ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Reproducibility of Results ; Specimen Handling/methods
Departments: Centar za translacijska i klinička istraživanja
Katedra za internu medicinu
Depositing User: Kristina Berketa
Status: Published
Čipčić Paljetak, HanaUNSPECIFIED
Matijašić, MarioUNSPECIFIED
Vranešić Bender, DarijaUNSPECIFIED
Krznarić, ŽeljkoUNSPECIFIED
Verbanac, DonatellaUNSPECIFIED
Date: 23 March 2018
Date Deposited: 01 Oct 2019 10:31
Last Modified: 01 Oct 2019 10:31
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