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

(Imperial College London UK) Panek, Marina; Čipčić Paljetak, Hana; Barešić, Anja; Perić, Mihaela; Matijašić, Mario; Lojkić, Ivana; Vranešić Bender, Darija; Krznarić, Željko; Verbanac, Donatella (2018) Methodology challenges in studying human gut microbiota – effects of collection, storage, DNA extraction and next generation sequencing technologies. Scientific Reports, 8 (5143). pp. 1-13. ISSN 2045-2322

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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.

Item Type: Article
Additional Information: Genomic DNA from Microbial Mock Community B (Even, Low Concentration), v5.1 L, for 16 S rRNA Gene Sequencing, HM-782D was obtained through BEI Resources, NIAID, NIH as part of the Human Microbiome Project. The authors fully acknowledge the Croatian Science Foundation (Hrvatska zaklada za znanost, HRZZ) for the support of this work under the project MINUTE for IBD (HRZZ grant no IP-11-2013-5467) as well as for supporting Ma.P. under "Young Researchers' Career Development Project - Training of Doctoral Students" (DR-6-2014). This work was also funded by the Scientific Center of Excellence for Reproductive and Regenerative Medicine (project "Reproductive and regenerative medicine - exploration of new platforms and potentials", GA KK01. funded by the EU through the ERDF). The authors are grateful to Vladimir Trkulja for critical reading of the manuscript and Kristian Vlahovicek for assistance in bioinformatics.
Uncontrolled Keywords: gut microbiota; DNA extraction; next generation sequencing technologies; Illumina
Subjects: NATURAL SCIENCES > Interdisciplinary Natural Sciences
Project titleProject leaderProject codeProject type
Utvrđivanje sastava crijevne mikrobiote, upalnih markera, prehrambenog i endokrinog statusa u pacijenata s upalnom bolesti crijeva-MINUTE for IBDDonatella VerbanacIP-2013-11-5467HRZZ
UNSPECIFIEDUNSPECIFIEDNutritional and Endocrinological Status in IBD Patients - MINUTE for IBDUNSPECIFIED
Depositing User: Anja Barešić
Date Deposited: 28 Jul 2020 11:51
DOI: 10.1038/s41598-018-23296-4

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