title: Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor.
abstract: Next-generation sequencing (NGS) is currently the method of choice for analyzing gut microbiota composition. As gut microbiota composition is a potential future target for clinical diagnostics, it is of utmost importance to enhance and optimize the NGS analysis procedures. Here, we have analyzed the impact of DNA extraction and selected 16S rDNA primers on the gut microbiota NGS results. Bacterial DNA from frozen stool specimens was extracted with 5 commercially available DNA extraction kits. Special attention was paid to the semiautomated DNA extraction methods that could expedite the analysis procedure, thus being especially suitable for clinical settings. The microbial composition was analyzed with 2 distinct protocols: 1 targeting the V3-V4 and the other targeting the V4-V5 area of the bacterial 16S rRNA gene. The overall effect of DNA extraction on the gut microbiota 16S rDNA profile was relatively small, whereas the 16S rRNA gene target region had an immense impact on the results. Furthermore, semiautomated DNA extraction methods clearly appeared suitable for NGS procedures, proposing that application of these methods could importantly reduce hands-on time and human errors without compromising the validity of results.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland;; Department of Clinical Microbiology and Immunology, Turku University Hospital, Turku, Finland.
authors.forename: Anniina
authors.initials: A
authors.lastname: Rintala
authors.affiliation: Turku Centre for Biotechnology, Turku, Finland.
authors.forename: Sami
authors.initials: S
authors.lastname: Pietilä
authors.affiliation: Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland;; Department of Clinical Microbiology and Immunology, Turku University Hospital, Turku, Finland.
authors.forename: Eveliina
authors.initials: E
authors.lastname: Munukka
authors.affiliation: Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland;; Department of Clinical Microbiology and Immunology, Turku University Hospital, Turku, Finland.
authors.forename: Erkki
authors.initials: E
authors.lastname: Eerola
authors.affiliation: Turku Clinical Sequencing Laboratory, University of Turku, Turku, Finland; and.
authors.forename: Juha-Pekka
authors.initials: JP
authors.lastname: Pursiheimo
authors.affiliation: Turku Centre for Biotechnology, Turku, Finland.
authors.forename: Asta
authors.initials: A
authors.lastname: Laiho
authors.affiliation: Unit of Health Sciences, Faculty of Sports, University of Jyväskylä, Jyväskylä, Finland.
authors.forename: Satu
authors.initials: S
authors.lastname: Pekkala
authors.affiliation: Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland.
authors.forename: Pentti
authors.initials: P
authors.lastname: Huovinen
mesh_terms: D000328:Adult; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology; D000069196:Gastrointestinal Microbiome; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D058889:Molecular Typing; D012336:RNA, Ribosomal, 16S / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: fecal samples; microbial diversity; next-generation sequencing; sample preprocessing