title: 16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform.
abstract: Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA.
  
  
  
  The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar.
  
  
  
  Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.
authors.affiliation: Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland.
authors.forename: Fiona
authors.initials: F
authors.lastname: Fouhy
authors.affiliation: School of Microbiology, University College Cork, Cork, Ireland.|APC Microbiome Institute, University College Cork, Cork, Ireland.
authors.forename: Adam G
authors.initials: AG
authors.lastname: Clooney
authors.affiliation: Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland.|APC Microbiome Institute, University College Cork, Cork, Ireland.
authors.forename: Catherine
authors.initials: C
authors.lastname: Stanton
authors.affiliation: School of Microbiology, University College Cork, Cork, Ireland. m.claesson@ucc.ie.|APC Microbiome Institute, University College Cork, Cork, Ireland. m.claesson@ucc.ie.
authors.forename: Marcus J
authors.initials: MJ
authors.lastname: Claesson
authors.affiliation: Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland. paul.cotter@teagasc.ie.|APC Microbiome Institute, University College Cork, Cork, Ireland. paul.cotter@teagasc.ie.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Cotter
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D059014:High-Throughput Nucleotide Sequencing; D064307:Microbiota; D012336:RNA, Ribosomal, 16S / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA; Bias; DNA extraction; Gut microbiota; Ion PGM; MiSeq; Mock communities; Next-generation sequencing