Microbiota Analysis Using Sequencing by Synthesis: From Library Preparation to Sequencing

  • Andrew NelsonEmail author
  • Christopher J. Stewart
Part of the Methods in Molecular Biology book series (MIMB, volume 2121)


The highly parallel nature of sequencing by synthesis (SBS) allows millions of amplicons to be sequenced simultaneously, which has led to enormous interest in the investigation of bacterial communities (often referred to as the microbiota). In this protocol, we describe a method for the ‘universal’ amplification of the v4 region of the bacterial 16S rRNA gene from genomic DNA and prepare these amplicons so that they can be sequenced using the MiSeq system (Illumina). The protocol provides instruction on sequencing of 188 genomic DNA samples plus PCR positive and negative controls, which can be applied to any sample type where bacterial DNA may be of interest.

Key words

DNA sequencing 16S rRNA Microbiota MiSeq PCR 



This work was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 794771 to C.J.S.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Faculty of Health and Life SciencesNorthumbria UniversityNewcastle upon TyneUK
  2. 2.Institute of Cellular MedicineNewcastle UniversityNewcastle upon TyneUK

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