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

Andrew Nelson*, Christopher J. Stewart

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

2 Citations (Scopus)

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages165-184
Number of pages20
DOIs
Publication statusPublished - 2020

Publication series

NameMethods in Molecular Biology
Volume2121
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • 16S rRNA
  • DNA sequencing
  • Microbiota
  • MiSeq
  • PCR

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