16S Illumina Library Preparation Protocol

Dave Baker

Historically in the early days of DNA sequencing technologies, sequencing whole genomes was expensive and impractical. Also, to enable determination of bacterial species of unknown origin, or where a mixture of different species is anticipated, a targeted PCR (Polymerase Chain Reaction) is needed. The approach also needs to use a region of the genome that is common or conserved in most if not all species. The region must also contain enough diversity to distinguish one species or genus from another. With the development of NGS (Next Generation Sequencing) in the late 2000s, and superseding single-read Sanger sequencing, came Illumina technology which can generate millions of different reads from a single sample. Also, over time more individual species have been isolated, cultured and sequenced individually, improving the size and accuracy of reference databases. Here we describe how to take metagenomic DNA (a single sample although a mixture of species) and process it into a viable Illumina library for sequencing. In principle, this method can be applied to any conserved target gene e.g. yeast ITS, animal and fish Co1, or plant RbcL. Here we present the targeting of the prokaryotic 16S ribosomal RNA gene (16S rRNA). This target can be present in multiple copies across the genome and is another consideration when trying to quantitatively determine the proportion of different species present.

5. 16S Illumina Library Preparation Protocol v1.0[pdf]

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Quadram Institute Best Practice in Microbiome Research: 16S Illumina Library Preparation v1.0 by David Baker is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.