Small-Scale Illumina Library Preparation Using the Illumina Nextera XT DNA Library Preparation Kit

Abstract

Here, we describe a protocol for a scaled-down version of a genomic DNA (gDNA)-fragmentation and tagmentation reaction using the Illumina Nextera XT DNA Library Preparation Kit. Using <i>Staphylococcus aureus</i> as an example<i>,</i> which has a genome size of ∼3 Mb, we show how 24 different samples can be pooled for a typical paired-end Illumina high-throughput sequencing run using the MiSeq Reagent V2 300-cycle kit, with which it is possible to sequence 5.1 Gb of DNA. As part of the protocol, a DNA size-selection method using a standard DNA agarose gel-extraction procedure and a final sample quality-control step using a Bioanalyzer are described.