Nextera XT DNA Library Preparation for Bacterial Genomic Sequencing

Genomic DNA was isolated and purified using the Wizard Genomic DNA Purification Kit (Promega) following the protocol for Gram-negative bacteria at one-third volume. Double-stranded DNA was quantified using SYBR Green I Nucleic Acid Stain (Invitrogen) in a SpectraMax M5 Fluorescence Microplate Reader (Molecular Devices). Libraries were created following the Nextera XT DNA Library Prep Kit protocol, at one-quarter volume, with Nextera XT Index Kit v2 adapters (Illumina). Libraries were individually quantified using the Qubit dsDNA High Sensitivity Assay with a Qubit 2.0 Flourometer (ThermoFisher) and fragment size determined using the Agilent 2100 BioAnalyzer with a High Sensitivity DNA Analysis Kits (Agilent). Libraries were pooled and sequenced on an Illumina NextSeq, producing 150 bp, paired-end reads. The Breseq computational pipeline was used to align reads to the reference sequence and identify mutations (Deatherage and Barrick 2014 (link)).