Nextera XT DNA Library Preparation for MiSeq

Preparation of MiSeq library was performed using Illumina Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) as previously described with minor modifications.¹⁹ (link) In brief, 1 ng of genomic DNA was fragmented in 5 µl of Amplicon Tagment Mix and 10 µl of Tagment DNA buffer. Tagmentation reaction was performed by incubation at 55 °C for 5 min followed by neutralisation with 5 µl of Neutralise Tagment Buffer for 5 min. Tagmented DNA (25 µl) was indexed in a 50 µl limited-cycle PCR (12 cycles) as outlined in the Nextera XT protocol and subsequently purified using 25 µl of AMPure XP beads (Beckman Coulter Inc, Australia). The fragment size distribution of the purified DNA was analysed utilising a 2100 Bioanalyser with a High Sensitivity DNA assay kit (Agilent Technologies, Santa Clara, CA). DNA libraries were adjusted to 2 nM, pooled in equal volumes and then denatured with 0.2 N NaOH according to the Nextera protocol. The libraries were sequenced using 2 × 300 paired-end protocols on an Illumina MiSeq instrument (MiSeq Reagent Kit v3 for 600 cycles).