Qubit dsdna high sensitivity and broad range assay kit

Whole-metagenome shotgun libraries were prepared in accordance with the Nextera XT DNA Library Preparation Guide from Illumina, with the exception that the tagmentation time was increased to 7 min. After indexing and clean-up of the PCR products, as described in the protocol, each sample was run on an Agilent bioanalyser high sensitivity chip (Agilent Technologies Ireland Ltd., Cork, Ireland) to determine the size range of the fragments obtained. The concentration of the samples was also determined at this point using a Qubit^TM dsDNA High Sensitivity and Broad Range assay kit (Invitrogen by Thermo Fisher Scientific, Dublin, Ireland). Samples were then pooled equimolarly, and the final concentration of the pooled library was determined by quantitative PCR using the Kapa Library Quantification kit for Illumina (Roche diagnostics Ltd., West Sussex, UK). The pooled library was then sequenced on the Illumina NextSeq 500 using the 2 × 150 High Output kit according to standard Illumina sequencing protocols.

Sample pellets were resuspended in 200 μL molecular grade water and bead beaten using 0.3 g sterile acid washed glass beads (Sigma-Aldrich, Merck Life Science Ltd., Wicklow, Ireland) for 180 s at 5.5 m/s [74 (link)]. A 200 µL volume of binding buffer and 40 µL proteinase K were added and incubated at 70 °C for 10 min. Following incubation, Roche high pure PCR template preparation kit (Roche diagnostics Ltd., West Sussex, UK) instructions were followed. For each batch of extractions, a negative extraction control was performed where the protocol was carried out from start to finish on 200µL molecular grade water. DNA was quantified on the Qubit fluorometer (Life Technologies, Thermo Fisher Scientific, Bleiswijk, Netherlands) employing the Qubit^TM dsDNA High Sensitivity and Broad Range assay kit (Invitrogen by Thermo Fisher Scientific, Dublin, Ireland). Then, 16S rDNA gene amplification was performed on negative extraction controls. Microbial DNA was enriched using the NEBNext^® Microbiome DNA Enrichment Kit (New England Biolabs Ltd., Hitchin, UK) [75 (link)] as per the manufacturer’s instructions. Enriched DNA then underwent ethanol precipitation and was stored at −20 °C.