Qubit dna hs assay kit

The 16S rDNA fragments were indexed using the Nextera XT Kit (Illumina, San Diego, CA, USA) according to the Nextera DNA Sample Preparation Guide (protocol #15044223). Each index PCR reaction contained 5 µL of the i7 and i5 adapter, 10 µL of KAPA HiFi HotStart ReadyMix, 20 µL of template DNA (7.0 ng/µL), and 10 µL of H 2 O for a total reaction volume of 50 µL. The indexed PCR was cycled according to the Nextera DNA Sample Prep Guide, and the libraries were cleaned up using MagSi-NGS PREP PLUS (Steinbrenner Laborsysteme, GmbH, Wiesenbach, Germany). The DNA libraries were quantified using the Qubit 4.0 along with the Qubit DNA HS Assay Kit, and the quality was assessed on a TapeStation 4200 using the High Sensitivity D1000 SreenTape Assay Kit (Agilent, Santa Clara, CA, USA). Indexed libraries were normalised to 4 nM and pooled. The normalised, pooled 4 nM library was denatured using 0.2 N NaOH and diluted to 10 pM using prechilled HT1 buffer supplied in Nextera XT Kit (Illumina). The 10% of denatured PhiX library (Illumina, San Diego, CA, USA) was spiked into the denatured and indexed library, which was loading into Illumina Miseq v3 reagent cartridge, and 16S rDNA gene amplicons were sequenced on an Illumina MiSeq platform using the 600 cycles (2 × 300 bp) v3 chemistry.