metagonomic sequence of periodontitis stage IV
PCR amplicons with tailed adapters were barcoded by PCR using LongAmp Taq 2X master mix (NEB) and barcode primers. The barcoded samples were cleaned using 1X AmPure XP beads and quantified using qubit fluorimeter. The equimolar concentration of the barcoded sample was pooled, and the sequencing adaptor were ligated (SQK-LSK114) onto double- stranded DNA fragments using NEB Quick T4 DNA Ligase (New England Biolabs, MA, USA). After purification with AMPure XP beads, the prepared library was ready for library quality control. Sequencing was carried out using Nanopore method. The raw reads were changed to fastq format. The processed readings were aligned against the Greengenes database for bacterial taxonomy using the Centrifuge program. This tool uses the Ferragina-Manzini indexing, and the Burrows-Wheeler transform to classify the microbial DNA sequences.