Nanopore-based 16S rRNA metagenomic analysis

DNA extraction was performed using the Epicentre MasterPure™ DNA Purification Kit (Epicenter, Middleton, WI, USA), according to the manufacturer’s recommendations. The quality and quantity of the extracted DNA was checked using a NanoDrop (Colibri Microvolume Spectrometer; Titertek-Berthold, Germany). Amplification of the entire (~1500 bp) 16S rRNA gene was performed using the 16S Barcoding Kit (SQK-RAB204; Oxford Nanopore Technologies, Oxford, UK) and LongAmp™ Taq 2 × Master Mix (New England Biolabs, UK) with 1 µg of input DNA per sample. Purification of the PCR products was performed using AMPure XP (Beckman Coulter, CA, USA) followed by quantification using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Equimolar amounts of the amplification products were pooled together, then a total of 100 ng DNA of the pooled sample was used for library preparation. The microbiota was analyzed using third-generation sequencing with Nanopore technology on a MinION device (Oxford Nanopore Technologies, UK). MinION™ sequencing was performed using R9.4 flow cells (Oxford Nanopore Technologies, UK) according to the manufacturer’s instructions. MinKNOW version 2.0 (Oxford Nanopore Technologies, UK) was used for live base calling and data acquisition. The raw data were converted into FASTQ format using Guppy v3.4.4, followed by demultiplexing and removal of nanopore and adaptor sequences. The FASTQ files were analyzed on the Nanopore EPI2ME platform with a default minimum Q score of 7. Preliminary bacterial identification was performed via the ‘What’s in my Pot?’ (WIMP) workflow provided by Oxford Nanopore Technologies (UK). Reads assigned to all targets were re-analyzed by the Kraken taxonomic sequence classification system using Partek^® Genomics Suite^® software (Copyright^© 2022; Partek Inc., St. Louis, MO, USA). The numbers of reads assigned per taxon were counted and the relative abundance of reads per taxon were used for separate downstream analysis, as described in our previous publications [27 (link),28 (link)].