16s library preparation protocol

Genomic DNA was extracted using a GENE STAR PI-480 automated DNA isolation system (Kurabo Industries, Ltd., Osaka, Japan). After extraction, the amount of DNA in the samples was measured using the Qubit^TM dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Polymerase chain reaction (PCR) analysis was performed using primers 16S-27Fmod (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGRGT TTGATYMTGGCTCAG-3′) and 16S-338R (5′-GTCTCGTGGGCTCGGAGATGTGTATA AGAGACAGTGCTGCCTCCCGTAGGAGT-3′) for the V1-V2 region of bacterial 16S rRNA and the KAPA HiFi Hot Start Ready Mix (Roche, Basel, Switzerland). PCR reactions were performed based on the 16S library preparation protocol (Illumina Inc., San Diego, CA, USA). The size of the mixed libraries was confirmed by agarose gel electrophoresis and the concentration was quantified by real-time PCR. Paired-end sequencing (250 bp) was performed using a MiSeq Reagent Kit v2 (500 cycles). The acquired sequence data were analyzed using Qiime2-2019.10 software [8 (link)]. Paired-end reads were merged, sequence errors removed, and sequence clustering performed. Operational taxonomic unit (OTU) clustering with a threshold of 97% was applied in our study. For taxonomic analysis, each OTU was aligned with q2-feature-classifier and the Greengenes 13_8 99% OTUs reference database.

The Illumina 16S Library preparation protocol was adopted to generate the libraries by amplifying the V3 and V4 regions of the 16S rRNA. Sequencing was performed on the Illumina MiSeq platform using the MiSeq Reagent kit v2 500 cycles.
The bioinformatic analysis was performed using a pipeline based on QIIME2 (http://qiime.org/ (accessed on 13th January 2022)). First, the dada2 algorithm was applied in order to denoise, merge forward, and reverse sequences per each pair. Then, the taxonomic classification was performed by applying the Sklearn classifier implemented in QIIME and adopting the Greengenes 13_8 97% OTU dataset as a reference.

Genomic DNA was extracted from gastric biopsy samples using MasterPure^TM DNA purification kit (Epicentre, Madison, WI, USA) according to manufacturer’s instructions. The V3–V4 region of 16S rRNA gene was amplified using S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 primers designed to include the Illumina-compatible adaptors [17 (link),18 (link)]. The 16S amplicon libraries were prepared according to Illumina 16S library preparation protocol [19 ]. Initial PCR amplification was performed using NEBNext High-Fidelity Master Mix (New England Biolabs, Ipswich, MA, USA) with the following conditions: An initial denaturation at 98 °C for 30 s, followed by 30 cycles consisting of denaturation (98 °C for 10 s), annealing (60 °C for 2 min) and extension (72 °C for 20 s), and a final extension step at 72 °C for 1 min. Automated cluster generation and a 2 × 250 bp paired-end sequencing (MiSeq 500-cycle reagent kit V2) was carried out on the MiSeq platform (Illumina, San Diego, CA, USA) at the Monash University Malaysia Genomics Facility. Metagenomics data reported in this paper are available for public access through the MG-RAST server. The accession number and demographic characteristics of each sample are listed in Table S1.