Sequencing and base-calling for mock community

For nanopore sequencing, genomic libraries for two target regions (full-length 16S rRNA gene and 16S-ITS-23S rRNA operon) were prepared by using the four-primer PCR method protocol, version FFP_9038_v108_revN_14Aug2019 (Oxford Nanopore Technologies [ONT], Oxford, UK) with slight modifications. The four-primer PCR uses two target-specific inner primers with a 5′ tail and two universal outer primers which prime off the tail on the 5′ end of the inner primers (Table ^S2), resulting in the generation of target amplicons with barcodes^15,18,19. PCR amplifications were conducted using either LongAmp™ Taq 2 × Master Mix (New England Biolabs, Ipswich, MA, USA) or the KAPA2G™ Robust HotStart Ready Mix PCR Kit (Kapa Biosystems, Wilmington, MA, USA). PCR was performed in a total volume of 25 µL containing the inner primers (50 nM each), the barcoded outer primer mixture (300 nM) from the PCR barcoding kit (SQK-PBK004; ONT), and the DNA cocktail (1 ng) as template. PCR conditions are shown in Table ^S3. The PCR amplicons were purified with the Agencourt AMPure XP purifier and quantified by a NanoDrop spectrophotometer (ThermoFisher Scientific), and the libraries were sequenced on a MinION sequencer using R9.4.1 flow cells (FLO-MIN106D; ONT) following the manufacturer’s instructions. Base-calling of raw fast5 data from the MinION was carried out in Guppy v. 3.6.1 software (ONT) with its “–trim_barcodes” option for removing sequencing adapters and barcodes. Details on each sample, including number of reads, median read lengths, and median read quality, are shown in Table ^S4.

The same genomic DNA was also sequenced on the Illumina MiSeq system targeting hypervariable region (V3-V4) by a commercial service (Oral Microbiome Center, Kagawa, Japan). This sequencing was conducted with a primer set of 341F and 806R following an Illumina protocol (16S Metagenomics Sequencing Library Preparation).