Fecal bacterial 16S rRNA gene sequence was analyzed by the MiSeq system (Illumina, San Diego, CA, USA) as previously described [43 (link)]. The
V3-V4 hypervariable regions of 16S rRNA were PCR amplified from microbial genomic DNA using universal primers for bacteria (341f/R806) [12 (link),
30 (link)] and the dual-index method [21 (link)]. Barcoded amplicons were sequenced using the paired-end method
with a 2 × 284-bp cycle run on the MiSeq system by MiSeq Reagent kit v.3 (600 Cycles) (Illumina, San Diego, CA, USA). After the alignment, overlapping regions within paired-end
reads were merged and primer regions were omitted, which resulted in a 430-bp sequence. Only reads with more than 99% of its sequence having quality value scores of ≥20 were
extracted for further analyses [43 (link)]. Chimeric sequences detected by Usearch6.1.544_i86 were precluded [15 (link)]. Based on these sequences, species were identified with an 97% confidence threshold using Metagenome@KIN analysis software (World Fusion, Osaka, Japan) and the
TechnoSuruga Lab Microbial Identification database DB-BA 10.0 (TechnoSuruga Laboratory, Shizuoka, Japan) [21 (link), 25 (link)]. The abundance of each taxon was calculated at both the phylum and genus levels (Supplementary Tables
3–6 ).
V3-V4 hypervariable regions of 16S rRNA were PCR amplified from microbial genomic DNA using universal primers for bacteria (341f/R806) [12 (link),
30 (link)] and the dual-index method [21 (link)]. Barcoded amplicons were sequenced using the paired-end method
with a 2 × 284-bp cycle run on the MiSeq system by MiSeq Reagent kit v.3 (600 Cycles) (Illumina, San Diego, CA, USA). After the alignment, overlapping regions within paired-end
reads were merged and primer regions were omitted, which resulted in a 430-bp sequence. Only reads with more than 99% of its sequence having quality value scores of ≥20 were
extracted for further analyses [43 (link)]. Chimeric sequences detected by Usearch6.1.544_i86 were precluded [15 (link)]. Based on these sequences, species were identified with an 97% confidence threshold using Metagenome@KIN analysis software (World Fusion, Osaka, Japan) and the
TechnoSuruga Lab Microbial Identification database DB-BA 10.0 (TechnoSuruga Laboratory, Shizuoka, Japan) [21 (link), 25 (link)]. The abundance of each taxon was calculated at both the phylum and genus levels (
3–6
