Additional file 1 of Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1–V2 and V3–V4 primer sets

Additional file 1: Figure 1S. Comparison of (A) quality plots of raw reads, (B,C) percentage of analyzable reads between (B) V12qI and V34qI and (C) V12qII and V34qII, and (D) quality plots and length distribution of analyzable reads. (A,D) The mean quality scores are plotted. The y-axis on the graph shows the quality scores. The higher the score, the better the base call. The background of the graph divides the Q-score into good: Q > 28 (green), passable: 28 > Q > 20 (orange) and poor: 20 > Q (red). (B,C) The ratio of the number of analyzable reads to the number of raw reads is shown. Double asterisks indicate statistical significance (p < 0.01). Figure 2S. Percentage of classified operational taxonomic units (OTUs) and amplicon sequence variants (ASVs). At each classification level, the results of calculating the percentage of OTUs/ASVs assigned to a taxonomy in relation to the total number of OTUs/ASVs are shown. Figure 3S. (A,B) Differences in the operational taxonomic unit (OTU) and amplicon sequence variant (ASVs) counts for the indicated phylum between the V12 and V34 regions. Double asterisks indicate statistical significance (p < 0.01). (C,D) Comparison of the total OTU/ASV numbers for the indicated phylum. Unclassified OTUs/ASVs are included in k__Bacteria;__, k__Bacteria;p__, k__Archaea;__, and Unclassified;__. Figure 4S. Bar chart of the individual bacterial compositions using V12 and V34 at the phylum level using (A) qI (upper panel: V12; lower panel: V34) and (B) qII (upper panel: V12; lower panel: V34). Figure 5S. Relative composition of Bacteroidetes, Firmicutes, and Proteobacteria using V12 and V34. Figure 6S. Bar chart of the individual bacterial compositions using V12 and V34 for the indicated phyla using (A) qI (upper panel: V12. lower panel V34) and (B) qII (upper panel: V12; lower panel: V34). Figure 7S. Bar chart of the individual bacterial compositions using V12 and V34 for the indicated genera using (A) qI (upper panel: V12; lower panel: V34) and (B) qII (upper panel: V12; lower panel: V34). Figure 8S. Bar chart of bacterial relative abundance using a DNA mock community kindly provided by NITE (National Institute of Technology and Evaluation, Tokyo, JPN). This community is made from an equal mix of genomic data from the 10 indicated strains. qPCR was performed targeting the rplL gene of each bacteria and normalized by total bacteria using the measured value of 16S rRNA gene. rRNA copy num indicates the percentage of copy number of the 16S rRNA gene. V12 and V34 indicate the results of 16S analysis. Figure 9S. Scatter plot of the indicated genera to compare the 16S analysis by (A,B) qI, (C,D) qII, and qPCR. The identity line (y = x) is indicated. The gray area indicates the 95% confidence interval for each regression line. Figure 10S. Scatter plot of the indicated genera to compare the 16S analysis by qII and qPCR. The identity line (y = x) is indicated. The gray area indicates the 95% confidence interval for each regression line. Figure 11S. Alignments and similarity of Cronobacter and OTU representative sequence with Akkermansia for the 16S rRNA gene. (A) V12 (B) V34 of 16S rRNA gene. Akkermansia indicates reference sequences, which are derived from Akkermansia muciniphila strain JCM 30893. Cronobacter sequences are derived from Cronobacter sakazakii strain cro360A2. Observed_OTU sequence is a representative sequence assigned to Akkermansia, which was matched to Cronobacterthrough BLAST. The values of similarity indicate percent identity between the reference sequence and the query sequence calculated by BLAST.