Table 1.
Designed mock community.
Fig 1.
Pipeline validation against mock communities.
(a) Stacked bar charts showing the actual relative abundance of bacterial families in the staggered and even BEI genomic mock communities and the measured relative abundance of the families obtained with the MiSeq sequencing pipeline. (b) The relative abundance as in a, but at the genus taxonomic level. (c) Relative abundance of the families obtained by sequencing genomic DNA of a single strain of bacteria or simple mix of bacterial genomic DNA. (d) Stacked bar charts showing the actual relative abundance of bacterial families in the plasmid based mock community and the measured relative abundance of the families obtained with the MiSeq sequencing pipeline. (e) The relative abundance as in d, but at the genus taxonomic level. The sequencing was performed in triplicate for all the samples (starting from the extracted DNA); the means of the triplicates are shown on the stacked bar charts.
Fig 2.
Pipeline sensitivity and assessment of the relative abundance accuracy.
(a) Stacked bar charts showing the relative abundance of bacterial families obtained by sequencing of a serial dilution of the plasmid-based mock community (from 10 and 107 copies of 16S gene for each of the 14 bacterial strains) compared to the actual relative abundance (right stacked bar). (b) Generalized UniFrac distance based PCoA analysis of the sequencing of a serial dilution of the plasmid-based mock community shown in a compared with the negative control generated by sequencing molecular biology-grade water with the same pipeline (H2O). UniFrac weight parameter (Alpha) was set to 0.6 for this analysis. (c) Stacked bar charts showing the relative abundance of bacterial families obtained by sequencing 7 mixes with different ratios of two of the bacterial plasmids containing the 16S gene of Geofilum rubicundum (GR) and Eubacterium barkeri (EB). The sequencing was performed in triplicate for all the samples (starting from the extracted DNA); the means of the triplicates are shown in a and individual triplicates are shown in b and c.
Fig 3.
Replicability and reproducibility.
(a) Stacked bar charts showing the actual relative abundance of bacterial families in the staggered and even BEI genomic mock communities and the measured relative abundance in triplicates of the families obtained with the MiSeq sequencing pipeline. (b) Stacked bar charts showing the relative abundance of bacterial families obtained by sequencing of triplicates of fecal samples and ileum mucosa samples collected from three different mice (Sample 1, 2 and 3). (c) Stacked bar charts showing the relative abundance of bacterial families obtained by sequencing of three samples of mouse ileum mucosa in six to seven runs each with different parameters described in the legend at the bottom left: different runs, new libraries from same extracted DNA or the same libraries already prepared, different MiSeq kit generation and different sequencers. Two different experimenters performed the different runs and reagent batch numbers (including Taq polymerase) varied from run to run.
Fig 4.
16S metagenomics on diverse tissue samples.
(a) Heatmap of the relative abundance of each bacterial family from sequencing of different mouse tissue samples performed in triplicate (three different mice for each tissue). Each line corresponds to a bacterial family; each one of the three columns for a tissue corresponds to a different mouse. (b) Generalized UniFrac distance-based PCoA analysis of sequencing data from the samples shown in a compared with the negative control generated by sequencing molecular biology-grade water with the same pipeline (H2O). UniFrac weight parameter (Alpha) was set to 0.2 for this analysis. (c) Rarefaction curve of the sequencing of the samples shown in a and b. For each tissue, only the sample with the median number of OTU is displayed. (d) Stacked bar charts showing the relative abundance of bacterial phyla obtained by sequencing of the mouse samples shown in a, b and c. (e) The relative abundance as in d, but at the family taxonomic level. MAT: Mesenteric adipose tissue. OTU: Operational Taxonomic Unit.