Extended Data Fig. 4: Characterization of the 11 strains.
From: A defined commensal consortium elicits CD8 T cells and anti-cancer immunity
a, DNA was extracted from each of the 26 strains and 16S rRNA gene sequences were determined by PCR and Sanger sequencing. Genome sequencing was conducted for 21 strains using the Illumina MiSeq or the MinION nanopore sequencer. To identify the closest reference species or strain, 16S rRNA gene and genes encoding 42 ribosomal proteins predicted from the assembled draft genome of each strain were blasted to the RDP and NCBI genome databases (RefSeq genome representative), respectively. Top-hit strains were defined as those with the highest 16S rRNA sequence similarity or those with the highest ribosomal gene sequence similarity for the maximum percentage of the 42 queried genes (strains for which this is less than 37 out of 42 are listed in parentheses). Percentage similarity refers to average sequence similarity between ribosomal genes of the isolated strain and those of the top-hit reference strain. b, A phylogenetic tree was constructed by comparing 42 concatenated ribosomal gene sequences of each isolate using the MEGAv7.0 package and the neighbour-joining method with a bootstrap of 1,000 replicates. c, Relative expression of the indicated genes in colonic epithelial cells (ECs) of germ-free mice colonized with or without the 11-mix for 1 week, as determined by qPCR. d, Frequencies of TCR Vβ gene usage among IFNγ+ CD8 T cells (left) and IFNγ− CD8 T cells (right) from the colons of GF (grey) or GF+11-mix (red) mice, as determined by flow cytometry. 2-way ANOVA interaction p-values: 0.0001 (IFNγ+ subset), 0.31 (IFNγ− subset). e, Luminal contents from the indicated anatomical positions of the gut as well as faecal samples were collected from three GF+11-mix mice 3-4 weeks post-colonization. The relative abundance of each of the 11 strains’ DNA was determined by qPCR. f, MLNs were collected from GF+11-mix mice 1 week after colonization and bacterial DNA was extracted. The relative abundance of each of the 11 strains’ DNA was determined by qPCR. Each circle represents an individual animal (c, e) or a pool of mice (d), and the height of each bar indicates the mean, and the number of mice in each group is shown. n.d., not detected. Error bars, s.d. ***P < 0.001; **P < 0.01; *P < 0.05; two-tailed unpaired t-test. See Source Data for exact P values.
