Additional file 1 of CRISPR arrays as high-resolution markers to track microbial transmission during influenza infection

Additional file 1: Fig. S1. Analysis pipeline. (a) (yellow and red paths) The metagenomics reads post-quality filtering and removal of human reads were assembled into contig—metagenome assembled genomes (MAGs)—using metaSPAdes. Viral MAGs were identified using CheckV and VirSorter2 and taxonomic assignments were done using vConTACT2. Bacterial MAGs were binned with vamb and taxonomic assignment was done using GTDB-Tk. We then mapped the reads back to the taxonomically assigned viral or bacterial MAGs to generate the bacterial and viral profiles for downstream differential abundance analyses. (b) (brown paths) We identified and extracted the spacers from the metagenomic reads using Crass. Spacers with 90% sequence identity were clustered together. We then used these spacers to identify the shared spacers between individuals and within and across households. Reads with spacers shared between individuals were mapped to the bacterial MAGs that had taxonomic assignments. Bacterial species containing shared spacers were identified as being shared between individuals. Fig. S2. Top 30 most abundant bacterial taxa. Bacterial taxa were ranked by their mean relative abundance across samples. The top 30 bacterial taxa are shown in the boxplot with the x axis representing the relative abundance for each sample. Fig. S3. Viral MAGs identified. (a) Top viral MAG taxa identified as sorted by median relative abundance. The x-axis shows the relative abundance of the viral taxa of all samples while the y-axis indicates the viral taxa, listed as family/genus and phage species included within MAG clusters. (b) Viral MAGs differentially abundant between the high flu infection household vs control or low flu infection household vs control, identified with FDR cut-off as 0.05. The log2 fold changes are shown on the x axis with the blue/turquoise indicating flu infection household groups and gray the control household group. Fig. S4. Shared bacteria between flu infection households. The bar plots show the bacteria shared between individuals and how many pairs of individuals from the high flu infection, low flu infection and no flu infection households shared bacteria. Fig. S5. Mapping of the spacers to the bacteria shared between individuals for each sample. For all panels, the x axis represents the spacers mapped to the specific bacteria as indicated by the plot titles while the y axis represents the subject ID and timepoint of the sample. The colored dots mark households, as per Fig. 3. The shaded boxes indicate which family members had a direct connection based on the sharing of bacteria. Fig. S6. Sharing of Bacteria and flu infection. (a) Density and boxplot plot for percent of spacers shared at the individual level within and between households. The red line on the density plot indicates the cut-off where all the “between household” individual pairs were removed. (b) The connection network was generated based on the percent of shared spacers between individuals for the data above the cut-off in (a). The nodes represent individuals and the edges represent percent of shared spacers. Same color nodes represent individuals from the same household. (c) Dotplot for proportion of individuals in each household that were connected. Number of individuals in (b) in each household were divided by the total number of individuals in the households and compared across flu infection groups. The x axis indicates household code and the panels show the household from high, low, or no flu infection groups. Fig. S7. Proportion of shared spacers between individuals with different flu infection status. The box plot shows the proportion of shared spacers between any two individuals that were: (1) both positive for flu, (2) one positive for flu, one negative for flu or (3) both negative for flu. We compared the proportion of shared spacers between the three groups and Kruskal-Wallis test p values are shown between any two groups. * indicates p values <2.22e-16.