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.

附加文件1：图S1 分析流程。
(a)（黄色与红色通路）经质量过滤并去除人类读段后的宏基因组读段，通过metaSPAdes工具组装为重叠群——即宏基因组组装基因组（metagenome assembled genomes, MAGs）——。使用CheckV与VirSorter2识别病毒MAGs，并通过vConTACT2完成分类学注释；细菌MAGs则通过vamb进行分箱，并使用GTDB-Tk完成分类学注释。随后将读段比对至已完成分类学注释的病毒或细菌MAGs，以生成细菌与病毒谱用于后续差异丰度分析。
(b)（棕色通路）使用Crass工具从宏基因组读段中识别并提取间隔序列（spacers）；将序列一致性≥90%的间隔序列进行聚类。随后利用这些间隔序列识别个体间、家庭内部及家庭间的共享间隔序列。将携带个体间共享间隔序列的读段比对至已完成分类学注释的细菌MAGs，携带共享间隔序列的细菌物种即被判定为个体间共享的细菌。

图S2 丰度排名前30的细菌分类群。细菌分类群按其在所有样本中的平均相对丰度进行排序。本箱线图展示了丰度排名前30的细菌分类群，横轴代表各样本的相对丰度。

图S3 已鉴定的病毒MAGs。
(a) 按中位相对丰度排序的已鉴定病毒MAG分类群。横轴展示所有样本中病毒分类群的相对丰度，纵轴标注病毒分类群，格式为MAG簇所包含的科/属及噬菌体物种。
(b) 以错误发现率（false discovery rate, FDR）阈值0.05筛选得到的高流感感染家庭vs对照家庭、低流感感染家庭vs对照家庭间差异丰度的病毒MAGs。横轴展示log₂倍变化值，蓝色/青绿色代表流感感染家庭组，灰色代表对照家庭组。

图S4 流感感染家庭间的共享细菌。本条形图展示了个体间共享的细菌，以及高流感感染家庭、低流感感染家庭与无流感感染家庭中，分别有多少对个体共享细菌。

图S5 各样本中间隔序列比对至个体间共享细菌的情况。所有子图中，横轴代表比对至图标题所标注特定细菌的间隔序列，纵轴代表样本的受试者ID与采样时间点。彩色圆点代表家庭分组，与图3一致。阴影框标注了基于细菌共享关系存在直接关联的家庭成员。

图S6 细菌共享与流感感染。
(a) 家庭内部与家庭间个体水平共享间隔序列比例的密度图与箱线图。密度图中的红色线条代表阈值，用于移除所有“家庭间”的个体对。
(b) 基于(a)中阈值以上的个体间共享间隔序列比例数据生成连接网络。节点代表个体，边代表共享间隔序列比例。颜色相同的节点代表来自同一家庭的个体。
(c) 展示各家庭中存在连接的个体比例的点图。将(b)中每个家庭的个体数除以该家庭总个体数，随后在不同流感感染分组间进行比较。横轴代表家庭编码，各子图分别对应高流感感染组、低流感感染组与无流感感染组的家庭。

图S7 不同流感感染状态个体间的共享间隔序列比例。本箱线图展示了任意两两个体间的共享间隔序列比例，分组为：(1) 均为流感阳性、(2) 一方流感阳性一方阴性、(3) 均为流感阴性。我们对三组间的共享间隔序列比例进行了比较，任意两组间的Kruskal-Wallis检验P值均已标注。*代表P值<2.22×10^-16。