Soft windowing application to improve analysis of high-throughput phenotyping data.

MOTIVATION: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors. RESULTS: Here we introduce \u27soft windowing\u27, a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources. AVAILABILITY AND IMPLEMENTATION: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

Hyper-locomotive phenotype of female germ-free mice is only rescued by complex microbiota.

<p><b>(A)</b> Bars show mean (±SEM) cumulative distance travelled (centimeters) in the open field arena by germ-free (G), conventionalized (C), and <i>Bifidobacterium</i>-colonized (B) mice during the 30-minute test session. <b>(B)</b> Bars show mean (±SEM) cumulative time (seconds) that mice in each treatment group spent in active movement during the test session. <b>(C)</b> Bars represent mean rearing activity (average number of rears ±SEM) of each group of mice during the test session. <b>(D)</b> Bars represent average time spent rearing (seconds) during the test session. All data (A-D) are presented as means ±SEM. Significant treatment effects (T), sex effects (S), and interaction between treatment and sex (TxS) as determined by 2-way ANOVA are indicated under the title of each graph. Data are shown as sexes combined and sexes separated in the same graph for visualization purposes. Tests used to determine statistical significance notated in graphs are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196510#pone.0196510.s001" target="_blank">S1 Table</a>. *<i>p</i> < 0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001. Results presented as mean ± SEM. GF = germ-free (n = 9m/13f), CONV = Conventionalized (n = 11m/8f), BIF = <i>Bifidobacterium</i>-colonized (n = 8m/9f).</p

Postnatal colonization with human "infant-type" <i>Bifidobacterium</i> species alters behavior of adult gnotobiotic mice

<div><p>Accumulating studies have defined a role for the intestinal microbiota in modulation of host behavior. Research using gnotobiotic mice emphasizes that early microbial colonization with a complex microbiota (conventionalization) can rescue some of the behavioral abnormalities observed in mice that grow to adulthood completely devoid of bacteria (germ-free mice). However, the human infant and adult microbiomes vary greatly, and effects of the neonatal microbiome on neurodevelopment are currently not well understood. Microbe-mediated modulation of neural circuit patterning in the brain during neurodevelopment may have significant long-term implications that we are only beginning to appreciate. Modulation of the host central nervous system by the early-life microbiota is predicted to have pervasive and lasting effects on brain function and behavior. We sought to replicate this early microbe-host interaction by colonizing gnotobiotic mice at the neonatal stage with a simplified model of the human infant gut microbiota. This model consortium consisted of four “infant-type” <i>Bifidobacterium</i> species known to be commensal members of the human infant microbiota present in high abundance during postnatal development. Germ-free mice and mice neonatally-colonized with a complex, conventional murine microbiota were used for comparison. Motor and non-motor behaviors of the mice were tested at 6–7 weeks of age, and colonization patterns were characterized by 16S ribosomal RNA gene sequencing. Adult germ-free mice were observed to have abnormal memory, sociability, anxiety-like behaviors, and motor performance. Conventionalization at the neonatal stage rescued these behavioral abnormalities, and mice colonized with <i>Bifidobacterium</i> spp. also exhibited important behavioral differences relative to the germ-free controls. The ability of <i>Bifidobacterium</i> spp. to improve the recognition memory of both male and female germ-free mice was a prominent finding. Together, these data demonstrate that the early-life gut microbiome, and human “infant-type” <i>Bifidobacterium</i> species, affect adult behavior in a strongly sex-dependent manner, and can selectively recapitulate the results observed when mice are colonized with a complex microbiota.</p></div

Sociability impairments of germ-free mice are rescued by colonization with a complex microbiota.

<p><b>(A)</b> Bars show total time (seconds) that the test subject spent interacting with either the novel object (O) or novel partner mouse (M) cups during the 10-minute test session. Males and females are presented separately (combined sexes not shown) <b>(B)</b> Bars represent Sociability Index (% time spent interacting with the mouse cup/total time interacting with cups) by each group of mice during the test session. <b>(C)</b> Representative heatmaps from male and female mice in each treatment group demonstrating time spent interacting with empty cups (E) during the habituation trial, and interaction time around the object (O) or mouse (M) cups during the test trial. The scale of the heatmap ranges between 0 seconds (blue) and 60 seconds (red). Significant treatment effects (T), sex effects (S), and interactions between treatment and sex (TxS) as determined by 2-way ANOVA are indicated under the title of each graph. Data are shown as sexes combined and sexes separated in the same graph for visualization purposes. Tests used to determine statistical significance notated in graphs are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196510#pone.0196510.s001" target="_blank">S1 Table</a>. *<i>p</i> < 0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001. Results presented as mean ± SEM. GF = germ-free (n = 9m/13f), CONV = Conventionalized (n = 11m/8f), BIF = <i>Bifidobacterium</i>-colonized (n = 8m/9f).</p

Longitudinal analysis of the gut microbiome in mice from each treatment group.

<p><b>(A)</b> Representative micrographs from Carnoy’s-fixed and paraffin-embedded intestinal tissue. The left panel image is from the colon of a <i>Bifidobacterium</i>-colonized mouse and shows the result of an <i>in situ</i> hybridization with the Texas Red-labeled, <i>Bifidobacterium</i> genus-specific probe Bif164. The right panel shows colon tissue from a conventionalized mouse that was hybridized with the FITC-labeled universal bacterial probe, Uni519. Nuclei of host intestinal epithelial cells are counterstained in both images using DAPI. Inset in left panel demonstrates “bifid” morphology typical of <i>Bifidobacterium</i> species. White arrows indicate what was considered positive signal versus background in both panels, and white stars highlight the diversity of bacterial species present in the conventionalized mice as shown by the varying morphologies (rod vs. cocci) and localization. Images taken at 60x; scale bar = 20μm. <b>(B)</b> Alpha diversity of microbiota in each treatment group as measured by the Shannon Diversity Index (right panel) and Operational Taxonomic Unit (OTU) richness as measured by number of observed OTUs (left panel) at 24, 48, and 72 hours in each of the cohort groups. <b>(C)</b> Longitudinal relative abundance of OTUs. Phylum—level (Top panel) and Genus-level (Bottom panel) comparisons distributed by mouse group and timepoint post-transfer from gnotobiotic isolators. (D-E) Principal Coordinates Analyses (PCoA) showing <b>(D)</b> stability of the CONV fecal microbiome over time after transfer from gnotobiotic isolators, and <b>(E)</b> similarity between male and female fecal microbiome profiles in the conventionalized cohort. The percentage variation explained by each of the three primary principal factors is indicated on each axis. Coordinates representing individual samples are colored according to group, with distance to other coordinates indicating similarity/dissimilarity. (Data from 24h, 48h, and 72h timepoints in CONV group is combined in this analysis. Additional data detailing diversity by timepoint and sex in CONV group is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196510#pone.0196510.s003" target="_blank">S1 Fig</a>. Data also shown for other treatment groups parsed by timepoint in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196510#pone.0196510.s003" target="_blank">S1 Fig</a>). Results presented as mean ± SEM. *<i>p</i> < 0.05, **<i>p</i><0.01, ***<i>p</i><0.001. (fecal microbiome analysis: n = 3m/3f per timepoint,t totaling n = 9m/9f per treatment) GF = germ-free, CONV = Conventionalized, BIF = <i>Bifidobacterium</i>-colonized.</p

Neonatal <i>Bifidobacterium</i> colonization restores functional recognition memory in both males and females.

<p><b>(A)</b> Representative heatmaps from female mice in each treatment group demonstrating time spent (seconds) around each object in the arena (F = familiar object, N = novel object). The scale of the heatmap ranges between 0 seconds (blue) and 5 seconds (red). <b>(B)</b> Bars represent the Recognition Index (time spent interacting with novel object / total time spent interacting with objects). Significant treatment effects (T), sex effects (S), and interactions between treatment and sex (TxS) as determined by 2-way ANOVA are indicated under the title of each graph. Data are shown as sexes combined and sexes separated in the same graph for visualization purposes. Tests used to determine statistical significance notated in graphs are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196510#pone.0196510.s001" target="_blank">S1 Table</a>. *<i>p</i> < 0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001 Results presented as mean ± SEM. GF = germ-free (n = 9m/13f), CONV = Conventionalized (n = 11m/8f), BIF = <i>Bifidobacterium</i>-colonized (n = 8m/9f).</p

Anxiolytic behaviors of female germ-free mice in the elevated plus maze are normalized by <i>Bifidobacterium</i> colonization.

<p><b>(A-B)</b> Bars show time (seconds) spent in the open and closed arms of the elevated plus maze by germ-free (GF), Conventionalized (CONV), and <i>Bifidobacterium</i>-colonized (BIF) mice during the 10-minute test session. <b>(C)</b> Bars represent total distance traveled (centimeters) on the open arms by each group of mice during the test session. <b>(D)</b> Bars represent total number of entries to the open arms of the maze during the test session <b>(E)</b> Representative track plots from female mice in each treatment group. The open arm of the maze is designated. <b>(F)</b> Representative heatmaps from female mice in each treatment group indicating how much time the animal spent (seconds) in different parts of the apparatus. The open arm is as designated. The scale of the heatmap ranges between 0 seconds (blue) and 60 seconds (red). All data (A-D) are presented as means ±SEM (n = 16–22 per treatment group, males and females). Significant treatment effects (T), sex effects (S), and interactions between treatment and sex (TxS) as determined by 2-way ANOVA are indicated under the title of each graph. Data are shown as sexes combined and sexes separated in the same graph for visualization purposes. Tests used to determine statistical significance notated in graphs are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196510#pone.0196510.s001" target="_blank">S1 Table</a>. *<i>p</i> < 0.05, **<i>p</i><0.01, ***<i>p</i><0.001. Results presented as mean ± SEM. GF = germ-free (n = 9m/13f), CONV = Conventionalized (n = 11m/8f), BIF = <i>Bifidobacterium</i>-colonized (n = 8m/9f).</p