Gut microbiome community changes in SCD patients (n=98) compared to healthy controls (n=46).

Sample whole community sequencing was profiled using the MetaPhlAn taxa marker database 20. (a) Distribution of sample alpha diversity measured as Shannon diversity for patients and controls. (b) A generalized linear model was used to determine the effect size and association of bacteria taxa abundance with sample metadata including SCD status, age, race, ethnicity, and gender, all modeled as fixed effects. Taxa with q-value < 0.05 for any effect are shown. (c) Distribution of Firmicutes to Bacteroidetes (F:B) ratio in the gut microbiome of patients and controls. (d) Scatter plot of health and disease indicator scores per sample. (e) Sample whole community sequencing was profiled using the HUMAnN pathway marker database 20. A generalized linear model was used to determine the effect size and association of pathway abundance with sample metadata including SCD status, age, race, ethnicity, and gender, all modeled as fixed effects. Additionally, the F:B was also included as fixed effect. Pathways with q-value < 0.05 for any effect are shown. In a and c, samples with values +/-3 s.d. from mean of all samples were removed. M.W.W.-Mann-Whitney-Wilcoxon test.

Aged neutrophil percentage (n=57) and gut microbiome feature (n=98) correlations with clinical measures and blood molecular immune markers.

Correlations were measured with Spearman ρ and significance was measured using permutation testing. Value of correlation is colored for strength of correlation where p < 0.05, otherwise comparison cell is gray. For the number of samples assayed for each clinical and molecular measure, see Supplemental Tables 4-5.

Gut microbiome viral population is altered in SCD.

Sample whole community sequencing was assembled and viral sequences were identified using marker genes and sequence features. (a-b) The number of viral sequences (a) and the fraction of provirus sequences (b) between patients and controls. (c) Comparison of viral population and bacterial population community-level metrics. (d) Histogram of active provirus fraction for SCD samples. (e) Correlation of viral community-level metrics with clinical and molecular markers as in 3b and 3c. Value of correlations is shown for comparisons with p < 0.05 otherwise comparison cell is gray. M.W.W = Mann-Whitney-Wilcoxon test.

Highly conserved prophage sequences are shared by gut microbiomes of SCD patients.

(a) Patient microbiomes sharing a highly conserved prophage sequence visualized as fully connected networks. Conserved prophages had 99% sequence identity over at least 70% of bidirectional sequence coverage. Node color indicates predicted prophage activity: active (blue), dormant (orange), and undetermined (gray). (b) Prophage sequence distribution of high-level viral protein functions compared between clustered (n=1,249) and singleton (n=3,713) prophages. Significance tested with a Mann-Whitney-Wilcoxon test: **** = p < 0.0001.

A model of interactions between the gut microbiome and markers of inflammation in the blood of SCD patients.

The gut microbiome can influence and is influenced by the immune system. Using multiple data modalities, we begin to understand the complex interaction of gut microbiome changes and immune system activation in the pathology of SCD.

SCD patient blood neutrophil profiling.

Neutrophil and AN counts were assayed by flow cytometry.

Correlation of flow cytometry and EHR measures of patient white blood cell populations.

Correlation measured with Pearson correlation coefficient. Abbreviations: WBC-white blood cells, ANC-absolute neutrophil count.

Enrichment in provirus or lysogenic virus prediction across multiple labeling methods.

The determination of a lysogenic virus can be made by either looking for the virus sequence integrated into a host genome or by predicting that a viral sequence has lysogenic potential. We utilized multiple methods that rely on different approaches to determine that the provirus enrichment observed is robust to method.

Provirus fraction but not non-integrated lysogenic virus fraction correlate with immune cytokines.

Using the vibrant viral calling method, the fraction of non-integrated, lysogenic predicted viruses was quantified and compared to the provirus fraction for correlation with molecular cytokines.

Provirus length distribution with kernel density estimation.

Prophage cluster sequence homology with bacterial hosts.

Representative cluster sequences with blast sequence homology to bacterial species sequences were aggregated to the genus level. Count represents the number of clusters homologous to species in the genus.

Genome content comparison between predicted active (n=136) and dormant (n=4739) prophages in the SCD gut microbiome.

Significance tested with a Mann-Whitney-Wilcoxon test: * = 0.01 <= p < 0.05; ** = 0.001 < p <= 0.01.

Study participant demographics by cohort.

SCD patient (n=98) medical history.

SCD patient (n=98) treatment history.

Clinical measures for SCD patients (n=98).

Molecular inflammatory measures for SCD patients (n=98).