Supplementary file: sensitivity analyses
Supplement to: Borgdorff H, Gautam R, Armstrong SD, Xia D, Ndayisaba GF, van Teijlingen NH,
Geijtenbeek TBH, Wastling JM, van de Wijgert JHHM. Cervicovaginal microbiome dysbiosis is associated with proteome changes related to alterations of the cervicovaginal mucosal barrier.
Results
Hormonal contraception
Twenty-one women were assigned to the hormonal contraception group (they used oral contraception
(n=4), injectable contraception (n=14), or hormonal implants (n=3)), 28 women were assigned to the non-hormonal group (they used condoms only (n=25) or no methods at all (n=3)), and one women who used an intrauterine device (IUD) was excluded from both groups. Heatmaps for the two hormonal groups are shown in Figures S1 and S2.
In the hormonal contraception group, the number of samples per vaginal microbiome (VMB) group was as follows: group 1 two samples, group 2 four samples, group 3 six samples, and group 4 nine samples.
Groups 1 and 2 were combined in the untargeted analysis (for explanation, see main manuscript) because of the small sample sizes. No differentially abundant proteins were identified using ANOVA and the Bonferoni-adjusted p-value cut-off of 9.1*10 -5 . However, the 82 proteins that were differentially abundant in the original analysis did show similar trends in this sensitivity analysis, and 88% of them were significant at a p<0.05 level. The main difference with the original analysis was that women using hormonal contraception who were in VMB group 3 (moderate dysbiosis) had a more similar protein profile to women in VMB groups 1 and 2 (lactobacilli-dominated) than to women in VMB group 4

(severe dysbiosis; Figure 8 in the main manuscript and Figure S1). The hypothesis-driven analyses showed similar trends as the original analyses (data available on request).
In the non-hormonal group, the number of samples per VMB group was as follows: group 1 four samples, group 2 seven samples, group 3 eight samples, and group 4 nine samples. Fifty-nine differentially abundant proteins were identified using ANOVA with Bonferroni correction, of which 44 proteins were also differentially abundant in the original analysis and the remaining 12 proteins had a pvalue of <0.03 in the original analysis (results not shown). All 38 proteins that were not differentially abundant in this sensitivity analysis but were in the original analysis had a p-value<0.02 in the sensitivity analysis. The hypothesis-driven analyses showed similar trends as in the original analyses (data available on request).
HIV-status
When the analysis was restricted to HIV-negative women, group 1 contained six samples, group 2 seven samples, group 3 six samples, and group 4 eight samples. Comparison of relative protein abundance between microbiome groups of the HIV-negative women using ANOVA with Bonferroni correction resulted in 27 differentially abundant proteins (results not shown). All of these proteins were also differentially abundant in the original analysis. The remaining 55 proteins that were not differentially abundant in HIV-negative women using the Bonferroni-adjusted p-value cut-off of 9.1*10^-5 had pvalues of <0.03 and showed similar trends as in the original analysis (Figure S3). The hypothesis-driven analyses showed similar trends as in the original analyses (data available on request).

Additional data available on request. Requests can be submitted to:
Professor Janneke van de Wijgert
Department of Clinical Infection, Microbiology and Immunology
Institute of Infection and Global Health, University of Liverpool
8 West Derby Street
Liverpool L69 7BE, UK.
Email: j.vandewijgert@liv.ac.uk
.
Tel: 44-151-795-9613
Fax: 44-151-795-5529

Figure S1. Hierarchical clustering, heatmap and classification of the 82 proteins that were differentially abundant in the original analysis of all 50 women, now restricted to women using hormonal contraception (n=21).
(A) Hierarchical clustering of the range-scaled ion intensities of 82 proteins that were differentially abundant in the original analysis. (B) Heatmap of normalized ion intensities per protein. Each row represents a protein and the color indicates the range-scaled (0 to 1) ion intensity per sample as indicated in the color bar below the heatmap. Each column represents a sample, with samples being ordered on microbiome composition as indicated on the top. (C) Molecular functions and biological processes represented in the group of proteins. A black box indicates that the protein is assigned into the particular classification. (D) p-value of the ANOVA performed of each protein among the microbiome groups, as indicated in the color bar below the bar. Each row represents a protein, in the same order as the heatmap.

Figure S2. Hierarchical clustering, heatmap and classification of the 82 proteins that were differentially abundant in the original analysis of all 50 women, now restricted to women not using hormonal contraception (n=28).
(A) Hierarchical clustering of the range-scaled ion intensities of 82 proteins that were differentially abundant in the original analysis. (B) Heatmap of normalized ion intensities per protein. Each row represents a protein and the color indicates the range-scaled (0 to 1) ion intensity per sample as indicated in the color bar below the heatmap. Each column represents a sample, with samples being ordered on microbiome composition as indicated on the top. (C) Molecular functions and biological processes represented in the group of proteins. A black box indicates that the protein is assigned into the particular classification. (D) p-value of the ANOVA performed of each protein among the microbiome groups, as indicated in the color bar below the bar. Each row represents a protein, in the same order as the heatmap.

Figure S3. Hierarchical clustering, heatmap and classification of the 82 proteins that were differentially abundant in the original analysis of all 50 women, now restricted to HIV-negative women (n=27).
(A) Hierarchical clustering of the range-scaled ion intensities of 82 proteins that were differentially abundant in the original analysis. (B) Heatmap of normalized ion intensities per protein. Each row represents a protein and the color indicates the range-scaled (0 to 1) ion intensity per sample as indicated in the color bar below the heatmap. Each column represents a sample, with samples being ordered on microbiome composition as indicated on the top. (C) Molecular functions and biological processes represented in the group of proteins. A black box indicates that the protein is assigned into the particular classification. (D) p-value of the ANOVA performed of each protein among the microbiome groups, as indicated in the color bar below the bar. Each row represents a protein, in the same order as the heatmap.