(A) The boxplot on the top panel shows the distribution of AUC values obtained when classifiers trained on different age-groups (YM: Young/Middle-aged; E: Elderly) in three cohorts of the curatedMetagenomicData (Training_Set1: ZellerG_2014, FengQ_2015 and VogtmannE_2016) are tested on the three datasets of the validation cohort (ThomasAJ_Cohort1, ThomasAJ_Cohort2 and WirbelJ_2019). The lower panel shows the same, but with age-group specific classifiers trained from within the validation cohort (Training_Set2). Both the classification models generated the same trends of classification, indicating age-group specific reproducibility of the disease signatures. The description of the point colors is the same as for Figure 2. (B) Age-group dependent associations of the known CRC markers in the two independent cohorts, namely Training_Set1 (curatedMetagenomicData) and Training_Set2 (Validation Cohort). Shades of blue indicate higher feature importance scores in the young/middle-aged and red indicates higher feature importance scores in the elderly. FDR p<0.15 indicates features identified as being high either in elderly of young/middle with Benjamini-Hochberg corrected Mann-Whitney test p-value<0.15. FDR p<0.25 indicates features identified as being high either in elderly of young/middle with Mann-Whitney test p-value<0.25. Out of the 19 known and validated CRC-markers (obtained from Thomas et al., 2019), 13 showed significant differences in their feature importance scores across the two age-groups (in the curatedMetagenomicData cohorts). For nine of these 13 markers, the pattern of associations could be reproduced in the Validation cohort, further indicating the replicability of the obtained results. The feature ranks of the top 10 markers obtained in Thomas et al. (2019) are also shown. Six of the top 10 markers show increased association, but only within the young/middle-aged. Only one of the markers associated with the elderly. This indicates a loss of disease-signature in the elderly. (C) Across cohort Spearman distances of feature rank profiles obtained for the disease classifiers trained on the different age-groups (See Materials and methods). A stable disease signature would result in reproducible species rank profiles across cohort and consequently lower Spearman distances. While this is the case for young/middle-aged, the elderly signatures obtained for the different cohorts show significantly high Spearman distances (showing significant variations and lack of disease signature). (D) The log ratios of the prevalence rates of the top six CRC-associated markers in elderly controls with respect to the young/middle-aged controls (in both the curatedMetagenomicData and CRC-specific cohorts). A positive value indicates higher prevalence rates in elderly controls. The significance of the increase is also indicated (p-values of fishers’ exact test combined using Fisher method) as ***: p<0.001, **: p<0.01, *: p<0.05. The increase in the elderly is characterized by a significant decrease in the effect-size differences between the controls and diseased in elderly, leading to masked signatures.