Functional diversity analysis
In order to investigate the functional potential, we compared the abundance of specific pathways and functional processes across the samples at H2, H3 and H4 in comparison to H1, and inferred using MOCAT approach. FMAP software was used to identify differentially abundant (DA) genes using Kruskal-Wallis rank-sum test (fold change > 2, p­value cutoff 0.05). Pathway enrichment analysis was used to determine differentially abundant pathways using Fishers exact test49 , with average log2 (fold change) and enriched significance (p value cut-off of 0.01), to identify significant differentiators between the heights. The analysis identified a set of 27 different functional processes (Figure 7).
FMAP generated significantly noticeable differences between functional potential encoded at different heights, mainly down regulated Carbohydrate; Amino Acid; Energy; Vitamins & Cofactor, biosynthesis of secondary metabolites & antibiotics and other Metabolic Pathways at H2 and H4 as compared to H1 (Figure 7). Incidentally, these pathways were upregulated at H3, which was expected, as the sojourners descended from H2 to H3, a bit close to H1. Samples at H2 and H3 appeared to be more enriched with genera than at H4 that encoded functions associated with four other pathways namely Biosynthesis of Amino acids and Fatty acid metabolism, both at H2 and H3, whereas, Xenobiotic degradation & metabolism and endocrine and metabolic diseases are more at H2, and Lipid metabolism and Glycan biosynthesis & metabolism are overrepresented at H3. Interesting to note that the microbial metabolism in diverse environments was markedly reduced at all three heights. The pathways detected in three subjects by WGS were reproduced by 16s rRNA in another 16 subjects at H1 and H2. The most common six sub-pathways were glucose seven phosphate isomerase; GDP mannose 4,6 dehydratase; 8-amino7-Oxononanoate Synthase; GMP synthase (glutamine hydrolysine); Phospho-ribosyl-formyl glycine amidinecycloligase; Preprotein translocase subunit fraction secA; Phospho-ribosyl-formyl-glycine–amidine-synthase (Supplementary Figure 8.) It is quite possible that these functional capabilities are related to high altitudes stress, however further studies are required.