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.