The taxonomic analysis by WGS at Genus level
Next, we analysed the frequency of most abundant genera in 12 fecal
samples at four different altitudes (Figure 4). The relative abundance
of around 20 genera was more than 1%. At H1 time point genus
Bacteroides was the most abundant (46%), followed by Prevotella (19%),
Parabacteroides and Alistipes (7%), Eubacterium (5%), Faecalibacterium
(3%) Ruminococcus (2%), Blautia, Coprobacillus, Clostridium,
Escherichia, Bifidobacterium, and unclassified (1%) (Figure 4 A).
Interestingly, after ascending to 4420m (H2) and staying for 6 months,
the richest genus was Prevotella (63%) with significant decrease in
Bacteroides to 2%, followed by Eubacterium, Faecalibacterium 3%, and
Parabacteroides 1%. There was a marginal increase in Coprococcus
contribution to 4%. Surprisingly even after getting adapted to
stressful environment at H2, on descending to H3, the scenario changed
markedly where Bacteroides reversed and increased significantly to 31%,
though still lower than Prevotella which was reduced to 43%.
Incidently, there was sudden appearance of Fusobacterium (5%),
Roseburia (4%) and Escherichia (2%). The pattern was similar to H1.
Interestingly, again the phylogeny structure was changed when they
ascended to 5800m (H4) and stayed for four months. Bacteroides
significantly reduced to 9%, Prevotella significantly increased to
65%, Eubacterium (3%), Faecalibacterium (2%), whereas
Parabacteroides, Alistipes, Coprococcus, Escherichia, and Coprobacillus
(1%) started reappearing. The pattern looked similar to H2. When
analyzing the overlap of different genera at four different altitudes,
it mostly belonged to Bacteroides and Prevotella, where Prevotella
covered the 63% of the total genera at H2 and H4 heights and
Bacteroides at H1 and H3. Although 3500m (H3) is defined as high
altitude, interestingly, the pattern of the metagenomic profile obtained
at H3 was quite similar to the base line profile (H1= 210m).
Further confirmation of the differences in the composition of fecal
microbiota of sojourners at different heights was achieved by LEfse
using the Greengene software. FDR correction of the p value was to
determine statistical significance (p < 0.05/number of tests)
and considered p < 0.05 to be statistically significant for
reproducibility (Figure 4D). Thus the genus abundance clearly indicated
the significant change in the microbiota mainly with abundance of
Bacteroides (p= 0.05) and Prevotella (p= 0.05). LEfse
analysis also showed concordant result with Wilcoxon rank test
(Supplementary FigureS4 with LDA score) indicating altitude or hypoxia,
could be the most important contributing factor to the diversity.
The WGS analysis of three subjects revealed the presence of 20 genera.
Amongst these, the relative abundance of genus Bacteroides, Prevotella,
Bifidobacterium and Fecalibacteria were significantly altered (with p
<0.05 Kruskal Wallis H test) (Figure 4 A). We reproduced and
correlated the significant presence of 20 genera in another 16 subjects
of the same cohort at H1 and H2 heights (p<0.05 Wilcoxon rank
test) with odds ratio of 95% (Figure 4B), by 16s rRNA analysis. The
second method reconfirmed the changes in the abundance of genus
Bacteroides, Prevotella, Faecalibacterium & Bifidobacterium as in WGS
(Figure4 C). Interestingly compared to WGS, the 16s rRNA also had higher
prevalence detection rate for other genus like Ruminococcus and
Akkermansia at H2 height.