Subject recruitment, enrolment and processing
Nineteen subjects were recruited from a group of HA sojourners, from North Indian Ethnicity who had not travelled above 3000m in past six months. Inclusion criteria were fasting, no antibiotic treatment, no known gastro-intestinal diseases and previous bariatric surgery or medication known to affect the immune system and metabolism. All the participants were males in the age group of 22-55 years, had normal weight (BMI = 20-24kg /m2). Everyone was subjected to undergo thorough medical and psychological examinations for any diseases to ensure the healthy population. Information on medication status was obtained by questionnaire and interview on the first day of examination.
The analysis of gut microbiota of 19 sojourners from North Indian Ethnicity, with similar dietary habits, who climbed from 210m base line (H1) to various heights of Korakuram range, was performed using Illumina Miseq platform. Three participants, after staying at 4420m (H2) for six months descended to 3500m (H3). After staying for two months now ascended to 5800m (H4) to stay for four months. Thus 12 fecal samples from three subjects at four heights were analyzed for WGS. Whereas 32 samples of remaining 16 participants from the same cohort were analyzed by 16S rRNA sequencing for two heights (H1 and H2), to illustrate microbial diversity at extreme altitudes.
Participants collected stool samples in 1 ml of RNA later early in the morning. The samples were immediately distributed in aliquots of 500 μl each, and stored at -80°C for sequencing analysis. Though there is evidence that storage of samples at low temperatures maintains the microbial community structure36 , but yet the time span from sampling to delivery was intended to be as short as possible37 .