Metagenomic Sequencing of Microbial Communities from Brackish Water of Pangong Lake of the Northwest Indian Himalayas

ABSTRACT Pangong is a brackish water lake having environmental conditions that are hostile to supporting life. This is the first report unveiling the microbial diversity of sediment from Pangong Lake, Ladakh, India, using a high-throughput metagenomic approach. Metagenomic data analysis revealed a community structure of microbes in which functional genetic diversity facilitates their survival.

S ediments are rich sources of microbial diversity and represent a special realm in aquatic environments (1). To overcome the limitations of the culture approach in studying these organisms, culture-independent approaches like metagenomics are applied to characterize microbial communities, discover novel genes, and analyze metabolic pathways directly from the environment (2, 3). There is very limited information available on the microbial diversity present at high-altitude cold habitats of the Himalayas (4). The present study investigates, through a metagenomic approach, the functional genetic diversity of microbes present in Pangong Lake, a large brackish water lake situated at a height of 4,250 m above mean sea level in the Himalayas. The microbes present there are halotolerant and cold adapted, and identifying the diversity of the novel cold-active enzymes and secondary metabolites assisting in the survival of these microbes may have great biotechnological potential.
The sediment samples were obtained from Pangong Lake (33°43=04.59ЉN: 78°53=48.48ЉE), Ladakh, J&K (Jammu and Kashmir) India, in September 2016 and stored at 4°C until further analysis. The DNA was extracted using the Exgene soil DNA kit (GeneAll Biotechnology Co., Ltd.), and sequencing was performed on the Illumina platform. The paired-end sequencing libraries (2 ϫ 150 bp) were prepared using the Illumina TruSeq Nano DNA library prep kit and were sequenced on the Illumina NextSeq500 platform. The raw data were processed to obtain high-quality clean reads (quality value Ͼ20) using Trimmomatic version 0.35 (5). The filtered high-quality reads of the sample were assembled into scaffolds using CLC Genomics Workbench, and genes were predicted using Prodigal version 2.6.3 with default parameters (6). Taxonomic analysis of the predicted genes was carried out using Kaiju (7), a program for sensitive taxonomic classification of high-throughput metagenomics sequencing data. Cognizer (8), which is a comprehensive stand-alone framework that simultaneously provides COG (9), KEGG (10), Pfam (11), GO (12), and SEED (13) subsystem annotations to individual sequences constituting metagenomics data sets, was used for performing the functional analysis of the genes.
Metagenomic analysis revealed a diverse domain of microbial communities thriving in harsh conditions, creating a base for further microbial exploration to improve the efficacy of bioprospecting metagenomics of soil and sediment, which may lead to the discovery of novel enzymes and bioactivities.
Accession number(s). The nucleotide sequences reported here have been submitted to the NCBI Sequence Read Archive (SRA) under accession number SRX2861366.

ACKNOWLEDGMENTS
R.R. is grateful to the UGC:NFSC, New Delhi, for a Junior Research Fellowship. I.S.T. extends thanks to the Shastri Indo-Canadian Institute (SICI), New Delhi, India, for financial support as a visiting scientist during the period in which the manuscript was prepared.
We have not received grants from other funding agencies. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.