Structural insights of microbial community of Deulajhari (India) hot spring using 16s-rRNA based metagenomic sequencing.

Insights about the distribution of the microbial community prove to be the major goal of understanding microbial ecology which remains to be fully deciphered. Hot springs being hub for the thermophilic microbiota attract the attention of the microbiologists. Deulajhari hot spring cluster is located in the Angul district of Odisha. Covered within a wooded area, Deulajhari hot spring is also fed by the plant litter resulting in a relatively high amount of total organic content (TOC). For the first time, Illumina sequencing based biodiversity analysis of microbial composition is studied through amplicon metagenome sequencing of 16s rRNA targeting V3-V4 region using metagenomic DNA from the hot spring sediment. Over 28 phyla were detected through the amplicon metagenome sequencing of which the most dominating phyla at the existing physiochemical parameters like; temperature 69 °C, pH 8.09, electroconductivity 0.025 dSm(- 1) and total organic carbon 0.356%, were Proteobacteria (88.12%), Bacteriodetes (10.76%), Firmicutes (0.35%), Spirochetes (0.18%) and chloroflexi (0.11%). Approximately 713 species were observed at the above physiochemical parameters. The analysis of the metagenome provides the quantitative insights into microbial populations based on the sequence data in Deulajhari hot spring. Metagenome sequence is deposited to SRA database which is available at NCBI with accession no. SRX1459736.

Direct link to deposited data

http:www.ncbi.nlm.nih.gov/sra/SRX1459736.

Experimental design, materials and methods

Thermal spring's microbiomes have been in attention of the scientific community since past few decades. These springs, being hub of diverse microflora, increase the probability of vast gene pool of uncultured microbiota [1]. Metagenomics have helped the microbiologist to reveal the genome of the rest of the 99% of non-cultivable microbes which further helped to better understand the global microbial ecology and also helped in meeting the current demand for novel enzymes [2]. Thus, with the advances in metagenomics all the hidden facts of the microbial ecology have been faced off.

Odisha being rich in bio-diversity due to seasonal/climatic variations possesses a variety of hot-springs located in different geographical locations and vary both in physio-chemical and microbial ecology parameters. Mainly four major hot-springs have been reported in Odisha i.e. Deulajhari hot spring in Angul, Taptapani hot spring in Ganjam, Atri hot spring in Khurda and Tarabalo in Nayagarh. Deulajhari hot spring is located at about 6 km from Athamallik and between 20°3′ North latitude and 84°49′ East longitude.

In our study, sediment sample from Deulajhari hot spring (Latitude 20.74199 N, Longitude 84.49206 E) was collected from the hot spring having 69 °C temperature. Metagenomic DNA was extracted from sediment as described by Kumar and Khanna 2014 [3]. The V3–V4 region of the 16s rRNA was amplified using primers 341F, 5′-CCTACGGGAGGCAGCAG-3′ and 518R, 5′-ATTACCGCGGCTGCTGG-3′ with 50 ng of metagenomic DNA. The amplified PCR product was purified by gel elution using minelute column (Qiagen, India) and further leads to 150 nucleotide paired end multiplex sequencing using Illumina GAIIX Sequencer at Genotypic Technology Pvt. Ltd. (Banglore, India). QIIME data analysis package was further used for bioinformatics analysis. Krona tool was used for plotting Krona graph as depicted in Fig. 1 [4]. 2,073,312 high quality reads obtained from the hot spring with temperature of 69 °C were used for further analysis. Out of the total, Proteobacteria (88.12%), Bacteriodetes (10.76%), Firmicutes (0.35%), Spirochetes (0.18%), Thermi (0.13%) and Chloroflexi (0.11%) were identified at the phylum level. Of the total 216 genera in this Deulajhari hot spring only 53.7% were identified and 46.29% were unidentified at the genus level.

Fig. 1

Community structure of Deulajhari hot spring metagenome.

Since all the phylotypes, retrieved through the sequencing, do not contribute to all the taxonomic groups known till date, it indicates the significance of the diversity of Deulajhari hot spring explored.

Specifications
Organism/cell line/tissue	Metagenome of Deulajhari hot spring
Sex	Not applicable
Sequencer or array type	Illumina Genome Analyzer IIx
Data format	Raw data: FASTAQ file
Experimental factors	Environmental sample
Experimental features	16S rRNA genes amplified from the metagenome using Illumina platform followed by bacterial community analysis using QIIME version 1.9.0
Consent	Not applicable
Sample source location	Sediment sample, Deulajhari hot spring, Odisha, India.