Whole Metagenome Analysis of Extreme Environment Samples by Quasi-Linear Whole Genome Amplification

Abstract

Microbiomes are gaining a lot of importance owing to its impact in the human health and other life processes. The significance of microbes in the deep sea is often undermined but it holds the key for the environmental changes that affects our planet. Recently scientists are investigating the diversity of microbial life in the sea to learn its role in the ocean processes, its interactions with other marine life, and its importance to humans. Emergence of cutting edge genomics and molecular biology tools has propelled the field of metagenomics to study the microbial populations without the need to culture them. In spite of these growth studies of extreme environmental samples with very low nucleic acid concentration remains still a major challenge. A whole genome amplification method with less bias is required to get over this problem. Commercially available single cell whole genome amplification kits don’t perform well with the low concentration of samples. A minimum of 100ng of DNA is the input requirement for these kits and some of the extreme environment samples can only yield 1-10ng of DNA. Our aim was to devise an efficient and cost effective workflow that could work with very low concentration of metagenomic DNA. We have adopted a quasi-linear whole genome amplification methodology, Multiple Annealing and Looping Based Amplification Cycles (MALBAC) that was earlier used in the single cell genomic studies.MALBAC methodology was carried out using 4ng of the deep sea sediment DNA and it was compared against the NEB Next Ultra DNA Library prep kit using 125ng of deep sea sediment DNA. The data generated were analysed and compared for the microbial diversity and gene function annotations. The results obtained through MALBAC methodology were comparable to that of the commercial kit based procedure utilizing nearly 125ng of DNA.The MALBAC based whole metagenome analysis of deep sea sample from Arabian Sea revealed the immense microbial diversity of unexplored extreme environment. The study also identified the functional genes coding for valuable enzymes and proteins that could be used in the biotech industry. This study has showed that MALBAC is a cost effective procedure for extreme environment samples containing very low concentration of DNA.