An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples.

Satyabrata Bag,Oluf Pedersen,Takashi Kurakawa,Naveen Kumar,Venkatesan Radha,Mayanka Dayal,Archana Pant,Nitya Wadhwa,Visvanathan Gnana Prakash,Shruti Saxena,Henrik Vestergaard,Kristine H Allin,Reva Tripathi,Kiyoshi Takeda,Shinjini Bhatnagar,Bipasa Saha,Ojasvi Mehta,G Balakrish Nair,Philip Abraham,D Anbumani,Manimozhiyan Arumugam,Bhabatosh Das,Verima Pereira,Torben Hansen,Promod Kumar ,Viswanathan Mohan ,R M Anjana

doi:10.1038/srep26775

Abstract

To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed.

Highlights

Rare representatives of each taxonomic groups possessing different thickness of cell wall and different layer of cell membranes with different embedded components casing their genomic contents
Several laboratories working on metagenomics reported different methods of community DNA extraction depending on the type of samples they used for analysis[2,3,4,5,6,7,8]
Environmental and human samples contain large numbers of microbial cells belonging to different phyla and they are reasonably heterogeneous in terms of their genomic contents, morphology and architecture of their cell wall (Fig. 1)

Summary

Introduction

Rare representatives of each taxonomic groups possessing different thickness of cell wall and different layer of cell membranes with different embedded components casing their genomic contents. We developed a highly sensitive method, by combining physical, mechanical and chemical lysis approaches, to isolate community bacterial DNA from different human and environmental samples (Fig. 2). All the selected samples harbor culturable and uncultured bacteria belonging to closely or distantly related taxonomic groups and having different thickness of cell wall and different layer of cell membranes (Fig. 1). We compared both the quality and quantity of isolated community DNA with existing methodologies and observed that this approach worked best compared to currently available approaches. The isolated DNAs are suitable for all types of high-resolution downstream applications including shotgun metagenomics sequencing where HMW genomic DNA is preferable

Methods

Results

Conclusion