Biotechnology Journal DOI 10.1002/biot.200800201 Biotechnol. J. 2009, 4, 480–494 480 © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 Introduction The first life forms, small microorganisms, have been found in fossils that are about 3.5 billion years old. At present, the total number of prokaryotic cells on earth has been estimated at 4 × 10 30 –6 × 10 30 [1], comprising 10 6 and 10 8 separate genospecies (distinct taxonomic groups based on gene se- quence analysis) [2]. This diversity presents an enormous but largely unexplored genetic and bio- logical pool and can be exploited for the recovery of novel genes, entire metabolic pathways and their products [3]. These microorganisms can be ac- cessed primarily by a classical approach, involving culturing the microorganism by preparing a solid or liquid growth medium containing appropriate carbon, energy and electron acceptor sources de- pending on the physiological conditions under which the organism is to be isolated. However, gen- eral routine conditions provided in the laboratory tend to impose selective pressure, thereby prevent- ing the growth of large number of microorganisms [4], but studies have shown that only 1–15% of mi- crobial genomes are cultivable under laboratory conditions and more than 85% have never studied [2]. Further, simple morphological and physiologi- cal traits of most microbes provide few identifica- tion clues [5]. This problem can be rectified by the use of phylogenetically directed isolation strate- Review Metagenomics: Concept, methodology, ecological inference and recent advances Jagtar Singh 1 , Arvind Behal 2 , Neha Singla 1 , Amit Joshi 3 , Niti Birbian 1 , Sukhdeep Singh 1 , Vandana Bali 2 and Navneet Batra 2 1 Department of Biotechnology, Panjab University, Chandigarh, India 2 Department of Biotechnology, GGDSD College Sector-32, Chandigarh, India 3 Department of Biotechnology, SGGS College Sector-26, Chandigarh, India Microorganisms constitute two third of the Earth’s biological diversity. As many as 99% of the mi- croorganisms present in certain environments cannot be cultured by standard techniques. Cul- ture-independent methods are required to understand the genetic diversity, population structure and ecological roles of the majority of organisms. Metagenomics is the genomic analysis of mi- croorganisms by direct extraction and cloning of DNA from their natural environment. Protocols have been developed to capture unexplored microbial diversity to overcome the existing barriers in estimation of diversity. New screening methods have been designed to select specific functional genes within metagenomic libraries to detect novel biocatalysts as well as bioactive molecules ap- plicable to mankind. To study the complete gene or operon clusters, various vectors including cos- mid, fosmid or bacterial artificial chromosomes are being developed. Bioinformatics tools and databases have added much to the study of microbial diversity. This review describes the various methodologies and tools developed to understand the biology of uncultured microbes including bacteria, archaea and viruses through metagenomic analysis. Keywords: Genomic library · Metagenomics · Metaproteomics · Microbial community · rRNA Correspondence: Dr. Navneet Batra, Department of Biotechnology, GGDSD College Sector 32, Chandigarh 160 030, India E-mail: batranavneet@gmail.com Fax: +91-172-2613656 Abbreviations: BAC, bacterial artificial chromosome; DD, differential display Received 12 September 2008 Revised 11 February 2009 Accepted 16 February 2009