Next Generation Sequencing (NGS)/Metagenomics
Metagenomics methods use genomic DNA from many different organisms, usually within a microbiome. Metagenomic analyses present different challenges than single species studies (for a more complete review, see)as microbiome samples can contain thousands of species, often novel and closely related.
Metagenomics is another useful bioinformatic tool to access the genetic information from entire community of organisms. It is also a powerful tool for generating novel findings about microbial functions. For example, new finding of proteorhodopsin-based photoheterophy or ammonia-oxidizing Archaea using metagenomics were amazing . Using metagenomics, functional gene composition of microbial communities can be accessed. The review article on metagenomics –a guide from sampling to data analysis gives us a flow diagram of a typical metagenome project. Metagenomics workflow involves following steps experimental design, sampling, sample fractionation, DNA extraction, DNA sequencing, assembly, binning, annotation, statistical analysis, data storage, metadata and data sharing . Metagenomics is useful in studying DNA of uncultured organisms. More than 99% of all microbes cannot be cultured. Metagenome is the entire genetic information of a group of organisms. Metagenomics can be done on samples collected from soil, sea water, sea bed, air, animal waste, etc. For example, Venter et al  show how environmental genome shotgun sequencing of the Sargasso sea water samples was done. About 1.2 million new genes were identified. The two main questions are how many microbes exist in the given sample and what are the functions provided by them. The metagenomic processing pipeline involves sample collection; DNA read sequencing, sequence comparison to the reference genome, comparison file and interactive analysis and visualization. Sequence comparison is done using BLAST, Megablast, BLAT and SSAHA. Identification of species by DNA by using BLAST. Then analysis based on NCBI taxonomy is done. Megan metagenome analyzer is the functional analysis using the SEED classification. IMG/M and MG-RAST are different metagenomic analysis tools. Various uses of metagenomic analysis researches are recently done. A core gut microbiome in obese and lean twins studies done using metagenomics indicates that there is phylogenetic diversity of microbiota of lean and obese individuals . Metagenomics involves binning. Binning is process in which DNA sequences are sorted into groups that might represent an individual genome or genomes from closely related organisms. Binning can be composition based or similarity based. Metagenomic sequencing is more challenging in soil samples than water samples, primarily due to uneven distribution of microorganisms in soil.
1. Tringe, S.G., et al., Comparative metagenomics of microbial communities. Science, 2005. 308(5721): p. 554-7.
2. Qin, J., et al., A human gut microbial gene catalogue established by metagenomic sequencing. Nature, 2010. 464(7285): p. 59-65.
3. Thomas, T., J. Gilbert, and F. Meyer, Metagenomics - a guide from sampling to data analysis. Microb Inform Exp, 2012. 2(1): p. 3.
4. Venter, J.C., et al., Environmental genome shotgun sequencing of the Sargasso Sea. Science, 2004. 304(5667): p. 66-74.
5. Turnbaugh, P.J., et al., A core gut microbiome in obese and lean twins. Nature, 2009. 457(7228): p. 480-4.