09-08-2013, 04:09 PM
Tools to unveil the agricultural treasure: Next Generation Sequencing
ABSTRACT
Though the structure of DNA was established in 1953, but RNA sequencing was the earliest forms of nucleotide sequencing. The major landmark of RNA sequencing is the sequence of the first complete gene and the genome of Bacteriophage MS2, while the first full DNA genome sequenced was that of bacteriophage φX174 in 1977 and in same year two methods of DNA sequencing were published, first by Maxam andGilbert and another by Frederick Sanger. Automated Sanger method is referred to as ‘first generation technology’ and NGS technologies are grouped into second generation (2G) and third generation (3G) approaches. Several 2G approaches are available (Roche-454, Illumina-Solexa, ABi-SOLiD) whilst all 3G platforms are near market, with the exception of the HelicosHeliScope and Pacific Bioscience which is already available.All 2G technologies are engineered to be massively parallel in terms of operations and output and are subject to on-going refinements in key areas to improvements include read-length, accuracy, parallelisationand time-per-run. 3G technologies deploy a single molecule template approach and thereby enable further massive parallelisation. Read-out methods include differential conductance across nanopores (ONT) and single molecule real-time sequencing using Fluorescence Resonance Energy Transfer (ABi) or zero-mode waveguide detectors (PacBio). 3G technologies offer greatly increased read-lengths, the opportunity to use very small sample sizes, faster outputs and significant improvements in sensitivity, and accuracy. Major areas for the application of 3G methodologies are anticipated to be genotyping, transcriptome sequencing and re-sequencing. NGS is currently being used to De novo sequencing of the genomes and also being used to re-sequence to provide a better understanding of genomic diversity and evolution.NGS is extensively being used in metagenomicsto identify genomic signatures from mixed populations, (environmental monitoring) or be analysed for useful leads (bioprospecting, Human Microbiome project, Earth Microbiome project, gut, rumen and soil microbial ecosystems). Looking forward, metagenomics (and metatranscriptomics) was considered to be an area with great potential to unveil the treasure of nature for the betterment of agriculture and humans.