NGS y Metagenómica
Cabrera-Mulero, Amanda,Alba-Bernal, Alfonso
- Published
- 2016-06-07
- Language
- en
Abstract
The Next Generation Sequencing (NGS) allows to sequence the whole genome of an organism, compared to Maxam and Gilbert and Sanger sequencing that only allow to sequence, hardly, a single gene. Removing the separation of DNA fragments by electrophoresis, and the development of techniques that let the parallelization (analysing simultaneously several DNA fragments) have been crucial for the improvements of this process. The new companies in this ambit, Roche and Illumina, bet for different protocols to achieve these goals. Illumina bets for the sequencing by synthesis (SBS), requiring the library preparation and the use of adapters. Likewise, Illumina has replaced Roche because its lower rate of misincorporation, making it ideal for studies of genetic variability, transcriptomic, epigenomic, and metagenomic, in which this study will focus. However, it is noteworthy that the last progress in sequencing is carried out by the third generation sequencing, using nanotechnology to design small sequencers that sequence the whole genome of an organism quickly and inexpensively. Moreover, they provide more reliable data than current systems because they sequence a single molecule, solving the problem of synchronisation. In this way, PacBio and Nanopore allow a great progress in diagnostic and personalized medicine. Metagenomics provide to make a qualitative and quantitative analysis of the various species present in a sample. The main advantage of this technique is the no necessary isolation and growth of the species, allowing the analysis of nonculturable species. The Illumina protocol studies the variable regions of the 16S rRNA gene, which contains variable and not variables regions providing a phylogenetic classification. Therefore, metagenomics is a topic of interest to know the biodiversity of complex ecosystems and to study the microbiome of patients given the high involvement with certain microbial profiles on the condition of certain metabolic diseases.
Full text
METAGENOMIC AND NGS Next generation sequencing metagenomic 454 - roche solid illumina Ion torrent strategy 16S specific PCR Randomly shear DNA BIBLIOGRAPHY NGS library is prepared by fragmenting a gDNA sample and ligating specialized adapters to both framgents ends Library is loaded into a flow cell where framents hybridizes. Each bound fragment is amplified to a clonal cluster by bridge amplification Fluorescent labeled nucleotides are added. The flow cell is imaged and the emissioin from each cluster is recorded. Library is prepared. An universal adaptor P1 is used so that the starting sequence of every fragment is both known and identical. Emulsion PCR. Primers hybridizing to the adapter sequence mold library is added Four types of probes fluorescently labeled two bases compete for binding to the first used for sequencing . Specificity is achieved by interrogating each first and second base in the ligation reaction . several cycles are performed . The product is removed and the mold is reused with a first complementary to the n-1 position to a second round of cycles 1. Mardis, E. R. A decade’s perspective on DNA sequencing technology. Nature 470, 198–203 (2011). 2. Liu, L. et al. Comparison of Next-Generation Sequencing Systems. Journal of Biomedicine and Biotechnology 2012, 1–11 (2012). 3. Dewey, F. E., Pan, S., Wheeler, M. T., Quake, S. R. & Ashley, E. A. DNA Sequencing: Clinical Applications of New DNA Sequencing Technologies. Circulation 125, 931–944 (2012). 4. Simon, C. & Daniel, R. Metagenomic Analyses: Past and Future Trends. Applied and Environmental Microbiology 77, 1153–1161 (2011). 5. Hugenholtz, P. & Tyson, G. W. Microbiology: Metagenomics. Nature 455, 481–483 (2008). 6. Sultana, H., Neelakanta, G. & Sultana, H. The Use of Metagenomic Approaches to Analyze Changes in Microbial Communities. Microbiology Insights 37 (2013). doi:10.4137/MBI.S10819 7. Michael L. Metzker. Sequencing technologies — the next generation. Nature Reviews Genetic, 11 , 31 – 46 (2010) TIMELINE 1. Meta genomics allows to identify most microorganisms cannot be grown in laboratory. 2. Meta genomics provides a view not only of the community structure (species richness and distribution) but also of the functional (metabolic) potential of a community. 3. Meta genomics can analyze any environment as long as nucleic acids can be extracted from sample material. However, most interest has centred on the marine environment (the largest meta genomic study to date is the Global Ocean Sampling Expedition) and in medicine (of particular note is an international initiative, the Human Microbiome Project, which aims to map human-associated microbial communities) 4. Metagenomics can be used for viruses, eukaryotes and prokaryotes. 5. Meta genomics has a high potential for serendipitous discovery. For instance, discoveries such as proteorhodopsin proteins or archaeal ammonia oxidizers. 6. Meta genomics can reconstruct whole genomes from an environmental sample by means random sequencing. application Prepare Genomic Library Prepare Template on Bead Sequence on Ion Chip Signal processing and Base Calling First, DNA is isolated and cut into smaller fragments , which bind small balls called bead. Each , together with millions of DNA fragments enter the microwells of a microchip. Then the microchip with a solution of one of nucleotides bathes. If the nucleotide is incorporated into the DNA chain ,a proton is released. This process is repeated every 15 seconds with a new solution of each nucleotide. Amanda Cabrera Mulero, Alfonso Alba Bernal. Genómica Estructural y Funcional. Máster en Biología Celular y Molecular.
