Next-generation methods

High demand for inexpensive sequencing produce thousands or millions of sequences at a time, and has promoted the development of the (next generation sequencing) or high-throughput sequencing technique called parallel sequencing. High-throughput sequencing technology, we aim to reduce the cost of DNA sequences beyond what is possible in the dye terminator standard methods. You can ultra-high throughput sequencing 500 000, to be run in parallel by combining maximum operation. There is a (NGS) technology three major next-generation sequencing today; 454 Illumina, and ion Trent. Sanger sequencing is used in the laboratory almost all still, it remains an important tool for creating a clone of the PCR-based sequence verification and simply.

Next-generation methods

DNA sequence in each system is very different, but the technology of all three, have many common features, they usually when you bind the oligonucleotide adapter, they are adapter butyric acid molecule of single or millions of people, starting with genomic DNA preparation of parallel sequence is highly amplified clonally, was fragmented several billion reading. (HGP) technology is finished Designed by Human Genome Project, has been developed. Sanger sequencing, which requires 15 years international efforts, and several billion U.S. dollars in order to complete was the power of rude equipment, a single genome HGP. It was developed by 454 Life Sciences Corporation is publishing in 2005 of the first NGS technology. Genomic DNA and adapter oligonucleotides that have been fragmented is connected, is amplified by PCR cloned emulsion (EPCR) then Library. Bead of 1 micrometer size, each drop emulsion containing the PCR reagents and ligation adapter molecule. EPCR beads after they are loaded into the pyrosequencing picotitre plate.

Pyrosequencing works by converting the release pyrophosphate when the sequence is read, it generates a “pyrogram”, incorporated dNTP in the optical signal, the polymerase. Approximately one million sequences can generate a plan for each sequence. GS-FLX system sold Roche 454 to generate a sequence of 700 bp 1M, NGS sequencer most common GS Junior, desktop version.The is generated from Illumina HiSeq a sequence of 600 million to 200 bp reading of length. They also, DNA sequencer personal that you can generate MiSeq sale, up to 17M reads of 500 base pairs in length. To form a “cluster” of about 1000 copies of a molecule library of the original, since the clones amplified in the “cell” using a bridge amplification, fragmentation Illumina, using a DNA ligation adapter to create a NGS library to. The Illumina, after being presented ready for the cycle and then the flow cell, a terminator, the following is removed, the synthesis of cyclic sequences blocked using a dNTP fluorescent reversible terminator in (SBS) chemistry.

Important factor in the spread of Illumina technology is the ease of process development and simplicity of sample preparation. Illumina, combined with a short piece of DNA and oligo adapter “type character Y” for the creation of a fragment library was PCR amplified. Is 35bp generation sequencer early Solexa base from Illumina, in 2007, reads as 1GB generated the data from the cell or 30M sequence. Lead length has increased to 100 bp of the HiSeq 2000 system-generated data of 300 gigabytes and 2.4b sequence as of December 2011. Cost of sequencing the human genome HiSeq 2000 is estimated at $ 6.500 only in 2011. The most common application of DNA sequencing NGS, exomes, amplicons, and transcriptome, the entire genome of today: protein interaction.

read can NGS sequence contains only one end of the fragment, is prepared by either of the edge is a molecule that was sequenced at both ends, having two ends, or the sequence. The format of the sequencing plan, prior to the preparation of the library, it is selected instances typically one side 36bp of default common for some of the 100 bp of paired-end cancer genome and chip array has been there. The structure of the data sequence is determined by a question is. The study of the chip and then, a large number of short, you need to read to be designed for a quick run them short. WGS, which must be long lead, as if they were used, the actual genome coverage is possible. Of RNA-seq. Option and surface structure. The length of the read sequence different, use the data for the study of many of the paired-end that uses both.