454 pyrosequencing

Parallelized version of pyrosequencing was developed by 454 Life Sciences, which was acquired by Roche Diagnostics then. Oil solution (emulsion PCR), followed by the method for amplifying a DNA in the aqueous droplet within each droplet containing beads primed single coating to form a clone colonies template DNA associated. Sequencing of the machine, includes picoliter volume many wells, including the sequencing enzyme beads and a single each. Pyro sequence of using a luciferase to generate light for detection of individual nucleotides used to fault generating read data sequence is added to the nascent DNA, was collected. This technology provides the value of the base and the length of the reading of the intermediate as compared to the Sanger sequence in solid Solexa and one end, from another.

454 Thing use the possible scale parallel pyrosequencing system is about 400-600 Mega-based sequencing of the DNA of 10 hours per run of GS FLX Titanium series reagents FLX genome sequencer. The system relies on determining were sprayed DNA capture bead small water-in-oil, a DNA fragment was ligated adapter. I was amplified by PCR DNA was fixed to these beads. To 29 meters – and I was placed PicoTiterPlate, the optical on-chip DNA binding strip any. It is a mixture of enzymes such as are packaged in the well DNA polymerase, ATP sulfurylase, luciferase and such. I will be placed in the GS FLX system sequence PicoTiterPlate then. The acquisition by the release of GS20 sequencing machine and Roche Diagnostics in 2005, next-generation DNA sequencing market since the first 454 has experienced rapid growth. 454 sequence run-GS FLX Titanium series reagents for use in genome sequencer FLX device capable of 400 to 600 million base pairs sequence of motion and 400-500 base pair 2008 reads a length. Sequencing, the company plans to release a kit that can read up to a length of 1000 bps to 2010. The end of 2009, 454 Life Sciences, announced the desktop version GS Junior system, the Genome Sequencer FLX system.

454 array can be analyzed as a FASTA file and bioinformatics standard tools, but the information can not be used in a pure nucleotide sequence flow value is included. Thus, the use of the flow makes a direct value algorithm several groups proposed. Are called to operate in, “flowspace” “Unlike nucleotide space”, this approach is to suppress the loss of information. For example, the set of flowgrams, the method using a maximum likelihood PyroNoise to determine whether there is a possibility that lead to sequence biological basic one or more separate. In a similar manner, the use of Bayesian statistics PyroBayes method, the number of base that is specified the flux values ​​are observed to determine the length of each run as perhaps homo. If the likelihood of the base exceeds a predetermined threshold, it is added to the execution of the homopolymer than the base. This is the number of the insertion error will be increased, as is unique to 454 pyrosequencing that may occur only for very undercalls substituted error consistently, it deletion and it is possible to reduce the number of replacement. To call centers more homopolymers, in order to allow higher recognition rates SNP, this trend will work.

For discovery mapping, a small RNA that flowgrams the target genome is an effective way directly been proven. It is also possible to achieve speeds higher accuracy of the nucleotide sequence of the assembly building flowspace consensus sequence of the data that is oversampled very much. Metagenomics is another area where the quality of the 454-pyrosequenced data has been attracting attention. The study for various purposes, and have a major impact on the processing of the data in a specific way that has not been used in sensitive characteristics of pyrosequencing data, but there is indicated the value of the direct current artifacts some are. For example, you can collapse the homo all sequences of length 1 of pyrosequencing that may introduce errors in homopolymer length when matching the index approach of the 454 series. For a long homo, also errors of many, there is work to improve the 454 series on the side of the chemical in particular, distribution of overlap is caused by a signal that leads to ambiguous base calls and wide. In addition to determining the exact length of the homopolymer, the base or the calling party (i.e., the noise flow rate) is particularly important low optical signal. There is a possibility to come from the most basic of conversation, but flow value of 0.49 is treated as noise from the 454 caller based it.