454 company was the first to market high-throughput genomic sequencing system--Genome Sequencer 20 System by the end of 2005, and this was reported by Nature as the first revolutionary  SBS (synthesis-based sequencing) technology. In 2007 next-generation sequencing system--Genome Sequencer FLX System was released. In October 2008, 454 launched all new GS FLX Titanium series including reagents and software, and they enable 5 times data output and increased accuracy and read length.  

Roche GS FLX System is a high-throughput genome sequencing platform based on the principle of pyrosequencing. It incorporates DNA polymerase, sulfurylase, luciferase and pyrophosphatase and couples the processes of dNTP polymerization and release of fluorescence signals.

A plate called "Pico TiterPlate" (PTP) containing more than 160 million fiber-optic wholes is applied in GS FLX sequencing. Each hole has one bead fused with clonal DNA templates and all the enzymes and substrates that chemiluminescence reactions need; during sequencing, four bases enter PTP sequentially. If one base complements the template, a PPi is released. Through a series of reactions, luciferin is oxidized to oxyluciferin by PPi, and optical signal is released to be captured by the highly sensitive CCD. When one base pairs the template, the CCD can capture a optical signal. By recording sensitively the signal and its strength proportional to the nucleotides incorporated, the DNA sequences are generated real-time. This technique does not require fluorescence labeling of nucleic acid primers or probes, nor the need for electrophoresis or cloning.

Workflow:

1.Library  Preparation

The DNA sample first is transformed into a library of DNA fragments for sequencing with the Genome Sequencer FLX System. The method for preparing this DNA library varies according to starting materials and objectives of the experiment, but it always includes the modification of each fragment with special sequences that facilitate later workflow steps.

2.Emulsion PCR

Fragments from the DNA library are immobilized onto microparticles (beads) with each bead carrying one single-stranded DNA library fragment. Each unique bead-bound library fragment is then emulsified with amplification reagents within its own microreactor for amplification. Amplification is carried out in bulk, resulting in beads that each is covered with tens of millions of copies of a single DNA fragment; each bead contains a different fragment. After amplification, the emulsion is broken with amplified fragments still bound to their specific beads.

3.Sequencing/Genome Sequencer FLX Operation

After amplification, the DNA-carrying beads are loaded into the wells of a PicoTiterPlate device (PTP device) such that each well contains no more than a single DNA bead. The loaded PTP device is then inserted into the GS FLX Instrument, and sequencing reagents sequentially flow over the plate. The GS FLX Instrument automatically performs and monitors sequencing reactions in all wells of the PTP device simultaneously.

4.Data Processing and Analysis

The GS FLX System can get more than 100 million reads, and read more than 4-6 million bases within 10 hours. By analyzing the raw output consists of a set of digital images (PIF files) using pipeline software, the sequence of the DNA library fragments (“reads”) can be determined, and is furthur subjected to a variety of analysis such as de novo assembly or genome re-sequencing dependingon the type of sample and objectives of your research.

Features:

 •  Fast. One run takes 10 hours and can read 4-6 million bases. It is about 100 times faster than traditional Sanger sequencing technique.

 •  Long read. Average read length exceeds 450 bp.

 • High-throughput. One run can generate more than 100 million reads with cost significantly reduced.

 •  High accuracy. When read length exceeds 400 bp, single read accuracy is over 99%.

 •  High consistency over 99.99%

 •  Paired-end sequencing

 •  Simple and highly effective. No need to clone, prepare plasmids or transform into vectors. One man can finish sequencing a microbe within a day.