2. sample DNA interruption: samples such as genomic DNA or BAC are interrupted into fragments of 300 ~ 800 BP; For small non-coding RNA, this step is not needed. Short PCR products can be amplified with GS fusion primers, and then directly proceed to step 4.
3. Linking: A and B linkers with specificity at 3' and 5' ends are linked to DNA fragments by a series of standard molecular biology techniques. The linker will also be used for subsequent purification, amplification and sequencing steps. Only single-stranded DNA fragments used in the following steps are shown in the figure.
4. A DNA fragment = a magnetic bead: the linker combines hundreds of DNA fragments onto a magnetic bead, and the magnetic bead is wrapped in a single oil-water mixed droplet and amplified independently in this droplet, which is not affected by other competing or polluting sequences, thus realizing parallel amplification (emPCR) of all DNA fragments.
5. One magnetic bead = one reading length: After emPCR amplification, there are thousands of identical copies of DNA fragments on each magnetic bead. After enrichment, these fragments are still bound to magnetic beads, and then they can be placed in microtiter plates for subsequent sequencing.
6. Data reading and analysis tool: FLX GS
The system provides three different bioinformatics tools to analyze sequencing data, which are suitable for different applications. For example, the resequencing of up to 3Gb sequences, the difference analysis between amplified products and known reference sequences, and the de novo sequencing work of 120Mb, etc.
Roche 454 sequencing system is the earliest new generation sequencing system, and its application results have been published in many papers in Nature and Science. The 454 system has been upgraded many times since its launch. The latest GS FLX sequence analyzer combined with titanium series sequencing reagents has higher sequencing throughput (400Mb per reaction), wider flexibility and accuracy, and maintains the advantages of the fastest running speed of the new generation sequencing technology (3-5 days) and the highest reading length of a single sequence (400bp). The accuracy rate of a single reading length can exceed 99.5%, and the accuracy rate of all sequencing results is greater than 99.5%. As a new member of Titanium series, the long-distance double-ended kit can double-end sequence 3-20kb inserts, which breaks through the bottleneck of poor repetitive sequences in the new generation sequencing technology. In addition, having a complete software package is a major feature of GS FLX, and its easy-to-use graphical interface can be used for drawing, splicing and detecting the differences of amplified products. At the same time, due to its ultra-high sequencing reading length, GS FLX can be integrated with traditional sequence analysis software, which is also impossible for other new generation sequencing technologies. Because of its excellent performance, GS FLX system can be widely used in genome-wide de novo sequencing and BAC library resequencing, amplification product resequencing, transcriptome analysis, gene regulation and other genomics research.