Principle of Ultra High Throughput Sequencing Technology for Roche 454 (GS FLX Titanium System)

At the end of 2005, 454 launched the revolutionary ultra-high-throughput genome sequencing system based on pyrosequencing, the Genome Sequencer 20 System, which was reported by Nature magazine as a milestone, creating a precedent for sequencing while synthesizing. In 2007, the Genome Sequencer FLX System, a second-generation genome sequencing system with better performance, was introduced. In October 2008, the 454 introduced the new GS FLX Titanium series of reagents and software, which increased the throughput of GS FLX by a factor of five, and further improved the accuracy and read length.

GS FLX sequencing principle

The sequencing principle of the GS FLX system is the same as that of the GS 20, and it is also a new technology for DNA sequence analysis by bioluminescence; under the synergistic action of DNA polymerase, ATP sulfurylase, luciferase and diphosphatase, the primers are used. The polymerization of each dNTP is coupled to a single fluorescent signal release (Figure 1). The purpose of determining the DNA sequence in real time can be achieved by detecting the presence or absence of fluorescence signal release. This technology does not require fluorescently labeled primers or nucleic acid probes, nor does it require electrophoresis; it has the characteristics of fast, accurate, sensitive, and automated analysis results.

The Roche GS FLX System is a high-throughput genome sequencing system based on the principle of pyrosequencing. At the time of sequencing, a plate called "Pico TiterPlate" (PTP) was used, which contained more than 1.6 million pores composed of optical fibers containing various enzymes and substrates required for chemiluminescence reactions. At the beginning of sequencing, the four bases placed in four separate reagent bottles are sequentially circulated into the PTP plate in the order of T, A, C, and G, entering only one base at a time. If base pairing occurs, a pyrophosphate is released. This pyrophosphate is subjected to a synthesis reaction and a chemiluminescence reaction under the action of various enzymes, and finally fluorescein is oxidized to oxyluciferin, and an optical signal is released. The light signal emitted by this reaction is captured in real time by the instrument's configured high sensitivity CCD. When one base is paired with a sequencing template, one molecule of the optical signal is captured; thus, one-to-one correspondence can accurately and quickly determine the base sequence of the template to be tested.

Figure 1: Schematic diagram of the GS FLX high-throughput sequencing method

Sequencing experiment process:
1. Library preparation: According to the type of the sample and the purpose of the experiment, the genomic DNA/cDNA is fragmented to 400-800 bp, and the single-stranded DNA (sstDNA) is recovered by denaturation treatment after modification by terminal repair and specific linker;

2. Emulsion PCR: A specific ratio of single-stranded DNA libraries is immobilized on specially designed DNA capture beads, allowing most of the magnetic beads to carry a unique single-stranded DNA fragment. The magnetic bead-bound library is emulsified by the amplification reagent to form a water-in-oil mixture, and each unique fragment is independently amplified in its own microreactor without being affected by other competing or contaminating sequences. Amplification of the entire fragment library was performed in parallel. Millions of identical copies were produced after amplification. Subsequently, the emulsion mixture is broken, and the fragments that are still bound to the magnetic beads after amplification can be recovered and purified for subsequent sequencing experiments;

3. Sequencing reaction: DNA-carrying beads were mixed with other reactants and subsequently placed in a PTP plate for subsequent sequencing. The diameter of the PTP hole (29um) can only accommodate one bead (20um). The PTP plate was then placed in GS FLX and sequencing was started. The addition of each nucleotide complementary to the template strand produces a chemiluminescent signal that is captured by a CCD camera;

4. Data analysis: The GS FLX system can obtain more than 1 million read lengths in a 10-hour run, read more than 400-600 million bases of information, and provide two different bioinformatics tools for sequencing through the GS FLX system. Data is analyzed.

Technical features:

• Fast, a sequencing reaction takes 10 hours and gets 400-600 million base pairs. 100 times faster than traditional Sanger sequencing methods;

• Long reading, the length of a single sequence is longer, with an average of 450 bases;

• High throughput, each reaction can get more than 1 million sequence read lengths, and the cost is greatly reduced;

• High accuracy, the read length can exceed 99% when the read length exceeds 400bp;

• Consistent, consistent with more than 99.99% sequencing results;

• Can perform Pair-End sequencing studies;

• Simple and efficient, no need to build a library, clone picking, plasmid extraction, etc., one person can complete the sequencing of a microbial species in one day.

GS FLX system application

Since the advent of the GS ultra-high-throughput genome sequencing system in late 2005, it has successfully settled in the world's major sequencing laboratories. The first "test article" of the technology came from James D Waston, who is known as the "father of DNA," who provided his blood sample to 454. At present, users of GS system have published more than 50 academic papers in the world's top journals such as Nature, Science and PNAS. (For a detailed list, please refer to https://). Compared with the GS 20 system, innovative improvements in hardware configuration and software systems make the GS FLX system a wide range of applications:

Whole genome sequencing

Sequencing up to 120 Mb of unknown genome

- Generating a genomic structure overview

- Study the organization, distribution and information of DNA sequences

-Gene screening: finding new genes, positioning and function

- Comparative study with other genomes

Whole genome sequencing by de novo shotgun sequencing, such as microbial genes, BAC and YAC clones.

Comparative genomic research

- Identify single base mutations

- Identify mutation hotspots and conserved regions

- Identify inserted or deleted genes

- Determine the relationship between genotype and phenotype (eg, study the genetic basis of drug resistance)

- Toxicity prediction based on genetic sequencing changes

- Conduct epidemiological analysis

-Understanding the differences in the sequence of industrially produced strains and their parental strains as the genetic basis for the development of industrially produced strains

- Conducting metagenomics studies

- Ancient fossil DNA sequencing study

Contigs are spliced ​​into Scaffolds using the Pair-End Tag method.