GS FLX, a new generation of ultra-high-throughput genome sequencing system, opens up new possibilities for scientific research

GS FLX, a new generation of ultra-high-throughput genome sequencing system, opens up new possibilities for scientific research

A new generation of ultra-high-throughput genome sequencing system GS FLX
Create new possibilities for scientific research
The GS FLX system is the most widely used ultra-high-throughput genome sequencing system launched by Roche 454 Life Sciences. Researchers use the high-accuracy, long-reading GS FLX system to quickly obtain high-quality biological data and obtain scientific research. Breakthrough progress. Since the advent of the GS system, more than 250 high-quality literature has been published in the world's top magazines, making the GS FLX system the fastest high-throughput sequencing instrument for publishing papers.
Application principle
The GS FLX sequencing system relies on bioluminescence for DNA sequence analysis, and the polymerization of each dNTP is coupled with the release of a chemiluminescent signal by the synergy of DNA polymerase, ATP sulfated enzyme, luciferase and diphosphatase. The purpose of real-time determination of DNA sequences is achieved by detecting the presence and intensity of chemiluminescent signals (). This technology does not require fluorescently labeled primers or nucleic acid probes, nor does it require electrophoresis. It has the characteristics of fast, accurate, high sensitivity and high automation. Each sequencing reaction takes only 10 hours to complete 500 million base data, and the average sequencing read length can reach 400 to 500 bases. When the read length exceeds 400 bp, the sequencing accuracy is greater than 99.99%.
The highly flexible nature of the GS FLX system enables groundbreaking scientific research in a wide range of fields, opening up new research directions for biological sciences and medical research.
Unknown genome sequencing
For unknown genomes such as wild flora, because there is no reference to compare, other sequencers (average reading length of 35bp) are difficult to accurately measure their sequence, while the GS FLX system has a throughput of 500Mb and a length of 450bp. The single read length capability makes it easy to perform a full-gen shotgun sequencing of unknown genomes from bacteria to viruses in one reaction. Standard unknown genomic stitching using GS FLX de novo The Assembler software stitches the long read sequence into contigs. By applying the Paired End technology, the contigs generated by sequencing can be sorted and their relative positions determined, resulting in a high quality, complete assembly. GS FLX is currently the sequencing system with the lowest Gap and the lowest cost of subsequent finishing.
There are many repeats in the bacterial genome, and the 3Kb repeat can be skipped using Paired End sequencing. At the beginning of next year, 454 will launch 20Kb Paired End sequencing technology, so that the longest repeat sequences in all genomes can be skipped, and bacterial genome splicing can be done at one time.
Genomic resequencing
    Microbial Genome: GS FLX was first applied to whole-genome sequencing of bacteria, which is characterized by very fast sequencing. The Johnson & Johnson Drug Development Center used 454 sequencing technology to detect drug-suppressed genes in samples. By screening drug-resistant strains by increasing the drug dose, the surviving strains are resistant to mutations, and the mutation sites may be drug targets. Since the bacterial mutation sites are usually more, two drug-resistant bacteria are selected for sequencing and compared with the original non-resistant bacteria. The measured tens of millions of bases showed that there were mutations at the four sites of the drug-resistant bacteria, and no strains with no drug resistance. Further research found that one of the enzymes is a drug target. In this experiment, a bacterium was measured in GS FLX in just one week, and the experimental results were obtained.
    Large animal and plant genome: 454 has cooperated with Australian and New Zealand laboratories to screen the genetic marker of the sheep genome by sequencing method. The sequencing coverage is 0.5 times and a total of 1.5G data is measured. Genetic polymorphism can then be obtained by splicing the genomic sketch of the cattle. Apply this method to quickly screen tens of millions of markers. In addition, in combination with Sanger and 454, the sequence of the grapes has been determined. Plants such as oranges, sorghum, and corn are also used for genome sequencing using 454.
    Human genome structure and mutations: Long-length reading and Paired End techniques are used to detect large-scale genomic changes, enabling greater genomic coverage and identification of more genetic variants, such as insertions, deletions or copy number variations of gene fragments. More than 25% of the information can be obtained compared to the short read length technique. The 454 company collaborated with a laboratory at Yale University to use the human genome as a Paired End library, sizing the library to approximately 3Kb, and sequencing both ends. It was found that the obtained data differed from the genomic data by 6 Kb, and it was speculated that the sample had a 3 Kb fragment deletion.
Amplicon sequencing
GX FLX amplicon sequencing has high sensitivity and can identify low frequency mutations and haploids, such as exons or viral quasispecies, which detect less than 0.5% of variation. For example, based on an amplicon ultra-deep sequencing method, rare somatic mutations can be identified in mixed tumor samples, or genetic differences can be analyzed in microbial, human, animal or plant populations to reveal SNP sites.
GS is rapidly and accurately identified from mixed samples with unprecedented sensitivity and speed and is used in the study of HIV resistance changes. Due to the high amount of error in HIV reverse transcriptase, HIV mutates very rapidly in the human body and causes mutations in the drug target, thereby rendering the drug ineffective. A pharmaceutical company uses GS FLX to sequence HIV in the patient's blood, and compares the sequencing results with a database of mutation-to-drug failures established by Stanford University to determine whether HIV is resistant, and if so, immediately change the drug.
Environmental genomics
Compared to short read length technology, GS FLX can more accurately predict the differences of species in different environments. For example, a marine biology research institute at MIT collects biological samples near the crater to study seabed organisms. By sequencing the bacterial samples, it was found that the bacteria living on the seabed were different from the terrestrial bacterial species. The bacteria in different sea areas also had completely different bacterial compositions, and the evolution was different after several years.
Rapid identification of new infectious diseases
GS FLX provides an efficient and accurate detection tool for rapid identification of new disease sources. In 2008, three Australian patients underwent a liver transplant. The donor was a 57-year-old man who died of a cerebral hemorrhage. However, after four to five weeks of surgery, the three recipients died of similar diseases. Columbia University researchers predict that unknown viruses were not detected by conventional methods during liver transplantation, so all cDNAs were sequenced using GS FLX. The measured results of more than 100,000 sequences showed that fourteen of the sequences were indeed similar to lymphocytic choriomeningitis virus, and it was also verified by PCR that the virus also appeared in the donor's organs.
Inspired by this research, oncology researchers used 454 sequencing to verify whether the virus has a direct effect on the onset of the tumor. By detecting skin tumor cDNA, we found multiple tumor T antigens and human receptor tyrosine phosphatase
A fusion protein is formed. The researchers then hypothesized that the virus may inhibit related proteins through this combination, which may lead to tumor development. By detecting more different tumor cells, it was found that different tumor cells have the same fusion protein, thus verifying that the virus can lead to tumor development, and the virus-producing mechanism is similar to that of tumors, and all of them are through mutation.
Gene methylation
Hypomethylation and hypermethylation of the promoter region CpG island are one of the important mechanisms for the regulation of activity of many genes. By sulfite treatment, GS FLX can detect the methylation status of each CpG island within the specified target genomic sequence and quantify each methylation. For example, samples of colorectal cancer and their adjacent normal tissues are analyzed, and the sample promoter is treated with sulfite. If there is no C to T conversion, the site is not methylated, otherwise the site occurs. Methylation. Each tumor can be mapped to a map. By comparing the maps, it can be found that some genes have different profiles in different tumors, which inspires new scientific predictions.
gene expression
Compared with short reading length technology, long reading length technology can find more gene annotations, more accurate prediction of pseudogenes, and obtain reliable allele-specific expression profiles, which can detect the level of gene expression more sensitively and better splicing. Genetic model. Some gene sequences that are very difficult to detect by Sanger are very detectable.
Break through the sequencing sample preparation bottleneck
With the continuous development of high-throughput sequencing technology, the preparation of a large number of resequencing samples using conventional PCR methods is costly and time consuming. In response to this problem, Roche NimbleGen () successfully developed the Sequence Capture Array technology, which can enrich any target genomic region up to 5Mb on a single chip, such as exons, diseases. Related areas, promoters, enhancers, etc. Compared to the PCR method, this technique can greatly reduce the time and cost of sample preparation. At the end of this year, NimbleGen will launch a 2.1M chip that can simultaneously enrich the 30Mb target sequence. The combination of NimbleGen sequence capture technology and GS FLX high-throughput sequencer is dedicated to enabling research in specific application areas with the lowest price and best performance, leading to a breakthrough in biomedical research.

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