Application and prospect of high-throughput sequencing technology

Application and prospect of high-throughput sequencing technology

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Application and prospect of high-throughput sequencing technology
(Author: Shanghai Biochip National Engineering Research Center Tengxiao Kun, Xiao Huasheng)

High-throughput sequencing technology is a revolutionary change to traditional sequencing, sequencing hundreds of thousands to millions of DNA molecules at a time, so in some literature it is called next generation sequencing. A epoch-making change, while high-throughput sequencing makes it possible to perform detailed analysis of the transcriptome and genome of a species, so it is also known as deep sequencing. The representative of the high-throughput sequencing platform is Roche. 454 Sequencer (Roch GS FLX sequencer), Illumina's Solexa Genome Analyzer and ABI's SOLiD se-quencer. April 2008 Helico BioScience's Timothy et al. Science reported on the true single-molecule sequencing technology they developed and used it to re-sequence a M13 viral genome. This technique is called true single-molecule sequencing because it completely crosses the above 3 A high-throughput sequencing-dependent PCR amplification-based signal amplification process that truly achieves the reading of a single fluorescent molecule Ability, to $ 1,000 a measured target the human genome has taken a big step.

The common feature of these platforms is the extremely high sequencing throughput. Compared to the 96 sequencing capillary sequencing of traditional sequencing, high-throughput sequencing can read 400,000 to 4 million sequences in one experiment. The read length varies from 25 bases depending on the platform. Up to 450 bases, different sequencing platforms can read the number of bases ranging from 1G to 14G in one experiment, so the huge sequencing ability is unmatched by traditional sequencers.

High-throughput sequencing applications

High-throughput sequencing can help researchers cross the library to construct this experimental step, avoiding the bias introduced in the subcloning process. Relying on a powerful bioinformatics analysis capability, a reference genome high-throughput sequencing Genomic resequencing is very easy to perform. In 2007, van Or-souw et al. [56] re-sequencing the maize genome with improved AFLP technology and 454 sequencing technology. The resequencing experiment found more than 75 The % SNP site can be verified by SNPWave technology, providing a technical route for polymorphism analysis of complex genomes, especially plant genomes containing highly repetitive sequences. In 2008, Hillier re-sequences Solexa on the CB4858 line of nematodes to find the nematode genome. Deletion or amplification of SNP sites and single sites in the middle. However, it should also be noted that due to the limitation of high-throughput sequencing reads, its use in de novo sequencing of unknown genomes is limited. This part of the work still requires the assistance of traditional sequencing (reading lengths up to 850 bases). But this does not affect Throughput sequencing mRNA expression profiles in the full set of genes, expression profiling of microRNA, ChIP-chip applications and other aspects of DNA methylation.

In 2008, Mortazavi et al. performed RNA deep sequencing of mouse brain, liver and skeletal muscles. This work demonstrates two major advances in transcriptome research in deep sequencing, expression counting and sequence analysis. The sequence is counted to obtain the expression level of each specific transcript. It is a digital expression profiling that can detect very low abundance transcripts. Analysis of the measured sequences, more than 90% of the data shows that it has Among the known exons, the information outside the known sequence is revealed by data analysis from the unreported RNA splicing form, the 3' end untranslated region, the altered promoter region and the potential small RNA precursors have found that at least 3,500 genes have more than one splicing form. This information cannot be discovered using either chip technology or SAGE library sequencing. In the same year, Sugarbaker used mRNA deep sequencing for malignant pleural tumors and control samples. In comparison, 15 different point mutations in the tumor were found.

High-throughput sequencing Another widely used field is small-molecule RNA or non-coding RNA (ncRNA) research. Sequencing methods can easily solve the technical problems encountered by chip technology in detecting small molecules (short sequence, highly homologous) And the short sequence of small RNAs coincides with the length of high-throughput sequencing, making the data “no waste”, and the sequencing method can also find new small RNAs in experiments. In Chlamydomonas, Zebrafish, Drosophila, New small RNAs have been successfully found in nematodes, humans and chimpanzees. 400,000 sequences have been obtained in the nematode, and 18 new small RNA molecules and a new class of small RNAs have been discovered through analysis. Analysis of human embryonic stem cells before and after development, 334 small RNA expression bands were obtained, including 104 newly discovered small RNAs.

In the study of DNA-protein interactions, chromatin immunoprecipitation-depth sequencing (ChIP-seq) experiments have also shown great potential. The DNA after chromatin immunoprecipitation is directly sequenced, and the protein can be directly obtained by comparing ref seq. Site information binding to DNA, ChIP-seq can detect smaller binding segments, unknown binding sites, mutations within the binding site, and segments with lower protein affinity than ChIP-chip. In the year, Johnson et al. used ChIP-seq to screen the binding site of the transcription factor NRSF on the DNA, and obtained 1946 binding sites. The zui small-resolved binding site is 50 bases. High-quality ChIP-seq results provide insight into new DNA-protein interactions, including important transcription factors in the islet developmental regulatory network. In the same year, Robertson et al. used the same method to detect the binding of transcription factors to genomic DNA. Both studies simultaneously validated the binding sites previously detected using the ChIP-chip assay and found new binding sites. Robertson et al. found that ChIP-seq has a resolution of up to 40 bases. Chen et al. published a paper on Cell, using ChIP-seq to detect the binding of 13 sequence-specific transcription factors such as Nanog, Oct4, STAT3, Smad1, Sox2 and genomic DNA. These transcription factors are both LIF and BMP pathways. Important regulatory molecules. The binding sites of these transcription factors in ES cells reveal a regulatory network within ES cells that determines the direction of ES cell development.

5 application prospects of gene chip and high-throughput sequencing technology

Although high-throughput sequencing technology has not been established for a long time, it has shown its extraordinary charm in various research fields of the genome, and it has increasingly shown its embarrassing situation of “replacement” of gene chips. So where does the gene chip go? What?
After nearly 15 years of development, gene chip technology has formed a systematic platform, from sample preparation, chip fabrication, chip hybridization, data scanning to post-data management, storage and deep data mining, with standardized processes and solid theories. With the support of experiments, it has become a very stable and credible experimental technology, which is used by a large number of researchers, and has also accumulated a huge public database. Deep sequencing to establish such a system also takes several years to complete. Chip hybridization results Intuitive, fast analysis, suitable for the detection of known information on a large number of biological samples, while the chip data analysis has mature and complete theory, providing strong support for later data analysis.

The downside of the gene chip is that it is a "closed system" that can only detect features (or limited variations) of known sequences. The strength of deep sequencing is that it is an "open system". The ability to discover capabilities and find new information is inherently higher than chip technology. Researchers can fully appreciate the comparative advantages of these two platforms, and use the strengths of the chip, ie, the known information, based on new information. High-throughput, low-cost (relative) detection capability for rapid detection of large numbers of samples, with a large amount of valid data available in a short period of time.

As two high-throughput genomics research techniques, there are overlaps and competitions in some aspects of the application, but in many respects, the advantages are complementary. The combination of the two methods will solve the problems that were difficult to solve in the previous single technology. For example, Euskirchen et al. used both ChIP-chip and ChIP-seq to detect the binding site of STAT1. The results are very interesting. The two techniques have a very good correlation for strongly positive segments, while for some weak binding sites. Point, ChIP-chip and ChIP-seq will lose some information, and the information lost by one method can be detected by another method. The complete data is from the integration of the two parts. The same situation also occurs in mRNA. In expression profiling, one technique can make up for another missing part of the technology. So the answer to a biological question requires the synergy of different experimental techniques. For example, the current emerging Target sequencing or sequence capture, Sequence Capture, technology, It combines chip and deep sequencing, uses chip probes to capture the fragments to be tested, and then uses deep sequencing technology to analyze nucleic acid sequences, using high-density chips. The 454 sequencer successfully captured 6726 500-base exons and 200 kb to 5 Mb DNA segments. The sequencing results showed that most of the captured DNA was designed to meet the design requirements. The specificity and feasibility of sequence capture, the sequence capture technology of the chip may replace the multiplex PCR process in the study of sequencing of genomic segments in the future. This high-throughput technology of the chip shows its advantages in sample selection and enrichment. And potential.

With the advancement of science and technology, it can continuously bring new growth points to a technology. Gene chip and deep sequencing are high-throughput revolutions of dot hybridization technology and sequencing. Both classic experimental techniques of molecular biology have developed to high. The era of flux, just as they have previously contributed to life science research, these two technologies will continue to work together to promote life science research into a new era.

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