Application and prospects of high-throughput sequencing technology (Author: Shanghai Biochip National Engineering Research Center Tengxiao Kun, Xiao Huasheng)
High-throughput sequencing technology is a revolutionary change from traditional sequencing, which can sequence hundreds of thousands to millions of DNA molecules at a time, so it is known in some literature as next generation sequencing technology (next generation sequencing). An epoch-making change, while high-throughput sequencing makes it possible to analyze the transcriptome and genome of a species in detail, so it is also called deep sequencing. The representative of the high-throughput sequencing platform is Roche (Roche) ) 454 sequencer (Roch GS FLX sequencer), Illumina ’s Solexa Genome Analyzer (Illumina Genome Analyzer) and ABI ’s SOLiD sequencer (ABI SOLiD se-quencer). In April 2008, Timothy et al. Of Helico BioScience Science reported on the true single-molecule sequencing technology they developed and used it to re-sequence a M13 viral genome. The reason why this technology is called true single-molecule sequencing is because it completely crosses the above 3 A high-throughput sequencing-based signal amplification process based on PCR amplification 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 with the 96-channel capillary sequencing of traditional sequencing, high-throughput sequencing can read 400,000 to 4 million sequences in one experiment. The reading length varies from 25 bases according to the platform Up to 450 bases, different sequencing platforms can read bases ranging from 1G to 14G in one experiment, such a huge sequencing capacity is unmatched by traditional sequencers.
High-throughput sequencing applications
High-throughput sequencing can help researchers build this experimental step across the library, avoiding the bias introduced during the subcloning process. Relying on strong bioinformatics analysis capabilities at a later stage, a reference genome (reference genome) high-throughput sequencing The technology can easily complete genome re-sequence (re-sequence). In 2007, van Or-souw et al. [56] combined improved AFLP technology and 454 sequencing technology to re-sequence the maize genome. The re-sequencing experiment found more than 75 % Of the SNP loci 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 performed a Solexa re-sequencing of the nematode CB4858 strain to find the nematode genome The deletion or amplification of SNP sites and unit points in the system. However, it should also be seen that due to the limitation of the read length of high-throughput sequencing, its application in de novo sequencing of unknown genomes is limited, This part of the work still needs the assistance of traditional sequencing (read length reaches 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 deep sequencing of the mouse brain, liver, and skeletal muscle. This work demonstrated the two major advances in deep sequencing in transcriptome research, expression counting and sequence analysis. For each item measured The sequence is counted to obtain the expression of each specific transcript. It is a digital expression profiling test that can detect transcripts with very low abundance. Analysis of the measured sequence shows that more than 90% of the data shows that it has fallen In known exons, those that are outside the known sequence show through data analysis RNA cleavages that have never been reported, the 3′-end untranslated region, the changed promoter region, and potentially small Precursor RNA, found that at least 3500 genes have more than one splicing form. And this information can not be found whether using chip technology or SAGE library sequencing. In the same year, Sugarbaker used deep sequencing of mRNA to perform malignant pleural tumors and control samples. In comparison, 15 different point mutations were found in the tumor.
High-throughput sequencing Another widely used area is small molecule RNA or non-coding RNA (ncRNA) research. Sequencing methods can easily solve the technical problems encountered by chip technology when detecting small molecules (short sequence, high homology) And, the short sequence of small molecule RNA just matches the length of high-throughput sequencing, making the data "not wasted", and the sequencing method can also find new small molecule RNA in the experiment. In Chlamydomonas, zebrafish, fruit flies, Nematodes, humans, and chimpanzees have all successfully found new small-molecule RNAs. 400,000 sequences were obtained in C. elegans, and 18 new small RNA molecules and a new class of small-molecule RNAs were found through analysis. After 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 interaction, the chromatin immunoprecipitation-deep sequencing (ChIP-seq) experiment also showed its great potential. The DNA after chromatin immunoprecipitation was directly sequenced, and the protein can be directly obtained by comparing ref seq Compared with the DNA binding site information, compared to ChIP-chip, ChIP-seq can detect smaller binding segments, unknown binding sites, mutations within the binding sites, and segments with lower protein affinity. 2007 In 1995, Johnson et al. Used ChIP-seq to screen the binding site of the transcription factor NRSF on DNA, and obtained 1946 binding sites. The smallest resolvable binding site was 50 bases. These high The quality ChIP-seq results provide new DNA-protein interactions, including important transcription factors in the islet development regulatory network. In the same year, Robertson et al. Used the same method to detect the binding of transcription factors and genomic DNA. These two studies also verified the binding sites detected in the previous ChIP-chip experiment and found new binding sites. Robertson et al. Found that the resolution of ChIP-seq can reach 40 bases. 2008 Chen et al. Published a paper on Cell and used 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 all LIF and BMP pathways. Important regulatory molecules. The binding sites of these transcription factors in ES cells reveal the regulatory network in ES cells that determines the direction of ES cell development.
5 Application prospects of gene chips and high-throughput sequencing technology
Although the 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 aggressive state of "replacement" of gene chips. So, where does the gene chip go? What?
Gene chip technology has formed a systematic platform after nearly 15 years of development, from sample preparation, chip manufacturing, chip hybridization, data scanning to later data management, storage and deep data mining have standardized processes and solid theory With the support of the experiment, it has become a very stable and reliable experimental technology, used by the majority of researchers, and has also accumulated a huge public database. It is also necessary for deep sequencing to establish such a system for several years. Chip hybridization results Intuitive and fast analysis, it is suitable for the detection of known information on a large number of biological samples.At the same time, the chip data analysis has a mature and complete theory, which provides strong support for later data analysis.
The disadvantage of the gene chip is that it is a "closed system", which can only detect the characteristics (or limited variation) of people's known sequences. The strength of deep sequencing is that it is an "open system", its The ability to discover and find new information is essentially higher than that of chip technology. Researchers can fully enjoy the comparative advantages of these two platforms, and on the basis of obtaining new information, take advantage of the strengths of the chip, that is, the known information High-throughput, low-cost (relative) detection capabilities, rapid detection of a large number of samples, a large amount of effective data obtained in a short time.
As two high-throughput genomics research technologies, there are overlaps and competitions in some aspects of application, but they are complementary in more aspects.The joint use of the two methods will solve the problems that were difficult to solve with 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 technologies have a very good correlation for strong positive segments, while for some weak binding sites Point, ChIP-chip and ChIP-seq will lose part of the information, and the information lost by one method can be detected by another method, the complete data comes from the integration of the two parts. The same situation also occurs in mRNA In expression profiling, one technology can make up for the missing part of another technology. Therefore, answering a biological question requires the cooperation of different experimental technologies. For example, the currently emerging Target sequencing or sequence capture, sequence capture, technology, It is a combination of chip and deep sequencing, using chip probes to capture the fragments to be tested, and then using deep sequencing technology to analyze the nucleic acid sequence, using high-density chips The 454 sequencer has successfully captured 6726 exons of 500 bases in length and DNA segments from 200 kb to 5 Mb. The sequencing results show that most of the captured DNA is the target fragment that meets the design requirements. This experiment verified The specificity and feasibility of sequence capture. The sequence capture technology of the chip may replace the multiplex PCR process in the study of genomic segment sequencing. The 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 continue to bring new growth points to a technology. Gene chip and deep sequencing are high-throughput revolutions of point hybridization technology and sequencing, and the two classic molecular biology experimental technologies have developed to high In the era of flux, just as they have previously contributed to the Institute of Life Sciences, in the future these two technologies will continue to work together to advance the life science research into a new era.
Stainless Steel Handle,Foot Rasp,Heel File,Professional Foot File
SZ CHENGYANG BEAUTY TOOLS CO.,LTD , https://www.cyfootfile.com