Second-generation sequencing and third-generation sequencing are two common sequencing technologies in current genomic research, and there are significant differences in principles, performance, and applications. With the continuous deepening of genomic research, the use scenarios of second-generation sequencing and third-generation sequencing are becoming more and more extensive, each with its own advantages and disadvantages.
The difference between second-generation sequencing and third-generation sequencing
Overview of first- and second-generation sequencingSecond-generation sequencing (also known as high-throughput sequencing or next-generation sequencing) is the most widely used genome sequencing technology at present. It uses chemical reaction-based technology to complete large-scale genomic data acquisition by fragmenting DNA and amplifying it into large-scale clones through parallel sequencing. 1. The advantages of second-generation sequencing are: (1) High throughput: able to sequence millions or even billions of DNA fragments simultaneously, which is very efficient. (2) Low cost: Due to the relatively mature technology, the cost of second-generation sequencing platforms on the market has been greatly reduced, becoming the first choice for most scientific research institutions and clinical laboratories. (3) High accuracy: Second-generation sequencing has high accuracy, especially within the short read range, which allows more accurate genomic data to be obtained. 2. The shortcomings of second-generation sequencing are: (1) Short read length: The length of the read segment of second-generation sequencing is generally shorter (usually 100-300bp), which makes it have limitations in the analysis of some complex regions (such as repeat sequences, structural variations). (2) Difficulty in analyzing repeat sequences: Due to the short sequencing fragments, second-generation sequencing is difficult to deal with long-distance genomic structures, and it is impossible to effectively reconstruct complex genomic structures and large repeat regions.
Overview of second and third generation sequencing Unlike second generation sequencing, third generation sequencing technology is a method of direct sequencing of single molecules, and there is no need to fragment DNA. . The biggest feature of third-generation sequencing is that it can read DNA fragments of up to thousands or even tens of thousands of base pairs, which is called "long read and long sequencing". 1. The advantages of third-generation sequencing include: (1) Long read length: Third-generation sequencing can provide longer read lengths (can reach thousands to tens of thousands of bases), allowing it to better deal with complex genomic structures, repeat sequences and other difficulties. (2) No fragmentation is required: Third-generation sequencing uses single-molecule real-time sequencing technology, which can directly sequence a single DNA molecule, avoiding the process of fragmentation and amplification in second-generation sequencing. (3) Structural variation analysis can be achieved: Due to the long read and long characteristics, third-generation sequencing can help more accurately identify structural variations in the genome, such as gene rearrangement, insertion and deletion. 2. The disadvantages of third-generation sequencing include: (1) High cost: At present, the technology and equipment for third-generation sequencing are relatively new, the cost is high, and the output of sequencing data is relatively limited. (2) Accuracy problem: Although third-generation sequencing can obtain long read data, due to the different sequencing principles, the error rate of single sequencing is high, so it is usually necessary to improve accuracy by combining second-generation sequencing data.
Three. The main differences between second-generation sequencing and third-generation sequencing1. Sequencing principle: Second-generation sequencing adopts the principle of "synthetic sequencing", by fragmenting DNA into high-density clones, and then performing parallel sequencing. Third-generation sequencing uses single-molecule real-time sequencing technology to directly determine the sequence of a single DNA molecule without fragmentation. 2. Read length: The read length of the second-generation sequencing is short, usually between 100-300bp, while the read length of the third-generation sequencing can reach several thousand to tens of thousands of bases. Long read length makes three-generation sequencing have obvious advantages in genome assembly and complex region analysis. 3. Accuracy: The second-generation sequencing has high accuracy, especially in the case of short reads and long genomic sequences can be accurately obtained. However, due to its fragmentation characteristics, it is difficult to deal with complex regions such as repeating sequences. Although third-generation sequencing can provide the advantage of long reads, its single sequencing has a high error rate and usually requires correction in combination with other data. 4. Application scenarios: Second-generation sequencing is usually used for genome sequencing, transcriptome analysis and small-scale genome analysis, etc., and is suitable for scenarios that require large-scale data generation and require low read length. Third-generation sequencing is more used in structural variation analysis, genome assembly, and research on complex genomes, and is especially suitable for research needs that require long readings.
Second-generation sequencing and third-generation sequencing each have their own unique advantages and disadvantages in genomic research. Second-generation sequencing is suitable for most conventional genomic studies with its high throughput, low cost and high accuracy. With its long read and long characteristics, the third-generation sequencing has unparalleled advantages in dealing with complex genomic structures, repeat sequences and structural variation analysis. According to research needs, choosing the right sequencing technology can more efficiently promote the research process of genomics.
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