A library construction method suitable for simplified genome sequencing
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A genome sequencing and library technology, applied in the field of simplified genome sequencing library construction, can solve the problems of large differences in genome regions, poor data quality, and small fragments, and achieve high-quality library, easy transplantation and promotion, and simple and fast library construction process Effect
Active Publication Date: 2020-06-16
CHINA AGRI UNIV
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The biggest disadvantage of this method is that the steps of ultrasonic interruption and gel recovery cannot guarantee the repeatability between each sample, which makes the genomic regions measured by each individual vary greatly, which increases the difficulty of subsequent data analysis
2b-RAD technology is similar to the method of RAD-Seq technology library construction, the difference is that the restriction endonuclease used is type II type B endonuclease (RAD-Seq is type II type A restriction endonuclease), this The endonuclease cuts at 8-15bp on both sides of the recognized sequence to produce a cleavage product of about 30bp in size. After connecting a specific linker, the fragment is recovered by gel recovery; the disadvantage is that the enzyme cleavage produces The fragments are too small (approximately 30bp), requiring sequencing of extremely high-density enzyme-cut fragments to detect the appropriate amount of genetic variation
GBS technology was developed by Elshire et al. of Cornell University. Its library construction method is the simplest. After the DNA is digested, it is connected to a specific adapter. By controlling the extension time during the PCR process (usually 30 seconds), To select the 100-500bp part of the digested product, so as to achieve the purpose of simplifying genome sequencing; however, the disadvantage of this method is that the amplification efficiency of small fragment digested products is higher in the library construction process, and the template amplification efficiency of the sequencer is higher. The growth rate also grows faster, which is easy to make the sequencing data less available and the data quality is poor
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[0041] Example 1 is used to illustrate the effect of magnetic beads purification
[0077] 2. Design and synthesize the sequence in Table 1 below. In the barcode adapter sequence, the short sequence with a length of 5-9bp in italics and black is used to distinguish the sequencing data of 24 samples, so that 24 samples Can be mixed together for sequencing.
[0078] Table 1 Sequences required for simplified genome sequencing of 24 pigs
[0079]
[0080]
[0081] 3. Preparation of barcode connector and universal connector
[0082] The oligo1 sequence and oligo2 sequence are annealed to make barcode linker and universal linker. The annealing system is: 100μM oligo1 5μL, 100μM oligo2 5μL, 5×Annealing Buffer 10μL, nuclease-free water 30μL; annealing procedure: heating to 95℃, and cooling at a rate of 1℃ / min, until it drops to 25℃, 25 Incubate a...
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Abstract
The invention provides a library construction method applicable to reduced-representation sequencing. The method comprises the following steps: respectively linking two ends of an enzyme-digested product with a general linker and a bar code linker so as to obtain a linked product and carrying out treatment in a mixing pool; adding magnetic beads accounting for 1.2 to 1.4 times of the volume of the linked product obtained after treatment in the mixing pool and carrying out primary purification so as to obtain a first purified product; adding magnetic beads accounting for 0.8 to 0.9 time of the volume of the first purified product and carrying out secondary purification so as to obtain a second purified product; subjecting the second purified product to PCR amplification so as to obtain a PCR product; adding magnetic beads accounting for 1.2 to 1.4 times of the volume of the PCR product and carrying out tertiary purification so as to obtain a third purified product; and adding magnetic beads accounting for 0.8 to 0.9 time of the volume of the third purified product and carrying out quaternary purification so as to obtain a reduced-representation sequencing library. With the method, a fragment with a specific length can be stably grabbed from genome and applied to library construction for high-throughputSNP genotyping; and the method reduces influence of a short enzyme-digested product to the process of PCR, and allows reduced-representation sequencing technology to be better transplanted and promoted to sequencing of different species under the conditions of different endonucleases.
Description
Technical field [0001] The present invention relates to the technical field of genome sequencing, and in particular, to a method for constructing a library suitable for simplified genome sequencing. Background technique [0002] Reduced-representation sequencing (reduced-representation sequencing) is a technology that stably recovers some of the digested products for sequencing after the genomic DNA is digested by restriction enzymes. Most of the recovered products only occupy the entire genome. Of 0.1-5%, so it is called simplified genome sequencing. The commonly used simplified genome sequencing methods mainly include RAD-Seq (Restriction-site Associated DNA Sequencing), GBS (Genotyping By Sequencing), 2b-RAD (IIB restriction endonucleases-siteAssociated DNA Sequencing) and other methods. Through the selection and combination of different restriction enzymes, these methods have been successfully applied in most animals and plants, making it possible to identify and type high-t...
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