32results about How to "Improve sequencing quality" patented technology

The invention provides a method for constructing a high-flux simplified genome sequencing library. The method comprises the following steps: digesting genome DNA of different samples by using restriction enzymes, thereby obtaining blunt-end DNA fragments with phosphorylation modification at the 5' end; adding the base A at the 3' end, connecting the DNA fragments of different samples with 5' phosphorylation and 3' cohesive ends A with joints containing different bar code sequences, connecting the products PCR, purifying and mixing the amplification products, performing low-cycle PCR amplification, thereby obtaining a target fragment amplification product of which the ratio of the joints to joint dimers is greatly reduced; and separating, purifying, and forming the high-flux simplified genome sequencing library from the purified products. The method disclosed by the invention has the advantages that the flux is high, the cost is low, and the generated library is high in sequencing quality and has very wide application prospects in researches such as high-flux SNP detection and marker development, genetic map drawing and functional gene location.

A solid phase substrate, its processing method and application, the solid phase substrate has at least one silanized surface, the surface is a surface exposed to a specific environment for a certain period of time, the specific environment is an inert gas environment, and the temperature of the specific environment is selected from 37°C to 60°C, a certain period of time is not less than 2 hours. The invention improves the distribution density and stability of the nucleic acid molecules fixed on the surface of the solid phase substrate, improves the chip quality, and can improve the sequencing quality when used in sequencing.

The invention relates to the technical field of nucleic acid chips, in particular to a solid-phase substrate and a treatment method and application thereof. The solid-phase substrate is provided with at least one silanized surface, the silanized surface is a second surface formed by exposing a silanized first surface to a specific environment for a certain time, the specific environment is an inert gas environment, the temperature of the specific environment is selected from 37 DEG C to 60 DEG C, and the certain time is not less than 2 hours. The performance of the solid-phase substrate is improved, so that the distribution density and the stability of nucleic acid molecules fixed on the surface of the solid-phase substrate are improved, and the quality of the nucleic acid chip is finally improved.

The invention relates to a method for detecting a PAS (Poly A Site) of an RNA (Ribose Nucleic Acid), and provides a method for analyzing APA (Alternative Polyadenylation) based on 3T-seq (TT-mRNA Terminal Sequencing) within a whole-genome range. The method for analyzing APA comprises the following steps: preparing magnetic beads containing oligo dT, i.e., mixing an oligo dT primer modified by 5' end biotin and the magnetic beads with streptavidin; mixing the magnetic beads with total RNA and screening RNA containing Poly A; performing reverse transcription, synthesizing the second strand of a double-stranded cDNA and breaking the double-stranded cDNA; removing the Poly A structure; after terminal modification, connecting a sequencing linker; and recovering libraries. According to the method for analyzing APA, RNA horizontal operation is reduced, a Poly A sequence is horizontally removed from the DNA, and the influence of the Poly structure on sequencing is eliminated; double-stranded DNA breaking enzyme is selected for treatment, and on the premise that the quality of the libraries is not affected, the DNA is broken horizontally; the experiment process is simplified, the experiment difficulty and cost are lowered and the method for analyzing APA is wide in application range.

The invention discloses a library construction method and reagent for pooling of large-sample-size PCR products based on high-throughput sequencing. The library construction method comprises the following steps: separately subjecting each of a plurality of PCR products derived from DNAs of different samples to annealing with a random primer having a tag sequence and carrying out an isothermal amplification reaction under the action of an isothermal amplification enzyme; mixing isothermal amplification products derived from different samples and carrying out piece selection on the mixed products; subjecting the products of the piece selection to repairing of 5' phosphorylated blunt ends and A addition reaction of 3' ends; connecting the products with linker sequences so as to obtain ligation products capable of distinguishing library information; and subjecting the ligation products to PCR amplification to obtain a library applicable to high-throughput sequencing. The method provided bythe invention realizes pooling without dependence on an ultrasonic breaking instrument, is lowered in library construction cost and complexity, reduces data waste, and improves the base randomness and coverage depth uniformity of sequencing data, thereby reducing the sequencing cost of a single sample.

The present application discloses a method for processing raw data of nucleic acid third-generation sequencing and its application. The method for processing the raw data of the third-generation nucleic acid sequencing of the present application includes comparing the second-generation short-sequence data to the third-generation self-error-correcting data, and counting the single bases of the third-generation self-error-correcting data in the comparison results Coverage depth, mask the area with a single base coverage depth lower than the threshold as N, and use the second-generation sequencing hole-filling software to fill holes in the N-shielded area to obtain nucleic acid third-generation sequencing with a low single-base error rate data. The method for processing the raw data of the third-generation nucleic acid sequencing of the present application uses the second-generation short-sequence data to compare with the third-generation long-sequence data, and uses the hole-filling software of the second-generation sequencing to compare the single base coverage in the comparison results The low-depth N-shielding region is complemented, which effectively reduces the single-base error rate in the third-generation sequencing data and improves the sequencing quality.