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

The invention discloses a method for simultaneously sequencing a plurality of nucleic acid samples, which comprises: respectively performing adaptor ligation, first PCR amplification, terminal phosphorylation, and DNA ligation in order of the plurality of nucleic acid samples; mixing the plurality of DNA ligation products; adding a base A at the 3'-terminal of the mixed DNA ligation products; connecting the ligation products with the 3'-terminal added with the base A to a sequencing interface; performing second PCR amplification of the sequencing interface-connected products to obtain a mixed sequencing library of the plurality of nucleic acid samples; sequencing the mixed sequencing library; and classifying the sequencing results to respectively obtain respective nucleic acid sequences of the plurality of nucleic acid samples. The method of the invention can simultaneously perform sequencing of a plurality of nucleic acid samples, is accurate in sequencing results, good in repeatability, low in cost, and high in efficiency, makes full use of sequencing flux, and is especially suitable for blood free DNA samples.

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 refers to a tubular member, such as a pipette tip, for binding nucleic acid molecules for a subsequent biomolecular reaction, to methods for at least partly performing a sequencing-by-synthesis reaction in a tubular member, to an automated system using the tubular member for performing the methods of the invention, and kits and computer programs for use in relation to the methods of the invention. By performing a sequencing-by-synthesis reaction in a tubular member, such as a pipette tip, a new format for enzymatic reactions of this type, i.e. sequencing by synthesis, is provided.

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 invention discloses a method and a detection kit used for nasal cavity flora detection. According to the method and detection kit used for nasal cavity flora detection, a whole set of primers usedfor sample flora detection are provided, and the primers comprise the primers for amplifying V4-V5 areas of bacterium 16S rDNA and the primers for amplifying V1-V9 areas of bacterium 16S rDNA. According to the method and detection kit used for nasal cavity flora detection, firstly, the V1-V9 primers which are high in amplification efficiency, high in specificity and low in humanized homology areadopted for enriching 16S rDNA fragments, and then nested PCR is used for amplifying target sections V4V5 double fragments, so that the problem that when the V4V5 sections are used for amplifying primers directly, the homology with a humanized genomic sequence is high, so that when a nasal cavity swab sample with high humanized gDNA content is amplified, a serious non-specific amplification phenomenon appears is solved.

The invention provides a high-fidelity Pfu DNA polymerase mutant. The high-fidelity Pfu DNA polymerase mutant is characterized in that on the basis of a wild type Pfu DNA polymerase, mutation of one, three or six amino acid sites is carried out, and the mutation sites are selected from the following sites: Y403A, P411H, V438K, A741S, R757Q and S768K. The invention also discloses coding DNA of the high-fidelity Pfu DNA polymerase mutant and application of the high-fidelity Pfu DNA polymerase mutant in NGS. The high-fidelity Pfu DNA polymerase mutant has ultrahigh fidelity and high amplification efficiency, can be stably and efficiently applied to the library enrichment process in next-generation sequencing, effectively improves the library sequencing quality, reduces the base mismatch rate, and can be used for precise analysis and interpretation of genetic information.

The invention discloses a multi-amplification database building method for trace DNA and a special kit. The multi-amplification database building method can realize multiple amplification sequencing with trace DNA or even single-cell-level DNA as a template, and the whole reaction process can be realized in a single tube. Experiments prove that the database building method achieves 207-fold one-tube amplifier amplification at the single cell level, has the fidelity higher than that of conventional commercial kits, not only has the advantages of convenient operation, low cost, high sequencing quality and high sensitivity, but also can meet the sequencing requirements of two sequencing platforms, and is suitable for high-throughput and low-frequency mutation detection fields such as the ctDNA mutation detection field and the low-initial-quantity DNA embryonic mutation detection field such as single cell mutation detection and screening.

The invention relates to the technical field of biological information, in particular to a library building connector and method suitable for simplified genome sequencing of a DNBSEQ technology. The linker comprises an upstream linker sequence and a downstream linker sequence; the upstream linker sequence comprises an upstream Barcode fragment, a Q1-1 fragment, an Index fragment and a Q1-2 fragment which are connected in sequence; the downstream linker sequence comprises a downstream Barcode fragment and a Q2 fragment; wherein the upstream linker sequence is reversely complementary, and the Q2 fragment part in the downstream linker sequence is reversely complementary; the upstream Barcode fragment and the downstream Barcode fragment are not equal in length. The invention provides a library building joint and designs a simplified genome sequencing method suitable for a DNBSEQ technology based on the library building joint, and genome sequencing can be accurately, quickly and effectively carried out.

The invention provides a gene sequencing chip and a packaging method thereof. The chip comprises a protecting frame, an aminated substrate and a cover plate, wherein the aminated substrate is arrangedin the protecting frame and the cover plate stacks on the substrate. The method is characterized in that the contact areas of the protecting frame, the substrate and the cover plate are adhered through the curing reaction of coated silica gel to form a sealed structure, wherein the silica gel comprises one or the combination of dealcoholized silica gel, deaminated silica gel and deamidized silicagel, and the silica gel and the curing reaction byproduct of the silica gel do not have reaction with amino. The method has the advantages that the silica gel series glue which does not have reactionwith the amino is used to perform adhering and sealing, the problem that the glue pollutes the surface of the chip during the packaging of the aminated chip is solved effectively, complete sequencingdata is guaranteed, and sequencing quality is increased.

The invention relates to a reduced genome library building method for millet. The method includes the steps of: 1. utilizing restrictive endonuclease ApeK I for enzyme digestion on the genome DNA of several millet samples respectively to obtain an enzyme-digested product; 2. connecting a bar code joint F and a universal joint R with the enzyme-digested product; 3. mixing the connection products ofthe several millet samples at an equal volume and performing purification to obtain a purified connection product; 4. taking the purified connection product, using a universal primer F and 1 or morebar code primers R for PCR amplification to obtain a PCR product; 5. purifying the PCR product to obtain a target fragment, thus obtaining a sequencing library; and 6. using a high-throughput sequencing platform for sequencing of the sequencing library. The method provided by the invention has the advantages of high detection flux, low cost, good library building stability, and accurate and reliable sequencing result, and at the same time simplifies the genome sequencing and library building steps.

The invention provides a method for rapid quantification of a high-throughput sequencing library of an MGI platform and a kit. The method comprises the following step of carrying out fluorescent quantitative PCR detection on a to-be-detected high-throughput sequencing library by using a composition containing a primer and a probe; wherein the composition containing the primers and the probes comprises a first primer and a first probe which are used for detecting an MGI library, and a second primer and a second probe which are used for detecting an arabidopsis PHYA gene, the first primer comprises sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2, and the first probe comprises a sequence shown as SEQ ID NO: 3. The kit comprises the first primer and the first probe, and further comprises the second primer and the second probe, the second primer comprises sequences as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and the second probe comprises a sequence as shown in SEQ ID NO: 6. The provided method or kit can be used for rapid quantification of the high-throughput sequencing library.

The invention provides a lysate which is used for plant cell nucleus extraction and ATAC-seq experiments based on a lysate of 10x Genomics Company. Under the condition that the basic components of the original formula are not changed as far as possible, the proper concentration of each component is found, and cell nucleuses of different tissue parts, development periods and stress states of plants can be extracted. It is found for the first time that the fixed time needs to be adjusted according to species, tissue parts, plant development periods and environmental states (such as stress of heat, cold, salt, drought and the like). It is found for the first time that extraction is carried out after plant materials are fixed, and beta-mercaptoethanol is added into original lysate components, so that plant cell nucleus extraction and plant ATAC-seq experiments can be carried out. Experiments prove that when the lysate provided by the invention is used for extracting the plant cell nucleus, the nuclear membrane of the obtained cell nucleus is complete, and the sequencing quality is high; the method is suitable for different tissue parts, different development periods and different environment states of different plant species.

The invention relates to the technical field of gene sequencing, in particular to a template polynucleotide paired terminal sequencing method and a kit. According to the invention, an extension primer for generating a complementary strand is specially designed to form an extension primer structure at least formed by connecting a first extension primer and a second extension primer into a whole, and a binding site of the first extension primer on template polynucleotide is designed to be located at the upstream of a binding site of the second extension primer. In an environment with strand displacement activity, the extension strand of the upstream extension primer displaces the extension strand of the downstream extension primer, and the displaced extension strand of the downstream extension primer is connected to the first extension primer through the second extension primer and then is combined to a template polynucleotide strand through the extension strand of the first extension primer, so that the template polynucleotide is obtained. The template polynucleotide chain is fixed on a solid support, so that the stability of the complementary chain in a cyclic sequencing environment is improved, and the sequencing quality is improved.

The present invention provides a primer composition for analyzing intestinal flora, a detection kit composed thereof, and an application of the same. The present invention employs two-step amplification to obtain a sequencing library, and the primer composition comprises a random base.

The invention provides a Y-shaped linker, a kit and a method for constructing an RRBS library. The Y-shaped linker comprises a methylation linker 1, namely 5'-acactctttccctacacgacgctcttccgatctn-3', and a methylation linker 2, namely 5'-n2n1' agatcggaagagcacacgtctgaactccagtcac-3', wherein the methylation linker 1 and the methylation linker 2 are annealed to form the Y-shaped linker; the bases c inthe methylation linker 1 and the methylation linker 2 are methylationmodified c; n1 and n1' are complementary paired protective base sequences; the number of bases of n1 or n1' is in a range of 6-12;n2 is a cohesive terminal base of an enzyme cutting site; and the number of bases of n2 is in a range of 1-5. The linker is helpful for improving sequencing data output, and also improves the evaluation and accuracy of enzyme digestion efficiency.