89 results about "Amplification/Sequencing" patented technology

The invention discloses a method for detecting mutation information in a multiplex amplification sequencing product of a genome. The method comprises steps as follows: sequencing data are subjected to quality assessment and preprocessing; a recognizable sequencing sequence is selected for sequence assembling; the recognizable sequencing sequence or a sequence obtained through assembling is compared with a reference gene sequence, and preliminary variation information is obtained; fine calibration of sequence variation is performed according to different types of conditions; a calibrated sequencing fragment is obtained; the homozygosis or heterozygosis state of a target fragment is obtained according to the type of the sequencing fragment with the highest abundance; finally, the mutation information in the multiplex amplification sequencing product of the genome is obtained. By means of the method, the amplification product can be rapidly, efficiently and accurately recognized, and the calculation resources are saved; the sequence assembling process is compatible, and the problem of reduction of the quality value of basic groups produced in the sequencing process can be effectively solved; the homozygosis/heterozygosis state of variation information can be more effectively and stably judged, and random errors introduced in the PCR (polymerase chain reaction) process and the sequencing process are eliminated.

The invention is directed to methods for increasing the sensitivity of high throughput sequencing, particularly for distinguishing true rare mutations from amplification, sequencing and other sample processing errors that occur in sequencing techniques. In one aspect, methods of the invention includes steps of (a) preparing templates from nucleic acids in a sample; (b) labeling by sampling the templates to form tag-template conjugates, wherein substantially every template of a tag-template conjugate has a unique sequence tag; (c) linearly amplifying the tag-template conjugates; (d) generating a plurality of sequence reads from the linearly amplified tag-template conjugates; and (e) determining a nucleotide sequence of each of the nucleic acids based on the frequencies, or numbers, of each type of nucleotide at each nucleotide position of each plurality of sequence reads having identical sequence tags.

The invention provides methods, apparatuses, and compositions for high-throughput amplification sequencing of specific target sequences in one or more samples. In some aspects, barcode-tagged polynucleotides are sequenced simultaneously and sample sources are identified on the basis of barcode sequences. In some aspects, sequencing data are used to determine one or more genotypes at one or more loci comprising a causal genetic variant.

The invention provides methods, apparatuses, and compositions for high-throughput amplification sequencing of specific target sequences in one or more samples. In some aspects, barcode-tagged polynucleotides are sequenced simultaneously and sample sources are identified on the basis of barcode sequences. In some aspects, sequencing data are used to determine one or more genotypes at one or more loci comprising a causal genetic variant.

The present invention relates to sequencing probes, methods, kits, and apparatuses that provide enzyme-free, amplification-free, and library-free nucleic acid sequencing that has long-read-lengths and with low error rate.

The invention provides methods, apparatuses, and compositions for high- throughput amplification sequencing of specific target sequences in one or more samples. In some aspects, barcode-tagged polynucleotides are sequenced simultaneously and sample sources are identified on the basis of barcode sequences. In some aspects, sequencing data are used to determine one or more genotypes at one or more loci comprising a causal genetic variant. In some aspects, systems and methods of detecting genetic variation are provided.

The invention discloses a method for fixed point knock-out of the second exon of rice OsPDCD5 gene by using a CRISPR/Cas9 system. The method comprises the following steps: selecting a target fragment in the second exon region of the rice OsPDCD5 gene, constructing a plant CRISPR/Cas9 recombinant vector, introducing the plant CRISPR/Cas9 recombinant vector to rice calluses through an Agrobacterium infection technology, carrying out regeneration to form seedlings, shearing double strands of the second exon of the OsPDCD5 gene under the combined action of guide RNA and Cas9 nuclease, and realizing random insertion or random deletion of the target gene fragment through the self DNA repairing function of cells. Nine strains with the target fragment having point mutation are obtained through a PCR amplification sequencing technology, and pass phenotype identification, so the purpose of the increase of the plant height and the yield of rice is realized. The method provides an efficient breeding way for cultivating high-yield and high-quality rice varieties.

The invention discloses a kit for extracting DNA in the egg shell membrane of Yangtze alligator and a use method thereof, and the kit comprises the following reagents: a reagent A, a reagent B, a reagent C, a reagent D, a reagent E and a reagent F. The template DNA which is extracted by the kit has good purity, and the kit can be used for gene amplification sequencing and typing. In the amplification of the 12S rRNA gene, the mitochondrial D-loop region and the Cyt b gene, all the extracted shell membrane DNA templates can be successfully amplified to obtain the fragment products with expected identical sizes, high concentration and high specificity. Microsatellite DNA marker specificity primers are utilized to obtain typing data through the PCR amplification to be used for the identification in disputed paternity of Yangtze alligator. In addition, the method also can be applied to the extraction of other ovipara such as the turtles, the birds and the like. The obtained template DNA can be widely applied to the studies of the ovipara on the fields such as population genetic analysis, molecular evolution, evolution of relationship among individuals and the like.

The invention belongs to the field of molecular genetics and relates to a novel broad-spectrum rice blast resistance allele pi21t and resistance application thereof. According to a cloned sequential design primer of the broad-spectrum rice blast resistance recessive allele pi21t, PCR amplification is performed on the wide compatibility variety 02428 which is used in high frequency in breeding, and sequencing comparative analysis is performed, so that the novel broad-spectrum rice blast resistance allele pi21t is identified in combination with a field rice blast inoculation identification result of the variety. The material has high resistance for popular microspecies of 6 rice blast groups in the Yangtze-Huaihe river basin, and provides new gene resources for breeding for rice blast resistance.

The invention discloses a construction method of an HDAC8 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC8 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC8, and performing separation to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method. After genome DNA is extracted, PCR amplification and sequencing are carried out, and cell clones of which two HDAC8 genes are subjected to homozygous frameshift mutation are successfully identified. In the HDAC8 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC8 knockout has no obvious influence on the cell growth rate, which shows that the HDAC8 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. A foundation is laid for further knocking out HDAC8 from suspension culture type BHK-21 cells and directly applying HDAC8 to production of foot-and-mouth disease vaccines.

The invention discloses a simple, economical and accurate mitochondria whole genome sequencing barcode amplification sequencing method based on second generation sequencing. Two groups of matched primers are designed, wherein the first group of primers is composed of 61 pairs of target fragment primer sequences and primer joint sequences which are overlaid and cover the mitochondria whole genome.The second group of primers is composed of positive and negative label primers which comprise the joint sequences and are matched two by two for marking samples of different sources. By matching the two pairs of primers in use, two pairs of the primers are added in a primary PCR reaction for amplification to obtain amplicons which have marks, are moderate in length and comprise mitochondria wholegenome. The amplicons are mixed and used for establishing a library and sequencing directly to achieve multi-sample mixed sequencing. Sequencing data identifies mark sequences at the head and tail ends at the same time by means of applying a bioinformatics assembling technology to different sequences of different sources to obtain all mitochondria genome sequence information of all samples. By employing the method, the sequencing experiment steps are simplified, the sequencing cost is lowered greatly, and the detection rate of low-frequency mutation is reduced, thereby providing probability for human mitochondria whole genome associated researches.

The invention discloses a primer group and a method for rapidly identifying influenza A virus subtypes through combination of DNA barcodes and second-generation high-throughput sequencing. The method comprises steps as follows: (1), genome extraction and inverse transcription; (2), primer design and PCR (polymerase chain reaction) amplification; (3), Ion Torrent sequencing; (4), bioinformatic analysis; and (5), phylogenetic analysis and genetic distance calculation. According to the primer group and the method, sequences of influenza A virus materials of 16 kinds of different areas and subtypes are analyzed, and the DNA barcodes which can be used for distinguishing the subtypes are found out; with the adoption of the technical idea of the DNA barcodes and a traditional RT-PCR (reverse transcription-polymerase chain reaction) detection method, whether the influenza virus exists can be rapidly judged with preference of a touch-down program, the subtype and a pathotype of the influenza A virus can be measured simultaneously, and one simpler and more rapid influenza typical diagnosis program is provided.

The invention discloses a construction method of an HDAC3 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC3 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC3, and separating the cells to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method; extracting the genome DNA, performing PCR amplification and sequencing, and successfully identifying the cell clones of which two HDAC3 genes are subjected to homozygous frameshift mutation. In the HDAC3 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC3 knockout has no obvious influence on the cell growth rate, so that the HDAC3 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. Therefore, the foundation is laid for further knocking out HDAC3 from suspension culture type BHK-21 cells and directly applying HDAC3 to foot-and-mouth disease vaccine production.

The invention discloses a spider mitochondria COIII gene complete sequence amplification primer and a method for rapidly identifying species of spiders by utilizing complete sequence sequencing information. The method comprises the following steps: extracting total DNA of spider genomes, designing the COIII gene complete sequence amplification primer, performing PCR amplification, sequencing and rapidly identifying the species of spiders based on the gene complete sequence. With the adoption of the universal amplification primer disclosed by the invention, the complete sequence of the multiple mitochondria COIII genes of spiders can be rapidly and efficiently amplified, a powerful tool is provided for researches such as rapid identification of the species of spiders, population diversity analysis and genetic resource conservation and utilization, and a foundation is laid for spider mitochondria whole genome sequencing and analysis and research on different taxonomic category system evolutions.

The invention relates to the technical field of milk powder detection, and particularly relates to a method for detecting probiotics in milk powder by an Illumina Miseq sequencing platform. The method comprises the following steps of: (1) extraction of DNA (deoxyribonucleic acid) of a milk powder genome, i.e., after brewing the milk powder, carrying out primary centrifugation to take supernatant, then carrying out secondary centrifugation to take precipitates, adding buffer solution to oscillate, and then after adding lysozyme to perform a reaction, carrying out extraction of the DNA of the genome; (2) PCR (Polymerase Chain Reaction) amplification, i.e., aiming at v1 to v3 regions of 16s rDNA of a bacterium, selecting a specific primer to carry out PCR amplification; (3) Illumina Miseq sequencing, i.e., carrying out Illumina Miseq sequencing on a PCR product. The Illumina Miseq sequencing method effectively avoids the defects of low flux, complex operation, low accuracy and the like by analysis, not only can simultaneous sequencing on a plurality of variable regions of a plurality of samples be implemented, but also both a sequencing speed and a sequencing flux are further, and a detection result is high in credibility.

The invention relates to a mycobacterium tuberculosis multi-line drug resistance gene identification method and device. The method comprises the following steps: acquiring a whole genome sequence obtained by metagenome sequencing of a sample or a sequence of a mycobacterium tuberculosis drug-resistant target region obtained on the basis of targeted amplification sequencing, and carrying out data quality control; comparing the sequence subjected to the data quality control to a mycobacterium tuberculosis genome, sequencing and screening out a comparison result meeting the quality requirement; carrying out local assembly and variation detection on the sequences of the gene regions where the variation sites of which the comparison positions are located in the mycobacterium tuberculosis drug resistance gene database are located; annotating a variation detection result to the mycobacterium tuberculosis drug resistance gene database according to the positions and mutation types of variation sites on the genome; and outputting a detection result of each drug resistance related mutation sites in the mycobacterium tuberculosis drug resistance gene database based on an annotation result. According to the invention, multiple drugs and corresponding drug resistance sites can be covered in one detection, and the detection precision is high.

The invention provides a new compound having medicinal value, a preparation method thereof and a new bacterial strain which can generate the compound. The compound with formula (I) is chosen and prepared by rapidly choosing bacillus from soil sample with specific primer, using 16Ss rRNA gene to preliminarily identify and separate bacterial strain, amplifying, sequencing and comparing partial sequence of NRPSs/PKSs gene cluster after choosing potential new resources, analyzing the diversity of metabolite thereof, acquiring bacterial strain F6-B70 which contains new NRPSs/PKSs gene cluster and has new potential after choosing a large amount, fermenting in a fermentation medium, extracting active substance from bacterial cell and fermenting liquid, and then separating and purifying by using multiple purification techniques. The compound is a polyene macrolides compound which has an obvious function of restraining drug-resisting staphylococcus and an activity of resisting tumor.

The invention relates to a nucleic acid aptamer specifically binding to P-glycoprotein (P-gp), and a preparation method and application of the nucleic acid aptamer, and belongs to the technical fieldof biology. The nucleotide sequence of the nucleic acid aptamer is selected from SEQ ID No.1 to SEQ ID No.14 in sequence tables; the nucleic acid aptamer is prepared by screening, amplifying and sequencing an original random oligonucleotide library through an SELEX technology in combination with surface modified magnetic beads; the nucleic acid aptamer is small in molecular weight, can be chemically synthesized, and is low in cost; the affinity and the specificity are high; marking is facilitated; and the repeatability and the stability are high, and storage is easy. The nucleic acid aptamer can be made into a fluorescent molecular probe, and fluorescent visualization and quantification of P-gp on in-vitro tissues and cells of an organism are realized; and the nucleic acid aptamer can be specifically bound with P-gp, inhibits the function of P-gp, and becomes a potential nucleic acid type inhibitor of the function of P-gp.

The invention discloses a construction method of an HDAC5 gene knockout BHK-21 cell line, which comprises the following steps: carrying out gene knockout on HDAC5 in a cell line BHK-21 for foot-and-mouth disease vaccine production by utilizing a CRISPR/Cas9 technology, transfecting BHK-21 cells by utilizing CRISPR plasmids for knocking out the HDAC5, and separating the cells to obtain a plurality of cell clones by utilizing antibiotic screening in combination with gradient dilution and a cloning ring method; extracting the genome DNA, performing PCR amplification and sequencing and successfully identifying the cell cloning of which one HDAC5 gene is subjected to homozygous frameshift mutation, wherein the HDAC5-KO-A2 has deletion of 13 basic groups at a Cas9 predetermined cutting position. In the HDAC5 knockout BHK-21 cell line, the replication rate of foot-and-mouth disease virus is obviously accelerated, the final virus titer is obviously improved, and the HDAC5 knockout has no obvious influence on the cell growth rate, so that the HDAC5 knockout BHK-21 cell line has the prospect of being used for foot-and-mouth disease vaccine production. Therefore, the foundation is laid for further knocking out HDAC5 from suspension culture type BHK-21 cells and directly applying HDAC5 to production of foot-and-mouth disease vaccines.

The invention belongs to the field of crops, and particularly relates to genetic transformation, gene editing and analysis methods of main soybean varieties. The invention discloses a gene editing andtransforming vector which is based on a CRISPR/Cas9 system and takes an agrobacterium transforming vector pCambia3301 as a skeleton, and a promoter is designed for improving expression of Cas9 and gRNA. According to the invention, rapid and effective genetic transformation of the main soybean varieties is realized for the first time, and a simple and rapid PCR amplification sequencing analysis method for identifying the gene editing effect is developed. Through genetic separation and screening by molecular biology analysis technology, gene editing offspring plants which do not carry exogenousgenes of the soybean varieties for production are successfully obtained, and editing sites are stably inherited.

The invention provides a molecular marker for identifying subspecies of northern sea track of sika deer, an identification method and application, and aims at accurately identifying the subspecies ofthe northern sea track of the sika deer. Four SNP sites S-1 to S-4 of the molecular marker are respectively shown in the subspecies of the northern sea track of the sika deer, and are respectively shown in non-subspecies of the northern sea track of the sika deer. The invention also provides a method for identifying the subspecies of the northern sea track of the sika deer. The method comprises the following steps of: performing PCR amplification and sequencing on any one or more pairs of primer pairs 1 to 4, and identifying whether a sample to be tested is the subspecies of the northern sea track of the sika deer according to SNP marker site bases. The four SNP sites are positioned on four gene segments, and have the characteristics of high specificity and high stability. The identification detection accuracy is 100 percent. The invention provides a stable and reliable molecular detection method for identifying subspecies of sika deer resources. The molecular detection method has important theoretical and application values in the aspects of DNA fingerprint drawing of the sika deer, wild subspecies protection management and the like.

The invention belongs to the field of biotechnology, and particularly relates to a fusarium oxysporum nucleotide sequence qualitative standard sample and a preparing method thereof. Fusarium oxysporum in the logarithmic phase is acquired, total DNA extraction is conducted, specific PCR amplification sequencing is conducted, and then plasmid preparation is conducted. The prepared nucleotide sequence standard sample does not have biological activity any more, biological pollution and infection are avoided, the source of molecular biology positive control is available, and the sample is stable, free of pollution and easy to store. Based on the fact that fusarium oxysporum has the nucleotide sequence technical index with taxonomical significance, the problems of fusarium oxysporum nucleotide sequence standard sample source tracing reference system and reference index are solved, the fusarium oxysporum nucleotide sequence standard sample with real source tracing significance is obtained through application in setting value analysis of the standard sample, and the fusarium oxysporum nucleotide sequence qualitative standard sample has great significance in development of nucleotide sequence classified gradation standard samples in China.