15 results about "Template switching" patented technology

The present application relates to the field of biotechnology and molecular detection, in particular to a plant and pathogenic bacteria interaction gene expression analysis method based on UMI tag, comprising: obtaining interaction sample total nucleic acid extract; and constructing a molecular hybridization system with host-specific chimeric blocking probes containing locked nucleic acid modification and 3' end dideoxy modification, and reverse transcription primers; through temperature rising and denaturation and temperature reduction and annealing, the blocking probes are combined with host high abundance nucleic acid to form a hybridization blocking complex, and the primers are combined with pathogenic bacteria nucleic acid; template switching oligonucleotides with unique molecular identifier sequences and reverse transcriptase are added, the host extension is blocked, and UMI is introduced into the pathogenic bacteria complementary chain, and an interaction expression library is generated; after sequencing, the pathogenic bacteria effective reads and the host invalid reads are counted, and the effective interaction signal signal-to-noise ratio is output; the present application can preferentially capture pathogenic bacteria transcripts in the high background of host RNA.

The invention provides a method which allows the separation of different workflow steps for barcoding of target nucleic acids and therefore providing optimal reaction conditions for each workflow step, especially for template switching reactions. Moreover this method provides the opportunity to perform the reactions such as barcoding reactions of two different nucleic acid molecules from one cell such as RNA and genomic DNA molecules in a single step. The method comprises the steps: (a) Providing a plurality of cells comprising target RNA molecules and at least one solid support comprising capture oligonucleotides for said target RNA molecules and barcode oligonucleotides; (b) Partitioning said plurality of cells and said solid supports such that each cell is included into a separate partition and each partition comprises a solid support; (c) Lysing said cell, thereby obtaining a mixture of target and non, target RNA molecules; (d) Hybridizing said target RNA molecules to the capture oligonucleotides for said target RNA molecules, thereby obtaining target RNA molecules attached to said solid support (e) Disrupting the partitions and separating the non-target RNA molecules from the target RNA molecules attached to said solid support (f) Generating double stranded nucleic acids from the target RNA molecules by nucleic acid synthesis, wherein the capture oligonucleotides serve as primer and the target RNA molecules serve as templates (g) Attaching the barcode oligonucleotides to the double stranded nucleic acids from target RNA molecules, thereby generating barcoded nucleic acids from target RNA molecules; Characterized in that in step a) said plurality of cells additionally comprise target genomic DNA molecules and said at least one solid support additionally comprise capture oligonucleotides for target genomic DNA molecules and in that the capture oligonucleotides for target RNA and target genomic DNA molecules are different.

Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end and are subsequently barcoded on the 5′ end following a template switching reaction and intermolecular and / or intramolecular hybridization and extension. 5′- and / or 3′-barcoded nucleic acid targets can serve as templates for amplification reactions and / or random priming and extension reactions to generate a sequencing library. The method can comprise generating a full-length sequence of the nucleic acid target by aligning a plurality of sequencing reads. Immune repertoire profiling methods are also provided in some embodiments.

A cDNA amplification method is disclosed that can comprehensively amplify mRNA from even extremely small cell samples with high efficiency and low bias. The complementary DNA chain amplification method of the present invention comprises the steps of: capturing target RNA and synthesizing a complementary DNA chain in a liquid or solid system using a reverse transcriptase having terminal transferase activity; adding a base such as CCC to the end of the complementary DNA chain; and performing homopolymer tailing by a TdT reaction in the presence of a chain-stopping nucleotide triphosphate. By further combining a template switching reaction, the complementary DNA chain can be amplified with even higher efficiency and low bias.

The application provides a full-length lncRNA absolute quantitative transcriptome library construction and sequencing method based on TSO-UMI marking and composite internal standard correction, and the library construction method steps comprise the following steps: S1, total RNA of a sample is extracted and rRNA is removed; S2, a composite internal standard is added; S3, a reverse transcription adapter primer is used to connect a product; S4, a TSO-UMI fusion primer is added, and full-length cDNA is synthesized through template switching; S5, cDNA is used as a template to perform PCR amplification; and S6, the amplified library is purified; the nucleotide sequence of the TSO-UMI fusion primer is shown in SEQ ID NO. 1 or SEQ ID NO. 2. Through TSO-UMI fusion primer sequence reconstruction, SIRV / ERCC specific composite internal standard ratio, full-process coupling experimental design, and supporting UMI+internal standard joint correction integrated bioinformatics algorithm, the problems such as PCR deviation, relative quantification, batch difference, isomer recognition, and polyA-free quantification are successfully solved, and reliable technical guidance is provided for lncRNA basic research, disease marker screening, and clinical efficacy monitoring.

The application discloses a method for constructing a single-cell transcriptome and chromatin accessibility double-omics single-cell sequencing library, which comprises the following steps: a) preparing a single-cell suspension; b) obtaining chromatin open sites; c) performing a reverse transcription reaction on the transcriptome of the cell by using a reverse transcriptase and a reverse transcription primer, so as to obtain the transcriptome information of the single cell; d) performing a template switching reaction by using a template switching oligo (TSO); e) subsequent coding; f) initial library amplification; and g) preparing a sequencing chromatin open site library and a transcriptome library. The application also discloses a method for sequencing a single-cell transcriptome and chromatin accessibility double-omics sequencing library, which comprises the steps of respectively sequencing the chromatin open site library and the transcriptome library prepared by the method.

The invention provides a cold air circulation rapid cooling system, which relates to the technical field of hot melt syrup and comprises a process parameter template switching module, a vibration suppression and dynamic parameter adjustment module, a neural network algorithm response module and an energy consumption linkage optimization module, the process parameter template switching module is used for storing cooling parameter sets of syrup with different viscosities and realizing rapid parameter switching, and the vibration suppression and dynamic parameter adjustment module dynamically adjusts cold air parameters based on vibration feedback so as to suppress carbon dioxide bubble breakage; in the aspect of improving the production efficiency, a plurality of groups of cooling parameter sets for syrup with different viscosities are pre-stored in the process parameter template switching module, and the cooling parameter switching can be completed through one-key template loading, so that the cooling remodeling efficiency is greatly improved, the repeated trial and error process of manual debugging is avoided, and the production cost is reduced. The syrup cooling device is particularly suitable for flexible cooling scenes of various kinds of syrup, and the turnover efficiency of a production line is remarkably improved.

The invention provides a dual-nucleic acid library co-construction method for synchronous detection of plasma ecDNA and related RNA markers. The method comprises the following steps: firstly, extracting total free nucleic acid from the same plasma sample; then carrying out linear DNA digestion treatment; then removing ribosome RNA (Ribose Nucleic Acid); then carrying out reverse transcription reaction by using a chimeric primer with 6-9 random basic groups at the 3'end and a template conversion oligonucleotide sequence at the 5 'end to obtain single-stranded cDNA with universal joints at the two ends; next, the single-stranded cDNA is subjected to cyclization, and annular cDNA is formed; then carrying out rolling circle amplification on the ecDNA and the annular cDNA; and carrying out fragmentation, terminal repair, linker connection and PCR enrichment on the amplification product to obtain the dual-nucleic acid library for high-throughput sequencing. By adopting the method, the collaborative analysis of the DNA level and the RNA level of the trace sample is realized.

The utility model relates to the technical field of clamp switching equipment, in particular to a drawer type clamp template switching device which comprises a body, an upper die sealing mechanism and a template switching mechanism, the upper die sealing mechanism and the template switching mechanism are arranged on the body, the template switching mechanism is located behind the upper die sealing mechanism, and a template mounting position is arranged on the upper die sealing mechanism. The template switching mechanism comprises a lifting rack and drawing assemblies arranged on the two sides of the lifting rack, and the drawing assemblies extend to the two sides of the upper die sealing mechanism. A plurality of layers of templates are arranged on the lifting rack in the vertical direction, the drawing assembly is used for drawing any template to a template mounting position of the upper die sealing mechanism, and the upper die sealing mechanism is used for driving the template on the template mounting position of the upper die sealing mechanism to move downwards to block the upper surface of a workpiece. The utility model can solve the technical problem that the existing clamp template is complicated to replace, disassemble and assemble.

Disclosed is a cDNA amplification method capable of comprehensively amplifying mRNA with high efficiency and low deviation from a very small amount of a cell sample. The complementary DNA chain amplification method according to the present invention comprises the steps of capturing a target RNA and synthesizing a complementary DNA chain in a liquid phase system or a solid phase system using a reverse transcriptase having terminal transferase activity, adding a base such as CCC to the terminal of the complementary DNA chain, and then performing homopolymer tailing by a TdT reaction in the presence of chain-terminated nucleotide triphosphate. By further combining the template conversion reactions, the complementary DNA strand can be amplified with higher efficiency and lower deviation.

The present disclosure relates to a coding control method and device, a storage medium and an electronic device, and relates to the field of silicon rod machining production, and is applied to a silicon rod machining control system. The method comprises the following steps: determining a target coding template from a plurality of preset coding templates according to the size of a silicon rod, the coding template being used to indicate a coding position. A template switching instruction is generated according to the target coding template, and the template switching instruction is sent to a coding machine. The template switching instruction is used to instruct the coding machine to retrieve the target coding template, so that the silicon rod is coded according to the coding position indicated by the target coding template. In the present disclosure, the target coding template matched with the size of the silicon rod is selected from the plurality of preset coding templates, so that the coding machine codes the silicon rod according to the coding position indicated by the target coding template, which can ensure that the coding content is kept at the center of the silicon rod, and improves the automation production efficiency of the silicon rod.

Methods are described for enhancing complementary DNA (cDNA) synthesis from a ribonucleic acid (RNA) template during reverse transcription by an enzyme having template switching activity. The methods provide for enhancing the processivity of a reverse transcriptase.

Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end and are subsequently barcoded on the 5′ end following a template switching reaction and intermolecular and / or intramolecular hybridization and extension. 5′- and / or 3′-barcoded nucleic acid targets can serve as templates for amplification reactions and / or random priming and extension reactions to generate a sequencing library. The method can comprise generating a full-length sequence of the nucleic acid target by aligning a plurality of sequencing reads. Immune repertoire profiling methods are also provided in some embodiments.

Provided are a method for synthesizing double-stranded cDNA and the use thereof. The method comprises: performing a reverse transcription reaction on an mRNA template by means of using a reverse transcription primer and a reverse transcription mixture, so as to obtain double-stranded cDNA, wherein the reverse transcription mixture comprises a reverse transcriptase having a template switching function, a template switch oligo and dNTPs; in the reverse transcription mixture, the final concentration of the reverse transcriptase is 5-20 U / μL, the final concentration of the template switch oligo is 2-12.5 μM, and the final concentration of dNTPs is 1-10 mM. The reverse transcription reaction is performed by means of using the reverse transcription mixture. By means of optimizing the amounts of the dNTPs, the template switch oligo and the reverse transcriptase involved in the reaction in the reverse transcription reaction system, the efficiency of the reverse transcription reaction is improved, enabling the production of a double-stranded cDNA product in a one-step reaction, thereby greatly shortening the reverse transcription reaction time and thus improving the quality of the double-stranded cDNA product.

Disclosed herein, are compositions, methods, and kits comprising engineered reverse transcription enzymes that exhibit several desired properties such as thermal stability, processive reverse transcription, non-templated base addition, and template switching ability. The engineered reverse transcription enzymes described herein demonstrate unexpectedly higher resistance to cell lysate inhibition, greater ability to capture full-length mRNA transcripts, and demonstrate improved results in small reaction volumes as compared to other engineered reverse transcription enzymes.