49results about How to "Reduce the cost of building a library" patented technology

The invention discloses a building method of a library for detecting non-small cell lung cancer gene mutation and a kit. The method includes: using tubular reaction to complete genome DNA breaking and connector connection, performing hybrid capture on connection products after amplification and non-small cell lung cancer related gene target area probes, and performing BGISEQ-500 / 1000 platform sequencing and data analysis to obtain mutation conditions. The method has the advantages that the experiment flow is optimized greatly by the tubular reaction, operation complexity and time are reduced, and the requirements on clinical sample initial amount are lowered; multiple genes and multiple sites can be detected in one step, point mutation, insertion and deletion, structural variation and copy number variation are covered, the detecting result is accurate and overcomes the defect that a PCR capture method cannot detect the structural variation in one step, and the effectiveness of the high-throughput sequencing applied to the detection of the non-small cell lung cancer gene mutation; the method is wide in coverage, high in cost performance, capable of providing a reference basis for the diagnosing, treatment and drug use performed by doctors, and the method is suitable for being popularized and used in a large-scale manner.

The invention relates to an mRNA fragmentation method and a method for constructing a sequencing library based on the mRNA fragmentation method. The mRNA fragmentation method provided by the invention employs forward and reverse bridge-type probes, realizes breakage of an mRNA sample through one-step reverse transcription and connection reaction, and introduces two terminal linkers during reverse transcription so as to obtain a cDNA library with linkers at two terminals. The cDNA library with the linkers at the two terminals can directly undergo cyclization reaction or undergo PCR amplification before cyclization reaction, so a sequencing library for single-stranded cyclic nucleic acids can be obtained; or a sequencing library for single-stranded nucleic acids can be obtained by directly subjecting the cDNA library to amplification. When the mRNA fragmentation method is applied to construction of the sequencing library, tedious steps like restoration of a tail terminal and arrangement of a linker on one end at first and another linker on the other end next in traditional construction of libraries can be omitted; experimental flow is greatly simplified; a library construction period is shortened; and library construction cost is greatly reduced.

A method for constructing banks of human dental pulp stem cells comprises the steps of acquiring basic information, obtaining dental pulp stem cells, cryopreserving and putting dental pulp stem cells into cell banks and recording and checking information. When the banks of dental pulp stem cells are used, recovery and multiplication culture are carried out in accordance with the actual demands. In the method, the dental pulp stem cells are obtained from dental pulp of waste healthy human teeth and a reserve bank for effective resources is established by using system engineering. A great quantity of dental pulp stem cells with functional activities can be obtained through short-term culture of the human dental pulp stem cells stored in the banks and can be stored for long term without losing the activities. Compared with the prior art, the invention is characterized in that the construction operation specification is simple, easy to grasp, safe and feasible; the bank construction cost is low and the standardization degree is high; and the cells from different age groups adopt different cryopreservation solution so that the activities of the recovered cells are enhanced, thus expanding the sources of the cells and enhancing the activities of the cells. The teeth used in the method are those which are extracted for various reasons and are waste, thus greatly saving the medical resources; therefore, the construction has wide application prospect.

The invention discloses a human placenta, umbilical cord mesenchyme stem cell bank and its making method. The method includes the following steps: the first is taking human placenta, umbilical cord to test; smashing after cleaning by phosphate buffer; adding phosphate buffer to dilute; the second is adding collagenase to digest; the third is adding phosphate buffer; the fourth is adding trypsinization; the fifth is mixing the cell from the second and fourth, centrifuging, removing supernatant, cleaning by the phosphate buffer, centrifuging, and removing supernatant; the mesenchyme stem cell are gained; the sixth is freezing them in liquid nitrogen; saving by ABO / Rh subtype and HLA subtype; setting recallable cell information file. The invention is newborn storage placenta and umbilical cord mesenchyme stem cell. It can offer mesenchyme stem cell to treat the disease for personal, family and others.

The invention discloses a method for rebuilding a gas storage reservoir and a layered injection and recovery system. The method for rebuilding the gas storage reservoir comprises the following steps of acquiring formation parameters of an oil and gas reservoir; respectively arranging an gas injection and recovery well and an observation well on the oil and gas reservoir; tripping a layered water drainage tubular column in the observation well; performing perforation on different sections of the layered water drainage tubular column to form perforation sections; tripping a gas injection and recovery tubular column in the gas injection and recovery well to perform gas injection; performing a single layer test in the observation well to obtain a single layer test result; determining a gas production layer position according to the single layer test result; and closing the gas production layer position. By the method for rebuilding the gas storage reservoir and the layered injection and recovery system, the content of water in the oil and gas reservoir can be reduced effectively, and the storage capacity of the gas storage reservoir is increased.

The invention discloses a method for constructing an umbilical cord mesenchymal stem cell bank using the umbilical cord of a newborn as a resource, aiming at fewer steps, simpler and easier operations when constructing the umbilical cord mesenchymal stem cell bank. The present invention consists of the following steps: (1) taking a human umbilical cord for detection, washing with a buffer to remove residual blood, and cutting into pieces to obtain human umbilical cord tissue; (2) adding collagenase to digest; (3) adding buffer to dilute and digest (4) Discard the supernatant, add buffer to resuspend the pellet, sieve, collect the filtrate, centrifuge, and repeat twice to obtain human umbilical cord mesenchymal stem cells; (5) The obtained human umbilical cord The mesenchymal stem cells are stored in liquid nitrogen, and stored according to their ABO / Rh typing and HLA typing, and a cell information file for retrieval is established, that is, a human umbilical cord mesenchymal stem cell bank is constructed. Compared with the existing method, the method of the present invention is faster, more convenient, and less damage to cells.

The invention discloses a construction method of an amplicon library for detecting low-frequency mutation of a target gene. The construction method of the amplicon library for detecting the low-frequency mutation of the target gene, provided by the invention, comprises the following steps: 1) designing and synthesizing a Barcode primer F1, an upstream primer F2, a downstream outer primer R1 and adownstream inner primer R2; 2) carrying out further PCR (Polymerase Chain Reaction) amplification on a cfDNA (cell-free Deoxyribonucleic Acid) of a sample to be detected by utilizing the Barcode primer F1, the upstream primer F2, the downstream outer primer R1 and the downstream inner primer R2, so as to obtain an amplified product, namely a DNA library for amplicon sequencing. By adopting the method disclosed by the invention, a tissue sample can be detected and different regions of free DNAs in samples including blood, urine, cerebrospinal fluid and the like can be rapidly, conveniently, sensitively and specifically subjected to target amplification and mutation which is as low as a 0.1 percent level is efficiently detected; the experiment operation is greatly simplified, the library loss and pollution are effectively avoided, the cost is remarkably reduced and the efficiency is improved.

The invention provides a primer for amplicon sequencing and a two-step PCR database building method. The method comprises the following steps of: 1) extracting genomic DNA of a species sample to be tested; 2) respectively connecting universal primer sequences to 5' ends of forward and reverse primers of the multiplex PCR, and taking the genomic DNA as a template to carry out a first round of multiplex PCR to obtain a PCR product with universal primer sequences at both ends; 3) purifying the product of the first round of PCR; 4) respectively connecting primer sequences matched with the first round of PCR universal primer sequences at both ends of the sequencing label sequence to obtain a PCR product carrying the label sequence, and using the PCR product as a template to carry out a second round of PCR; and 5) purifying the PCR product, and sequencing on the machine after passing the quality inspection. The invention adopts the two-step PCR method to build a database, so that the amplicon sequencing cost is reduced, the detection cost is reduced by more than 50%, and the quality index of the amplicon is effectively improved. That is to say, the detection rate of the site and the datauniformity are improved, and the method has extremely high economic value.

The invention discloses a kit for DNA library building and an application of the kit, and in particular relates to a kit for peripheral blood cfDNA library building and an application of the kit. According to the invention, the kit for DNA library building is firstly provided, wherein the kit comprises a component A and a component B; the component A consists of the following ingredients at a ratio as follows: 1-3U of T4 PNK to 1-4.5U of Klenow Fragment to 0.25-0.5U of LA Taq DNA Polymerase to 0.3-0.9U of T4 DNA Polymerase to 0.5-2.5 nmol of dNTP to 0.5-2.5nmol of dATP; and the component B consists of the following ingredients at a ratio as follows: 2.5-8nmol of ATP to 26.5 132U of T4 DNA Ligase to 0.213 0.534[mu]L of PEG 8000. According to the kit and the application method provided by the invention, DNA loss in a library building process can be reduced to the greatest extent, the usage amount of plasma can be reduced, library building cost can be reduced, library building time can beshortened, operations can be simplified and more stable and reliable results can be obtained. The kit provided by the invention is significant for improving library building efficiency and reducing labor cost.

The invention discloses a method for constructing an absolute quantitative transcriptome library based on a unique recognition sequence. Using the fragmented mRNA as a template, the first cDNA strand is synthesized under the action of reverse transcriptase using a primer pool with a universal linker sequence, and a unique recognition UID sequence is added to the 3' end of the synthesized cDNA by an enzymatic reaction. Build a library linker so that each cDNA has a unique sequence tag; finally, use a universal library linker to perform PCR amplification to obtain an RNA library. For the first time, the present invention uses the splint ligation method to construct an RNA library based on single-stranded cDNA. At the same time, the UID sequence is used to accurately restore the cDNA composition before PCR amplification, which can realize accurate quantification of transcripts; the present invention uses single-stranded cDNA as a raw material for library construction. The step of second-strand synthesis is omitted, the template loss rate is reduced, cost and time are saved, and the existing technology can only relatively quantify transcripts.

The application discloses a method for constructing a sequencing library, a reagent for building a library and an application thereof. The sequencing library construction method of the present application includes using degenerate primers to perform constant temperature amplification on the enriched target gene, and then performing end repair, adding "A" bases, and adapters to the constant temperature amplification products, and purifying to obtain a sequencing library; And the 5' end of the primer has a tag sequence, and the 3' end is a random sequence. The sequencing library construction method of the present application uses degenerate primers to amplify the target gene at a constant temperature to obtain fragmented DNA suitable for sequencing, which omits the fragmentation step and avoids the loss of tag sequences and useless sequencing data. The sequencing library construction method of the present application simplifies the library construction process, shortens the library construction cycle, reduces the cost of library construction, improves data utilization, and provides a new library construction scheme for high-throughput sequencing.

The present invention provides a simplified genome next-generation sequencing library construction method and kit based on double-enzyme digestion. The present invention addresses the shortcomings of existing double-enzyme-digestion simplified genome sequencing library construction methods, expands the scope of double-enzyme-digestion combinations, and reduces the number of simplified Genomic library construction is overly dependent on expensive instruments, which simplifies the library construction process, reduces the cost of library construction and improves sequencing efficiency. At the same time, the technology is simple, flexible and easier to be mastered by researchers and can be implemented in ordinary molecular laboratories. . It is especially suitable for micro or medium-scale laboratories that need to conduct SNP molecular marker development, genetic map construction, population genetics research and phylogenetic biology research on a large number of species with incomplete reference genomes. The invention has good practical application value and application prospect in the fields of agricultural molecular breeding, conservation biology and evolutionary biology.

The invention relates to a method for constructing a human amniotic mesenchymal stem cell bank. The method is to take human amniotic membrane for detection, wash it with phosphate buffer solution, crush it, add phosphate buffer solution to dilute it, digest it with trypsin, digest it with collagenase IV and deoxyribonuclease I, and filter it to make a single cell suspension; Then add human serum albumin, transferrin, insulin and sodium selenite to VDMEM:VF12=1:1 DMEM / F12 basal medium, so that human amniotic mesenchymal stem cells can be placed in the incubator under serum-free conditions , medium change and passaging. The mesenchymal stem cells obtained from in vitro culture and expansion were frozen in liquid nitrogen, stored according to the neonatal sex, ABO / Rh type and HLA type, and the cell information files were established to construct a human amniotic mesenchymal stem cell bank. The invention has the characteristics of no other animal origin, wide sources and no ethical restrictions. Mesenchymal stem cells can be provided for cell therapy and other applications.