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

The invention discloses a construction method of an umbilical cord mesenchyma stem cell bank utilizing newborn umbilical cords as resources, aiming at causing the steps to be less and the operation to be simpler and easier when constructing the umbilical cord mesenchyma stem cell bank. The invention comprises the following steps: (1), taking a human umbilical cord for detection, using a buffer solution to wash and remove residual blood, shearing to pieces, thus obtaining human umbilical cord tissue; (2), adding collagenase for digestion; (3), adding the buffer solution for diluting the digested tissue followed by organizing, mixing evenly and centrifuging; (4), abandoning the supernatant, adding the buffer solution for heavy suspension precipitation, screening, collecting filtrate and centrifuging, and repeating twice to obtain the human umbilical cord mesenchyma stem cell; (5), saving the obtained human umbilical cord mesenchyma stem cell in liquid nitrogen according to ABO / Rh parting and HLA parting thereof, establishing a cell information archive for retrieval, thus constructing the human umbilical cord mesenchyma stem cell bank. Compared with the existing method, the invention is more fast and convenient, and has less damage to the cell.

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 construction method of a small-fragment DNA (deoxyribonucleic acid) library based on an Illumina Hiseq 2500 sequencing platform, which comprises the following steps: blood free DNA extraction, fragment size screening, terminal repair, 3' terminal linker addition, PCR (polymerase chain reaction) amplification, magnetic bead purification and the like. Compared with the library construction process based on a sequencing platform in the past, the method disclosed by the invention simplifies the experiment process, shortens the library construction time, optimizes the reaction system, greatly reduces the reagent consumption, lowers the library construction cost, lowers the library loss, and is suitable for constructing an artificially-fragmented small DNA library.

The invention provides a construction method for a simplified genome next generation sequencing library based on double enzyme digestion and a kit. Aiming at defects of an existing construction method for the double enzyme digestion simplified genome next generation sequencing library, the double enzyme digestion combined range is expanded, and excessive dependence on expensive instruments of constructing the simplified genome library is reduced, the library construction flow path is simplified, library construction cost is reduced, the sequencing efficiency is improved, and meanwhile the technology is easy and flexible to operate and easier for researchers to master and can be realized in a common molecule lab. The construction method is particularly suitable for miniature or medium-scale labs needing to conduct SNP molecular marker development, genetic map construction, population genetics research, phylogeny biological research and the like on a great number of species with incomplete reference genomes. The construction method has good practical application value and application prospects in the fields of molecular breeding of agriculture, conservation biology and evolutionary biology.

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 relates to the field of gene sequencing, and in particular to a DNA library kit for sequencing and a construction method of a DNA library for sequencing. The construction method of the DNA library for sequencing comprises the following steps: DNA fragmentation is preformed for terminal repair and addition of A, an obtained product is ligated to a linker and purified, the DNA fragmentis subjected to size screening; amplification and enrichment, an obtained product is purified to obtain the DNA library. The method simplifies the process of DNA library construction, the steps of DNA sequence terminal repair and addition of A at 3' end are completed in one step, and the product can be directly used for ligation reaction without intermediate purification, which greatly shortens the experimental time consumed by the process of library construction; the unique linker design greatly saves the synthesis cost of the linker, which provides great convenience for the experimental staff of the library construction, reduces the consumption of reagents, and reduces the overall library construction cost.

The invention discloses a lung cancer related gene mutation detection method, primer and reagent based on a high-flux sequencing platform, which are used for detecting 50 mutant sites of four lung cancer related genes of EGFR, KRAS, BRAF and PIK3CA. The detection method comprises the steps of sample DNA (deoxyribonucleic acid) extraction, multiplex PCR (polymerase chain reaction) target segment enrichment, primer elimination, linker connection, library amplification, library detection, high-flux sequencing and the like. According to the invention, the library building cost is lowered, and the possibility of mutation introduction from PCR amplification is reduced, so that the sequencing result is more accurate, and the detection flux and detection sensitivity are improved; and meanwhile, reference can be provided for molecular diagnosis and individualized medication of lung cancer.

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 relates to a next generation sequencing detection method of a microsatellite instability state. The method includes: performing DNA fragmentation on the cancer tissue sample and normal tissue reference substance of a tumor patient, polishing and adding tail A to tail ends, and performing joint connection; designing primers and probes for target area capturing and primers for amplification, the probes are one-way DNA extension probes aiming at five microsatellite loci, and using the probes and the primers to perform target area capturing and then performing amplification to obtaina library; sequencing to obtain the repeated sequence length distribution data of the five microsatellite loci of the cancer tissue sample and the normal tissue reference substance, and comparing thedata of the five microsatellite loci of the cancer tissue sample and the normal tissue reference substance to judge the stability of the microsatellite loci. The invention further relates to a next generation sequencing primer probe set of the microsatellite instability state. The next generation sequencing detection method can reduce influence of sequencing depth on result judgement and is simple, practicable, high in accuracy, high in sensitivity and low in cost.

The invention relates to an electricity-saving economical civil-engineering multiple-layer fixed goods shelf all-in-one novel refrigeration house which successively comprises an internal system, a totally-sealed waterproof moisture barrier system, a thermal insulation system and an external system from inside to outside and causes the refrigeration house to be systematized. The invention fills an intact net-type totally-sealed waterproof moisture barrier system outside an intact net-type insulation layer in the ground, and a totally-sealed waterproof moisture barrier system in a wall / roof insulation layer and compounds an I-type magic cube, an II-type magic cube and a insulation layer material required to be matched in the insulation layer so as to build an intact totally-sealed waterproof moisture barrier system and the thermal insulation system. The steel pipe column supported multiple-layer fixed rack is in a multi-purpose support load bearing structure as a whole in the house, steel pipe columns are welded on a steel plate embedded part in the reinforced concrete load bearing ground in the house, the specifications from bottom to top of lateral, longitudinal and diagonal rectangle steels among steel pipe columns are matched with load bearing requirements on the steel pipe columns and connection thereof is in standard welding.

The invention relates to a whole genome methylation library single-chain library building method and the obtained whole genome methylation library. The method comprises the following steps: (a) carrying out bisulfite treatment on broken or unbroken genome DNA to convert a non-methylated C base into a U base, and forming a random AP site on the DNA; (b) carrying out 5' end dephosphorylation treatment on the reaction product in the previous step by adopting dephosphorylase to remove the phosphate group at the 5' end; (c) amplifying the denatured single strand of the reaction product in the previous step under the action of DNA polymerase and random primers to synthesize a second strand; and (d) removing the AP site by using endonuclease with an AP site removal effect, wherein the step (d) iscarried out at any stage after the step (a). The method has the advantages of low initial quantity, high library quality, high data utilization rate and high data reliability.

The invention discloses an absolute quantitative transcriptome library construction method based on a specific recognition sequence. Fragmented mRNA is used as a template, a first cDNA chain is synthesized under the action of reverse transcriptase by a primer pool with a general joint sequence, a library construction joint with a special recognition UID sequence is added to cDNA 3' end through anenzymatic reaction, so that each cDNA carries a unique sequence label; and finally, PCR amplification is performed by the general library construction joint to obtain an RNA library. The method constructs the RNA library on the basis of single-stranded cDNA by a splint connection method for the first time, at the same time, a UID sequence is used to precisely reduce the cDNA composition before PCRamplification and can realize precise quantification of transcripts. Library construction is performed by the single-stranded cDNA as a raw material, the step of second strand synthesis is omitted, the template loss rate is reduced, the cost and time are saved, and the defect that the prior art can relatively quantify transcripts is thoroughly solved.

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 discloses a double-end tag-specific linker for a blood micro cfDNA library, a kit thereof, and a construction method of the library. The sequence of the double-end tag-specific linker isrepresented by SEQ ID NO.1-5. The sequence of the primer set is represented by SEQ ID NO.6-9. The invention also discloses the construction method for constructing the cfDNA double-end tag-specific genome library based on random sequences. The construction method of the library is simple to operate, and does not need tube replacement or purification in preliminary PCR, so the loss of cfDNA in thelibrary construction process is avoided, and the linkage efficiency of the library is greatly improved; and experimental steps are simplified, and restriction enzyme cutting sites and biotin labelingsites are added to the linker sequence, so the efficiency of the linker is greatly improved, thereby the sensitivity of the library construction is enhanced.

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 provides a semi-specific amplification primer group, a method and a kit for quickly detecting chromosome number abnormality. The invention relates to two groups of amplification primers. The kit provided by the invention consists of the following components: a 10*PCR buffer solution, ddH2O, a dNTP solution, PlatinumPf*DNA polymerase (2.5U / mu L), primer pairs of amplification primer 1 with concentration of 25 mu m / mu l, an amplification primer 2 and purified buffer (QIAGENMinElutePCRPurificationKit). Another object of the invention is to provide a method for quickly detecting chromosome number abnormality through the semi-specific amplification primer group. By adopting the invention, the chromosome number abnormality can be detected quickly.

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 relates to a method and kit for preparing a library of free miRNAs in blood, and the method mainly comprises the following steps: (a) separating serum or plasma from blood, and extracting free RNAs; (b) mixing the free RNA with exogenous RNA, wherein any part of an exogenous RNA sequence is not overlapped with a known miRNA sequence of a human / rat / mouse; (c) adding a linker to the RNA mixture in the step (b), carrying out reverse transcription on the first strand and the second strand for synthesis and amplification, and recovering the target DNA fragment to obtain the free miRNAsequencing library. The invention further relates to a miRNA expression quantification method. The method comprises the steps (a), (b) and (c), the miRNA sequencing library is subjected to next-generation sequencing and data analysis, in the data analysis, the S2 random tag sequence of the adaptor RA5 can be used as a quantification tag, the PCR repetitive sequence is removed, and the detection accuracy is improved.

The invention provides an injection-production gas well variable-diameter well completion method and an injection-production string. The method includes the steps: firstly, mounting an upper casing (1) and a lower casing (2) for well completion in a well; secondly, tripping a first injection-production string into the casings at a gas-drive oil production stage; thirdly, taking out the first injection-production string from the casings and then tripping a second injection-production string into the casings at a gas storage stage. The external diameter of the upper casing (1) is larger than that of the lower casing (2), and the external diameter of an oil tube used in the second injection-production string is larger than that of an oil tube used in the first injection-production string. Without drilling the well again, the requirement of gas-drive early low injection allocation rate can be met, and the requirement of high injection allocation rate at a later injection-production stage can also be met.

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 provides a DNA library construction kit based on an illumina sequencing platform, a library construction method and application. The kit comprises a P7 linker, a P5 linker, an enzyme composition or a buffer solution, wherein the enzyme composition comprises any one or a combination of at least two of a terminal repair enzyme, a PCR amplification enzyme or a T4DNA ligase; and the buffer solution comprises a tail end repairing buffer solution and / or a connecting buffer solution. According to the kit disclosed by the invention, a reaction solution is prepared by adopting commercially available reagents, so that the library construction cost is reduced, the reaction procedure is simplified, a terminal repair reaction system and an A-adding reaction system are combined in one system, the reaction time is shortened, and the enzyme connection efficiency is improved.

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 invention relates to a construction method of a medical human amniotic membrane tissue reserve bank, comprising the following steps of: 1, selecting materials of human amniotic membranes and establishing a file; 2, taking a fetal membrane with a donor meeting medical standard cesarean section or normal labor; separating camisia foetus and maintaining amniotic membranes; 3, repeatedly rinsing the amniotic membranes by using a phosphate buffering solution and cutting into membrane sheets; 4, utilizing a phosphate buffering solution containing gentamicin with the concentration of 1000 U / ml and amphotericin B with the concentration of 2.5 micrograms / millimeter; 5, placing the membrane sheets into a sterile cell culturing bottle or plastic bag of a phosphate buffering solution containing 4-100% of glycerol and freezing and storing at a temperature in a range of 20 DEG C below zero to 80 DEG C; 6, carrying out radiation sterilization treatment on human amniotic membrane sheets by cobalt 60 to obtain medical human amniotic membrane sheets; and 7, placing amniotic membrane tissue membrane sheets to be frozen and stored at 80 DEG C below zero to establish an amniotic membrane tissue membrane sheet file capable of being searched, namely establishing the human amniotic membrane tissue membrane sheet reserve bank which has long storage time and can be effectively used for clinical treatment in time.

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.