39results about How to "Increased sequencing depth" patented technology

The present invention relates to a method for detecting PKD1 gene mutation. The method utilizes sequence information of the PKD1 gene, designs nine pairs of PCR tag primers, and uses a long-chain PCR technology for targeting amplification of PKD1 gene; while amplification products are labeled, PCR products are purified and mixed for direct construction of a single SMRTBell library; a PacBio RSII sequencer is employed for sequencing; and bioinformatic analysis is carried out to obtain PKD1 gene mutation information. The invention has the beneficial effects that a simplified pair of primers is designed and combined with tag primer technology to realize respective marking of PCR products of multiple DNA samples; the amplified PCR products do not need to perform knockout and can be directly used for the library construction, and the library construction does not need extra PCR, so that a plurality of samples are mixed into one library and processed simultaneously by a PacBio RSII sequencing library construction link, and the experimental operation is greatly simplified.

The invention relates to a mitochondrial genome library based on high-throughput sequencing and a building method thereof. The building method comprises the following steps of S1, performing one-step PCR (polymerase chain reaction) amplification of mitochondria full-length DNA (deoxyribonucleic acid) in total DNA; S2, crushing the mitochondria full-length DNA to obtain DNA fragments; S3, performing tail end restoration on the DNA fragments to obtain flat tail end DNA fragments; S4, adding A to the end 3' of the flat tail end DNA fragments to obtain the A-added DNA fragments; S5, adding a connector to the end 3' of the A-added DNA fragments to obtain connector-added DNA fragments; S6, performing front LM-PCR on the connector-added DNA fragments; S7, performing post PCR on the LM-PCR product. The building method of the mitochondrial genome library has the advantages that the mitochondrial full-length DNA is obtained through further PCR amplification from the total DNA, so that the precise amplification of the human mitochondrial genome can be guaranteed; the pollution mixing by the homologous sequence of the human genome is avoided, so that the mutation detection is more sensitive and accurate.

The invention provides a fetus chromosome detecting method based on DNA variation counting. The method comprises the steps of obtaining and sequencing free DNA of plasma in a peripheral blood sample of a pregnant woman, comparing the free DNA of the plasma with human reference sequences, namely DNA long sequences on 24 chromosomes, counting the number of variation in the free DNA of the plasma andconducting comparison. The invention further discloses a system for achieving the detecting method. The system comprises a DNA short sequence data input module, a module for positioning short sequences on the long sequences, a module for founding sequence difference, a difference screening module, a first counting module and a second counting module, wherein the modules are sequentially and electrically connected. According to the fetus chromosome detecting method based on DNA variation counting and the system for achieving the method, by detecting chromosomes in peripheral blood of the pregnant woman, whether there is chromosome abnormality or not in a fetus is judged, thus harmful effects on the pregnant woman and the fetus in a detecting process are greatly reduced, and by calculatingthe variation number to judge the chromosome abnormality of the fetus, compared with a method that a sequence number is calculated simply, the fetus chromosome detecting method based on DNA variationcounting is more accurate.

The invention relates to the field of biotechnology, and specifically provides a solid-phase-carrier-membrane-based target nucleic acid enrichment method for high-throughput sequencing. The solid-phase-carrier-membrane-based target nucleic acid enrichment method comprises: 1) combining a nucleic acid probe library and a solid phase carrier membrane to form a probe-solid phase carrier membrane complex; 2) adding target nucleic acid to a hybridization solution containing the probe-solid phase carrier membrane complex, and enriching the target nucleic acid; 3) washing the obtained probe-solid phase carrier membrane complex with the target nucleic acid by using a washing solution, eluting from the solid phase carrier membrane by using an eluent, and purifying the enriched target nucleic acid library; and 4) applying the target nucleic acid library enriched in the step 3) in high-throughput sequencing. According to the present invention, with the method, the high-multiple coverage and single-base displacement nucleic acid probe for enriching the target nucleic acid can be rapidly produced, and is immovably fixed on the solid phase carrier membrane; and the method has advantages of simple operation, flexible probe acquisition and low cost.

The present invention discloses probes and uses thereof. The group of the probes specifically recognize at least one part of a FLG gene coding region, and meet at least one condition selected from the following conditions that (1) the length of the probe is 75 bp; (2) the probe specifically recognizes the sequence between the upstream 10 bp and the downstream 10 bp of the FLG gene coding region; (3) the multiplier of the probe specifically recognizing the region having the GC content of higher than 0.6 and lower than 0.3 is more than 2; (4) the melting temperatures of the probe and the target sequence is 60-10 DEG C, preferably 80 DEG C; (5) the probe does not contain a hairpin structure; (6) the probe is matched with two sites on a reference genome at the most; and (7) the window sliding size is 10 bp during the probe selection. According to the present invention, the group of the probes provides good capture specificity, high sensitivity and high coverage for at least one part of the specifically-recognized FLG gene coding region, and can be effectively used for the capture detection of the FLG gene coding region.

The invention provides a symmetric encryption method for DNA storage, and the method comprises the following steps: obtaining a to-be-encrypted file, encrypting the binary information of the to-be-encrypted file according to an encryption key, and obtaining a DNA storage sequence; carrying out confusion operation processing on the DNA storage sequence; synthesizing the plurality of DNA storage sequences processed by the confusion operation to obtain a DNA molecular sequence, storing the DNA molecular sequence, sequencing the DNA molecular sequence to obtain a read length, and decrypting according to the read length and the encryption key to obtain binary information. According to the method, under different error rates, the sequencing depth is increased, the data recovery rate is in a rising trend, the information redundancy in the encryption and decryption process can be effectively reduced, the robustness is higher, the malicious cracking difficulty is large, the confidentiality effect is better, and the method can be widely applied to the technical field of system biology research.

The invention belongs to a technology of medical in vitro diagnosis, and in particular relates to an amplification primer for detecting mutation of autosomal dominant polycystic kidney disease-causing gene PKD2 as well as a detection method. The amplification primer, in accordance with the sequence information of the PKD2 gene, designs four pairs of PCR primers; the PKD2 is subjected to targeted amplification by virtue of long-chain PCR; and an amplification region includes all exon sequences and most intron sequences, as well as partial 5' untranslated region sequences and 3' untranslated region sequences. According to the method, by combining long-chain PCR targeted amplification to the newest developed next generation of sequencing technology, a targeted sequencing process is simplified, the scope and flux of detecting the PKD2 gene mutation are improved, a detection cycle is shortened and cost is reduced.

A mitochondrial genomic library based on high-throughput sequencing and a method for constructing the library. The method comprises the following steps: S1. performing a one-step PCR amplification of total mitochondrial DNA from total DNA; S2. fragmenting the total mitochondrial DNA to obtain a DNA fragment; S3. performing end repair on the DNA fragment to obtain a blunt-end DNA fragment; S4. A-tailing a 3' end of the blunt-end DNA fragment to obtain an A-tailed DNA fragment; S5. adding a linker on the 3' end of the A-tailed DNA fragment to obtain a DNA fragment with linkers; S6. performing LM-PCR on the DNA fragment with linkers; and S7. performing PCR on an LM-PCR product. The method for constructing the mitochondrial genomic library performs PCR amplification on total DNA to obtain total mitochondrial DNA, thereby ensuring accurate amplification of a human mitochondrial genome and improving the accuracy and precision of mutation detection by preventing contamination or mixing of a homologous sequence from a human genome.

The present invention relates to the field of biomedical technology, in particular to a method for detecting tumor mutation load and a prediction model; it includes the following steps: S1: extracting DNA from tumor tissue and blood samples to be tested; S2: constructing target regions for tumor-related genes Targeted capture sequencing library; S3: Using a high-throughput sequencing platform to obtain the original off-machine data; S4: Obtain the tumor mutation load of the sample through the detection process. The purpose of the present invention is to provide a method for detecting tumor mutation load and a prediction model, through which the detection and calculation of gene mutations can achieve the same results as the TMB detection of whole exome sequencing, so as to improve Accuracy of Tumor Mutational Burden Estimation.

The invention provides a fetus chromosome detecting method based on DNA variation counting. The method comprises the steps of obtaining and sequencing free DNA of plasma in a peripheral blood sample of a pregnant woman, comparing the free DNA of the plasma with human reference sequences, namely DNA long sequences on 24 chromosomes, counting the number of variation in the free DNA of the plasma andconducting comparison. The invention further discloses a system for achieving the detecting method. The system comprises a DNA short sequence data input module, a module for positioning short sequences on the long sequences, a module for founding sequence difference, a difference screening module, a first counting module and a second counting module, wherein the modules are sequentially and electrically connected. According to the fetus chromosome detecting method based on DNA variation counting and the system for achieving the method, by detecting chromosomes in peripheral blood of the pregnant woman, whether there is chromosome abnormality or not in a fetus is judged, thus harmful effects on the pregnant woman and the fetus in a detecting process are greatly reduced, and by calculatingthe variation number to judge the chromosome abnormality of the fetus, compared with a method that a sequence number is calculated simply, the fetus chromosome detecting method based on DNA variationcounting is more accurate.

The invention relates to the field of molecular biology detection, in particular to a primer, a kit, a model and a construction method for the methylation of cervical cancer-related genes. The methylation primer is designed, and cervical cell DNA is extracted, and the cervical cell DNA is methylated. Methylation treatment, using a PCR amplification kit to perform the first PCR amplification and purification on the cervical cell DNA after methylation treatment, and performing the second PCR amplification and purification on the treated product to obtain a library. The library was subjected to high-pass sequencing on the next-generation sequencing platform for bioinformatics analysis. Based on cervical cancer samples and normal samples, a methylation score was calculated for each PCR amplification region as an independent variable, and the correlation between cervical cancer and cervical cancer was constructed by logistic regression. Gene methylation evaluation model; the present invention builds a database through multiple PCR methylation, and obtains the methylation status of the sample through high-throughput sequencing. The sequencing depth is high, the time is short, and the cost is low. According to the assessed risk, it is decided whether to treat or observe , can effectively reduce overtreatment.