62 results about "Amplicon sequencing" patented technology

The invention discloses a primer applied to amplicon sequencing library construction and a method for constructing an amplicon sequencing library. The primer consists of a forward primer and a reverse primer, and the forward primer and the reverse primer comprise a joint sequence, a sequencing primer sequence and a target fragment amplification primer sequence from a sequence from the 5' end to the 3' end. According to the primer provided by the invention, the joint sequence, the sequencing primer sequence and the target fragment amplification primer sequence are integrated on a pair of primers, so that library construction can be finished in one step by utilizing the primer. The sequencing library quality and library construction efficiency are improved, the constructed amplicon sequencing library can perform high-throughput sequencing due to the joint sequence and the sequencing primer sequence used on a conventional sequencing platform by utilizing a common reagent for on-board sequencing, and the phenomena that an extra sequencing primer is provided and the mixed sequencing primers of the PHiX library are changed are avoided.

The invention discloses a primer applicable to amplicon sequencing library construction, a construction method, an amplicon library and a kit comprising the amplicon library. The primer comprises a target fragment amplification primer sequence, a sequencing primer sequence and an adaptor sequence, wherein at least one end of a target fragment amplification primer sequence, obtained from the target fragment amplification primer sequence, the sequencing primer sequence and the adaptor sequence by virtue of an amplification step, in the amplicon library is provided with an internal label sequence; the internal label sequence is formed by using 6-10 basic groups in different permutation and combination patterns. The primer enables the constructed amplicon library to have the internal label, and therefore, the number of the mixed samples of the library can be increased and the flux of sequencing can be increased, and besides, contaminated data generated in the library construction process can be differentiated and removed and the accuracy of late data analysis can be improved; as a result, the abundance ratio of different floras in an original sample can be reflected more really.

The invention discloses a plant endophyte 16S rRNA gene amplification method and application. The amplification method includes the following steps of plant pretreating; plant-sample total DNA extracting; sample 16S rRNA gene amplifying, wherein sample 16S rRNA gene amplifying comprises 16S rRNA gene total-length emulsion PCR amplifying and 16S rRNA gene hypervariable-region/conserved-region amplifying sub-amplifying; high-throughput sequencing and biological information analyzing based on a Illumina platform, wherein high-throughput sequencing and biological information analyzing based on the Illumina platform comprises plant endophyte 16S rRNA gene amplicon purifying and recycling, amplicon sequencing library establishing, Illumina HiSeq sequencing and sequencing data bioinformatics analysis. The gene amplification method is used for high-throughput-sequencing plant disease detection and phytophagous animal enteric microorganism detection. Sequencing analysis of plant endophytic bacteria is carried out with the high-throughput sequencing technology, the data size is larger, the detection result is more complete, pollution of plant hosts is reduced to the maximum degree, the result multiformity is higher, and the cost is low.

The invention discloses amplicon next-generation sequencing based small fragment insertion and deletion detection method and device. The amplicon sequencing based small fragment insertion and deletion detection method includes the steps of S1, respectively extracting DNA of a to-be-detected sample and a negative control sample, and amplifying a target region of a mutation related to the target trait by multiple amplicons; S2, carrying out the next-generation sequencing to obtain sequence of the target region; S3, comparing the sequence of the target region with a reference genome, wherein bases which are not matched with the reference genome are subjected to mispairing; S4, processing to obtain background noise of hotspot small fragment insertion and deletion related to the target trait according to the negative control sample, modeling the background noise by means of binomial distribution, distinguishing the hotspot short fragment insertion and deletion and the background noise, on each base, of the to-be-detected sample, and determining the mispairing bases as the small fragment insertion and deletion through positive detection if the proportion of the mispairing bases of the to-be-detected sample to the reference genotype is much different from that of the background noise. By the amplicon next-generation sequencing based small fragment insertion and deletion detection method and device, accuracy of small fragment insertion and deletion detection is improved.

Some embodiments of the invention include a method of preparing a sample for sequencing that includes receiving a sample and amplifying at least one marker within the sample. In some embodiments, amplification of the first marker may include mixing the sample with a first oligonucleotide that comprises a first universal tail sequence and a second oligonucleotide that comprises a second universal tail sequence. In some aspects of the invention, the first universal tail sequence and the second universal tail sequence are different sequences.

The invention discloses a DNA library construction method for high-throughput sequencing. The DNA library construction method includes the steps of carrying out multiple PCR (polymerase chain reaction) to a DNA template obtained from a to-be-tested sample through multiple pairs of fusion primers, then recycling PCR products to obtain a sequencing library. The multiple pairs of fusion primers respectively aim at different target fragments of the DNA template, and each fusion primer comprises specificity primer sequence aiming at the target fragment and linker sequence for sequencing. The multiple PCR is carried out on reaction conditions: reacting at 95 DEG C for 2 minutes; reacting within 38 cycles, wherein each cycle includes processes of preserving at the temperature of 95 DEG C for 30 seconds, cooling to the temperature from 76 DEG C to 55-58 DEG C slowly at the speed of 0.1 DEG C each second, holding the temperature for 20 seconds once cooling to the temperature of 58 DEG C, and then preserving at 72 DEG C for 30 seconds; after that, preserving at 72 DEG C for 2 minutes, and finally preserving at 4 DEG C. The invention further discloses application of the library construction method in testing STR locus and paternity identification on the basis of high-throughput sequencing.

The invention discloses a quadruple-tag primer set for building a 16S rRNA gene amplicon sequencing library. The quadruple-tag primer set is composed of a first-step PCR double-tag primer pair and a second-step PCR double-tag primer pair, the sequence of an upstream primer of the first-step PCR double-tag primer pair is one of SEQ ID NO:1-8 while that of a downstream primer of the same is one of SEQ ID NO:9-20, and the sequence of an upstream primer of the second-step PCR double-tag primer pair is one of SEQ ID NO:21-44 while that of a downstream primer of the same is one of SEQ ID NO:45-68. Abalanced base sequence is added into the quadruple-tag primer, so that diversity of the library is improved, and sequencing quality is improved; during online sequencing, only a small amount of PhiXlibrary needs to be mixed, and staggered sequencing needs to be performed, so that waste of sequencing flux is reduced, and sequencing quality is ensured.

The invention discloses an absolute quantitative method for microflora based on a multi-internal standard system, which belongs to the field of biotechnology and omics analysis. The present inventionprovides a method for efficiently quantifying microbes during amplicon sequencing, the method is characterized in that the target plasmid with a corresponding gradient is added according to the copy number of the total amount of microbes in a sample, the steps of DNA separation, quality control, and sequencing are carried out to obtain an OTU classification unit, and the absolute number of corresponding microbes in the sample is calculated based on a proportional relationship between the amount of target substance added and the detection amount. The method can realize the absolute quantification of the sample to-be-tested, greatly improves the accuracy of the measurement result, and has the advantages of low cost and the like, and plays an important role in promoting the progress of a sequencing technology.

The invention discloses a modeling method of a non-rodent animal model for studying intestinal flora. A male rhesus monkey of 12-months old is taken as a laboratory animal and fed with mixed antibiotic for 21 days to clear symbiotic intestinal flora in the body. The antibiotic includes neomycin, streptomycin, ampicillin sodium, kanamycin, metronidazole and vancomycin; different antibiotic formulasare adopted for the first 10 days and later 11 days of feeding. The modeling result is verified by combining a Realtime-PCR method and an amplicon sequencing method. The method disclosed by the invention has the advantages of simplicity and easiness in implementation, short time, high repeatability, low death rate and stable model. The method has an important application value in the treatment offlora-related metabolic diseases, inflammatory bowel diseases and infectious diseases, evaluation of intestinal flora in the immune system, development of new medicines and the like.

A method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.

The invention discloses a fast construction primer and method for a water/mud sample DNA amplicon sequencing library in a sewage treatment system, and belongs to the field of high-throughput sequencing. The method comprises the steps of firstly, sample DNA extraction, secondly, target fragment amplification, thirdly, amplification product purification, fourthly, amplification product quality determination, fifthly, amplification product mixing, sixthly, mixed library quality determination and seventhly, mixed library preparing and sequencing. The primer comprises a connector sequence, an index sequence, a heterogeneity spacer sequence and a target fragment amplification sequence. The primer is used for further amplification, library construction can be fast completed, the sequencing cost is reduced, and a large amount of library construction time is saved. According to the fast construction primer and method, the problem about high diversity bacterial flora structure analysis in complex sewage/mud samples is solved, and the technical requirement that the fast construction primer and method are applied to water/mud sample bacterial flora deep analysis in the sewage treatment system based on the high-throughput DNA sequencing technology is met.

The invention relates to a rapid full-length amplicon library construction method and primers applicable to PacBio platform and a full-length amplicon sequencing method based on the library construction method. According to the characteristic that PCR is required for full-length amplicon sequencing, an amplicon primer with a general binding sequence and a dumbbell-shaped primer matched with the general binding sequence are designed in combination with a dumbbell-shaped library joint structure of the PacBio sequencing platform, the rapid full-length amplicon library construction method applicable to the PacBio platform is provided based on the two primers with special structures, through library construction by the method, addition of sequencing joints can be realized by increasing two cycles of annular primer amplification only after conventional amplification, and meanwhile, a PCR product becomes a standard library applicable to the PacBio sequencing platform under the action of damage repair enzyme. The method optimizes an original PacBio standard library construction process into only one-step amplification and repair, the library construction process of a full-length amplicon is greatly simplified, and the sequencing cost is significantly reduced.

The present invention discloses a universal primer for plant-derived component identification and an application of the universal primer. An upstream primer sequence of the universal primer is shown as SEQ No.1 and a downstream primer sequence of the universal primer is shown as SEQ No.2. An amplicon sequencing analysis method is used to identify at least 10 plant-derived components of walnuts, peanuts, soybeans, sesame seeds, hazelnuts, apricot kernels, coconuts, pine nuts, pumpkin seeds, Badam, etc. and can solve a problem that current PCR and ELISA detection methods can only conduct one-to-one targeted screening of the targeted components, only one experiment is needed to conduct non-targeted screening of the plant components of the product, intensity of detection work is reduced, costof synthezing primers and probes is saved, and in the case of saving time, labor and material costs, the universal primer can be flexibly used in a variety of food containing allergenic ingredients tomeet screening work of large quantities of plant component-containing samples, realizes development from target detection to target-free screening, improves a level of adulteration detection and provides an effective technical means for supervision.

The invention relates to a method for detecting whether fusion mutation of target gene occurs, a primer combination, a kit and an application thereof. A plurality of primers are sequentially designedaccording to the possible gene fusion occurrence direction by the primer design method of the invention, forming a primer combination, wherein the primers of the primer combination extend along the direction in which gene fusion occurs, can carry out directional enrichment on the target gene in which unknown fusion occurs, overcomes the disadvantage that the existing amplicon sequencing method cannot enrich the unknown fusion gene targetedly, and is conducive to the discovery and detection of the unknown fusion type of the gene; At the same time, the interval between two adjacent primers is about 0 - 130 bp, which is good for highly fragmented samples (such as cfDNA, FFPE samples). The invention also designs primer combinations for detecting fusion mutations of lung cancer genes (includingALK gene, NTRK1 gene, RET gene, ROS1 gene, FGFR3 gene and NTRK3 gene), After multiple rounds of detection and optimization of the primer sequence, the homogeneity of the sequencing data was more than85%, and the capture efficiency was more than 92%. Finally, the effective detection of unknown fusion mutation of various lung cancer genes was successfully achieved.

The invention belongs to the field of gene engineering and particularly relates to construction and application of a mutation detection system for detecting artificial nuclease induced insertion and deletions (Indels). According to the adopted specific technical scheme, the ara-mFabI detection system is provided and comprises three detection carriers, and each carrier is separately responsible forquantitative and qualitative reveal of one type of insert fragment. The three carriers are distinguished in that the types of open reading frames (ORFs) of mFabI genes are different; and nine basic groups, ten basic groups and eleven basic groups are introduced through upstream primers of amplification primers of the mFabI genes correspondingly so as to change the ORFs of the mFabI genes. By using the detection system, quantitative reveal of the mutation frequency of all the types of nuclease induced Indels can be realized; and the mutation frequency of the Indels close to amplicon sequencingand type distribution can be realized.