CN116377046A - Quality control product and kit for parallel detection of tag primer sequences - Google Patents
Quality control product and kit for parallel detection of tag primer sequences Download PDFInfo
- Publication number
- CN116377046A CN116377046A CN202211734834.6A CN202211734834A CN116377046A CN 116377046 A CN116377046 A CN 116377046A CN 202211734834 A CN202211734834 A CN 202211734834A CN 116377046 A CN116377046 A CN 116377046A
- Authority
- CN
- China
- Prior art keywords
- sequence
- primer
- quality control
- seq
- detected
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000003908 quality control method Methods 0.000 title claims abstract description 73
- 238000001514 detection method Methods 0.000 title claims description 15
- 238000012163 sequencing technique Methods 0.000 claims abstract description 78
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 44
- 108020004414 DNA Proteins 0.000 claims abstract description 38
- 239000012634 fragment Substances 0.000 claims abstract description 35
- 230000000295 complement effect Effects 0.000 claims abstract description 25
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 16
- 238000012864 cross contamination Methods 0.000 claims abstract description 16
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 16
- 238000012408 PCR amplification Methods 0.000 claims abstract description 11
- 102000053602 DNA Human genes 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 18
- 125000003729 nucleotide group Chemical group 0.000 claims description 17
- 230000003321 amplification Effects 0.000 claims description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 13
- 239000002773 nucleotide Substances 0.000 claims description 13
- 108091081021 Sense strand Proteins 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 230000000692 anti-sense effect Effects 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 5
- 239000000047 product Substances 0.000 description 42
- 238000003752 polymerase chain reaction Methods 0.000 description 21
- 108090000623 proteins and genes Proteins 0.000 description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- 238000002156 mixing Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 230000004544 DNA amplification Effects 0.000 description 6
- 238000012165 high-throughput sequencing Methods 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 239000012264 purified product Substances 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000011324 bead Substances 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 238000007834 ligase chain reaction Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 241000589989 Helicobacter Species 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000011978 dissolution method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000011807 nanoball Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000007671 third-generation sequencing Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Evolutionary Biology (AREA)
- Medical Informatics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Theoretical Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a quality control product for detecting a label primer, which is a double-stranded DNA fragment and comprises a segment of non-natural oligonucleotide fragment with a known sequence, wherein the non-natural oligonucleotide fragment is completely different from any position sequence on the genome of any existing known species. The two ends of the DNA fragment are provided with linker sequences which are complementary sequences capable of being specifically combined with the 3' -end of the label primer pair to be detected. The quality control product can carry out PCR amplification reaction with the label primer pair to be detected, the amplified product is used as a sequencing library to carry out second generation sequencing, the quality control product is used for splitting the data of the next machine, and the cross contamination condition and/or the synthesis error condition of different label primers can be obtained by analysis.
Description
Technical Field
The invention belongs to the field of high-throughput gene sequencing, and relates to a quality control product and a kit for large-scale parallel detection of a tag primer sequence.
Background
High throughput sequencing technology (High-throughput sequencing), also known as "Next generation" sequencing technology ("Next-generation" sequencing technology, NGS), is marked by the ability to sequence hundreds of thousands to millions of DNA molecules in parallel at a time, and by the general short read lengths. High throughput sequencing techniques can sequence millions of DNA molecules simultaneously, detecting hundreds or even thousands of samples in parallel at a time. In high throughput sequencing, a large number of tag sequences are required to label different sample libraries to facilitate distinguishing between different sample sequences in high throughput sequencing results.
In second generation sequencing, tag primers (index primers) are used to add tags for sequencing data resolution in different libraries. Each library needs to strictly correspond to a unique index sequence, so that the accuracy of sequencing data obtained by splitting is ensured, and no cross contamination exists among the libraries. However, in the actual use process, the phenomenon of cross contamination among different label primers (namely, mixing a small amount of primer B into primer A) can be found out due to large-scale automation operation and the like. This causes cross-contamination of sequencing data from different libraries. In addition, the synthesis method of the label primer has certain limitations, so that the synthesis error phenomenon exists in the label primer. If high-proportion cross contamination exists among the label primers, the accuracy of the sequencing result may be reduced, false positive and false negative data results are reported, and the accuracy of the sequencing result is greatly influenced. If a high proportion of synthesis errors occur in the tag primer, the proportion of undeployed data in the next machine data is increased, and the high proportion of sequencing cost is increased.
For NGS tag primers, in the prior art, primer synthesis companies often employ strict process flows to control, for example, by isolating different batches of primers to reduce the likelihood of cross-contamination between primers. In the aspect of quality control, quality control means such as nanodrop concentration detection, capillary electrophoresis or mass spectrometry for detecting the number of nucleotides are mostly adopted. However, these quality control means cannot effectively calibrate the accuracy of NGS tag primer sequences, and it is difficult to meet the actual quality control requirements and sequencing requirements during large-scale parallel sequencing in downstream experiments.
Disclosure of Invention
Aiming at the defects of the prior art and the requirements in practical production experiments, the invention provides a quality control product and a kit which can be used for large-scale parallel detection of a tag primer sequence. The quality control product is a known sequence oligonucleotide segment with one end complementary with the label primer to be detected, PCR amplification reaction can be carried out on the oligonucleotide segment and the label primer to be detected, the amplified product is used as a sequencing library to carry out second generation sequencing on a machine, the machine-down data is split through the quality control product, and the cross contamination condition and/or the synthesis error condition of different label primers can be obtained through analysis.
Specifically, the invention adopts the following technical scheme:
1. a quality control product for detecting a label primer is characterized in that,
the quality control product is a double-stranded DNA fragment;
the DNA fragment comprises a segment of non-natural oligonucleotide with a known sequence, and is characterized in that the non-natural oligonucleotide segment is completely different from any position sequence on the genome of any known species.
The two ends of the DNA fragment are provided with linker sequences, and the linker sequences are complementary sequences which can be specifically combined with the 3' end of the label primer pair to be detected.
2. The quality control product according to item 1, characterized in that,
the 3' ends of the sense strand and the antisense strand of the linker sequence have the same sequence and comprise a sequence specifically complementary to any one of the tag primer pairs to be detected;
the 5' ends of the sense strand and the antisense strand of the linker sequence have the same sequence and comprise a sequence specifically complementary to the other one of the pair of tag primers to be detected;
3. the quality control product according to item 1, wherein the quality control product can be PCR amplified with a pair of tag primers to be detected, and the amplified product is a tag primer detection library.
4. The quality control product according to item 2, wherein the label primer pair to be detected is a second generation sequencing label primer pair;
the label primer pair to be detected comprises a forward primer sequence and a reverse primer sequence, wherein the two primer sequences have complementary sequences which are specifically recognized with the 3' end of the DNA fragment with the known sequence respectively; the oligonucleotide sequence exists on any one of the forward primer sequence and the reverse primer sequence and is positioned between the 5' end sequence and the end sequence;
preferably, the 5' end sequences of the forward and reverse strand primer sequences are provided with a linker sequence complementary to the sequencing platform, respectively.
5. The quality control product according to item 4, wherein the sequence of the label primer to be detected is any pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.1 and SEQ ID NO. 4.
6. The quality control product according to item 5, wherein any one of the chain sequences of the quality control product is shown as SEQ ID NO. 55.
7. The quality control according to item 1, characterized in that the known non-natural oligonucleotide fragment has a length of 50-1000 bp, preferably 150-500 bp.
8. The quality control article of item 1 wherein the non-native oligonucleotide chip sequences of known sequence are used to split the tag primer detection library data.
9. A kit for detecting a tag primer, wherein the kit comprises the quality control article of items 1-8.
10. The kit according to item 9, further comprising a pair of adaptor primers, a PCR amplification reagent, and a purification reagent.
11. A quality control method of a second generation sequencing tag primer, which is characterized by comprising the following steps:
A. carrying out PCR amplification on the label primer pair to be detected and the quality control product;
B. obtaining an amplification product, and sequencing the amplification product on a machine to obtain sequencing data;
C. confirming the sequence consistency of the oligonucleotides to be detected according to the sequencing data;
wherein the quality control product is selected from any one of the quality control products in items 1-8;
the label primer pair to be detected comprises a forward primer sequence and a reverse primer sequence, wherein the two primer sequences have complementary sequences which are specifically recognized with the 3' end of the DNA fragment of the known sequence respectively; the oligonucleotide sequence exists on any one of the forward primer sequence and the reverse primer sequence and is positioned between the 5' end sequence and the end sequence;
preferably, the 5' end sequences of the forward primer sequence and the reverse primer sequence are respectively provided with a connector sequence which is complementarily matched with the sequencing platform;
preferably, the sequence of the label primer to be detected is any pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.1 and SEQ ID NO.4; any chain sequence of the quality control product is shown as SEQ ID NO. 5.
12. The quality control method according to item 11, wherein,
the step of confirming the sequence consistency of the oligonucleotide to be detected according to the sequencing data is to split the original sequencing data through the sequence data of the DNA fragments with known sequences, split the data containing the DNA fragments with the same known sequences into the same data set, and confirm the sequence consistency of the oligonucleotide to be detected in the data set.
13. The method of quality control according to item 12, wherein,
the sequence consistency of the oligonucleotide to be detected comprises the cross contamination rate and the synthesis error rate of the nucleotide to be detected;
preferably, the cross contamination rate and the synthesis error rate are calculated for counting the types and the number of tag sequences in the sequencing read length of the original sequencing data.
Effects of the invention
When the quality control product is applied by means of a second-generation sequencing technology, a set of complete label primer sequence quality control experimental flow can be established, and the sequence accuracy of the label primer to be detected is analyzed and detected by detecting the known sequence carried on the quality control product. The quality control product is used for detecting the sequence of the label primer, the DNA fragment with the known sequence in the quality control product is different from the known genome of the existing species, and the detection process is not polluted by other libraries sequenced in the same batch.
Compared with the prior art, the quality control product can be used for detecting the consistency of batch label primers in large scale in parallel, is not influenced by the homology of exogenous DNA, and can be used for establishing a standardized quality control flow. By means of the second generation sequencing analysis technology, the sequencing result of the label primer can be accurately analyzed, and the quality inspection process is completed in high throughput. The cross contamination condition and the synthesis error condition of the label primer are analyzed through specific sequences, and a high-throughput quality inspection method conforming to practical test application is provided.
Drawings
FIGS. 1-2 show the quality control detection flow of quality control products for single-label primer pairs and bidirectional label primer pairs, respectively.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that certain terms are used throughout the specification and the claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The description and the items do not differ by the terms of their components, but rather by the functional differences of the components. As referred to throughout the specification and claims, the terms "comprise" or "include" are open-ended terms, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the invention, but is not intended to limit the scope of the invention, as the description proceeds with reference to the general principles of the description. The scope of the invention is defined by the appended claims.
The term "oligonucleotide" as used herein refers to a linear polynucleotide fragment in which 2 to 10 nucleotide residues are linked by phosphodiester bonds, but the number of nucleotide residues is not strictly defined when this term is used, and in many documents, polynucleotide molecules containing 30 or more nucleotide residues are also referred to as oligonucleotides. The oligonucleotide can be automatically synthesized by an instrument, and can be used as a Primer (Primer), a gene Probe (Probe) and the like for DNA synthesis.
The term "primer" as used herein refers to a macromolecule, which stimulates synthesis at the initiation of nucleotide polymerization, having a specific nucleotide sequence, which is hydrogen-bonded to a reactant, such a molecule being referred to as a primer. Primers are typically two oligonucleotide sequences synthesized, i.e., primer pairs, one primer complementary to one strand of the DNA template at one end of the target region and the other primer complementary to the other strand of the DNA template at the other end of the target region, and function as a starting point for nucleotide polymerization, from the 3-terminus of which a nucleic acid polymerase can begin to synthesize a new strand of nucleic acid.
The term "amplification" as used herein refers to gene amplification, i.e., the process whereby the copy number of a particular gene is selectively increased while the copy number of other genes is not increased to scale.
Natural gene amplification, also known as chromosomal replication, or gene replication, is the primary mechanism by which new genetic material is produced during the evolution of biomolecules. It refers to the replication of any DNA fragment containing a gene.
Gene amplification may also be performed manually by:
polymerase Chain Reaction (PCR): the method of replicating the target DNA fragment is repeated by polymerizing the nucleotides.
Ligase Chain Reaction (LCR): a gene amplification method of a nucleic acid amplification probe. For each of the two DNA strands, the ligase ligates the two partial probes into the actual one. Thus, LCR uses two enzymes: DNA polymerase (for initial template amplification) and thermostable DNA ligase.
Transcription mediated amplification: an isothermal gene amplification method, which utilizes two enzymes, namely RNA polymerase and reverse transcriptase, to rapidly amplify target RNA/DNA.
In the present invention, the amplification method is not particularly limited, and a polymerase chain reaction, i.e., a PCR amplification method, is preferably used.
The term "sequencing" as used herein refers to gene sequencing, a novel gene detection technique capable of analyzing and determining the complete sequence of a gene from blood or saliva, and predicting the possibility of suffering from various diseases, and having reasonable behavior characteristics and behaviors of individuals. The gene sequencing technology can lock the individual lesion gene and prevent and treat the individual lesion gene in advance. The existing gene sequencing technology can be divided into 3 generations according to the technical characteristics, and mainly comprises the step of first generation sequencing, which is also called Sanger sequencing and capillary sequencing; second generation sequencing (NGS) is also known as high throughput sequencing, large-scale parallel sequencing; third generation sequencing is also called single molecule sequencing, and includes the technologies of Heliscope sequencing technology, SMRT (Single Molecule Real Time, single molecule real-time sequencing) Ion semiconductor sequencing technology (Ion Torrent), and the like. More sophisticated is the SMRT sequencing technology.
The invention is not limited to the method of sequencing, and preferably, second generation sequencing (NGS) is employed.
The sense strand, also called the coding strand, is generally located at the upper end of the double-stranded DNA, and the direction is 5 '-3' from left to right, and the base sequence is basically the same as that of the mRNA of the gene; the primer combined with the strand is a positive strand primer; which is extended along the plus strand.
The negative strand, i.e., the nonsense strand, also called the non-coding strand, is complementary to the positive strand, and the primer that binds to this strand is the reverse strand primer, which is a primer that extends uninterrupted along the negative strand, upstream of the DNA duplex.
The term "5 'end" herein means that the phosphate group of the last nucleotide and the hydroxyl group of the next nucleotide form a phosphodiester bond when DNA is ligated, and the phosphate end of one more phosphate group is added to both ends of the nucleotide chain, and the term "3' end" herein means the hydroxyl end of one more hydroxyl group is added to both ends of the nucleotide chain.
The term "sequencing platform" as used herein refers to the apparatus or equipment or software used in gene sequencing, including but not limited to Sanger, 2.454, solid, hiSeq2000, helicobacter, DNA Nanoball array, the PacBio RS system, PGM, miSeq, illuminate, etc., and the present invention is not limited to the type of sequencing platform, preferably, the second generation sequencing platform from the company illuminate is used
The term "complementary pairing" as used herein, i.e., base complementary pairing, refers to the phenomenon whereby the bases of nucleotide residues in a nucleic acid molecule are hydrogen bonded to each other in a corresponding relationship of A to T, A with U and G to C.
The invention specifically comprises a quality control product for detecting a label primer, which is characterized in that,
the quality control product is a double-stranded DNA fragment, and the DNA fragment comprises a non-natural oligonucleotide fragment with a known sequence, and is characterized in that the non-natural oligonucleotide fragment is completely different from any position sequence on the genome of any existing known species.
The two ends of the DNA fragment are provided with linker sequences, and the linker sequences are complementary sequences which can be specifically combined with the 3' end of the label primer pair to be detected.
The 3' ends of the sense strand and the antisense strand of the linker sequence have the same sequence and comprise a sequence specifically complementary to any one of the tag primer pairs to be detected; the sense strand and the antisense strand of the linker sequence have the same sequence at their 5' ends and comprise a sequence specifically complementary to the other of the pair of tag primers to be detected
The quality control product can be subjected to PCR amplification with the label primer pair to be detected, and the amplified product is a label primer detection library. The label primer pair to be detected is a second generation sequencing label primer pair;
the label primer pair to be detected comprises a forward primer sequence and a reverse primer sequence, wherein the two primer sequences have complementary sequences which are specifically recognized with the 3' end of the DNA fragment with the known sequence respectively; the oligonucleotide sequence exists on any one of the forward primer sequence and the reverse primer sequence and is positioned between the 5' end sequence and the end sequence;
preferably, the 5' end sequences of the forward and reverse strand primer sequences are provided with a linker sequence complementary to the sequencing platform, respectively.
Specifically, the sequence of the label primer to be detected is any pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.1 and SEQ ID NO.4; any chain sequence of the quality control product is shown as SEQ ID NO. 55.
Further, the quality control product comprises a non-natural sequence of a known sequence, and is characterized in that the non-natural sequence is completely different from any sequence at any position on the genome of any known species. The non-native sequence may be obtained by any method, such as artificial synthesis.
The length of the known DNA sequence is 50-1000 bp, preferably 150-500 bp, for example 150bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp or 500bp.
The length of the label in the label primer to be detected is 6-20 bp, preferably 6-12 bp, for example, 6bp, 7bp, 8bp, 9bp, 10bp, 11bp or 12bp.
The tag may be located in the forward primer and/or reverse Yan Zu sequence of the tag primer.
The non-natural oligonucleotide chip sequences of known sequence can be used to split the tag primer detection library data.
The invention also relates to a kit for detecting the label primer, which comprises the quality control product.
Further, it may further comprise a pair of adaptor primers, a PCR amplification reagent, and a purification reagent.
The invention also relates to a quality control method of the second-generation sequencing tag primer, which is characterized by comprising the following steps of:
A. carrying out PCR amplification on the label primer pair to be detected and the quality control product;
B. obtaining an amplification product, and sequencing the amplification product on a machine to obtain sequencing data;
C. confirming the sequence consistency of the oligonucleotides to be detected according to the sequencing data;
wherein the quality control product is selected from any one of the quality control products;
the label primer pair to be detected comprises a forward primer sequence and a reverse primer sequence, wherein the two primer sequences have complementary sequences which are specifically recognized with the 3' end of the DNA fragment of the known sequence respectively; the oligonucleotide sequence exists on any one of the forward primer sequence and the reverse primer sequence and is positioned between the 5' end sequence and the end sequence;
preferably, the 5' end sequences of the forward primer sequence and the reverse primer sequence are respectively provided with a connector sequence which is complementarily matched with the sequencing platform;
preferably, the sequence of the label primer to be detected is any pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.1 and SEQ ID NO.4; any chain sequence of the quality control product is shown as SEQ ID NO. 55.
The step of confirming the sequence consistency of the oligonucleotide to be detected according to the sequencing data is to split the original sequencing data through the sequence data of the DNA fragments with known sequences, split the data containing the DNA fragments with the same known sequences into the same data set, and confirm the sequence consistency of the oligonucleotide to be detected in the data set.
The sequence consistency of the oligonucleotide to be detected comprises the cross contamination rate and the synthesis error rate of the nucleotide to be detected;
preferably, the cross contamination rate and the synthesis error rate are calculated for counting the types and the number of tag sequences in the sequencing read length of the original sequencing data.
Further, the quality control method is characterized in that the sequence consistency of the oligonucleotide to be detected is confirmed according to the sequencing data, namely, the original sequencing data are split through the sequence data of the DNA fragments with known sequences, the data containing the DNA fragments with the same known sequences are split into the same data set, and the sequence consistency of the oligonucleotide to be detected in the data set is confirmed.
The sequence consistency of the oligonucleotide to be detected means that the sequence of the oligonucleotide to be detected is consistent with the sequencing data of a preset sequence and/or the sequencing data only contains a single sequence data result.
In a specific embodiment, the quality control sequence is selected from any one or more of SEQ ID NO.5-SEQ ID NO. 29.
In one embodiment, the forward and reverse strand primer sequences are:
SEQ ID NO:1
5’-CAAGCAGAAGACGGCATACGAGATNNNN…NNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGA TCT-3’
SEQ ID NO:2
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
or (b)
SEQ ID NO:3
5’-CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’
SEQ ID NO:4
5’-AATGATACGGCGACCACCGAGATCTNNNN…NNNNACACTCTTTCCCTACACGACGCTCTTCCGA TCT-3’
SEQ ID NO:55
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-NNNNN........NNNNN-AGATCGGAAGAGCACACGTCT-3’
Example 1
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The 25 quality control products are shown in SEQ ID NO.6-30, and table 1.
TABLE 1 25 known DNA sequences
The sequences of 25 tag primers to be detected are shown in SEQ ID NO.31-55, see Table 2
TABLE 2 25 oligonucleotide sequences to be detected
| SEQ ID NO.30 | 1 | CATTGCTT |
| SEQ ID NO.31 | 2 | TTCGGATT |
| SEQ ID NO.32 | 3 | TCATCATT |
| SEQ ID NO.33 | 4 | CAACAGGT |
| SEQ ID NO.34 | 5 | TTCAAGGT |
| SEQ ID NO.35 | 6 | CCTAACGT |
| SEQ ID NO.36 | 7 | CACGTAGT |
| SEQ ID NO.37 | 8 | TACCTTCT |
| SEQ ID NO.38 | 9 | CCAGCGCT |
| SEQ ID NO.39 | 10 | ACCAGACT |
| SEQ ID NO.40 | 11 | CTATAACT |
| SEQ ID NO.41 | 12 | CTAGTTAT |
| SEQ ID NO.42 | 13 | TCTTATAT |
| SEQ ID NO.43 | 14 | AATAAGAT |
| SEQ ID NO.44 | 15 | TATGCCAT |
| SEQ ID NO.45 | 16 | ATTCTAAT |
| SEQ ID NO.46 | 17 | TAATGTTG |
| SEQ ID NO.47 | 18 | ATTCACTG |
| SEQ ID NO.48 | 19 | ATCATATG |
| SEQ ID NO.49 | 20 | CTTGATGG |
| SEQ ID NO.50 | 21 | TTAACCGG |
| SEQ ID NO.51 | 22 | CTAAGTCG |
| SEQ ID NO.52 | 23 | TATTCGCG |
| SEQ ID NO.53 | 24 | CCTGTGAG |
| SEQ ID NO.54 | 25 | CAACTAAG |
1. Preparation of quality control with known DNA sequences (known sequences)
(1) Artificial synthesis of 25 known DNA sequences with PCR linker (known sequence is shown as SEQ ID NO. 5-29)
(2) 25 known sequences can be amplified using the adaptor primers 1 and 2 described below, and a large number of available known sequences can be obtained continuously and stably;
the sequence of the adaptor primer 1 is shown in SEQ ID NO. 55:
SEQ ID NO.55：GACTGGAGTTCAGACGTGTGCTCTTCCGATCT
the sequence of the adaptor primer 2 is shown in SEQ ID NO. 56:
SEQ ID NO.56：ACACTCTTTCCCTACACGACGCTCTTCCGATCT
(3) Artificial synthesis of known sequence dilutions: artificially synthesized known DNA sequence (known sequence) concentrations were detected using a qubit HS and diluted to 1ng/ul using an dilution buffer;
(4) The PCR amplification system is shown in Table 3, 2.
TABLE 3 Table 3
| Sequence number | Total system 50. Mu.L.times.1 tube | Single use amount (mu L) |
| 1 | Known DNA sequences | 1 |
| 2 | HiFi Mix | 25 |
| 3 | Adapter primer 1 (10 pmol/. Mu.L) | 4 |
| 4 | Adapter primer 2 (10 pmol/. Mu.L) | 4 |
| 5 | ddH 2 O | 16 |
(5) PCR amplification procedure 94℃for 2min; (94 ℃ C. 15s,62 ℃ C. 30s,72 ℃ C. 30 s) 17cycles;72 ℃ for 10min;4 ℃ forever;
(6) Purifying magnetic beads: purification with 1.5-fold beads after amplification was completed, elution with 50ul elution buffer
2. Preparation of label primer to be detected
(1) Primer solubilization
And dissolving the dry powder of the label primer to be detected into working solution.
Wherein, the primer sequence of the forward chain of the primer pair is shown as SEQ ID NO.1 or SEQ ID NO.3;
the reverse strand primer sequence is shown as SEQ ID NO.2 or SEQ ID NO.4;
the NNNN … NNNN part of the shown sequence was replaced with the sequence obtained by the oligonucleotide sequences to be examined shown in Table 2. A combination of SEQ ID NO.1 and SEQ ID NO. 2.
Selected for use in the present embodiment
The dissolution method comprises the following steps: the dry label primer powder was placed on a high-speed centrifuge and centrifuged at 12000rpm for 5min. Diluting the primer dry powder to 10 pmol/mu L with sterilized purified water, wherein the added volume of the sterilized purified water is 100 times of the nmol number of the primer, adding the sterilized purified water, shaking and mixing uniformly, and placing the mixture in a palm centrifuge for short centrifugation; standing for 5min, repeating shaking, mixing, and centrifuging in palm centrifuge for short time.
Note that: the effective period of the label primer working solution is 14 months, and the storage temperature is below-15 ℃.
PCR reaction
(1) Taking out the known DNA sequence, melting at room temperature, shaking, mixing, centrifuging instantaneously, and placing on an ice box. By fluorescence quantifier
dsDNA HS Assay Kit the concentration of the template of the known DNA sequence after thawing was measured and the sample was measured at 1. Mu.L. mu.L of the known DNA sequence was taken and the known DNA sequence template was diluted to 1 ng/. Mu.L with sterile purified water.
(2) Taking out KAPA HiFi Hotstart Ready Mix and the reverse strand primer, melting at room temperature, shaking, mixing, centrifuging, and placing on an ice box. The preparation of the premix according to the PCR reaction system was performed on an ice bin, and the premix was as shown in Table 4 below. And (3) oscillating and uniformly mixing the prepared PCR reaction premix, and performing instantaneous centrifugation.
TABLE 4 Table 4
| Reagent name | Single reaction quantity (mu L) |
| KAPA HiFi Hotstart Ready Mix | 25 |
| Reverse strand primer (10 pmol/. Mu.L) | 4 |
| Sterilizing purified water | 16 |
(3) mu.L of the PCR reaction premix was aspirated separately and added to 23 wells (or PCR tubes) of a 96-well PCR plate.
(4) And respectively sucking 4 mu L of the label primer to be detected, adding the label primer to a PCR plate (or a PCR tube) filled with the PCR reaction premix, oscillating and uniformly mixing, and performing instantaneous centrifugation.
(5) 1. Mu.L of the known DNA sequences diluted in 1) were each aspirated, and the 96-well PCR plate (or PCR tube) added to 4) was correspondingly used according to the following table. Shaking and mixing evenly, and carrying out instantaneous centrifugation.
(6) The sample was placed on a PCR gene amplification apparatus, and the PCR conditions were as shown in Table 5 below.
TABLE 5
Note that: the PCR instrument was capped at 105℃and a volume of 55. Mu.L.
PCR reaction product purification
Purification of 0.9 Xmagnetic beads, elution with 50ul elution buffer
Amplification of purified products
Using a fluorescent quantifier
dsDNA HS Assay Kit the concentration of the amplified and purified product was measured, and the sample measurement was 1. Mu.L.
The sample volumes (volumes) of the amplified and purified products were calculated separately, and according to the sample volumes (volumes), 23 amplified and purified products were pipetted into 1 new 1.5ml centrifuge tubes and mixed into a one-tube library.
5. Library quality detection
The pooling library concentration was determined using a fluorescent quantitative PCR analyzer.
6. Sequencing on machine
The library was subjected to on-machine sequencing, sequencing type: SE40+8,8M reads,75cycles.
7. Information analysis
And (3) splitting and analyzing the data after the machine is started, and checking whether each label primer to be checked is 'free from data output' and the cross contamination rate. The Nextseq550/500 platform processes the reads dataset and counts the ratio of corresponding nucleotide sequence reads to be tested to corresponding single dataset reads in the split dataset.
The results are shown in Table 6
TABLE 6
Analysis conclusion:
of the 25 sets of oligonucleotides tested,
group 8, group 15 had cross contamination with contamination rates of 0.34% and 0.37%, respectively; group 22 had a synthetic error.
Claims (13)
1. A quality control product for detecting a label primer is characterized in that,
the quality control product is a double-stranded DNA fragment;
the DNA fragment comprises a segment of non-natural oligonucleotide with a known sequence, and is characterized in that the non-natural oligonucleotide segment is completely different from any position sequence on the genome of any known species.
The two ends of the DNA fragment are provided with linker sequences, and the linker sequences are complementary sequences which can be specifically combined with the 3' end of the label primer pair to be detected.
2. The quality control article according to claim 1, wherein,
the 3' ends of the sense strand and the antisense strand of the linker sequence have the same sequence and comprise a sequence specifically complementary to any one of the tag primer pairs to be detected;
the 5' ends of the sense strand and the antisense strand of the linker sequence have the same sequence and comprise a sequence specifically complementary to the other of the pair of tag primers to be detected.
3. The quality control product according to claim 1, wherein the quality control product can be amplified by PCR with a pair of tag primers to be detected, and the amplified product is a tag primer detection library.
4. The quality control article of claim 2, wherein the pair of tag primers to be detected is a pair of second generation sequencing tag primers;
the label primer pair to be detected comprises a forward primer sequence and a reverse primer sequence, wherein the two primer sequences have complementary sequences which are specifically recognized with the 3' end of the DNA fragment with the known sequence respectively; the oligonucleotide sequence exists on any one of the forward primer sequence and the reverse primer sequence and is positioned between the 5' end sequence and the end sequence;
preferably, the 5' end sequences of the forward and reverse strand primer sequences are provided with a linker sequence complementary to the sequencing platform, respectively.
5. The quality control article according to claim 4, wherein the tag primer sequences to be detected are SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.2
NO 1 and SEQ ID NO 4.
6. The quality control product according to claim 5, wherein any one of the chain sequences of the quality control product is shown as SEQ ID NO. 55.
7. The quality control according to claim 1, wherein the known non-natural oligonucleotide fragment has a length of 50-1000 bp, preferably 150-500 bp.
8. The quality control of claim 1 wherein the sequence-known unnatural oligonucleotide chip sequence is used to resolve tag primer detection library data.
9. A kit for detecting a tagged primer, comprising the quality control article of claims 1-8.
10. The kit according to claim 9, further comprising a pair of adapter primers, PCR
Amplification reagent, purification reagent.
11. A quality control method of a second generation sequencing tag primer, which is characterized by comprising the following steps:
A. carrying out PCR amplification on the label primer pair to be detected and the quality control product;
B. obtaining an amplification product, and sequencing the amplification product on a machine to obtain sequencing data;
C. confirming the sequence consistency of the oligonucleotides to be detected according to the sequencing data;
wherein the quality control product is selected from any one of the quality control products of claims 1-8;
the label primer pair to be detected comprises a forward primer sequence and a reverse primer sequence, wherein the two primer sequences have complementary sequences which are specifically recognized with the 3' end of the DNA fragment of the known sequence respectively; the oligonucleotide sequence exists on any one of the forward primer sequence and the reverse primer sequence and is positioned between the 5' end sequence and the end sequence;
preferably, the 5' end sequences of the forward primer sequence and the reverse primer sequence are respectively provided with a connector sequence which is complementarily matched with the sequencing platform;
preferably, the sequence of the label primer to be detected is any pair of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.1 and SEQ ID NO.4; any chain sequence of the quality control product is shown as SEQ ID NO. 5.
12. The method of claim 11, wherein,
the step of confirming the sequence consistency of the oligonucleotide to be detected according to the sequencing data is to split the original sequencing data through the sequence data of the DNA fragments with known sequences, split the data containing the DNA fragments with the same known sequences into the same data set, and confirm the sequence consistency of the oligonucleotide to be detected in the data set.
13. The method of claim 12, wherein,
the sequence consistency of the oligonucleotide to be detected comprises the cross contamination rate and the synthesis error rate of the nucleotide to be detected;
preferably, the cross contamination rate and the synthesis error rate are calculated for counting the types and the number of tag sequences in the sequencing read length of the original sequencing data.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2021116750667 | 2021-12-31 | ||
| CN202111675066 | 2021-12-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116377046A true CN116377046A (en) | 2023-07-04 |
Family
ID=85890819
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211733871.5A Pending CN115948522A (en) | 2021-12-31 | 2022-12-30 | Method for detecting oligonucleotide sequence consistency |
| CN202211734834.6A Pending CN116377046A (en) | 2021-12-31 | 2022-12-31 | Quality control product and kit for parallel detection of tag primer sequences |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211733871.5A Pending CN115948522A (en) | 2021-12-31 | 2022-12-30 | Method for detecting oligonucleotide sequence consistency |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN115948522A (en) |
-
2022
- 2022-12-30 CN CN202211733871.5A patent/CN115948522A/en active Pending
- 2022-12-31 CN CN202211734834.6A patent/CN116377046A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CN115948522A (en) | 2023-04-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3371309B1 (en) | Combinatorial sets of nucleic acid barcodes for analysis of nucleic acids associated with single cells | |
| CA2697640C (en) | Tools and methods for genetic tests using next generation sequencing | |
| CN111808854B (en) | Balanced joint with molecular bar code and method for quickly constructing transcriptome library | |
| CN110129415B (en) | NGS library-building molecular joint and preparation method and application thereof | |
| CN108138175B (en) | Reagents, kits and methods for molecular barcode encoding | |
| WO2012037881A1 (en) | Nucleic acid tags and use thereof | |
| KR20170133270A (en) | Method for preparing libraries for massively parallel sequencing using molecular barcoding and the use thereof | |
| CN113308526A (en) | Fusion primer direct amplification method human mitochondrial whole genome high-throughput sequencing kit | |
| CN108932401B (en) | Identification method of sequencing sample and application thereof | |
| CN112795654A (en) | Method and kit for organism fusion gene detection and fusion abundance quantification | |
| CN116377046A (en) | Quality control product and kit for parallel detection of tag primer sequences | |
| CN116287161A (en) | Oligonucleotide sequence consistency detection method | |
| CN110892079A (en) | Assay methods and compositions for detecting nucleic acid identifier contamination | |
| CN114854825A (en) | Library building joint and method for simplified genome sequencing suitable for DNBSEQ technology | |
| CN111793623A (en) | Typing genetic marker composition, kit, identification system and typing method of 62 multi-allelic SNP-NGS | |
| CN116515977B (en) | Single-ended-adaptor-transposase-based single-cell genome sequencing kit and method | |
| CN116103383B (en) | Method for identifying false base of NGS linker oligo and library thereof | |
| Flood | Novel Fragmentation Method for Automated Next Generation Sequencing Exome Library Preparation | |
| CN112176041A (en) | Detection method, reagent and application of epigenetic modification | |
| WO2022125100A1 (en) | Methods for sequencing polynucleotide fragments from both ends | |
| CN112646809A (en) | Nucleic acid sequence, method and kit for detecting enzyme end repair capacity | |
| KR20220122095A (en) | Composition for improving molecular barcoding efficiency and use thereof | |
| CN110656159A (en) | Method for detecting copy number variation | |
| CN114622286A (en) | High-throughput transcriptome sequencing library construction method and application thereof | |
| CN114686562A (en) | Compositions, kits, methods, and systems for nucleic acid sample amplification |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |