CN115710751A - Method for preventing pollution by multiple PCR library construction - Google Patents
Method for preventing pollution by multiple PCR library construction Download PDFInfo
- Publication number
- CN115710751A CN115710751A CN202211399951.1A CN202211399951A CN115710751A CN 115710751 A CN115710751 A CN 115710751A CN 202211399951 A CN202211399951 A CN 202211399951A CN 115710751 A CN115710751 A CN 115710751A
- Authority
- CN
- China
- Prior art keywords
- sample
- pcr
- label
- amplification
- sequence
- 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
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000010276 construction Methods 0.000 title claims abstract description 11
- 238000012163 sequencing technique Methods 0.000 claims abstract description 23
- 238000012408 PCR amplification Methods 0.000 claims abstract description 6
- 239000000523 sample Substances 0.000 claims description 62
- 239000011324 bead Substances 0.000 claims description 33
- 230000003321 amplification Effects 0.000 claims description 27
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 27
- 239000002773 nucleotide Substances 0.000 claims description 18
- 125000003729 nucleotide group Chemical group 0.000 claims description 18
- 238000011109 contamination Methods 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 6
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 239000000539 dimer Substances 0.000 claims description 5
- 238000007403 mPCR Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 238000011176 pooling Methods 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 13
- 238000001514 detection method Methods 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000003766 bioinformatics method Methods 0.000 abstract 1
- 238000003752 polymerase chain reaction Methods 0.000 description 33
- 239000000047 product Substances 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000007664 blowing Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for preventing pollution by multiple PCR library construction, which relates to the technical field of molecular biology, and the specific technical scheme is as follows: adding one or more artificially synthesized error-proof label sequences at the first time after a sample is taken, wherein the label must be amplified by at least one pair of primers in a primer pool, then carrying out subsequent PCR amplification and sequencing along with the sample, and finally distinguishing whether the detected label in each sample is consistent with the label added during library establishment through bioinformatics analysis of off-line data, thereby determining that the sample is not confused and the accuracy of a detection result is ensured, and simultaneously analyzing the label composition in each sample to prevent the accurate judgment of whether the sample is polluted and the pollution degree. The method can prevent sample pollution caused by unavoidable factors in the library building process, and can distinguish samples through different labels in subsequent biological information analysis.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for preventing pollution by multiple PCR (polymerase chain reaction) library building.
Background
The multiplex PCR bank building is an efficient technology capable of simultaneously amplifying and increasing pairs of primers, can simultaneously add a plurality of pairs of primers to carry out PCR reaction, and can theoretically efficiently and accurately detect and identify a plurality of microorganisms. However, in practice, when the number of samples is too large, the samples may be contaminated due to PCR aerosol, uncertainty of manual operation, and the like, and even the samples may be confused and the sample information may be wrongly recorded, which may affect the experimental result.
Disclosure of Invention
The invention provides a method for preventing pollution in multiple PCR library construction, which solves the problems of large sample number, easy pollution and easy confusion in the background technology.
The invention provides the following technical scheme: a method for preventing pollution by multiple PCR library building comprises the following steps:
designing and synthesizing a nucleotide tag sequence, diluting, subpackaging and numbering;
step two, adding at least one nucleotide tag sequence into a sample;
thirdly, performing nucleic acid extraction, PCR targeted amplification and product purification on the sample;
step four, adding a sequencing linker and barcode into the PCR product obtained in the step three, and performing a second round of PCR amplification;
step five, purifying the amplification product in the step four, and finally, mixing the library and operating the machine;
and step six, identifying whether the reads contain the tag sequences through sequencing, judging whether the actually detected tag sequences are consistent with the added tag sequences, if so, indicating that the sample is not mixed up, if the sample only detects the probes added into the sample, indicating that the sample is not polluted, and if the sample not only detects the own tag but also can detect the tags of other samples, indicating that the sample is polluted.
Preferably, the concentration of tags added per sample is between 10^1 and 10^9 copies, and the nucleotide tag sequences added between samples are different.
Preferably, the artificially designed and synthesized nucleotide tag sequence can be amplified by at least one pair of primers, the tag sequence in the nucleotide sequence can be read by sequencing reads, and the reads at least one end of the nucleotide tag sequence cover the corresponding tag sequence on the sample.
Preferably, each nucleotide tag sequence has a difference of 2bp or more.
Preferably, when the sample is contaminated, contamination filtration may be performed in proportion.
Preferably, after the first round of PCR targeted amplification, magnetic beads are used for purifying the amplification product to remove the excess primers and dimer non-specificity, and after the second round of PCR targeted amplification is performed, part of the excess primers and non-specific amplification are removed.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the method for preventing pollution in the multiple PCR library construction, one or more artificial label sequences are respectively added into a sample, each pair of labels can be amplified by at least one pair of primers in a primer pool along with a sample, sample pollution caused by factors which are difficult to avoid in the library construction process can be prevented, the sample can be distinguished through different labels in subsequent biological information analysis, and sample detection results are effectively prevented from being wrong due to sample confusion or sample pollution.
2. The method for preventing the pollution of the multiple PCR library establishment can distinguish whether the label detected in each sample is consistent with the label added during the library establishment, thereby determining that the samples are not confused and the accuracy of the detection result is ensured, and meanwhile, the label composition in each sample can be analyzed to prevent the accurate judgment of whether the samples are polluted and the pollution degree.
Drawings
FIG. 1 is a schematic illustration of sequencing in an embodiment of the invention;
FIG. 2 is a diagram showing the conditions of a second round of PCR reaction in the example of the present invention;
FIG. 3 is a schematic diagram showing exemplary conditions of a PCR reaction according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of an amplification system according to an embodiment of the present invention;
FIG. 5 is a graph showing the results of tests in which different anti-contamination tag sequences were added to different samples.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The present invention provides an embodiment: a method for preventing pollution by multiple PCR library building comprises the following steps:
firstly, designing and synthesizing at least one nucleotide tag sequence which can be amplified by at least one pair of primers according to a pair of primers found in a multiple primer set, diluting, subpackaging and numbering, wherein each nucleotide tag sequence has a difference of more than 2 bp;
adding at least one nucleotide tag sequence into the samples, wherein the concentration of the tags added into each sample is 10^1 to 10^9 copies, and the nucleotide tag sequences added among the samples are different, so that whether the samples are polluted or not can be conveniently judged;
thirdly, performing nucleic acid extraction, PCR targeted amplification and product purification on the sample, and removing excessive primers, dimer non-specificity and the like;
step four, adding adaptive sequencing joints and barcode to the PCR product obtained in the step three according to different sequencing platforms, performing second-round PCR amplification, and removing part of redundant primers and non-specific amplification;
step five, purifying the amplification product in the step four, removing part of redundant primers and non-specific amplification, and finally mixing the library and operating the machine;
and step six, identifying whether the reads contain the tag sequences through sequencing, judging whether the actually detected tag sequences are consistent with the added tag sequences, if the actually detected tag sequences are completely consistent with the added tag sequences, indicating that the sample is not mixed up, if the sample only detects the probes added into the sample, indicating that the sample is not polluted, and if the sample not only detects the own tag but also can detect the tags of other samples, indicating that the sample is polluted, and filtering the pollution according to the proportion when the sample is polluted.
In summary, the following steps: the principle of the method is as follows: adding one or more artificial tag sequences to the sample, each pair of tags being amplifiable with at least one pair of primers in the primer pool; by library construction, sequencing can finally determine whether contamination has occurred by whether the tag in the sample is identical to the added tag. If the sample is found to be contaminated, contamination filtering can be performed proportionally, and if the label sequence is not added, the analyzed result can be the result after sample confusion, and particularly, the judgment of some low-graduation signals can be greatly influenced. And the tag sequence is added, so that whether the sequencing result is consistent with the library building result can be detected, and the result is more accurate and credible.
Example (b):
in this embodiment, a sample is based on a second-generation sequencing platform, and multiple PCR amplification is performed, and the method is used to detect whether the sample is mixed up, the specific steps are as follows:
1. amplification system
0.2ml of PCR single tube/octal tube was used, and the reaction was configured according to the system shown in FIG. 4, and the cautions were: (1) After all reagents are unfrozen, vortex and centrifugation are carried out to prevent the components of the solution from being uneven after the reagents are unfrozen; (2) The labeled components are required to prepare a reaction mix in a reagent dispensing chamber; (3) two negative controls need to be added for each machine loading: the templates are respectively used for nucleic acid extraction water and reservoir building water; (4) The prepared PCR system is instantly centrifuged to centrifuge the liquid on the tube wall to the tube bottom before loading.
2. First round PCR reaction
Referring to FIG. 3, in the first round of reaction, if the nucleic acid of the sample to be detected contains the amplification target in the panel A mix, it will be amplified by the primer; if the amplification target in the panel A mix is not contained, the amplification target cannot be amplified; however, regardless of whether the sample nucleic acid to be tested contains amplification targets within the panel A mix, the IAC pool will be amplified by primers within the panel A mix. Through the amplification of the digestion primer, a large amount of dimers are reduced, and the quality of PCR amplification products is improved.
3. First round amplification product purification
After the first PCR reaction, the amplified product is purified by magnetic beads, excess primers are removed, dimer is non-specific, and the like. Certain exemplary purification conditions are as follows:
i, adding DNA purification magnetic beads with 0.5 times of the original PCR volume (if the PCR system is 30ul, 15ul magnetic beads are added) into the PCR reaction solution; and (3) blowing the mixture up and down for 10 to 15 times by using a pipette with the range of 50ul so as to fully and uniformly mix the amplification product and the magnetic beads. The mixture was allowed to stand at room temperature for 2 minutes.
And ii, adsorbing the magnetic beads by using a magnetic frame until the solution is clarified (about 2min is required). Transfer the supernatant to a new EP tube with a pipette, avoid attracting the magnetic beads, discard the magnetic beads.
Iii, adding DNA purification magnetic beads with 0.7 time of the original PCR volume (if the PCR system is 30ul, 21ul of magnetic beads are added) into the supernate; and (3) blowing the mixture up and down for 10 to 15 times by using a pipette with the range of 50ul so as to fully and uniformly mix the amplification product and the magnetic beads. Standing at room temperature for 2min.
Iv, adsorbing the magnetic beads by using a magnetic rack until the solution is clear. The supernatant was carefully removed with a pipette to avoid attracting to the beads.
V, 40ul BW11 was added to the beads, the beads were resuspended, and allowed to stand at room temperature for 2 minutes.
Vi, adsorb the beads with a magnetic stand until the solution is clarified (approximately 2 min). The supernatant was removed with a pipette to avoid attracting magnetic beads.
Vii, adding 100ul 80% ethanol into the magnetic beads, repeatedly adsorbing the magnetic beads back and forth on two different sides by using a magnetic rack to fully suspend and wash the magnetic beads, and adsorbing the magnetic beads by using the magnetic rack until the solution is clarified (about 2min is needed). The supernatant was carefully removed with a pipette to avoid pipetting into the beads.
4. Second round PCR reaction
After obtaining PCR products, different adapters can be selectively ligated according to different sequencing platforms. Exemplary PCR conditions are shown in FIG. 2.
5. Second round PCR purification
After ligation of the sequencing adaptors, the library needs to be purified to remove some of the excess primers and non-specific amplification. An example flow is as follows:
adding 0.9 times volume of DNA purification magnetic beads (for example, 27ul magnetic beads are added when the PCR system is 30 ul) to the PCR reaction solution, and blowing up and down with a pipette at a range of 50ul to sufficiently mix the recovery product with the magnetic beads. The mixture was allowed to stand at room temperature for 2 minutes.
Ii, using a magnetic rack to adsorb the magnetic beads until the solution is clarified (about 2min is needed).
Iii, carefully remove the supernatant with a pipette to avoid aspiration to the beads.
And iv, adding 40ul BW08 into the magnetic beads, uniformly swirling, and standing at room temperature for 2min.
V, adsorb the beads with a magnetic stand until the solution is clear (about 2 min). The supernatant was removed with a pipette.
Vi, adding 100ul 80% ethanol, repeatedly adsorbing the magnetic beads on different two sides by using a magnetic frame to fully suspend and wash the magnetic beads, and carefully removing the supernatant by using a pipette to avoid adsorbing the magnetic beads.
6. Sequencing on machine
The platform for sequencing on the computer can be selected from illumina or ionotorent, MGI and the like. However, the method needs the sequencing length to cover the label sequence corresponding to the sample so as to distinguish whether the detection result is from the IAC sequence or the real detection sequence; as shown in FIG. 1, reads at least one end of the sequencing overlay the tag on the IAC.
7. Data analysis
After the sequencing data are obtained, the sequencing data need to be distinguished, and whether the tag sequence detected by each sample is consistent with the tag sequence added during pretreatment is checked through comparison software, so that whether the samples are confused can be determined.
The using effect of the method is shown in figure 5: the label ID of a behavior initial adding in the sample is registered in the table, the actual label is the label actually detected by the sequencing technology, the other rows in the table are the reads number detected by each label in each sample, and the sample is considered not to be confused or polluted only when the actual label is completely consistent with the registered label. If the sample has other labels besides the registration label, the sample is indicated to be polluted, and then the sample is deducted according to the equal proportion principle.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A method for preventing pollution by multiple PCR library building is characterized by comprising the following steps:
designing and synthesizing a nucleotide tag sequence, diluting, subpackaging and numbering;
step two, adding at least one nucleotide tag sequence into a sample;
thirdly, performing nucleic acid extraction, PCR targeted amplification and product purification on the sample;
step four, adding a sequencing linker and barcode into the PCR product obtained in the step three, and performing a second round of PCR amplification;
step five, purifying the amplification product in the step four, and finally mixing the library and operating the machine;
and step six, identifying whether the reads contain the label sequence through sequencing, judging whether the actually detected label sequence is consistent with the added label sequence, if so, indicating that the sample is not confused, if the sample only detects the probe added into the sample, indicating that the sample is not polluted, and if the sample not only detects the label of the sample, but also can detect the labels of other samples, indicating that the sample is polluted.
2. The method for preventing contamination in multiplex PCR library construction according to claim 1, wherein: the concentration of the added tags per sample is between 10^1 and 10^9 copies, and the nucleotide tag sequences added between samples are different.
3. The method for multiple PCR pooling for contamination prevention according to claim 1, wherein: the artificially designed and synthesized nucleotide tag sequence can be amplified by at least one pair of primers, the tag sequence in the nucleotide sequence can be read by sequencing reading, and at least one end of the nucleotide tag sequence is covered with reads on the corresponding tag sequence on a sample.
4. The method for preventing contamination in multiplex PCR library construction according to claim 1, wherein: each nucleotide tag sequence has a difference of 2bp or more.
5. The method for preventing contamination in multiplex PCR library construction according to claim 1, wherein: when the sample is contaminated, contamination filtration can be performed in proportion.
6. The method for preventing contamination in multiplex PCR library construction according to claim 1, wherein: after the first round of PCR targeted amplification, magnetic beads are adopted to purify the amplification product, surplus primers and dimer non-specificity are removed, and after the second round of PCR targeted amplification product is purified, part of surplus primers and non-specific amplification are removed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211399951.1A CN115710751A (en) | 2022-11-09 | 2022-11-09 | Method for preventing pollution by multiple PCR library construction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211399951.1A CN115710751A (en) | 2022-11-09 | 2022-11-09 | Method for preventing pollution by multiple PCR library construction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115710751A true CN115710751A (en) | 2023-02-24 |
Family
ID=85232682
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211399951.1A Pending CN115710751A (en) | 2022-11-09 | 2022-11-09 | Method for preventing pollution by multiple PCR library construction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115710751A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116926213A (en) * | 2023-06-21 | 2023-10-24 | 迪飞医学科技(南京)有限公司 | Method for detecting drug-resistant gene mutation of mycobacterium tuberculosis |
-
2022
- 2022-11-09 CN CN202211399951.1A patent/CN115710751A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116926213A (en) * | 2023-06-21 | 2023-10-24 | 迪飞医学科技(南京)有限公司 | Method for detecting drug-resistant gene mutation of mycobacterium tuberculosis |
| CN116926213B (en) * | 2023-06-21 | 2024-09-06 | 迪飞医学科技(南京)有限公司 | A method for detecting drug-resistant gene mutation of Mycobacterium tuberculosis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110904206A (en) | Liquid path system, biomolecule analysis system and nucleic acid sequence measuring system | |
| CN101278058A (en) | Improved strategies for sequencing complex genomes using high throughput sequencing technologies | |
| RU2011141128A (en) | DEVICE FOR INTEGRAL ANALYSIS OF NUCLEIC ACIDS IN REAL TIME AND METHOD FOR DETERMINING TARGET NUCLEIC ACID WITH ITS USE | |
| CN113308526B (en) | Fusion primer direct amplification method human mitochondrial whole genome high-throughput sequencing kit | |
| JP3752488B2 (en) | Nucleic acid analysis method | |
| CN113462749A (en) | High-sensitivity amplicon library construction kit, library construction method and application | |
| CN115710751A (en) | Method for preventing pollution by multiple PCR library construction | |
| Herbst et al. | Colorimetric RT-LAMP and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples | |
| CN115612746A (en) | Targeted capture sequencing chip for broiler genetic relationship identification | |
| US20210363517A1 (en) | High throughput amplification and detection of short rna fragments | |
| CN112592965B (en) | E.coli host DNA residue detection kit adopting TaqMan probe method | |
| US10941453B1 (en) | High throughput detection of pathogen RNA in clinical specimens | |
| WO2014001400A1 (en) | Method for improved quantification of mirnas | |
| EP3325697B1 (en) | Optimized clinical sample sequencing | |
| US20200181602A1 (en) | Assay methods and compositions for detecting contamination of nucleic acid identifiers | |
| CN111304309A (en) | Detection method for sequencing platform tag sequence pollution | |
| CN111944806A (en) | Molecular tag group for high-throughput sequencing pollution detection and application thereof | |
| CN105755129B (en) | STR typing method of gene locus D8S1179 based on next generation sequencing | |
| WO2017219511A1 (en) | Method for rapid homogenization or equal proportion of dna samples | |
| CN114277092A (en) | Method and application of RNA virus metatranscriptional library based on nanopore sequencing platform | |
| CN113930487B (en) | Novel multi-sample multi-fragment DNA methylation detection method | |
| JP7604530B2 (en) | Assay methods and compositions for detecting contamination of nucleic acid identifiers | |
| US20120035060A1 (en) | Highly multiplexed genotyping using leukoreduced blood samples | |
| CN216786143U (en) | Phosphorylation reaction's preliminary examination article preparation ware and quality testing equipment | |
| CN117821642B (en) | A primer set and kit for identifying Phalaenopsis varieties and their application |
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 |