CN117661123A - Single-cell library construction method for multiple sample mixing - Google Patents
Single-cell library construction method for multiple sample mixing Download PDFInfo
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- 238000002156 mixing Methods 0.000 title claims abstract description 17
- 238000010276 construction Methods 0.000 title abstract description 9
- 239000012634 fragment Substances 0.000 claims abstract description 41
- 238000012163 sequencing technique Methods 0.000 claims abstract description 35
- 230000004927 fusion Effects 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000011324 bead Substances 0.000 claims abstract description 10
- 239000000853 adhesive Substances 0.000 claims abstract description 6
- 230000001070 adhesive effect Effects 0.000 claims abstract description 6
- 238000012408 PCR amplification Methods 0.000 claims abstract description 4
- 239000011543 agarose gel Substances 0.000 claims abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 38
- 238000004458 analytical method Methods 0.000 claims description 17
- 238000000746 purification Methods 0.000 claims description 13
- 238000001914 filtration Methods 0.000 claims description 11
- 238000012512 characterization method Methods 0.000 claims description 10
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 238000010201 enrichment analysis Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000011176 pooling Methods 0.000 claims 4
- 238000013354 cell banking Methods 0.000 claims 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 3
- 230000003321 amplification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
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- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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- 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
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- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
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- 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
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- G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
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Abstract
The application relates to the technical field of bioinformatics and discloses a multi-sample mixed single-cell library construction method, wherein genomic DNA of each sample is broken, and DNA fragments with bands distributed in 500bp are obtained; adding an adhesive primer combined with an adhesive tail end into the obtained DNA fragment, and adding a tail end leveling reagent for mixing reaction; designing an initial primer according to the DNA fragment, and respectively connecting a plurality of labels with the designed initial primer to obtain a fusion primer; connecting the first fusion primer with the head end of the DNA fragment by adopting an A joint, and connecting the second fusion primer with the tail end of the DNA fragment by adopting a P joint; purifying magnetic beads; performing PCR amplification on DNA fragments with different fusion primers at the head and the tail; and (3) after quantitative and sample mixing, separating and purifying by agarose gel to obtain a single-cell sequencing document. The method and the device are beneficial to reducing cost and have higher reliability.
Description
Technical Field
The application relates to the technical field of bioinformatics, in particular to a single-cell library construction method for multi-sample mixing.
Background
Single cell sequencing is a very costly method, and therefore, the cost can be effectively reduced by adopting a mixed sample mode for sequencing. However, existing mixed sample protocols mix up to 12 at a time, while being subject to cellular conditions, adding more tags is detrimental to the activity of the sample and is not easily distinguishable, and thus a reliable mixed sample sequencing technique is needed to solve these problems.
Disclosure of Invention
The present application is directed to a single-cell library construction method for multiple sample mixing, so as to solve the technical problems set forth in the background art.
In order to achieve the above purpose, the present application discloses the following technical solutions:
a single-cell library building method for multiple sample mixing sequentially comprises the following steps:
breaking the genome DNA of each sample to obtain DNA fragments with bands distributed in 500 bp;
adding an adhesive primer combined with an adhesive tail end into the obtained DNA fragment, and adding a tail end leveling reagent for mixing reaction;
designing an initial primer according to the DNA fragment, and respectively connecting a plurality of labels with the designed initial primer to obtain a fusion primer;
connecting the first fusion primer with the head end of the DNA fragment by adopting an A joint, and connecting the second fusion primer with the tail end of the DNA fragment by adopting a P joint;
purifying magnetic beads;
performing PCR amplification on DNA fragments with different fusion primers at the head and the tail;
and (3) after quantitative and sample mixing, agarose gel separation and purification are carried out to obtain a single-cell sequencing library.
Preferably, the fusion primer includes an upstream general primer sequence sequentially arranged in a 5 'to 3' direction and a specific upstream primer sequence designed according to a target amplification region in the purified DNA fragment, and a downstream general primer sequence sequentially arranged in a 5 'to 3' direction and a specific downstream primer sequence designed according to a target amplification region in the purified DNA fragment.
Preferably, the upstream universal primer sequence and the downstream universal primer sequence are 16S rRNA sequencing primers, respectively.
Preferably, the magnetic bead purification includes: the DNA fragment was purified using 1.2 times of AMPure XP beads to remove redundant small fragment adaptors and primers from the DNA fragment.
Preferably, the A-and P-linkers are linkers of Ion Torrent, ion PGM, ion Proton or Ion S5/S5XL sequencing platforms.
Preferably, the concentration of the genomic DNA is 0.1 to 150 ng/. Mu.L.
Preferably, the method further comprises: designing a matched biological information analysis method based on a database building structure;
the biological information analysis method sequentially comprises the following steps:
acquiring sequencing data of a DNA sequence to be tested;
inputting the sequencing data into a purification model for purification according to a preset filtering rule to obtain purified sequencing data only retaining biological information;
filtering the purified sequencing data according to a preset tag filtering rule to obtain a characterization sequence corresponding to the purified sequencing data;
comparing the characterization sequence with preset tag sequence data to obtain fusion primer information;
respectively carrying out GO enrichment analysis, KO enrichment analysis and WGS analysis based on the fusion primer information to obtain sequence types, sequence distribution and data base distribution characteristics corresponding to the characterization sequence;
and (5) carrying out statistics on the characteristics of the purified sequencing data, the sequence types, the sequence distribution and the data base distribution, and outputting the statistics as a biological information analysis result.
Preferably, said WGS analysis is effected by employing one or more of Refseq, GEO Database and Expression Atlas.
The beneficial effects are that: according to the single-cell library construction method for the multi-sample mixing, the quantity of the labels is enriched by adopting the form of the fusion primer, and the corresponding labels are connected to the head end or the tail end of the DNA fragment by adopting the connector, so that the analysis is convenient, the cell state is kept, the cells are easy to distinguish, and the influence on the cell activity is reduced. Further, the real condition of the sample is conveniently restored through the later data filtering, and the related biological information is rapidly acquired.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic flow chart of a single-cell library construction method for multi-sample mixing according to an embodiment of the present application.
Detailed Description
The following description of the technical solutions in the embodiments of the present application will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
In this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising … …" does not exclude the presence of other like elements in a process, method, article or apparatus that comprises the element.
The applicant found that the existing mixed sample scheme mixes at most 12 at a time, and we can mix more while maintaining the sample quality, and the suspension quality simultaneously meets various single cell platform on-machine requirements. The blood sample is especially suitable, and the cost can be greatly reduced.
Based on the above, this embodiment discloses a single cell library construction method for multi-sample mixing as shown in fig. 1, which sequentially includes the following steps:
s101, breaking the genome DNA of each sample to obtain DNA fragments with bands distributed in 500bp in a centralized way; in this example, the concentration of the genomic DNA is 0.1-150 ng/. Mu.L;
s102, adding a cohesive primer combined with a cohesive terminal to the obtained DNA fragment, and adding a terminal filling reagent to carry out a mixing reaction;
s103, designing an initial primer according to the DNA fragment, and respectively connecting a plurality of labels with the designed initial primer to obtain a fusion primer;
s104, connecting the first fusion primer with the head end of the DNA fragment by adopting an A joint, and connecting the second fusion primer with the tail end of the DNA fragment by adopting a P joint;
s105-magnetic bead purification;
s106, carrying out PCR amplification on the DNA fragments with different fusion primers at the head and the tail;
s107-quantifying, mixing, and performing agarose gel separation and purification to obtain a single-cell sequencing library.
In this embodiment, the fusion primer includes an upstream general primer sequence sequentially arranged in the 5 'to 3' direction and a specific upstream primer sequence designed according to the target amplified region in the purified DNA fragment, and a downstream general primer sequence sequentially arranged in the 5 'to 3' direction and a specific downstream primer sequence designed according to the target amplified region in the purified DNA fragment. Wherein the upstream universal primer sequence and the downstream universal primer sequence are 16S rRNA sequencing primers, respectively.
In this embodiment, the magnetic bead purification comprises: the DNA fragment was purified using 1.2 times of AMPure XP beads to remove redundant small fragment adaptors and primers from the DNA fragment.
It is possible that the A-and P-linkers are linkers of Ion Torrent, ion PGM, ion Proton or Ion S5/S5XL sequencing platforms. It should be noted that Ion Torrent, ion PGM, ion proton or Ion S5/S5XL sequencing platforms are common sequencing platforms, and in order to make the library more compatible, the present embodiment uses a linker in the common sequencing platform.
As a preferred implementation of this embodiment, the method further includes: designing a matched biological information analysis method based on a database building structure;
the biological information analysis method sequentially comprises the following steps:
acquiring sequencing data of a DNA sequence to be tested;
inputting the sequencing data into a purification model for purification according to a preset filtering rule to obtain purified sequencing data only retaining biological information;
filtering the purified sequencing data according to a preset tag filtering rule to obtain a characterization sequence corresponding to the purified sequencing data;
comparing the characterization sequence with preset tag sequence data to obtain fusion primer information;
respectively carrying out GO enrichment analysis, KO enrichment analysis and WGS analysis based on the fusion primer information to obtain sequence types, sequence distribution and data base distribution characteristics corresponding to the characterization sequence;
and (3) carrying out statistics on the characteristics of the purified sequencing data, the sequence types, the sequence distribution and the data base distribution, and outputting the statistics as a biological information analysis result, wherein the real condition of the sample is the difference between the purified sequencing data and the characterization sequence.
In this embodiment, the WGS analysis is performed by using one or more of Refseq, GEO Database, and Expression Atlas.
The single-cell library construction method for mixing multiple samples, which is recorded in the embodiment, adopts the form of fusion primers, enriches the number of labels, and the corresponding labels are connected at the head end or the tail end of the DNA fragment by adopting a connector, so that the analysis is convenient, the cell state is kept, the cells are easy to distinguish, and the influence on the cell activity is reduced. Further, the real condition of the sample is conveniently restored through the later data filtering, and the related biological information is rapidly acquired.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present application, and although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof, and any modifications, equivalents, improvements or changes that fall within the spirit and principles of the present application are intended to be included in the scope of protection of the present application.
Claims (8)
1. The single-cell library building method for the mixture of multiple samples is characterized by comprising the following steps of:
breaking the genome DNA of each sample to obtain DNA fragments with bands distributed in 500 bp;
adding an adhesive primer combined with an adhesive tail end into the obtained DNA fragment, and adding a tail end leveling reagent for mixing reaction;
designing an initial primer according to the DNA fragment, and respectively connecting a plurality of labels with the designed initial primer to obtain a fusion primer;
connecting the first fusion primer with the head end of the DNA fragment by adopting an A joint, and connecting the second fusion primer with the tail end of the DNA fragment by adopting a P joint;
purifying magnetic beads;
performing PCR amplification on DNA fragments with different fusion primers at the head and the tail;
and (3) after quantitative and sample mixing, agarose gel separation and purification are carried out to obtain a single-cell sequencing library.
2. The multi-sample mixed single cell pooling method according to claim 1, wherein the fusion primer includes an upstream general primer sequence sequentially arranged in a 5 'to 3' direction and a specific upstream primer sequence designed according to a target amplified region in the purified DNA fragment, and a downstream general primer sequence sequentially arranged in a 5 'to 3' direction and a specific downstream primer sequence designed according to a target amplified region in the purified DNA fragment.
3. The multi-sample mixed single cell pooling method of claim 2, wherein the upstream universal primer sequence and the downstream universal primer sequence are each 16S rRNA sequencing primers.
4. The multi-sample mixed single cell banking method according to claim 1, wherein the magnetic bead purification comprises: the DNA fragment was purified using 1.2 times of AMPure XP beads to remove redundant small fragment adaptors and primers from the DNA fragment.
5. The multi-sample mixed single cell pooling method of claim 1, wherein the a-and P-junctions are junctions of Ion Torrent, ion PGM, ion proton or Ion S5/S5XL sequencing platforms.
6. The method for single cell banking of multiple sample mixtures according to claim 1, wherein the concentration of genomic DNA is 0.1 to 150 ng/. Mu.l.
7. The multi-sample mixed single cell banking method according to claim 1, further comprising: designing a matched biological information analysis method based on a database building structure;
the biological information analysis method sequentially comprises the following steps:
acquiring sequencing data of a DNA sequence to be tested;
inputting the sequencing data into a purification model for purification according to a preset filtering rule to obtain purified sequencing data only retaining biological information;
filtering the purified sequencing data according to a preset tag filtering rule to obtain a characterization sequence corresponding to the purified sequencing data;
comparing the characterization sequence with preset tag sequence data to obtain fusion primer information;
respectively carrying out GO enrichment analysis, KO enrichment analysis and WGS analysis based on the fusion primer information to obtain sequence types, sequence distribution and data base distribution characteristics corresponding to the characterization sequence;
and (5) carrying out statistics on the characteristics of the purified sequencing data, the sequence types, the sequence distribution and the data base distribution, and outputting the statistics as a biological information analysis result.
8. The multi-sample mixed single cell pooling method of claim 7 wherein said WGS analysis is achieved by employing one or more of Refseq, GEO Database and Expression Atlas.
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| CN109797437A (en) * | 2019-01-18 | 2019-05-24 | 北京爱普益生物科技有限公司 | A kind of construction method of sequencing library when detecting multiple samples and its application |
| US20190352711A1 (en) * | 2017-04-05 | 2019-11-21 | Genetron Health (Beijing) Co., Ltd. | Method for Rapidly Constructing Amplicon Library Through One-Step Process |
| CN115247281A (en) * | 2021-04-25 | 2022-10-28 | 中国科学院宁波材料技术与工程研究所慈溪生物医学工程研究所 | Preparation method and application of amplicon library |
| WO2023284768A1 (en) * | 2021-07-13 | 2023-01-19 | 北京爱普益生物科技有限公司 | Fusion primer direct amplification method-based human mitochondrial whole genome high-throughput sequencing kit |
| WO2023082070A1 (en) * | 2021-11-09 | 2023-05-19 | 京东方科技集团股份有限公司 | Dna terminal repair, linker reagent, kit and dna library construction method |
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- 2023-12-02 CN CN202311638029.8A patent/CN117661123A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190352711A1 (en) * | 2017-04-05 | 2019-11-21 | Genetron Health (Beijing) Co., Ltd. | Method for Rapidly Constructing Amplicon Library Through One-Step Process |
| CN109797437A (en) * | 2019-01-18 | 2019-05-24 | 北京爱普益生物科技有限公司 | A kind of construction method of sequencing library when detecting multiple samples and its application |
| CN115247281A (en) * | 2021-04-25 | 2022-10-28 | 中国科学院宁波材料技术与工程研究所慈溪生物医学工程研究所 | Preparation method and application of amplicon library |
| WO2023284768A1 (en) * | 2021-07-13 | 2023-01-19 | 北京爱普益生物科技有限公司 | Fusion primer direct amplification method-based human mitochondrial whole genome high-throughput sequencing kit |
| WO2023082070A1 (en) * | 2021-11-09 | 2023-05-19 | 京东方科技集团股份有限公司 | Dna terminal repair, linker reagent, kit and dna library construction method |
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