CN106591447B - Single cell whole genome sequencing method - Google Patents
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Abstract
The invention provides a single cell whole genome sequencing method, which comprises the following steps: separating and lysing the single cells; performing single cell whole genome amplification on the whole genome DNA of the single cell; carrying out quality detection on the amplification product; capturing the amplification products qualified by detection and constructing a DNA sequencing library; high-throughput sequencing; wherein, the quality detection of the amplification product comprises the following steps: and detecting the length of the DNA fragment in the amplification product, comparing the length of the maximum DNA fragment with a preset value, and judging whether the quality of the amplification product is qualified, wherein the quality of the amplification product with the length of the maximum DNA fragment larger than the preset value is qualified after the quality detection. The method for detecting the quality of the amplified product has simple steps, does not need to synthesize additional primers, has low cost, needs less initial amount for detecting the amplified product, has accurate and effective detection result and has strong practicability.
Description
Technical Field
The invention relates to the field of single cell sequencing, in particular to a sequencing method of a single cell whole genome.
Background
Single cell whole genome sequencing is a new technology for amplifying and sequencing whole genome at single cell level, and the method comprises the steps of amplifying separated trace whole genome DNA of a single cell, obtaining a complete genome with high coverage rate and then carrying out high-throughput sequencing for revealing cell population difference and cell evolution relation. An annual special report is published in Nature methodology in 1 month 2014, and the application of single-cell sequencing is the most important methodological development in 2013, so that the importance of a single-cell sequencing technology can be seen. A paper on Multiple Annealing and Looping-around Amplification technology (MALBAC), which can reduce PCR Amplification bias of the whole genome of a single cell, so that 93% of the genome in a single cell can be sequenced, so that it becomes easier to detect smaller DNA sequence variations in a single cell, and thus genetic differences between individual cells can be found, was published in 2012 by professor of harvard university, academy of sciences of usa, and dawn professor of sierra-basic Amplification Cycles. Such differences can help explain cancer exacerbation mechanisms, germ cell formation mechanisms, and even difference mechanisms of individual neurons, and have wide application prospects.
Because the DNA content of the genome of a single cell is picogram-level, and the current sequencing technology requires the initial DNA amount to be microgram-level, the genome of a single cell must be amplified by Whole Genome Amplification (WGA) to a sufficient amount for sequencing, which puts higher demands on the nucleic acid amplification technology (amplification technology). The quality of single cells can greatly affect the pre-amplification of single cells and the coverage rate of a whole genome, and in the case of trace DNA, any degradation, sample loss or pollution can bring very serious influence on a sequencing result, so that the success rate of amplification reaching 100% cannot be ensured. In view of the difficult quality of genomic DNA of single cells (-6 pg) and the expensive cost of sequencing, accurate estimation of the coverage of whole genome amplification of single cells before sequencing or even pooling is very important.
Chinese patent with publication date of 2014, 4, and 30, and publication number of CN102533960B discloses a method and kit for analyzing single cell genome. The patent adopts a Housekeeping Gene detection method to carry out quality detection on the amplification product of the single cell whole genome. The method needs to perform PCR amplification on 10 genes on 10 different chromosomes respectively, and each reaction needs to perform independent amplification and electrophoresis detection respectively; a template amount of 15ng is required for each reaction, and 150ng of genomic amplification product is consumed to complete quality testing of a single sample. This method is not only cumbersome, but also wastes reagents and single cell genome amplification samples.
The Chinese patent application with publication number CN104762405A, publication date of 2015, 7 and 8, discloses a method and a kit for identifying the quality of an amplified product after single cell genome amplification. The patent adopts 5 pairs of specific primers of conserved segments on a genome, puts the primers in an amplification system, performs PCR amplification by taking a whole genome amplification product as a template, performs electrophoresis detection on the 5 amplification products, and judges the quality of the whole genome amplification product according to a detection result. The method needs to perform PCR amplification on 5 genes positioned on 5 different chromosomes, the PCR is operated in the same reaction system, although the waste of templates can be reduced, a plurality of reactions in the same system are easy to cause nonspecific amplification. And the resolution ratio of electrophoresis is not high, which easily causes misjudgment and influences the accuracy of the subsequent sequencing result.
Disclosure of Invention
The invention mainly aims to provide a single cell whole genome sequencing method aiming at the defects in the prior art, the sequencing method can detect the quality of an amplification product before library construction and sequencing, the cost is low, the initial quantity required by quality control detection is low, and the operation is simple.
A single cell whole genome sequencing method comprises the following steps:
separating and cracking the single cell to obtain the whole genome DNA of the single cell;
performing single cell whole genome amplification on the whole genome DNA of the single cell to obtain a whole genome amplification product;
carrying out quality detection on the amplification product;
capturing the amplification products qualified by detection and constructing a DNA sequencing library;
performing high-throughput sequencing on the data after the database is built;
wherein, the quality detection of the amplification product comprises the following steps: and detecting the length of the DNA fragment in the amplification product, comparing the length of the maximum DNA fragment with a preset value, and judging whether the quality of the amplification product is qualified, wherein the quality of the amplification product with the length of the maximum DNA fragment larger than the preset value is qualified after the quality detection.
Preferably, the comparing the length of the largest DNA fragment with a preset value means: and (3) establishing a curve by taking the length of the DNA fragment as an abscissa and the abundance of the DNA fragment as an ordinate, and evaluating whether the quality of the amplification product is qualified or not by comparing the abscissa of the tail peak position with a preset value.
Depending on the method of detecting the length of the DNA fragment, the abundance of the DNA fragment can be expressed by selecting various indicators, for example, Fluorescence intensity (Fluorescence intensity) as an indicator for expressing the abundance.
Preferably, the preset value is 10380bp, and if the length of the maximum DNA fragment is more than 10380bp, the quality of the amplification product is qualified.
Preferably, when the length of the DNA fragment in the amplification product is detected, a low-fragment marker and a high-fragment marker are added to the amplification product, wherein the length of the high-fragment marker is the same as a preset value.
As is well known to those skilled in the art, when detecting the length of a DNA fragment, a low-fragment marker and a high-fragment marker are usually added to the DNA to be detected as reference standards. When the preset value is set, the preset value can be used as the size of the high-fragment marker and added into the DNA fragment to be detected, so that the length of the maximum DNA fragment and the size of the preset value can be observed and compared conveniently. Preferably, when a marker with a preset value of 10380bp is added, qualified amplification products are selected by comparing the length of the maximum DNA fragment with the size of the preset value, and then library construction and sequencing are carried out.
According to the general requirement of single cell high-throughput sequencing, the original sequencing depth of targeted sequencing needs to reach 3000x of the size of a targeted region, wherein the coverage rate of 100x is more than 70%, and the original sequencing depth of a whole exon needs to reach 10G, wherein the coverage rate of 1x is more than 70%. On the basis of the coverage rate, the quality of the single cell whole genome amplification product can be considered to be qualified.
The person skilled in the art can choose different ways to detect the length of the DNA fragment according to the known techniques in the art and the size of the predetermined value. Preferably, the length of the DNA fragment in the amplification product is detected by a DNA analysis chip based on capillary electrophoresis. More preferably, the length of the DNA fragment in the amplification product is detected by using an Agilent 2100 bioanalyzer and a corresponding kit, wherein the kit comprises a DNA analysis chip based on capillary electrophoresis. Preferably, the Kit may be an Agilent High Sensitivity DNA Kit.
More preferably, the amplification product is diluted to 1-4 ng/muL by nuclease-free water and then the length of the DNA fragment is detected. The diluted concentration can be selectively injected according to a specific chip for detecting the length of the DNA fragment.
More preferably, after the single cell whole genome is amplified, the amplified product is separated and recovered by using a magnetic bead purification kit or a gel cutting recovery kit, and then a part of the amplified product is diluted and then the length of the DNA fragment is detected.
Preferably, the single cell whole genome amplification is performed by using an MALBAC method.
The invention detects whether the amplification product of the whole genome of the single cell is qualified or not by detecting the length of the maximum DNA fragment, thereby detecting the quality of the single cell before the step of constructing the library of the high-throughput sequencing, carrying out the subsequent library construction on the qualified amplification product, eliminating a great amount of unqualified amplification samples without the step of subsequent high-throughput sequencing on the unqualified amplification product, controlling the quality of the downstream on-computer sequencing library and avoiding unnecessary waste to a great extent. The method for detecting the quality of the amplified product has simple steps, does not need to synthesize additional primers, has low cost, needs less initial amount for detecting the amplified product, has accurate and effective detection result and has strong practicability.
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FIG. 1 is a diagram of chip identification of qualified amplification products;
FIG. 2 is a diagram of chip identification of an unqualified amplification product;
in FIGS. 1 and 2, the abscissa represents the size of the DNA fragment and the ordinate represents the fluorescence intensity (fluorescence intensity);
in the figure, 1-low fragment marker peak, 2-high fragment marker peak, 3-tail peak;
FIG. 3 is a diagram of the chip identification of the single cell whole genome amplification products of cells 1-6;
FIG. 4 is a diagram of chip identification of single cell whole genome amplification products of cells 1-11;
FIG. 5 is a diagram of chip identification of single cell whole genome amplification products of cell 2-1;
FIG. 6 is a diagram of chip identification of the single cell whole genome amplification product of cell 2-2.
Detailed Description
The invention is further illustrated by the following specific examples: the experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1
The embodiment of the invention mainly comprises the following steps:
(1) cutting 1 CTC single cell from each CTC sample by using a laser micro-cutting device PALM (PALM MicroBeam, Zeiss), adding 5 microliter of lysate (from MALBAC kit) to cover the target cell, performing vortex centrifugation to precipitate the lysate and the cell to the bottom of the tube, heating at 50 ℃ for 50min, then heating at 80 ℃ for 10min, and obtaining the whole genome DNA of the single cell after complete lysis.
(2) Adding 30. mu.L of pre-amplification mixture (from MALBAC kit) to the whole genome DNA of each single cell, performing the first single cell whole genome amplification according to Table 1, adding 30. mu.L of pre-amplification mixture (from MALBAC kit), performing the second single cell whole genome amplification according to Table 2, and obtaining a whole genome amplification product:
TABLE 1 first Single cell Whole genome amplification
Table 2 second single cell whole genome amplification.
(3) After the whole genome amplification product is recovered, part of the recovered amplification product is diluted to (1-4) ng/mu L by using nuclease-free water, and then a chip is identified by using an Agilent 2100 bioanalyzer and a detection kit matched with the bioanalyzer, so that the length of the DNA fragment is detected. The matched detection kit comprises a capillary electrophoresis chip. In the embodiment of the invention, the preset value is 10380bp, the low fragment marker is 35bp, the High fragment marker is 10380bp, and the matched detection Kit is Agilent High Sensitivity DNA Kit (from Agilent). Comparing the length of the largest DNA fragment with the size of 10380bp, thereby performing quality detection on the amplified product. The detection results are shown in fig. 1 and fig. 2, and if the position of the tail peak 3 is before the peak 2 of the high fragment marker, the quality detection result is unqualified; the quality test result is passed if the position of the tail peak 3 is after the high fragment marker peak 2.
(4) The remaining amplification products were captured and a DNA sequencing library was constructed.
(5) Performing high-throughput sequencing on the data after library establishment by using an illumina on-machine kit, selecting a Hiseq4000PE150 double-end sequencing mode by using the on-machine sequencing, simultaneously running 2 flowcells, wherein each Flowcell is 8 lanes, each lane can be mixed with libraries with different indexes to perform sequencing according to requirements, the theoretical data of each lane is produced to 90G, and the total running time is less than 3.5 days.
Example 2 Targeted Single cell sequencing
14 single CTC cells were isolated and lysed according to step (1) of example 1 above to obtain whole genomic DNA of each single CTC cell. The whole genome DNA of each CTC single cell was then amplified according to step (2) of example 1 above to obtain a whole genome amplification product. The PCR amplification products were each run with 2% low melting point Gel, the amplification products of 350bp to 450bp size were excised, and the amplification products were recovered with QIAquick Gel Extraction Kit sol (from Qiagen), and then subjected to chip identification according to the procedure (3) of example 1 described above, and the DNA fragment lengths of the amplification products were examined, as shown in FIGS. 3 and 4, with the results similar to those shown in FIGS. 3 and 4. Each of the remaining amplification products was subjected to targeted capture amplification using a MygeneSeq amplification capture kit (from Hibiscus Biotech Co., Ltd.) following step (4) of example 1 above. The MygeneSeq amplification capture kit comprises the following primers (targeting region 105KB) specific to 36 common tumor mutant genes:
| ACVR2A | SMAD4 | TP53 | NRAS | p65(NFKB3) | ATM |
| PRKCB | AR | SUMO1 | PIK3CA | APC | MLH1 |
| BRAF | HDAC1 | ABL1 | IGF2 | TCF7L2 | MLH3 |
| MFS2 | KRAS | STAT1 | MYC | ERBB3 | MSH2 |
| ERBB2 | TGFBR1 | AMER1 | PTEN | JUN | MSH3 |
| TRIM63 | CDK1 | SOX9 | PMS2 | ARID1A | MSH6 |
after targeted capture amplification, a DNA sequencing library was constructed using the library construction Kit NEXTflex Rapid DNA-Seq Kit (from BiooScientific, Inc.), respectively.
High throughput sequencing was performed using the illumina machine kit according to step (5) of example 1 above, 3000x or more per library (0.5G raw machine-out data), the mean depth of the targeted region was 4000x to 20000x, statistically >100x coverage, and the results are shown in table 3, compared to the results of the corresponding chip identification in step (3).
TABLE 3 DNA fragment length vs. target sequencing coverage
As can be seen from Table 3, the single-cell amplification products with the maximum DNA fragment length of more than 10380bp are qualified amplification products, such as cells 1-11, cells 1-12 and cells 1-14, and the coverage rate of 100x after library construction and sequencing can reach more than 80%, which indicates that the cell quality is good and the amplification product quality is qualified.
Example 3 Whole exon Single cell sequencing
13 single CTC cells were isolated and lysed according to step (1) of example 1 above to obtain whole genomic DNA for each single CTC cell. The whole genome DNA of each CTC single cell was then amplified according to step (2) of example 1 above to obtain a whole genome amplification product. Each whole genome amplification product was recovered using Ampure xp beads kit (from Beckman Co.), the amplification product was recovered using 1.4 × volume separation, and then chip identification was performed according to the above step (3) of example 1, and the DNA fragment length of the amplification product was measured, and the results are shown in FIGS. 5 and 6, and the rest of the measurement results are similar to those in FIGS. 5 and 6. Each of the remaining amplification products was individually subjected to whole exon capture and DNA sequencing library construction according to the step (4) of example 1 above. The Exon banking kit is the SureSelect Human All Exon V5+ UTRs kit (from agilent).
High throughput sequencing was performed using the illumina machine kit according to step (5) of example 1 above, 9G raw off-machine data was measured for each library, the mean depth of the targeted region was 44x to 85x, 40x or more was measured for each library, and a coverage of >1x was counted and compared to the results of the corresponding chip identification in step (3) and the results are shown in table 4.
TABLE 4 DNA fragment length vs. Whole exon sequencing coverage
As can be seen from Table 4, the single cell amplification product with the maximum DNA fragment length of more than 10380bp is a qualified amplification product, such as cell 2-2, cell 2-8 and cell 2-10, and the 1x coverage rate can reach more than 70% after the library construction and sequencing, which indicates that the cytoplasm amount is good and the amplification product quality is qualified.
It should be noted that the above embodiments are designed to verify whether the quality detection result and the coverage result correspond to each other. Other libraries and high throughput sequencing kits commonly used by those skilled in the art may also be used in the actual sequencing process.
The other chip identification diagrams of the present invention are similar to those in FIGS. 3 to 6, and are not repeated herein. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the embodiments of the present invention and are intended to be within the scope of the embodiments of the present invention.
Claims (8)
1. A single cell whole genome sequencing method is characterized in that: the method comprises the following steps:
separating and cracking the single cell to obtain the whole genome DNA of the single cell;
performing single cell whole genome amplification on the whole genome DNA of the single cell to obtain a whole genome amplification product;
carrying out quality detection on the amplification product;
capturing the amplification products qualified by detection and constructing a DNA sequencing library;
performing high-throughput sequencing on the data after the database is built;
wherein, the quality detection of the amplification product comprises the following steps: detecting the length of the DNA fragment in the amplification product, comparing the length of the maximum DNA fragment with a preset value, and judging whether the quality of the amplification product is qualified or not, wherein the length of the maximum DNA fragment is greater than the preset value, and the quality of the amplification product is qualified after the quality detection;
the preset value is 10380bp, and if the length of the maximum DNA fragment is more than 10380bp, the quality of an amplification product is qualified.
2. The single cell whole genome sequencing method of claim 1, wherein: the comparison of the length of the largest DNA fragment with a preset value is as follows: and (3) establishing a curve by taking the length of the DNA fragment as an abscissa and the abundance of the DNA fragment as an ordinate, and judging whether the quality of the amplification product is qualified or not by comparing the abscissa of the tail peak position with a preset value.
3. The single cell whole genome sequencing method of claim 1, wherein: when the length of the DNA fragment in the amplification product is detected, adding a low-fragment marker and a high-fragment marker into the amplification product, wherein the length of the high-fragment marker is the same as a preset value.
4. The single cell whole genome sequencing method of claim 1, wherein: the length of the DNA fragment in the amplification product is detected by a DNA analysis chip based on capillary electrophoresis.
5. The method for sequencing the whole genome of the single cell according to claim 4, wherein: the length of the DNA fragment in the amplification product is detected by using an Agilent 2100 bioanalyzer and a corresponding kit, wherein the kit comprises a DNA analysis chip based on capillary electrophoresis.
6. The single cell whole genome sequencing method of claim 5, wherein: and diluting the amplification product to 1-4 ng/mu L by nuclease-free water, and then detecting the length of the DNA fragment.
7. The single cell whole genome sequencing method of claim 5, wherein: after the single cell whole genome is amplified, the amplified product is separated and recovered by using a magnetic bead purification kit or a gel cutting recovery kit, and then a part of the amplified product is diluted to detect the length of the DNA fragment.
8. The single cell whole genome sequencing method of claim 1, wherein: the single cell whole genome amplification adopts an MALBAC method for amplification.
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| CN105483115A (en) * | 2014-09-19 | 2016-04-13 | 深圳华大基因科技有限公司 | Kit for single cell genome-wide amplification and application thereof |
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| CN104762405A (en) * | 2015-04-22 | 2015-07-08 | 北京嘉宝仁和医疗科技有限公司 | Method and kit for quality appraisal for amplification products after single cell genome amplification |
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Inventor after: Zhang Xianglin Inventor after: Zhao Huiru Inventor after: Zhan Xuefeng Inventor after: Chen Changyue Inventor after: Li Jing Inventor after: Gan Guangli Inventor after: Yang Fei Inventor before: Chen Changyue Inventor before: Li Jing Inventor before: Gan Guangli Inventor before: Zhang Xianglin Inventor before: Yang Fei Inventor before: Zhan Xuefeng Inventor before: Zhao Huiru |
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| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220630 Address after: 201318 No.3, Lane 3399, Kangxin Road, Pudong New Area, Shanghai Patentee after: SHANGHAI MAJORBIO BIO-PHARM TECHNOLOGY CO.,LTD. Address before: 201321 building 3, Lane 3399, Kangxin Road, Pudong New Area, Shanghai Patentee before: SHANGHAI MAJORBIO BIO-PHARM TECHNOLOGY CO.,LTD. |