CN109957615B - Method for capturing target area of single cell genome - Google Patents
Method for capturing target area of single cell genome Download PDFInfo
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- CN109957615B CN109957615B CN201811596459.7A CN201811596459A CN109957615B CN 109957615 B CN109957615 B CN 109957615B CN 201811596459 A CN201811596459 A CN 201811596459A CN 109957615 B CN109957615 B CN 109957615B
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Abstract
The invention relates to a method for capturing target areas of a single cell genome. Specifically, the whole genome amplification is carried out on a single cell genome, the amplified genome is broken, the tail end is repaired, the 3' end is added with A, the joint is added, the PCR amplification is carried out, the target region is captured, and the PCR amplification and the sequencing are carried out on the DNA fragment captured by the target region. The method provided by the invention can improve the coverage of capturing the sequencing result and the specificity of the capturing result.
Description
Technical Field
The invention relates to a method for capturing target areas of single-cell genome, and belongs to the technical field of gene detection.
Background
Single cell sequencing was one of the most promising technologies in 2011 by Nature Methods and in 2013 by Science as the six most interesting fields of the year, which is increasingly favored by researchers, and related research results are also well-documented in top-level journals, which all show that single cell sequencing has gradually become a popular point of research.
Single cell sequencing techniques can dissect cell heterogeneity, revealing the variation that occurs in the tumor cell genome. Helping us describe the clonal structure of tumor cells and track the progression and spread of the disease. The technology has been widely used in the fields of assisted reproduction, cancer, neurology, immunology, etc.
The single cell target region capturing sequencing is a technical means of amplifying trace whole genome DNA of an isolated single cell, customizing a probe of a target genome region after obtaining a complete genome with high coverage rate, hybridizing with the genome DNA, enriching the target region DNA and then carrying out high-throughput sequencing. The single cell target region sequencing technology is used for revealing cell population difference and cell evolution relationship, is also helpful for finding and verifying relevant candidate genes or relevant sites of diseases, particularly cancers, and has great application potential in clinical diagnosis and drug development [1]. The advantage of target region capture sequencing is that only genomic regions of interest are studied, and the sequencing depth is deeper and the results are more accurate when the same amount of data is obtained; and compared with conventional genome sequencing, the method has the advantages of lower cost and simpler data interpretation.
Single cell genome target region capture sequencing still differs significantly from the effect of target region capture sequencing of the genome of a normal sample (a sample that does not require amplification of the genome, the amount of genome being sufficient to perform target region capture sequencing). First, the single cell genome has only two copies, with a total of about 6pg. During the amplification process, the complete genome may not be reproducible. Second, if the missing portion is just the area of the chip probe, then capture specificity is affected, resulting in a waste of data volume. Moreover, the coverage of single-cell target region capture sequencing is lower than that of common genome under the same sequencing depth.
Reference to the literature
[1]Wang,Y.et al.Clonal evolution in breast cancer revealed by single nucleus genome sequencing.Nature 512,155–160(2014).
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a single-cell genome target region capturing method, which improves the coverage of capturing sequencing results and the specificity of capturing results by optimizing a whole genome amplification flow, a library construction flow and a target region capturing flow.
1. A method of single cell genomic target region capture comprising:
step A: isothermal amplification is carried out on the single-cell genome by adopting a multiple displacement amplification method, so as to obtain an amplified genome;
and (B) step (B): breaking 3 mug amplified genome to obtain fragmented DNA fragments;
step C: performing end repair on the fragmented DNA fragment to obtain a blunt-end DNA fragment;
step D: carrying out 3 'end addition A on the blunt end DNA fragment to obtain a DNA fragment with 3' end addition A;
step E: the DNA fragment with the 3' -end added with A is subjected to joint adding, so that a DNA fragment with the joint is obtained;
step F: carrying out PCR amplification on the spliced DNA fragments to obtain amplified DNA fragments;
step G: capturing the amplified DNA fragment in a target area to obtain a captured DNA fragment;
step H: carrying out PCR amplification on the captured DNA fragments to obtain PCR amplification products;
step I: sequencing the PCR amplification product;
wherein, the isothermal amplification condition in the step A is 30 ℃ for 2 hours;
the cycle number of PCR amplification in the step F is 4-6 cycles;
the condition for capturing the target area in the step G is that the target area is reacted for 16 to 24 hours at the temperature of 65 ℃.
2. The method of item 1, wherein step a further comprises, after:
step A-1: the amplified genome is subjected to PCR amplification by using a primer with a nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 44.
3. The method according to any one of items 1 or 2, wherein the amount of the DNA fragment ligated in the step F is 100 to 250ng.
4. The method according to any one of items 1 or 2, wherein the amount of the DNA fragment ligated in the step F is 150 to 200ng.
5. The method according to any one of items 1 to 4, wherein the number of cycles of PCR amplification in the step F is 5 cycles.
6. The method according to any one of items 1 to 5, wherein the condition for capturing the target region in the step G is 65℃for 24 hours.
7. The method according to item 1, wherein the step of purifying the DNA fragment is comprised between step B and step C, between step C and step D, between step D and step E, between step E and step F, between step F and step G, between step G and step H, between step H and step I, and/or after step I.
The invention has the beneficial effects that: compared with the prior art, the invention provides a single-cell genome target region capturing method, which shortens the capturing sequencing time and improves the coverage and the specificity of capturing sequencing results by optimizing the whole genome amplification flow, the library construction flow and the target region capturing flow.
Detailed Description
The present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention provides a method for capturing a target area of a single cell genome, which comprises the following steps:
step A: amplifying the single cell genome by adopting a multiple displacement amplification method to obtain an amplified genome;
and (B) step (B): breaking 3 mug amplified genome to obtain fragmented DNA fragments;
step C: performing end repair on the fragmented DNA fragment to obtain a blunt-end DNA fragment;
step D: carrying out 3 'end addition A on the blunt end DNA fragment to obtain a DNA fragment with 3' end addition A;
step E: the DNA fragment with the 3' -end added with A is subjected to joint adding, so that a DNA fragment with the joint is obtained;
step F: carrying out PCR amplification on the spliced DNA fragments to obtain amplified DNA fragments;
step G: capturing the amplified DNA fragment in a target area to obtain a captured DNA fragment;
step H: carrying out PCR amplification on the captured DNA fragments to obtain PCR amplification products;
step I: sequencing the PCR amplification product;
wherein the amplification conditions in the step A are that the reaction is carried out for 2 hours at 30 ℃;
the cycle number of PCR amplification in the step F is 4-6 cycles;
the condition for capturing the target area in the step G is that the target area is reacted for 16 to 24 hours at the temperature of 65 ℃.
Such multiplex displacement amplification methods can be found, for example, in U.S. Pat. No. 3,182,62. The multiplex displacement amplification method generally includes contacting a set of primers, a DNA polymerase, and a target nucleic acid in solution; and amplifying the solution such that an amplification reaction of the target nucleic acid is performed to replicate the target nucleic acid; in the case of single cell genome amplification, it is generally considered that a longer amplification reaction time is required, for example, 8 hours as specified in the operating instructions for REPLI-g Single Cell Kit (Qiagen). However, the inventors of the present invention have found that when the amplification reaction time exceeds 2 hours, the yield of the amplification reaction product has reached a substantial saturation, and preferably the amplification time is 2 hours.
Preferably, step A further comprises step A-1: the amplified genome is subjected to PCR detection by using the primers with nucleotide sequences shown as SEQ ID NO. 1-SEQ ID NO. 44, the integrity of the amplified genome is further detected, and the waste of time and cost caused by using the amplified genome with poor amplification effect for library construction and subsequent research is effectively avoided.
In conventional library construction methods, the initial amount of library construction is generally considered to be 100ng to 2 μg in order to meet the requirements of library quality control, on-machine sequencing, and other assays. For example, the library starting amount recommended in the TruSeq Nano DNA kit (Illumina) operating Specification is 100 to 200ng,TruSeq DNA PCR-the library starting amount recommended in the Free kit (Illumina) operating Specification is 1 to 2. Mu.g. In the present invention, the inventors found that coverage of a target region in a genome can be improved by increasing the library starting amount, preferably 3. Mu.g.
The step B is interrupted using an ultrasonic or enzymatic cutting method, and other interruption methods known to those skilled in the art may be used in this step.
And C, performing end repair by using a buffer solution, T4DNA Polymerase, klenow DNA Polymerase, T4 polynucleotide Kinase and dNTPs, wherein a reaction system and reaction conditions are performed according to conventional end repair conditions.
The step D is carried out by adding A at the 3 'end by using buffer solution, klenow Exo (-), dATP, and the reaction system and reaction conditions are carried out according to the conventional step of adding A at the 3' end.
The step E uses buffer solution, a linker and T4DNA Ligase for linker adding, wherein the linker is used in the conventional sequencing and library building step, and any linker capable of realizing the step is used without special limitation. The reaction system and reaction conditions are carried out according to the conventional grafting step.
In the present invention, the amount of the adaptor-added DNA fragment obtained in the step F is 100 to 250ng, and it is generally considered that the whole adaptor-added DNA fragment is directly subjected to PCR amplification in the step F, preferably the amount of the adaptor-added DNA fragment is 150 to 200ng, and the number of PCR amplification cycles is 4 to 6 cycles, preferably the number of PCR amplification cycles is 5.
The hybridization is performed in the step G using an existing hybridization kit, for example, a reaction condition defined in the instructions of SureSelect Target Enrichment System kit (Agilent Technologies Co.) is 65℃for 48 to 72 hours, and preferably, the condition for capturing hybridization in the step G is 65℃for 16 to 24 hours.
Preferably, the DNA fragment may be purified between the steps of the method of the invention for single cell genomic target region capture, e.g. between step B and step C, between step C and step D, between step D and step E, between step E and step F, between step F and step G, between step G and step, between step H and step I, and/or after step I.
Example 1
Library construction and hybridization capture were performed using SureSelectXT Reagent kit, HSQ,96 (alignment Co.).
1 Single cell Gene amplification
1.1 treatment of sorted cells:
cells were directly sorted into 3 μl PBS in an ultra clean bench without freeze thawing.
1.2 Single cell Whole Genome Amplification (WGA):
whole genome amplification was performed using MDA using the REPLI-g Single Cell Kit kit (Qiagen).
a) Preparing DLB Buffer: to the supplied tube was added 500. Mu.L of water, thoroughly mixed and centrifuged briefly. DLB Buffer can be stored at-20deg.C for 6 months.
b) All buffers and reagents were vortexed prior to use.
c) The temperature of the thermal cover of the PCR instrument was adjusted to 70 ℃.
d) A sufficient amount of Buffer D2 (denatured Buffer) was prepared, and Buffer D2 was allowed to stand at-20℃for a maximum of 3 months. Each reaction requires 3. Mu.L of Buffer D2
e) The cell volume was made up to 4 μl with kit-supplied PBS.
f) Add 3. Mu.L Buffer D2, gently mix and briefly centrifuge.
g) Amplification was carried out at 65℃for 10 minutes.
h) Add 3. Mu.L of reaction stop solution, gently mix, briefly centrifuge and place on ice.
REPLI-g sc DNA Polymerase was placed on ice, the other reagents melted at room temperature, vortexed, and centrifuged briefly.
i) Mix is prepared and centrifuged briefly after preparation.
Add 40. Mu.L mix to 10. Mu.L denatured DNA in step h) and amplify for 2 hours at 30 ℃. Left at 65℃for 3 minutes to inactivate REPLI-g sc DNA Polymerase. mu.L of water (40. Mu.L) was added thereto, and the mixture was purified by 1X Ampure beads, and the purified product was dissolved in 40. Mu.L of EB to obtain amplified genome.
1.3 WGA product quality inspection
Concentration and fragment size were measured using Qubit BR and agilent 2100.
1.4 detection of amplification efficiency
Primer list
The reaction system:
the reaction conditions were as follows:
PCR product amplification was detected using 2% gel electrophoresis.
2 library construction
2.1 breaking
3. Mu.g of amplified genome was taken, 80. Mu.L of disruption system was used, and 200bp fragmentation was performed. 1.8XAmpure beads were added for purification, and the resulting mixture was dissolved in 50. Mu.L of water to obtain a fragmented DNA fragment.
2.2 terminal repair
Reaction conditions: reaction at 20℃for 30 min, purification by addition of 180. Mu.L Ampure beads (1.8X), 70% alcohol (Tvice), 32. Mu.L ddH 2 O elution to obtain blunt-ended DNA fragments.
2.3 3' -end is added with A
Reaction conditions: reaction at 37℃for 30 min, purification by addition of 90. Mu.L Ampure beads (1.8X), 70% alcohol (Tvice), 29.5. Mu.LddH 2 O elution to obtain DNA fragment with 3' end added with A.
2.4 add-on connector
Reaction conditions: reaction at 20℃for 15 min, purification by addition of 90. Mu.L Ampure beads (1.8X), 70% alcohol (Tvice), 32. Mu.LddH 2 O elution to obtain the ligated DNA fragment.
2.5 PCR amplification
Reaction conditions:
after the reaction was completed, 45. Mu.L of Ampure beads (0.9X) purified, 70% alcohol (Tvice), 26. Mu.L of ddH were added to each sample 2 O elution to obtain DNA library.
2.6 library quality detection
The constructed DNA library was examined for fragment size using an agilent 2100 bioanalyzer.
3 target area capture
The chip used was a 1.4M tumor chip customized at Agilent, designated SureSelect Hyb3.
3.1 library preparation
750ng of DNA library was concentrated in vacuo to a volume of 3.4. Mu.L.
3.2 Block Mix preparation
3.3 blocking reactions
The reaction was carried out at 95℃for 5 minutes and at 65℃for 5 minutes with a hot cover to 105 ℃.
3.4 Hybridization Mix preparation
After being ready, the mixture is placed on an ice box for standby, and is preheated to be clear at 65 ℃ before being used.
3.5 hybrid Capture
Keeping the product in the step 3.4 at 65 ℃ all the time, adding 5 mu L Capture Library, then adding 15 mu L of hybridization Mix rapidly, blowing and sucking uniformly, covering and preheating to 105 ℃, and reacting at 65 ℃ for 24 hours to obtain a hybridization product.
3.6 SureSelect Wash Buffer preparation
600 mu L SureSelect Wash Buffer 2 was placed in an EP tube and preheated at 65℃for further use. 200 mu L SureSelect Wash Buffer 1 was mounted on an EP tube for further use.
3.7 MyOne Streptavidin T1 magnetic bead preparation
The supernatant was removed by magnetic rack, washed with 200. Mu. L SureSelect Binding Buffer for every 50. Mu.L of beads, repeated three times, resuspended with 200. Mu. LSureSelect Binding Buffer for every 50. Mu.L of beads, and mounted in a 1.5mL EP tube.
3.8 Binding DNA
200 mu L of the magnetic beads prepared in the step 3.7 are added into the hybridization product obtained in the step 3.5, uniformly mixed, transferred into the rest magnetic beads, and reacted for 30 minutes at 25 ℃ at a constant temperature mixer at 160 rpm.
3.9 washing of magnetic beads
Taking down the sample in the step 3.8, centrifuging by using a palm centrifuge, removing the supernatant by using a magnetic rack, adding 200 mu L SureSelect Wash Buffer 1, uniformly mixing, and then placing the mixture on a constant temperature mixing instrument for reaction for 15 minutes at 25 ℃;
taking down a sample, removing the supernatant on a magnetic rack, adding 200 mu L SureSelect Wash Buffer 2 (65 ℃) for reaction for 10 minutes, and repeating for three times;
after adding 20. Mu.L of nucleic-free water and mixing, the hybridization product was obtained and placed on ice for use.
3.10 PCR reaction and product purification
The reaction conditions were as follows:
after the reaction, the supernatant was purified by 0.9XAmpure beads, 70% alcohol (Tvice), and 22. Mu.L EB eluted.
3.11 Capture library quality control
The capture library was assayed for fragment size using an Agilent 2100 bioanalyzer.
Comparative example 1
Comparative example 1 differs from the example in that: single cell amplification k) step amplification temperature was 8 hours and library construction and target region capture was performed using SureSelectXT Reagent kit, HSQ,96, the procedure being identical to that described in the product specification.
Comparative example 2
Comparative example 2 differs from the examples in that: library construction and target region capture were performed using normal samples using SureSelectXT Reagent kit, HSQ,96, and the procedure was identical to that described in the product specification.
Where M1 is the sample number of comparative example 1, M2 is the sample number of the example (using the method of the present invention), and M3 is the sample number of comparative example 2. The quality control results of sequencing are shown in the following table, under the condition of the same sequencing data quantity, the data quality control of the samples M1, M2 and M3 can be qualified, wherein the data values of the samples M2 and M3 in various data indexes are basically consistent through the comparison of quality control data, the data indexes of the sample M1 are poorer, particularly, the capturing efficiency of target areas of the samples M2 and M3 is 77.59 percent and 80.7 percent respectively in the aspect of capturing specificity, the capturing efficiency of the sample M1 is 64.2 percent, and the capturing specificity of the sample M2 is basically consistent with that of the sample M3 and higher than that of the sample M1. For the coverage of the target area, the coverage of the target area of the sample M1 is 85.39%, the coverage of the target area of the sample M2 and the target area of the sample M3 are 99.31% and 99.31% respectively, the sample M2 basically covers the target area and is obviously higher than that of the sample M1, so that the single-cell target area capturing sequencing result is obviously improved in the aspects of the coverage of the target area and the capturing specificity and is close to the genome capturing sequencing result of a normal sample by the method.
Raw Reads: the number of Reads originally off the machine;
clear Reads: the number of the filtered Reads;
clean Reads Rate (%): the clear Reads obtained after filtering accounts for the ratio of Raw Reads;
ns Reads: the number of Reads with the removed N content being more than 5%;
ns Reads Rate (%): the proportion of the removed Reads with the N content being more than 5 percent is the proportion of the Raw Reads;
raw Q30Bases Rate (%): a ratio of Bases with a sequencing quality value greater than 30 (error rate less than 0.1%) to total Bases (Raw Bases) in Raw Reads;
clean Q30Bases Rate (%): the proportion of Bases with a sequencing quality value greater than 30 (error rate less than 0.1%) in clear Reads to total Bases (clear Bases);
target Region: genomic target region capture region size
Reads Mapped To Genome: the number of Reads aligned to the reference genome;
map Rate (%): percentage of bases aligned to the reference genome;
capture Specificity (%): the number of Reads aligned to the genomic target capture region is the ratio of the number of Reads aligned to the genomic Reads;
duplex Rate (%): the number of Reads removed from PCR repeats is a proportion of the Mapped Reads;
uniq Rate (%): the ratio of Reads aligned to the unique position of the genome to Reads aligned after removal of PCR repeats;
coverage Of Target Region (%): average sequencing depth of genomic target capture region Uniq Reads, i.e., data depth for subsequent analysis.
While the foregoing description illustrates and describes the preferred embodiments of the present invention, as noted above, it is to be understood that the invention is not limited to the forms disclosed herein but is not to be construed as excluding other embodiments, and that various other combinations, modifications and environments are possible and may be made within the scope of the inventive concepts described herein, either by way of the foregoing teachings or by those of skill or knowledge of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Industrial applicability
According to the present invention, a method of single cell genomic target region capture is provided.
Sequence listing
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Claims (6)
1. A method of single cell genomic target region capture comprising:
step A: isothermal amplification is carried out on the single-cell genome by adopting a multiple displacement amplification method, so as to obtain an amplified genome;
and (B) step (B): breaking 3 mug amplified genome to obtain fragmented DNA fragments;
step C: performing end repair on the fragmented DNA fragment to obtain a blunt-end DNA fragment;
step D: carrying out 3 'end addition A on the blunt end DNA fragment to obtain a DNA fragment with 3' end addition A;
step E: the DNA fragment with the 3' -end added with A is subjected to joint adding, so that a DNA fragment with the joint is obtained;
step F: carrying out PCR amplification on the spliced DNA fragments to obtain amplified DNA fragments;
step G: capturing the amplified DNA fragment in a target area to obtain a captured DNA fragment;
step H: carrying out PCR amplification on the captured DNA fragments to obtain PCR amplification products;
step I: sequencing the PCR amplification product;
wherein, the isothermal amplification condition in the step A is 30 ℃ for 2 hours;
the step A further comprises the following steps:
step A-1: PCR amplification is carried out on the amplified genome by using a primer with a nucleotide sequence shown as SEQ ID NO. 1-SEQ ID NO. 44;
the cycle number of PCR amplification in the step F is 4-6 cycles;
the condition for capturing the target area in the step G is that the target area is reacted for 16 to 24 hours at the temperature of 65 ℃.
2. The method according to claim 1, wherein the amount of the ligated DNA fragments in step F is 100 to 250ng.
3. The method according to claim 1, wherein the amount of the ligated DNA fragments in step F is 150 to 200ng.
4. A method according to any one of claims 1 to 3, wherein the number of cycles of PCR amplification in step F is 5 cycles.
5. The method according to any one of claims 1 to 4, wherein the condition for capturing the target region in step G is 65 ℃ for 24 hours.
6. The method according to claim 1, wherein the steps between step B and step C, between step C and step D, between step D and step E, between step E and step F, between step F and step G, between step G and step H, between step H and step I, and/or after step I comprise a step of purifying the DNA fragment.
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| CN101967684A (en) * | 2010-09-01 | 2011-02-09 | 深圳华大基因科技有限公司 | Sequencing library, preparation method thereof, and terminal sequencing method and device |
| CN102952855A (en) * | 2011-08-26 | 2013-03-06 | 深圳华大基因科技有限公司 | Genetic map construction method and device, haplotype analytical method and device |
| WO2015171656A1 (en) * | 2014-05-06 | 2015-11-12 | Baylor College Of Medicine | Methods of linearly amplifying whole genome of a single cell |
| CN106591447A (en) * | 2016-12-09 | 2017-04-26 | 上海美吉医学检验有限公司 | Sequencing method of single cell whole genome |
| CN107400705A (en) * | 2016-05-20 | 2017-11-28 | 深圳华大基因研究院 | A kind of high-throughout unicellular whole genome amplification method |
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| CN101967684A (en) * | 2010-09-01 | 2011-02-09 | 深圳华大基因科技有限公司 | Sequencing library, preparation method thereof, and terminal sequencing method and device |
| CN102952855A (en) * | 2011-08-26 | 2013-03-06 | 深圳华大基因科技有限公司 | Genetic map construction method and device, haplotype analytical method and device |
| WO2015171656A1 (en) * | 2014-05-06 | 2015-11-12 | Baylor College Of Medicine | Methods of linearly amplifying whole genome of a single cell |
| CN107400705A (en) * | 2016-05-20 | 2017-11-28 | 深圳华大基因研究院 | A kind of high-throughout unicellular whole genome amplification method |
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