CN109957615A - A kind of method of unicellular genome target region capture - Google Patents
A kind of method of unicellular genome target region capture Download PDFInfo
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Abstract
The present invention relates to a kind of methods of unicellular genome target region capture.Specifically by carrying out whole genome amplification to unicellular genome, the genome after amplification is interrupted, end is repaired, the end 3' adds A, adjunction head, PCR amplification, target area capture, carries out PCR amplification and sequencing to the DNA fragmentation of target area capture.The coverage of capture sequencing result can be improved in the method provided through the invention, improves the specificity of capture result.
Description
Technical field
The present invention relates to a kind of methods of unicellular genome target region capture, belong to technical field of gene detection.
Background technique
Unicellular sequencing was classified as year one of technology worthy of expecting by " Nature Methods " in 2011,2013
Year is classified as the six big field umber ones most to merit attention in year by " Science ", increasingly by the favor of researcher, phase
The research achievement of pass is also published in blowout on each top periodical, these all show that unicellular sequencing has been increasingly becoming scientific research
Hot spot.
Unicellular sequencing technologies can dissect the heterogeneity of cell, disclose the variation occurred in tumor cell gene group.Have
Help us and describe the clonal structure of tumour cell, and tracks the progress and range of scatter of disease.The technology is widely at present
It applies in fields such as supplementary reproduction, cancer, neurology and immunologys.
Unicellular target area capture sequencing is to expand the micro complete genome DNA of isolated individual cells, is obtained
The complete genome for obtaining high coverage rate customizes the probe of target genome area later, is hybridized with genomic DNA, by mesh
Mark the technological means that high-flux sequence is carried out after regional DNA is enriched with.Unicellular target area sequencing technologies are for disclosing cell mass
Body difference and Cellular evolution relationship, it helps the correlation candidate gene or relevant bits of discovery and verifying disease especially cancer
Point has huge application potential [1] in terms of clinical diagnosis and drug development.The advantage of target area capture sequencing is only
Interested genome area is studied, and sequencing depth is deeper when obtaining same quantity of data, it is as a result more acurrate；And
And compared with the sequencing of conventional gene group, not only expense is lower, and data interpretation is also more simple.
Unicellular genome target region capture sequencing (is not necessarily to amplification gene group, the amount of genome compared to normal sample
Enough carry out target area capture sequencing sample) genome target area capture sequencing effect still have significant difference
's.Firstly, only there are two copy, total amount about 6pg for unicellular genome.In amplification procedure, possibly complete base can not be reappeared
Because of group.If, then just will affect capture specificity, causing data volume secondly, the part lost just is the region of chip probe
Waste.And under identical sequencing depth, the more common genome of target region coverage of unicellular target area capture sequencing
Want low.
Bibliography
[1]Wang,Y.et al.Clonal evolution in breast cancer revealed by single
nucleus genome sequencing.Nature 512,155–160(2014).
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provides a kind of sides of unicellular genome target region capture
Method captures process by optimization whole genome amplification process, library construction process and target area, to improve capture sequencing
As a result the specificity of coverage and capture result.
1. a kind of method of unicellular genome target region capture, comprising:
Step A: isothermal duplication is carried out to unicellular genome using multiple displacement amplification method, the gene after being expanded
Group；
Step B: the genome after taking 3 μ g to expand is interrupted, and the DNA fragmentation of fragmentation is obtained；
Step C: the DNA fragmentation of fragmentation is subjected to end reparation, obtains flat terminal DNA fragments；
Step D: the flat terminal DNA fragments progress end 3' is added into A, obtains the DNA fragmentation that the end 3' adds A；
Step E: add the DNA fragmentation of A to carry out adjunction head at the end 3', obtain the DNA fragmentation of adjunction head；
Step F: the DNA fragmentation of adjunction head is subjected to PCR amplification, obtains the DNA fragmentation of amplification；
Step G: the DNA fragmentation of amplification is subjected to target area capture, obtains the DNA fragmentation of capture；
Step H: the DNA fragmentation of capture is subjected to PCR amplification, obtains pcr amplification product；
Step I: pcr amplification product is sequenced；
Wherein, the condition of the step A isothermal amplification is 30 DEG C of reactions 2 hours；
The recurring number of PCR amplification is 4~6 circulations in the step F；
The condition that target area captures in the step G is 65 DEG C and reacts 16~24 hours.
2. according to 1 the method for item, wherein after the step A further include:
Step A-1: after nucleotide sequence primer pair amplifies as shown in SEQ ID NO:1~SEQ ID NO:44
Genome carries out PCR amplification.
3. according to the described in any item methods of item 1 or 2, wherein the amount of the DNA fragmentation of adjunction head is 100 in the step F
~250ng.
4. according to the described in any item methods of item 1 or 2, wherein the amount of the DNA fragmentation of adjunction head is 150 in the step F
~200ng.
5. according to the described in any item methods of item 1~4, wherein the recurring number of PCR amplification is followed in the step F for 5
Ring.
6. according to the described in any item methods of item 1~5, wherein the condition that target area captures in the step G is 65 DEG C
Reaction 24 hours.
7. according to method described in item 1, wherein between the step B and step C, between step C and step D, step D
Between step E, between step E and step F, between step F and step G, between step G and step H, step H and step I it
Between, and/or after step I include the steps that purifying DNA fragmentation.
Beneficial effects of the present invention: compared with prior art, invention provides a kind of unicellular genome target region and catches
The method obtained captures process by optimization whole genome amplification process, library construction process and target area, shortens capture
The time of sequencing improves the coverage of capture sequencing result and the specificity of capture result.
Specific embodiment
With reference to embodiments, the present invention will be described in further detail.It should be appreciated that specific reality described herein
Example is applied only to explain the present invention, is not intended to limit the present invention.
The present invention provides a kind of method of unicellular genome target region capture, comprising:
Step A: unicellular genome is expanded using multiple displacement amplification method, the genome after being expanded；
Step B: the genome after taking 3 μ g to expand is interrupted, and the DNA fragmentation of fragmentation is obtained；
Step C: the DNA fragmentation of fragmentation is subjected to end reparation, obtains flat terminal DNA fragments；
Step D: the flat terminal DNA fragments progress end 3' is added into A, obtains the DNA fragmentation that the end 3' adds A；
Step E: add the DNA fragmentation of A to carry out adjunction head at the end 3', obtain the DNA fragmentation of adjunction head；
Step F: the DNA fragmentation of adjunction head is subjected to PCR amplification, obtains the DNA fragmentation of amplification；
Step G: the DNA fragmentation of amplification is subjected to target area capture, obtains the DNA fragmentation of capture；
Step H: the DNA fragmentation of capture is subjected to PCR amplification, obtains pcr amplification product；
Step I: pcr amplification product is sequenced；
Wherein, the condition expanded in the step A is 30 DEG C and reacts 2 hours；
The recurring number of PCR amplification is 4~6 circulations in the step F；
The condition that target area captures in the step G is 65 DEG C and reacts 16~24 hours.
The multiple displacement amplification method, may refer to such as United States Patent (USP) US6, and 124,120.The multiple displacement amplification side
Method generally includes to contact one group of primer, archaeal dna polymerase and target nucleic acid in the solution；And the solution is expanded, make
The amplified reaction of the target nucleic acid must be carried out to replicate the target nucleic acid；For unicellular genome amplification, it is generally recognized that need
Longer amplified reaction time, such as REPLI-g Single Cell Kit (the Qiagen public affairs for unicellular genome amplification
Department) operational manual specified in the amplified reaction time be 8 hours.But the inventors of the present invention discovered through researches that when amplification is anti-
When between seasonable more than 2 hours, the yield of amplified reaction product, which has reached, to be substantially saturated, it is preferred that the proliferation time is 2 small
When.
Preferably, further include step A-1 after step A: using nucleotide sequence such as SEQ ID NO:1~SEQ ID NO:
Genome after primer pair amplifies shown in 44 carries out PCR detection, further the integrality of the genome after detection amplification, effectively
Genome after avoiding the amplification using expanding effect difference build library and follow-up study generates bring time and cost
Waste.
In conventional library constructing method, in order to meet the demand of library Quality Control, upper machine sequencing and other detections, lead to
The initial amount for often thinking library construction is the μ of 100ng~2 g.Such as TruSeq Nano DNA kit (Illumina company) behaviour
The library initial amount for explaining book suggestion is 100~200ng, TruSeq DNA PCR-Free kit (Illumina company)
The library initial amount suggested in operational manual is 1~2 μ g.In the present invention, inventor's discovery is by improving library initial amount
It can be improved the coverage of target area in genome, it is preferable that the library initial amount is 3 μ g.
The step B using ultrasound or enzymatic cleavage methods interrupted and other skilled in the art known to interrupt
Method can use in this step.
The step C uses buffer, T4 DNA Polymerase, Klenow DNA Polymerase, T4
Polynucleotide Kinase, dNTP carry out end reparation, reaction system and reaction condition and repair item according to conventional end
Part carries out.
The step D carries out the end 3' using buffer, Klenow Exo (-), dATP and adds A, reaction system and reaction condition
Step A is added to carry out according to the conventional end 3'.
The step E carries out adjunction head using buffer, connector, T4DNA Ligase, and the connector is that conventional sequencing is built
Connector used in the step of library, no specifically limited use can be realized any connector of this step.Reaction system and reaction condition
It is carried out according to conventional adjunction head step.
In the present invention, take the amount of the DNA fragmentation of adjunction head in 100~250ng in the step F, it is generally recognized that F-step is straight
It connecing and the DNA fragmentation of adjunction head is all subjected to PCR amplifications, it is preferable that the amount of the DNA fragmentation that adjunction head is added is 150~
200ng, the PCR amplification recurring number are 4~6 circulations, it is preferable that PCR amplification recurring number is 5.
Hybridized in the step G using existing hybridization kit, such as SureSelect Target
Provide that reaction condition is 65 DEG C anti-in Enrichment System kit (Agilent Technologies company) specification
It answers 48~72 hours, it is preferable that the condition of hybrid capture is 65 DEG C of reactions 16~24 hours in the step G.
Preferably, such as step between each step for the method that unicellular genome target region of the invention captures
Between B and step C, between step C and step D, between step D and step E, between step E and step F, step F and step G
Between, between step G and step, between step H and step I, and/or DNA fragmentation can be purified after step I
Step.
Embodiment 1
The present embodiment carries out library by using SureSelectXT Reagent kit, HSQ, 96 (Aligent company)
Building and hybrid capture.
1 unicellular gene magnification
1.1 processing sorting cells:
Cell is directly sorted into superclean bench in 3 μ L PBS, without freeze thawing.
1.2 single cell whole genome amplifications (WGA):
Whole genome amplification is carried out using MDA, (Qiagen is public using REPLI-g Single Cell Kit kit
Department).
A) prepare DLB Buffer: the water of 500 μ L being added into the pipe of offer, thoroughly mix, of short duration centrifugation.DLB
Buffer can be saved 6 months at -20 DEG C.
B) all Buffer and reagent will be vortexed mixing before the use.
C) the hot lid temperature of PCR instrument is adjusted to 70 DEG C.
D) prepare the Buffer D2 (denaturation Buffer) of sufficient amount, Buffer D2 can save 3 in -20 DEG C of longests
Month.Each reaction needs the Buffer D2 of 3 μ L
E) cell volume is supplied as 4 μ L with the PBS that kit is provided.
F) 3 μ L Buffer D2 are added, mild to mix, of short duration centrifugation.
G) 65 DEG C of 10 clocks of amplification.
H) 3 μ L reaction terminating liquids are added, mild to mix, of short duration centrifugation is placed on ice.
REPLI-g sc DNA Polymerase is placed on ice, other reagent room temperatures melt, be vortexed mix, it is of short duration from
The heart.
I) mix is prepared, rear of short duration centrifugation is prepared.
It is added in the DNA that 10 μ L are denaturalized in 40 μ L mix to step h), 30 DEG C expand 2 hours.65 DEG C place 3 minutes so that
REPLI-g sc DNA Polymerase inactivation.It takes 10 μ L to add 40 μ L water, is purified with 1 × Ampure beads, with 40 μ L EB
Dissolution, the genome after being expanded.
1.3 WGA product quality inspections
With Qubit BR and 2100 detectable concentration of Agilent and clip size.
The detection of 1.4 amplification efficiencies
List of primers
Reaction system:
Reaction condition is as follows:
Utilize 2% detected through gel electrophoresis PCR product amplification situation.
2 library constructions
2.1 interrupting
Genome after taking 3 μ g to expand, 80 μ L interrupt system, carry out 200bp fragmentation.1.8 × Ampure is added
Beads purifying, is dissolved with 50 μ L water, obtains fragmentation DNA fragmentation.
It repairs 2.2 ends
Reaction condition: 20 DEG C are reacted 30 minutes, and 180 μ L Ampure beads purifying (1.8 ×), 70% alcohol is added
(Twice), 32 μ L ddH2O elution, obtains flat terminal DNA fragments.
2.3 ends 3' add A
Reaction condition: 37 DEG C are reacted 30 minutes, and 90 μ L Ampure beads purifying (1.8 ×), 70% alcohol is added
(Twice), 29.5 μ LddH2O elution obtains the DNA fragmentation that the end 3' adds A.
2.4 adjunction heads
Reaction condition: 20 DEG C are reacted 15 minutes, and 90 μ L Ampure beads (1.8 ×) purifying, 70% alcohol is added
(Twice), 32 μ LddH2O elution, obtains the DNA fragmentation of adjunction head.
2.5 PCR amplification
Reaction condition:
Each sample is added 45 μ L Ampure beads (0.9 ×) and purifies after reaction, 70% alcohol (Twice), and 26
μL ddH2O elution, obtains DNA library.
The detection of 2.6 Library Qualities
The DNA library of building detects the clip size in library using 2100 biological analyser of Agilent.
The capture of 3 target areas
The chip used is the tumour chip of the 1.4M customized in Agilent company, is named as SureSelect Hyb3.
3.1 libraries prepare
The library 750ngDNA is taken to carry out being concentrated in vacuo to 3.4 μ L of volume.
3.2 Block Mix prepare
3.3 capping
For heat lid to 105 DEG C, 95 DEG C are reacted 5 minutes, 65 DEG C 5 minutes.
3.4 Hybridization Mix prepare
It is placed on after being ready to spare on ice chest, is preheated to clarification using first 65 DEG C.
3.5 hybrid capture
It keeps the product of step 3.4 to be constantly in 65 DEG C, 5 μ L Capture Library is first added, are then rapidly added
15 μ L hybridize Mix, and pressure-vaccum mixes, and lid is preheated to 105 DEG C, and 65 DEG C are reacted 24 hours, obtain hybrid product.
3.6 SureSelect Wash Buffer prepare
600 μ L SureSelect Wash Buffer 2 are taken to manage loaded on EP, 65 DEG C of constant temperature preheatings are spare.Take 200 μ L
SureSelect Wash Buffer 1 is managed loaded on EP, spare.
3.7 MyOne Streptavidin T1 magnetic beads prepare
It mixes, removes supernatant with magnetic frame, every 50 μ L magnetic bead is washed with 200 μ L SureSelect Binding Buffer, weight
After backwashing is washed three times, and every 50 μ L magnetic bead is resuspended with 200 μ LSureSelect Binding Buffer, is managed loaded on 1.5mL EP.
3.8 Binding DNA
It takes the 200 ready magnetic beads of μ L step 3.7 to be added in the hybrid product of step 3.5 acquisition, mixes, be transferred to surplus
In residual magnetism pearl, constant temperature blending instrument, 1600rpm, 25 DEG C are reacted 30 minutes.
The washing of 3.9 magnetic beads
Sample in step 3.8 is removed, is centrifuged with palm centrifuge, supernatant is removed using magnetic frame, 200 μ L is added
SureSelect Wash Buffer 1, mixing, which is placed on constant temperature blending instrument, reacts 15 minutes for 25 DEG C；
Remove sample, magnetic frame gets on supernatant, and 200 μ L SureSelect Wash Buffer, 2 (65 DEG C) reactions are added
10 minutes, in triplicate；
After 20 μ L nuclease-free water mixing is added, hybrid product is obtained, is placed in spare on ice.
3.10 PCR reaction and product purification
Reaction condition is as follows:
After reaction, supernatant is taken, 0.9 × Ampure beads magnetic beads for purifying, 70% alcohol (Twice), 22 μ L EB wash
It is de-.
3.11 capture library Quality Controls
Library is captured using the clip size in 2100 biological analyser of Agilent detection library.
Comparative example 1
Comparative example 1 and the difference of embodiment are: unicellular amplification k) step amplification temperature is 8 hours and uses
SureSelectXT Reagent kit, HSQ, 96 carry out library construction and target area capture, operating procedure and the description of product
The step of recording in book is consistent.
Comparative example 2
Comparative example 2 and the difference of embodiment are: normal sample is used, using SureSelectXT Reagent kit,
HSQ, 96 progress library constructions and target area capture, operating procedure are consistent with the step of record in product description.
Wherein M1 is the sample number of comparative example 1, and M2 is the sample number (using method of the invention) of embodiment, and M3 is
The sample number of comparative example 2.The Quality Control result such as following table of sequencing, under identical sequencing data amount, it can be seen that sample M1,
The data Quality Control of M2, M3 are qualified, wherein by the comparison of Quality Control data, sample M2 and sample M3 are in each data target
Numerical value is almost the same, and the data target of sample M1 is poor, and especially in terms of capturing specificity, the target area of M2, M3 are caught
Obtaining efficiency is respectively 77.59%, 80.7%, and the capture rate of M1 is 64.2%, the capture specificity and sample M3 of sample M2
It is almost the same and be higher than M1.For the coverage of target area, the target area coverage of sample M1 is 85.39%, M2, M3
Target area coverage be respectively 99.31%, 99.31%, sample M2 covers target area substantially, hence it is evident that be higher than M1 sample
This, therefore by means of the present invention, the result of unicellular target area capture sequencing is special in target area coverage and capture
Property aspect be significantly increased, it is close with normal sample genome capture sequencing result.
Raw Reads: the Reads number of original lower machine；
Clean Reads: the number of filtered Reads；
Clean Reads Rate (%): the Clean Reads obtained after filtering accounts for the ratio of Raw Reads；
Ns Reads: the ratio containing N of removal is greater than 5% Reads number；
Ns Reads Rate (%): Reads of the ratio containing N of removal greater than 5% accounts for the ratio of Raw Reads；
Raw Q30Bases Rate (%): sequencing quality value is greater than 30 (error rate is less than 0.1%) in Raw Reads
The ratio of the total base of base Zhan (Raw Bases)；
Clean Q30Bases Rate (%): greater than 30, (error rate is less than sequencing quality value in Clean Reads
0.1%) ratio of the total base of base Zhan (Clean Bases)；
Target Region: genome target region capture region size
Reads Mapped To Genome: it compares to Reads number on reference genome；
Map Rate (%): it compares to the base percentage on reference genome；
Capture Specificity (%): compare to genome target capture region Reads number accounting to arrive gene
The ratio of group Reads；
Duplication Rate (%): the PCR of removal duplicate Reads number accounts for the ratio of Mapped Reads；
Uniq Rate (%): the Reads compared to genome unique positions is accounted for except the Reads compared after PCR repetition
Ratio；
The average sequencing of Coverage Of Target Region (%): genome target capture region Uniq Reads
Depth is used for the data depth of subsequent analysis.
The preferred embodiment of the present invention has shown and described in above description, as previously described, it should be understood that the present invention is not office
Be limited to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification and
Environment, and can be changed within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge
It is dynamic.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, then it all should be appended by the present invention
In scope of protection of the claims.
Industrial applicibility
According to the present invention, a kind of method of unicellular genome target region capture is provided.
Sequence table
<110>peace promise is excellent reaches Gene science (Beijing) Co., Ltd
<120>a kind of method of unicellular genome target region capture
<130> 1704SSCN
<150> 2017114314506
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<213>artificial sequence ()
<400> 43
hrgcaccatt caaccaatct ga 22
<210> 44
<211> 22
<212> DNA
<213>artificial sequence ()
<400> 44
hrtgccattc cctctaatcc tg 22
Claims (7)
1. a kind of method of unicellular genome target region capture, comprising:
Step A: isothermal duplication is carried out to unicellular genome using multiple displacement amplification method, the genome after being expanded；
Step B: the genome after taking 3 μ g to expand is interrupted, and the DNA fragmentation of fragmentation is obtained；
Step C: the DNA fragmentation of fragmentation is subjected to end reparation, obtains flat terminal DNA fragments；
Step D: the flat terminal DNA fragments progress end 3' is added into A, obtains the DNA fragmentation that the end 3' adds A；
Step E: add the DNA fragmentation of A to carry out adjunction head at the end 3', obtain the DNA fragmentation of adjunction head；
Step F: the DNA fragmentation of adjunction head is subjected to PCR amplification, obtains the DNA fragmentation of amplification；
Step G: the DNA fragmentation of amplification is subjected to target area capture, obtains the DNA fragmentation of capture；
Step H: the DNA fragmentation of capture is subjected to PCR amplification, obtains pcr amplification product；
Step I: pcr amplification product is sequenced；
Wherein, the condition of the step A isothermal amplification is 30 DEG C of reactions 2 hours；
The recurring number of PCR amplification is 4~6 circulations in the step F；
The condition that target area captures in the step G is 65 DEG C and reacts 16~24 hours.
2. method according to claim 1, which is characterized in that after the step A further include:
Step A-1: the gene after nucleotide sequence primer pair amplifies as shown in SEQ ID NO:1~SEQ ID NO:44 is used
Group carries out PCR amplification.
3. the method according to any one of claims 1 and 2, which is characterized in that the DNA fragmentation of adjunction head in the step F
Amount be 100~250ng.
4. the method according to any one of claims 1 and 2, which is characterized in that the DNA fragmentation of adjunction head in the step F
Amount be 150~200ng.
5. method according to any one of claims 1 to 4, which is characterized in that the recurring number of PCR amplification in the step F
It is recycled for 5.
6. described in any item methods according to claim 1~5, which is characterized in that the item that target area captures in the step G
Part is 65 DEG C and reacts 24 hours.
7. the method according to claim 1, wherein between the step B and step C, step C and step D it
Between, between step D and step E, between step E and step F, between step F and step G, between step G and step H, step H
Include the steps that purifying DNA fragmentation between step I, and/or after step I.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
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