CN106835291A - The preparation method and kit in DNA libraries - Google Patents
The preparation method and kit in DNA libraries Download PDFInfo
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- CN106835291A CN106835291A CN201611154433.8A CN201611154433A CN106835291A CN 106835291 A CN106835291 A CN 106835291A CN 201611154433 A CN201611154433 A CN 201611154433A CN 106835291 A CN106835291 A CN 106835291A
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Abstract
Prepared the invention provides a kind of DNA library and the special method for capturing and kit, methods described is applied to the structure and the hybrid capture of specific target genetic region of cfDNA, leucocyte gDNA and the DNA library from tissue samples.In the preparation method of DNA library of the present invention, end reparation and 3 ' ends plus A are carried out in same reaction system, and improve hybridization input amount, blocking preparation in hybrid capture is optimized, thereby is achieved capture rate higher, library diversity higher and more homogeneous coverage, the structure in the sample library of micro and low abundance mutation is especially suitable for, the saving on techniqueflow is advantageously implemented.The method of the present invention can be easy to implement all types of variations of target gene, such as the detection of mutation, missing, insertion, fusion and copy number amplification, overcome the method at present by PCR on DNA level and be enriched with target-gene sequence, but mutation and the missing of target gene can only be detected, it is impossible to the shortcoming of detection fusion.
Description
Technical field
Preparation and target capture method and kit the present invention relates to DNA library.More particularly to plasma DNA text
Storehouse, the preparation in leucocyte gDNA libraries or tissue-derived DNA library and target capture method and kit.
Background technology
Research finds there is a small amount of dissociative DNA, i.e. plasma DNA (cfDNA) in cancer patient's blood, and these
Some comes from tumour cell in dissociative DNA, and the Oncogenome fragment that this part is free in blood is referred to as following
Ring Tumour DNA (ctDNA).CtDNA is detected, recurs and treated as a kind of new tumor markers in the personalized medicine of tumour
The aspects such as effect monitoring play an important role.Be discharged into the ctDNA in blood from tumor tissues to obtain cancer by detecting
The method of related genes variants information, as " liquid biopsy ".The liquid biopsy concept based on ctDNA is gradually by doctor in recent years
Receive and approve.It not only with auxiliary diagnosis solid tumor, and can realize resistance recurrence monitoring, and curative effect evaluation is
Organize the strong supplement of biopsy.
The two generations sequencing law technology of probe capture behind storehouse is set up based on full genome, the parallel inspection of polygenes Mutiple Targets can be realized
Survey, and possess high sensitivity and specificity, the liquid biopsy of ctDNA can be realized.Therefore, based on two generation sequencing technologies
Auxiliary diagnosis, clinical guidance medication etc. of the structure in ctDNA libraries for realizing liquid biopsy and tumour all have important
Meaning.
The content of the invention
The invention provides the preparation method and kit of a kind of DNA library, methods described is applied to cfDNA, white thin
The structure of born of the same parents gDNA and the DNA library from tissue samples.The method of the present invention can be easy to implement all of target gene
The detection that variation type, such as mutation, missing, insertion, fusion and copy number are expanded, overcomes and passes through on DNA level at present
The method enrichment target-gene sequence of PCR, but mutation and the missing of target gene can only be detected, it is impossible to the shortcoming of detection fusion.
DNA library preparation method of the invention, be on the basis of the banking process of SureSelect XT, to pre- library with
And the details in whole library construction process is improved, such as end reparation and 3 ' ends plus A are in same reaction system
Carry out, the improvement and crossing breeding input amount and blocking preparation blocked to hybrid capture is optimized, thereby is achieved capture effect higher
Rate, library diversity higher, and more homogeneous coverage, are suitable for the structure in the sample library of micro and low abundance mutation
Build, and realize the saving on techniqueflow.One aspect of the present invention is related to the preparation method of DNA library, it include pre- library and
The preparation process in whole library, wherein described pre- library preparation process includes that the preparation of DNA, end are repaired and 3 ' ends plus A, joint
Connection, the purifying of joint connection product, pre- amplified library, the pre- library purifying of amplification, described whole library preparation process includes pre-
Prepared by Library hybridization, capture wash-out, whole library, and whole library purge process, wherein the DNA library is cfDNA libraries, white
Cell gDNA libraries or tissue-derived DNA library.
The preparation method of any one, adds A to be one wherein described end is repaired with 3 ' ends according to a first aspect of the present invention
Carried out in individual reaction system.
The preparation method of any one according to a first aspect of the present invention, wherein end are repaired and the reaction system of 3 ' ends plus A contains
There are ER＆AT buffer solutions, ER＆AT enzymes, and cfDNA, and ER＆AT buffer solutions：ER＆AT enzymes：The volume ratio of cfDNA is 7：3：
50。
The preparation method of any one according to a first aspect of the present invention, wherein end repair and 3 ' ends plus A reaction system bar
Part is：
20 DEG C 30 minutes (85 DEG C of heat lids)
65 DEG C 30 minutes (85 DEG C of heat lids)
4 DEG C of maintenances.
The preparation method of any one according to a first aspect of the present invention, wherein the blocking agent used in the process of pre- Library hybridization is
Two-way short chain blocking agent.
The preparation method of any one according to a first aspect of the present invention, wherein described two-way short chain blocking agent structure is as follows：
The preparation method of any one according to a first aspect of the present invention, the magnetic bead amount for wherein being added in purification step is more conventional
Reduce half.
The preparation method of any one, connects anti-in its center tap connection product purge process according to a first aspect of the present invention
It is 1 to answer system and magnetic bead volume ratio：0.8.
The preparation method of any one according to a first aspect of the present invention, wherein PCR volumes in the pre- library purge process for expanding
With magnetic bead volume 1：1.2.
The preparation method of any one according to a first aspect of the present invention, it applies also for leucocyte gDNA and tissue samples
The library construction of DNA.
The preparation method of any one according to a first aspect of the present invention, wherein carrying out leucocyte gDNA and tissue samples
DNA library further relates to the time of interrupting, interrupts dilution and the optimization of wash buffer piping and druming position when building.
The preparation method of any one according to a first aspect of the present invention, wherein the leucocyte gDNA and tissue samples
DNA library interrupts the time for 30s to 150s when building.
The preparation method of any one according to a first aspect of the present invention, wherein the leucocyte gDNA and tissue samples
The DNA library dilution that interrupts used when building is IDTE (10mM Tris 0.1M EDTA, pH8.0).
The preparation method of any one according to a first aspect of the present invention, wherein the leucocyte gDNA and tissue samples
Wash buffer blows and beats position for PCR instrument is carried out at for 65 DEG C when DNA library builds.
The preparation method of any one according to a first aspect of the present invention, wherein still further comprising the pre- library of purifying or text eventually
The clip size detection of the quality inspection, the pre- library of purifying or whole library in storehouse.
The second aspect of the present invention provides two-way short chain blocking agent.
The two-way short chain blocking agent of any one according to a second aspect of the present invention, its concrete structure is as follows：
The two-way short chain blocking agent, can produce more preferable blocking effect in crossover process so that hybridization is more thorough,
Result is more accurate.
The two-way short chain blocking agent of any one is preparing the kit for constructed dna library according to a second aspect of the present invention
In application, wherein the DNA library be cfDNA libraries, leucocyte gDNA libraries or tissue-derived DNA library.
Brief description of the drawings
Fig. 1 is pre- library total amount result figure.Figure 1A is the pre- library total amounts of cfDNA；Figure 1B is the pre- library total amounts of WBC gDNA,
Wherein sample number is 8.
Fig. 2 is KAPA-XT groups and the pre- library yield of XT groups under FFPE gDNA difference input amounts, and wherein sample number is 7.Its
Middle KAPA is following KAPA-XT groups, and QXT is XT-30 groups.
Fig. 3 is library concentration result figure.Fig. 3 A are cfDNA results, and Fig. 3 B are WBC gDNA libraries result, and wherein sample is same
Fig. 1.
Fig. 4 is clip size result figure in library.Fig. 4 A are cfDNA results, and Fig. 4 B are WBC gDNA libraries result.
Fig. 5 is the results contrast figure of capture rate, and wherein abscissa represents cfDNA the and WBC gDNA of above-mentioned 8 samples
Totally 16 libraries.
Fig. 6 is the result figure of library complexity and sequencing depth.Fig. 6 A and Fig. 6 B are the complexity in cfDNA libraries
(5000X) and sequencing depth results；Fig. 6 C and Fig. 6 D are the complexity (5000X) and sequencing depth results in WBC gDNA libraries.
Fig. 7 is KAPA+XT group experimental implementation flow charts.
Above-mentioned WBC gDNA are the gDNA in leucocyte source, and KAPA-30 is following KAPA-XT groups, and XT-30 is
Following XT-30 groups.
Specific embodiment
The present invention is expanded on further with reference to specific embodiment, it should be appreciated that following examples are merely to illustrate the present invention
Rather than limit the scope of the invention.
Embodiment
1. experiment packet
Experiment is divided into two groups to be carried out, and specific packet situation is as follows：
First group (XT-30 groups)：Standard Operating Procedure according to existing SureSelect XT methods is carried out, and conduct
The gDNA in the control group of the different subgroups below, cfDNA used or leucocyte source keeps one with each subgroup amount used
Cause.
Second group (KAPA-XT groups)：The group is according further to different samples sources (cfDNA in blood plasma source, leucocyte
The gDNA in the gDNA in source, FFPE (formalin fix FFPE) source) it is divided into three subgroups.Three subgroups are according to following
Described specific steps are carried out.
2. experimental procedure
2.1DNA is extracted and quality inspection
Foundation《CfDNA extraction standard flows》Extract 30ng cfDNA and eluted, elution requirement is room temperature, and volume is
55μL.50 μ L enter the following flow, and 3 μ L are used for carrying out quality control process.
On the basis of Qubit3.0HS Kit kit standard flows, following improvement has been carried out：When interrupting of leucocyte
Between 150s be adjusted to by 90s interrupt, interrupt dilution by ddH2O is adjusted to IDTE, and the piping and druming position of wash buffer is by operating
Table top is adjusted in PCR instrument, in being carried out at 65 DEG C, to extract 200ng gDNA, is then eluted, and elution requirement is room temperature, volume
It is 55 μ L.50 μ L enter below scheme, and 3 μ L are used for carrying out quality control process.
The gDNA in FFPE sources is extracted, and the gDNA ultrasounds that FFPE originates are interrupted into 200-250bp or so, interrupt the time
30s, 60s, and 150s are set to, are prepared to build storehouse.
2.2 ends are repaired, and 3 ' ends add A
1) this flow docking step 2.1 final step, opens KAPA Hyper Prep 96reaction Kit (- 20
DEG C), take out ER＆AT buffer solutions and ER＆AT enzymatic reagents are placed in thawed on ice.
2) PCR instrument (BioRad T100 or AB Veriti) is set, and specific procedure (program is entitled " ERA ") sets as follows：
20 DEG C 30 minutes (85 DEG C of heat lids)
65 DEG C 30 minutes (85 DEG C of heat lids)
4 DEG C of maintenances.
3) according to step 2.1 gDNA that quantitatively preparation 30ng blood plasma cfDNA, 200ng leucocyte are originated respectively, 5ng,
The gDNA in 10ng, 30ng FFPE sources, whole opisthosoma product is 50 μ L.
4) prepare end on ice to repair and add A reaction systems mixing liquid (table 1), finger is flicked 3-5 times, mixing of turning upside down
2-3 times, small table centrifuge 1-3 seconds.
5) with the P20 pipettors absorption 10 μ L mixing liquids above-mentioned 50 μ L DNA solutions of addition, soft piping and druming up and down is mixed for 10 times
It is even, small table centrifuge of short duration centrifugation 1-3 seconds.
Repair and 3 ' ends plus A reaction systems the end of table 1
| Reagent | Reaction volume |
| ER＆AT buffer solutions | 7μL |
| ER＆AT enzymes | 3μL |
| The gDNA of cfDNA or fragmentation | 50μL |
| Total amount | 60μL |
6) it is put into PCR instrument Bio-Rad T100 or AB Veriti, using program " ERA ", (85 DEG C of heat lids, 20 DEG C 30 points
Clock, 65 DEG C 30 minutes, 4 DEG C maintenance).Enter next step in 2 hours.
2.3 joints are connected
1) reagent as shown in table 2 below is prepared：
The joint of table 2 is connected and purified reagent
2) program is set：It is entitled " LIG " that (BioRad T100or AB Veriti) definition program is set
20 DEG C 15 minutes (85 DEG C of heat lids)
70 DEG C 10 minutes (85 DEG C of heat lids)
4 DEG C of holdings.
3) joint coupled reaction system mixing liquid (table 3) is prepared on ice, finger is flicked 3-5 times, turn upside down mixing 2-3
It is secondary, small table centrifuge 1-3 seconds.
4) with the P100 pipettors absorption 50 μ L mixing liquids above-mentioned 0.2mL pipes of addition, pipettor keeps 50 μ L to blow and beat 5 up and down
Secondary mixing, small table centrifuge 1-3 seconds.
The coupled reaction system of table 3 is set
| Reagent | Pre-reaction volume |
| Connection buffer solution | 30μL |
| DNA ligase | 10μL |
| Adaptor Oligo Mix | 10μL |
| Repair mixed liquor in end | 60μL |
| Cumulative volume | 110ul |
5) in PCR instrument (T100or AB Veriti) operation program " LIG " (20 DEG C 15 minutes, 70 DEG C 10 minutes, 4 DEG C
Maintain (85 DEG C of heat lids)).
2.4 joint connection products are purified
1) AMPURE XP magnetic beads are placed in room temperature at least 30 minutes, are turned upside down 2-3 times, be vortexed and mixed 5-10 seconds
The AMPURE XP magnetic beads of room temperature are replied, is made its homogenization.
2) 75% fresh ethanol is prepared according to each library 600 μ L.
3) Eppendorf LoBind 1.5mL centrifuge tubes are taken, by coupled reaction system and magnetic bead volume 1：0.8 ratio, first
The magnetic bead of 88 μ L homogenizations and the pre- library of 110 μ L adjunctions head are added afterwards.Pipettor P200 blows and beats 5 mixings, and gentle rotation is incubated
Educate 5 minutes.
4) centrifuge tube is placed in magnetic frame.Wait solution clarification (about 3-5 minutes).
5) centrifuge tube is placed in motionless on magnetic frame, opening lid, clarified supernatant is carefully siphoned away (about with pipettor P200
200 μ L), it is to avoid encounter magnetic bead.
6) centrifuge tube is still placed on magnetic frame, adds 75% second of 300 μ L Fresh in every pipe with pipettor P1000
Alcohol.
7) waiting makes magnetic bead fully precipitate in 1 minute, and period, slow rotating centrifugal pipe one was enclosed in the horizontal direction, siphoned away ethanol.
8) 6,7 steps are repeated once.
9) small table centrifuge 1-3 seconds, centrifuge tube is placed back in into magnetic frame and stands 30 seconds, use P10 liquid reliefs
The cleared residual ethanol of device, keeps lid to open.
10) room temperature dries magnetic bead in 2 minutes, non-reflective with magnetic bead surfaces, and magnetic bead block does not have crackle to be defined.
11) 28 μ L EB solution are added, fully piping and druming is mixed, is incubated at room temperature 3 minutes.
12) centrifuge tube is placed in magnetic frame 2 minutes, until solution is clarified.
13) the μ L of supernatant 27.5 to new 0.2mL PCR pipes are pipetted, it is standby on ice.
2.5 pre- amplified libraries
1) reagent needed for pre- amplified library and reaction condition are referring to table 4 and table 5:
Table 4PCR purifying prepares
The pre- amplified library PCR reaction conditions of table 5
2) to avoid cross pollution, the PCR platforms in special clean region or ultraviolet disinfection and with malleation air-flow are prepared
PCR (except all the components of library DNA) reaction solution.
3) reaction system mixing liquid (preparing on ice) is prepared according to table 6, finger is flicked 3-5 times, turn upside down mixing 2-3
It is secondary, small table centrifuge 1-3 seconds.
4) (the 27.5 linker Purified products of μ L are contained) per effective in the PCR pipe in " purifying of joint connection product " this step
P100 pipettors add 22.5 μ L reaction mixing liquids, up and down 10 mixings of soft piping and druming.
5) it is placed in operation " PRE " program in PCR instrument.
The preenrichment PCR system of table 6
| Reagent | Pre-reaction volume |
| 5xKAPA HiFi Buffer | 10μL |
| 10mM dNTP Mix | 1.5μL |
| SureSelect Primer | 5μL |
| Pre-Capture PCR Reverse Primer | 5μL |
| KAPA HiFi | 1μL |
| Clean connection mixed liquor | 27.5μL |
| Cumulative volume | 50μL |
The pre- library purifying of 2.6 amplifications
1) AMPURE XP magnetic beads are placed in room temperature at least 30 minutes.
2) 75% fresh ethanol is prepared according to each library 600 μ L.
3) turn upside down 2-3 times, be vortexed the AMPURE XP magnetic beads for mixing and having responded to room temperature for 5-10 seconds, makes its homogenization.
4) Eppendorf LoBind 1.5mL centrifuge tubes are taken, by PCR volumes and magnetic bead volume 1：1.2 ratios, successively add
Enter magnetic bead and the 50 pre- libraries of μ LPCR of 60 μ L homogenizations.Pipettor P200 is transferred to 100 μ L and blows and beats up and down 5 times, and gentle rotation is incubated
5 minutes.
5) centrifuge tube is placed in magnetic frame.Wait solution clarification (about 3-5 minutes).
6) to be still placed in magnetic frame motionless for centrifuge tube, opens lid, carefully siphons away supernatant (about 110 μ L), it is to avoid encounter magnetic bead.
7) centrifuge tube is still placed on magnetic frame, adds 75% second of 300 μ L Fresh in every pipe with pipettor P1000
Alcohol.
8) waiting makes magnetic bead fully precipitate in 1 minute, and period, slow rotating centrifugal pipe one was enclosed in the horizontal direction, siphoned away ethanol.
9) 7,8 steps are repeated once.
10) small table centrifuge 1-3 seconds, centrifuge tube is placed back in into magnetic frame and stands 30s, use P10 liquid reliefs
The cleared residual ethanol of device, keeps lid to open.
11) room temperature dries magnetic bead in 2 minutes, non-reflective with magnetic bead surfaces, and magnetic bead block does not have crackle to be defined.
12) 28 μ L ddH2O are added, fully piping and druming is mixed, is incubated at room temperature 3 minutes.
13) centrifuge tube is placed in magnetic frame 2 minutes, until solution is clarified.
14) the μ L of supernatant 27.5 to new 0.2mL PCR pipes are pipetted, it is standby on ice.
The pre- library quality inspection of 2.7 purifying
The pre- library dilution of purifying that above-mentioned steps are obtained.Take 1 μ L pre- libraries after purification to a new 1.5mL EP
Guan Zhong, adds 10 μ L ddH2O, P20 pipettors to blow and beat mixing 10 times up and down, wherein 1 μ L are quantitative as Qubit, under remaining is used as
The Labchip QC (being carried out using Labchip DNA HS Kit kits) of one step.
2.8 pre- Library hybridizations
1) reagent in table 7 is prepared.
2) program is set：It is entitled " FASTHYB " that (BioRad S1000) definition program is set
The pre- Library hybridization reagent of table 7 prepares
Wherein, the blocking agent BLK used in the process of pre- Library hybridization is two-way short chain blocking agent, and its concrete structure is as follows：
3) hybridization reaction needs at least 300ng pre- libraries, according to Qubit quantitative results, judges whether to carry out miscellaneous
Hand over.If pre- library yield is more than 1500ng, pipette 12 μ L after purification pre- library to 48 orifice plates.If pre- library yield is more than
750ng but less than 1500ng, pipette corresponding to 750ng after purification pre- library to 48 orifice plates, with water polishing volume to 12 μ L.If
Pre- library yield is less than 750ng, but more than 300ng, can attempt hybridization, all pre- libraries to 48 orifice plates is pipetted, with water polishing body
Accumulate to 12 μ L.
4) pre- library closed system is prepared by the normal temperature of table 8, probe system is configured by the normal temperature of table 9.
The pre- library closed system of table 8
| Pre- library closing | Pre-reaction volume |
| SureSelect QXT Fast Blocker Mix | 5μL |
| BLK | 1μL |
| Pre- library | 12μL |
| Cumulative volume (Hyb Buffer) | 18μL |
The probe configuration system of table 9
5) 48 orifice plates containing pre- library closed system are covered into 8 connecting leg lids, is put into PCR instrument.
95 DEG C of operation program " FASTHYB " program 5 minutes, to the 65 DEG C of beginnings of the 3rd step, suspends PCR programs, opens 8 connecting legs
Lid.
6) corresponding Probe components are quickly transferred to contain pre- library by the volume of each sample 12 μ L with the P20 volley of rifle fires
The hybridization plate hole of component, the μ L of cumulative volume 30 blow and beat 5 mixings, and each sample probe component need to change pipette tips.By eight connecting legs in plate
It is upper to cover tightly, then the micro seal B pad pastings for cutting into half are sticked to prevent from being evaporated.Run hybridization procedures to EP (end of program)
(65 DEG C of holdings).Overnight hybridization is such as needed, 65 DEG C of hybridization procedures can be also kept overnight to carrying out next step after 16 hours.
2.9 capture wash-outs
1) 65 DEG C of thermostat metal bath temperature is set.
2) SCB/T1 magnetic beads (are a kind of affine strepto- magnetic bead, selection comes from Thermo Fisher's 65601MyOneTMStreptavidin T1) equilibrium at room temperature more than 30 minutes.
3) SureSelect Wash Buffer 2 are placed in into metal in 15ml conical pipes by the consumption of 600 μ L/ samples to add
The 65 DEG C of incubations of hot device.
4) SCB/T1 magnetic beads are taken out, mixing 5 times of turning upside down is vortexed and mixes 10 seconds, is stored at room temperature more than half an hour, is vortexed
Mix 10 seconds, being distributed into 1.5ml LoBind by sample number manages, each sample needs 50 μ L, each Lobind pipes at most to put 250 μ L.
Magnetic frame upper 3 minute is stood, supernatant is abandoned.
5) the original magnetic beads of every 25 μ L add 200 μ L SureSelect Binding Buffer, are vortexed and mix 3 seconds, it is of short duration from
The heart, stands magnetic frame upper 3 minute, abandons supernatant.
6) above step is repeated 2 times, totally 3 times.
7) the original magnetic beads of every 25 μ L add 200 μ L SureSelect Binding Buffer, are vortexed and mix 3 seconds, resuspended
SCB/T1,200 μ L/ pipes of packing are in LoBind pipes.
8) the hybridization μ L P200 of sample about 28 are transferred in resuspended SCB/T1 magnetic beads, are placed on rotation blending instrument, room
Temperature rotation is incubated 30 minutes.
9) Magnetic bead sample is mixed into liquid pipe and is placed in standing 3 minutes on magnetic frame, about 230 μ L of supernatant are sucked with P1000.Add
500 μ L SureSelect Wash Buffer 1, are vortexed and mix 5 seconds, are placed on rotation blending instrument, and room temperature rotation is incubated 15 points
Clock.
10) Magnetic bead sample is mixed and stand 3 minutes on liquid pipe magnetic frame, about 500 μ L of supernatant are sucked with P1000.Inhaled with P20
Most residual liquid.
11) the SureSelect Wash Buffer 2 for adding 200 μ L to be preheated to 65 DEG C, are vortexed and mix 10 seconds, are placed in dry
On formula blending instrument, 65 DEG C of concussions are incubated 10 minutes, and rotating speed is 300rpm.3 minutes are stood on magnetic frame, about 200 are sucked with P1000
μ L of supernatant, then exhaust residual liquid with P20.
11) repeat step 10 twice, totally three times.
12) with residual liquid in the cleared sample tubes of P20, P100 pipettors add 30 μ L EB, and 10 mixings are blown and beaten up and down,
It is placed on ice.
It is prepared by 2.10 whole libraries
1) following reagent is prepared by table 10；According to table 11, PCR instrument (BioRad S1000) program " POST " is set.
Table 10 captures amplified library and purifying
Table 11PostPCR is set
2) magnetic bead in 20 μ L SCB/T1 combinations libraries is taken, vortex mixing is added in 0.2mL PCR pipes.
3) system as shown in table 12PostPCR systems, sequentially adds 0.2ml PCR pipes, and the μ L of range 30 are set with P100
10 mixings of piping and druming, of short duration centrifugation closes the lid.Table " POST " program in PCR instrument.
Table 12PCR systems set
2.11 whole library purifying
1) PCR primer is placed in micro centrifuge and 1s is quickly centrifuged, rested on for 96 hole magnetic frame upper 5 minutes, draw 50 μ L
Supernatant is added in new 1.5mL Eppendorf LoBind pipes.
2) 50 μ L AMPURE XP magnetic beads are added, is mixed, be incubated at room temperature 5 minutes on vortex blending instrument.
3) centrifuge tube is placed in magnetic frame.Wait solution clarification (about 2 minutes).
4) to be still placed in magnetic frame motionless for centrifuge tube, while carefully siphoning away the μ L of clarified supernatant about 100, should not encounter magnetic bead.
5) centrifuge tube is still placed on magnetic frame, and often pipe adds 75% ethanol of 300 μ L Fresh.
Wait makes magnetic bead fully precipitate in 1 minute, and period, slow rotating centrifugal pipe one was enclosed in the horizontal direction, siphoned away ethanol.
75% ethanol repeated washing is once.
6) centrifuge tube is placed back in into magnetic frame and stands 30s, use the cleared residual ethanol of P10 pipettors.
7) uncap, room temperature dries magnetic bead in 2 minutes, and next step is carried out at once.It is over-drying seriously to reduce dissolved efficiency.
8) 30 μ L EB are added, fully piping and druming is mixed, is incubated at room temperature 3 minutes.
9) centrifuge tube is placed in magnetic frame 2 minutes, until solution is clarified.
10) about 30 μ L of supernatant to new 1.5ml centrifuge tubes are pipetted, magnetic bead is discarded.
3 experimental results
3.1 pre- library total amounts and pre- library QC
In the case of with XT-30 group input amount identicals, the pre- libraries of cfDNA and WBC gDNA under KAPA+XT methods obtain
Rate is significantly higher than XT-30 groups, and concrete outcome is referring to Fig. 1.
Choose the different FFPE gDNA samples of quality to compare test, the method flow of KAPA+XT groups is substantially better than base
In the pre- library yield after the Library development flow of XT groups, 10 PCR cycles apparently higher than XT group flows, concrete outcome referring to table 13 with
And Fig. 2.And because the pre- library yield of XT groups is too low, therefore does not carry out follow-up sequencing and compare.
KAPA-XT groups and the pre- library yield of XT groups under table 13FFPE gDNA difference input amounts
3.2 library concentrations
Determine the concentration in library after purification.Concentration it is too low (<10nM) then indication PCR cycle number is not enough or purifying was lost
It is many, too high (>50nM) it is likely to indicate that undue amplification or non-specific capture composition are excessive.
The constructed cfDNA of KAPA+XT groups of the present invention and leucocyte gDNA library concentrations are all remarkably higher than XT-30 groups,
Concrete outcome is referring to Fig. 3.
3.3 library fragments size detections after purification
By detecting the clip size in library, judge whether library is normal.
Interpretation of result：The library fragments of the cfDNA of adjunction head are distributed between 160-170bp in KAPA+XT groups, leucocyte
The library fragments of gDNA are distributed in 200bp or so, referring to Fig. 4, meet the requirement of library construction, and library construction result is normal.
3.4 capture rates
The capture rate of KAPA+XT groups is average 67% or so and more more stable than XT-30 groups, referring specifically to Fig. 5.
3.5 complexities and sequencing depth
, at identical input amount (30ng), with being sequenced under depth (5000 ×), KAPA+XT groups and XT-30 groups are in complexity for cfDNA
Preferable uniformity is kept on degree, referring specifically to Fig. 6, and in two samples, either library complexity is still sequenced depth,
KAPA+XT groups are superior to XT-30 groups.
Time required for 3.6 whole library constructions
Construction method of the invention, saves experimental implementation flow, and the time is calculated by 8 samples, when manually operated
Between be 170 minutes, experimental period about 10 hours is amounted to, referring to Fig. 7.
According to the SureSelect XT methods of standard, the time is calculated by 8 samples, and the manually operated time is 230 hours,
Amount to experimental period about 26 hours.
Each index of method of the present invention comparison with standard method in library construction can be seen that by above-mentioned experimental result
Aspect generates more excellent technique effect, and this and the above-mentioned improvements to standard method for referring to, and used
For example two-way short chain blocking agent it is directly related.
Agents useful for same is referring specifically to table 14 in embodiment：
Claims (16)
- The preparation method in 1.DNA libraries, its preparation process for including pre- library and whole library, wherein prepared by described pre- library Journey includes that the preparation of DNA, end are repaired and 3 ' ends plus A, joint connection, the purifying of joint connection product, pre- amplified library, amplification Pre- library purifying, described whole library preparation process includes pre- Library hybridization, and prepared by capture wash-out, whole library, and whole library Purge process.
- 2. the preparation method described in claim 1, adds A to enter in a reaction system wherein described end is repaired with 3 ' ends OK.
- 3. the preparation method described in claim 2, wherein end are repaired and the reaction system of 3 ' ends plus A contains ER＆AT buffer solutions, ER＆AT enzymes, and cfDNA.
- 4. the preparation method described in claim 3, wherein end are repaired and the reaction system condition of 3 ' ends plus A is：20 DEG C 30 minutes (85 DEG C of heat lids)65 DEG C 30 minutes (85 DEG C of heat lids)4 DEG C of maintenances.
- 5. the preparation method described in any one of claim 2-4 claim, wherein end repair and 3 ' ends plus A reaction system Middle ER＆AT buffer solutions：ER＆AT enzymes：The volume ratio of cfDNA is 7：3：50.
- 6. the preparation method described in foregoing any one claim, wherein the blocking agent used in the process of the pre- Library hybridization is Two-way short chain blocking agent.
- 7. the preparation method described in claim 6, wherein described two-way short chain blocking agent structure is as follows：
- 8. the preparation method described in foregoing any one claim, wherein being caught with the affine magnetic bead of strepto- in capture elution step Obtain.
- 9. the more conventional method usage amount of the preparation method described in claim 8, the wherein affine magnetic bead amount of strepto- reduces half.
- 10. the preparation method described in claim 8, coupled reaction system and magnetic bead body in its center tap connection product purge process Product is than being 1：0.8.
- Preparation method described in 11. claim any one of 8-10, wherein PCR volumes and magnetic in the pre- library purge process for expanding Pearl volume ratio is 1：1.2.
- Preparation method described in 12. foregoing any one claims, DNA library therein is cfDNA libraries, leucocyte gDNA texts Storehouse or tissue-derived DNA library.
- Preparation method described in 13. foregoing any one claims, wherein still further comprising the pre- library or whole library of purifying Quality inspection, the pre- library of purifying or whole library clip size detection process.
- Preparation method described in 14. claims 12 or 13, wherein carrying out leucocyte gDNA or tissue-derived DNA library structure When building, the time of interrupting is further related to, interrupt dilution and the optimization of wash buffer piping and druming position.
- Two-way short chain blocking agent described in 15. claims 7 is preparing the purposes in being used for DNA library capture agent box.
- The purposes of 16. claims 15, wherein the DNA library is cfDNA libraries, leucocyte gDNA libraries or tissue-derived DNA library.
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