CN107190067A - A kind of improved two generations sequencing random tags joint preparation method - Google Patents
A kind of improved two generations sequencing random tags joint preparation method Download PDFInfo
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- CN107190067A CN107190067A CN201710443643.7A CN201710443643A CN107190067A CN 107190067 A CN107190067 A CN 107190067A CN 201710443643 A CN201710443643 A CN 201710443643A CN 107190067 A CN107190067 A CN 107190067A
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Abstract
The invention discloses the improved two generations sequencing random tags joint preparation method of one kind, it is characterised in that：The Spacer modifications that polymerase can be prevented to extend are introduced on the adapter-primer P7 that joint makes；Extend step in joint, polymerase terminates extension after running into the Spacer sites of P7 template strands, leaves one section of 5 ' protruding terminus；Ensuing digestion step, restriction endonuclease removes the protruding terminus including Spacer sites, produces complete joint；And the insufficient residual of digestion is not digested joint, still retain longer 5 ' protruding terminus.
Description
Technical field
The present invention relates to Nucleic acid sequencing techniques field, and in particular to one kind is connect for improved two generations sequencing with random tags
The process optimization of head.
Background technology
Maturation and the reduction of cost with technology, the sequencing of two generations because of the advantage in its sequencing throughput, in scientific research and
Clinical conversion field is gradually replacing traditional generation sequencing and conventional PCR molecular detecting methods.
Two generations sequencing be applied to clinic is also faced with present many problems with challenge, one of outstanding problem be exactly sequencing compared with
High error rate.Two wider generation microarray datasets of application mainly have Illumina platforms (HiSeq, MiSeq, NextSeq at present
Deng) and Life Ion Torrent platforms (PGM and Proton and derivative series), the sequencing error rate of the generation of two class two sequencing
Also higher (Illumina platforms 0.1%, Ion Torrent platforms 1%).
For somatic mutation (somatic mutation) detection using tumor cell mutations as representative, due to swollen
The ratio in mutational site may be very low (1%) in heterogeneity between oncocyte, tumor sample；And in liquid biopsy, swell
The mutation rate of knurl dissociative DNA is even below 0.1%.Due to higher sequencing error rate background, conventional two generation microarray datasets without
Method detects that these are mutated.The method by molecular label (UMI unique mouecule identifier) main at present come
Overcome this problem.
Molecular label is a bit of DNA fragmentation, can be combination or the random sequence of fixed sequence program, in sequencing
Preceding library preparatory phase, by molecular label mark is introduced in each original DNA template, to reach differentiation DNA profiling
Purpose；In the follow-up bioinformatic analysis stage, pass through the identification of molecular label, it is possible to using in library construction process
The repetitive sequence (duplication) of generation come realize sequencing base correction purpose.
Molecular label technology mainly includes PCR methods and random tags joint method, and random tags joint method is due to lower mistake
Miss rate (10-8) and preferably library construction is compatible, using more extensive.
But random tags joint, due to its manufacture craft, the joint finally made has partially digested incomplete
Flat end fitting, the presence of the flat end fitting in this part can cause the loss of sequencing library template and the wave of sequencing data amount
Take (Fig. 1, Fig. 2).And although the primer oligo methods prepared by biotin labeling joint can remove flat end fitting,
This method can increase in the preparation process of joint, and avidin magnetic bead purge process due to misoperation, easily cause double-strand and connect
Unwinding for head, causes the partial failure of joint.
Therefore, how effectively to remove the flat end fitting in random tags joint be still one urgently need solve
Problem.
The content of the invention
It is an object of the invention to provide optimize technique prepared by a kind of random tags joint.
Characterized in that, introducing Spacer modifications on the adapter-primer P7 that joint makes, the species of modification is included but not
It is limited to the nucleic acid modification (Fig. 3) that C6Spacer, Spacer 18, dSpacer etc. can prevent polymerase from extending.In joint extension
Step (Fig. 4), polymerase terminates extension after running into the Spacer sites of P7 template strands, leaves one section of 5 ' protruding terminus；Connect down
The digestion step come, restriction endonuclease removes the protruding terminus including Spacer sites, produces complete joint；And digestion is not filled
That divides residual is not digested joint, still retains longer 5 ' protruding terminus.
In the building process in library (Fig. 5), normal joint passes through the T bases of end and the A bases of template DNA end
Connection, forms complete library；Non- digestion joint is because the 5 ' ends with longer protrusion, it is impossible to and the DNA profiling with A tails
Connect, the structure in library is not influenceed.
Random tags joint is made up of random tags joint A, random tags joint B and random tags joint C, random tags
The adapter-primer that joint A, random tags joint B and random tags joint C are modified with Spacer respectively by adapter-primer P5 respectively
P7-A, adapter-primer P7-B and adapter-primer P7-C are synthesized into, wherein：
Adapter-primer P5 is SEQ ID NO：01
SEQ1：ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Adapter-primer P7-A is that FFFFF (Spacer) EEEEEJJJJJNNNNNNNNNNNN is connected to SEQ ID NO：02
5 ' end gained；
Adapter-primer P7-B is that FFFFF (Spacer) EEEEEKKKKKNNNNNNNNNNNN is connected to SEQ ID NO：02
5 ' end gained；
Adapter-primer P7-C is that FFFFF (Spacer) EEEEELLLLLNNNNNNNNNNNN is connected to SEQ ID NO：02
5 ' end gained；
SEQ2：AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC
The FFFFF is protruding terminus sequence, and Spacer is Spacer decorating sites, and EEEEE is restriction enzyme site, JJJJJ,
KKKKK and LLLLL is positioning sequence label and JJJJJ, KKKKK and LLLLL are different, and NNNNNNNNNNNN is random molecular
Sequence label.
Wherein：
FFFFF sequences are other base sequences (such as restriction enzyme site protection base sequence) of inhuman source genome, length
5 bases, such as 5-20 base need to be more than；6/7/8/9/10 base etc..
Spacer decorating sites are the Spacer chains between the base or base that Spacer is modified, such as dSpacer, C3
Spacer, C6 Spacer, C12 Spacer, Spacer 9, Spacer 18 etc.；Spacer modification number can for 1,2
It is individual, 3 etc.；The Spacer of more than 2 (including 2) can also be distributed in protruding terminus with first Spacer series connection
In sequence (FFFFF), Spacer is distributed in the pollution that the sequence can be avoided to bring in protruding terminus sequence；
JJJJJ includes but is not limited to 5 identical bases (such as 5-10 differ base)；Equally, KKKKK is included but not
It is limited to 5 identical bases (such as 5-10 differ base), LLLLL and includes but is not limited to 5 identical bases (such as 5-10
Differ base), include but is not limited to 5 identical bases (such as 5-10 differ base), NNNNNNNNNNNN with EEEEE
For 4 to 12 randomized bases, and without four consecutive identical bases.
Wherein, the NNNNNNNNNNNN is expressed as BDHVBDHV, and wherein B represents that the position is the base in addition to A, D
It is the base in addition to C to put the expression position, and H represents that the position is the base in addition to G, and V represents that the position is the base in addition to T.
Wherein, described JJJJJ, KKKKK and LLLLL sequence can be overlapping or complete with the Sequence of the EEEEE
Overlapping, when partially or completely overlapping when, the base of lap only occurs once.
A kind of preparation method of random tags joint group, it is characterised in that：Comprise the following steps：
(1) anneal：By adapter-primer P5, adapter-primer P7-A, adapter-primer P7-B, adapter-primer P7-C and buffer solution and
After appropriate amount of deionized water mixing, progress, which makes annealing treatment, obtains annealing joint A/B/C；
(2) extension annealing joint：Polymerase extension is carried out to gained annealing joint A/B/C and obtains extension joint A/B/C；
(3) precipitate for the first time：Ethanol is carried out respectively to gained extension joint A/B/C or isopropanol precipitating is purified
Extension joint A/B/C afterwards；
(4) digestion：Extension joint A/B/C after purification, which is separately added into, can produce the restricted interior of 3 ' T protruding terminuses
Enzyme cutting carries out digestion and obtains digestion joint A/B/C；
(5) second of precipitation：Gained digestion joint A/B/C is subjected to ethanol or isopropanol precipitating and obtains described after purification
Many positioning random tags joint groups.
Beneficial effects of the present invention
In the manufacturing process of joint (Fig. 4), by the P7oligo sequences digestion position made for random tags joint
Spacer modifications are introduced after point, so that the non-digestion joint remained in the joint prepared after digestion retains cohesive terminus,cohesive termini, from
And prevent it from being connected with library DNA template (the flat end with A tails) and normal joint (the flat end with T tails), make joint
The non-digestion joint remained in preparation process is no longer participate in the structure in library, so as to reduce the template loss for building storehouse and sequencing number
According to the waste of amount.Compared with the mode that biotin labeling removes the non-digestion joint of residual, the present invention need not change original connect
Head preparation process, with more preferable processing compatibility；And because biotin-avidin purification system need not be used, reduce
The cost of manufacture of joint.
Brief description of the drawings
Fig. 1：It is that joint manufacturing process is produced in the residual (Agilent 2100) of flat end fitting, square frame after prepared by joint
The flat end fitting not being digested.
Fig. 2：In influence of the flat end fitting of residual to library construction, template connection procedure, due to flat end fitting end
End can only be single-stranded connection without phosphate group, therefore with the connection of DNA profiling, and the connection product that part disconnects can not be expanded,
Ultimately cause the single-stranded loss or double-strand loss of template.
Fig. 3：What Spacer was modified is not digested joint design
Fig. 4：Spacer modifies joint manufacturing process
Fig. 5：The non-digestion joint of residual is not involved in library construction.
Embodiment
Technical scheme is further detailed and described below by way of embodiment combination accompanying drawing.
Embodiment 1：Joint after improvement is prepared (dSpacer modifies joint)
By adapter-primer P5, adapter-primer P7-A, adapter-primer P7-B, tetra- primer (adapter-primers of adapter-primer P7-C
P5 is SEQ ID NO：1；Adapter-primer P7-A is that FFFFF (Spacer) EEEEEJJJJJNNNNNNNNNNNN is connected to SEQ ID
NO：02 5 ' end gained；Adapter-primer P7-B is that FFFFF (Spacer) EEEEEKKKKKNNNNNNNNNNNN is connected to SEQ ID
NO：02 5 ' end gained；Adapter-primer P7-C is that FFFFF (Spacer) EEEEELLLLLNNNNNNNNNNNN is connected to SEQ ID
NO：02 5 ' end gained；Synthesize producer：Sangon Biotech (Shanghai) Co., Ltd.) it is dilute with ddH2O (or TE buffer solutions)
Release to 100uM；
Wherein FFFFF (Spacer) sequence is TCTTCTTC-dSpacer-T-dSpacer-TTCTTCT
Spacer decorating sites select dSpacer, EEEEE to be restriction enzyme site, and JJJJJ/KKKKK/LLLLL is positioning label
Sequence, NNNNNNNNNNNN is random molecular sequence label.
FFFFF/EEEEE/JJJJJ/KKKKK/LLLLL includes but is not limited to 5 identical bases simultaneously；NNNNNNNNNNNN
For 4 to 12 randomized bases, and without four consecutive identical bases.
The preparation method step of random tags joint is as follows：
Annealing：Following system is prepared in 15ml centrifuge tubes：Adapter-primer P5：1ml, adapter-primer P7 (A)：334ul, connects
Head primer P7 (B)：334ul, adapter-primer P7 (C)：334ul,NEB buffer2:300ul, ddH2O：700ul；Common 3ml.Will
This system is reacted as follows after mixing：95 DEG C, 5min in water-bath；Then it is put at once in the beaker equipped with 95 DEG C of hot water,
Slow cooling is to 24-27 DEG C under room temperature condition；
Fragment of annealing extension：Added in former 15ml centrifuge tubes：10×NEB buffer：200ul, 25mM dNTP mix：
200ul, 500mM DTT：6ul, Klenow exo- (5U/ul)：100ul, volume is supplied with ddH2O to 5ml, after mixing, 37 DEG C
Rotate and mix in insulating box, be incubated 1h.
Precipitate for the first time：The NaAC (3M) of 1/10 volume and the anhydrous second of 2.5 times of volumes are added into above-mentioned products therefrom
- 20 DEG C of 2h are placed in after alcohol, mixing；13000g centrifuges 30min；Supernatant is removed, the rinsing of the ethanol of 5ml 70% is added and precipitates, 4 DEG C,
13000g centrifuges 30min；Supernatant is removed, room temperature dries DNA 20-30min, DNA is resuspended with 3ml ddH2O, and surveyed with Quantus
Concentration.
Enzymolysis (by taking HpyCH4III restriction endonucleases as an example, restriction enzyme site：ACNGT, corresponding adapter-primer P7 sequences EEEEE changes
For ACAGT)：Above-mentioned gained fragment extension products are taken, 10 × NEB CutSmart buffer are added according to its quality x (ug)：2x
Ul, HpyCH4III (5U/ul)：2x ul, volume is supplied with ddH2O to 20x ul, after mixing, is rotated and is incubated in 37 DEG C of insulating boxs
Educate, digest 16h.
Second of precipitation：The absolute ethyl alcohol of NaAC and 2.5 times of volume of 1/10 volume is added into above-mentioned enzymolysis product, is mixed
- 20 DEG C of 2h are placed in after even；4 DEG C, 13000g centrifugations 30min；Supernatant is removed, the rinsing of the ethanol of 10ml 70% is added and precipitates, 4 DEG C,
13000g centrifuges 30min；Supernatant is removed, room temperature dries DNA 20-30min, DNA is resuspended with 1.5ml TE low buffer, be
Final random tags joint, joint through Quality Control it is qualified after dispensed, -20 DEG C freeze it is standby.
Embodiment 2：Joint after improvement is prepared (spacer18 modifies joint)
By adapter-primer P5, adapter-primer P7-A, adapter-primer P7-B, tetra- primer (adapter-primers of adapter-primer P7-C
P5 is SEQ ID NO：1；Adapter-primer P7-A is that FFFFF (Spacer) EEEEEJJJJJNNNNNNNNNNNN is connected to SEQ ID
NO：02 5 ' end gained；Adapter-primer P7-B is that FFFFF (Spacer) EEEEEKKKKKNNNNNNNNNNNN is connected to SEQ ID
NO：02 5 ' end gained；Adapter-primer P7-C is that FFFFF (Spacer) EEEEELLLLLNNNNNNNNNNNN is connected to SEQ ID
NO：02 5 ' end gained；Synthesize producer：Sangon Biotech (Shanghai) Co., Ltd.) it is dilute with ddH2O (or TE buffer solutions)
Release to 100uM；
Wherein FFFFF (Spacer) sequence is TCTTCTTCTTC (spacer18) TTCTTCT
Spacer decorating sites select spacer18, EEEEE to be restriction enzyme site, and JJJJJ/KKKKK/LLLLL marks for positioning
Sequence is signed, NNNNNNNNNNNN is random molecular sequence label.
FFFFF/EEEEE/JJJJJ/KKKKK/LLLLL includes but is not limited to 5 identical bases simultaneously；NNNNNNNNNNNN
For 4 to 12 randomized bases, and without four consecutive identical bases.
The preparation method step of random tags joint is as follows：
Annealing：Following system is prepared in 15ml centrifuge tubes：Adapter-primer P5：1ml, adapter-primer P7 (A)：334ul, connects
Head primer P7 (B)：334ul, adapter-primer P7 (C)：334ul,NEB buffer2:300ul, ddH2O：700ul；Common 3ml.Will
This system is reacted as follows after mixing：95 DEG C, 5min in water-bath；Then it is put at once in the beaker equipped with 95 DEG C of hot water,
Slow cooling is to 24-27 DEG C under room temperature condition；
Fragment of annealing extension：Added in former 15ml centrifuge tubes：10×NEB buffer：200ul, 25mM dNTP mix：
200ul, 500mM DTT：6ul, Klenow exo- (5U/ul)：100ul, volume is supplied with ddH2O to 5ml, after mixing, 37 DEG C
Rotate and mix in insulating box, be incubated 1h.
Precipitate for the first time：The NaAC (3M) of 1/10 volume and the anhydrous second of 2.5 times of volumes are added into above-mentioned products therefrom
- 20 DEG C of 2h are placed in after alcohol, mixing；13000g centrifuges 30min；Supernatant is removed, the rinsing of the ethanol of 5ml 70% is added and precipitates, 4 DEG C,
13000g centrifuges 30min；Supernatant is removed, room temperature dries DNA 20-30min, DNA is resuspended with 3ml ddH2O, and surveyed with Quantus
Concentration.
Enzymolysis (by taking HpyCH4III restriction endonucleases as an example, restriction enzyme site：ACNGT, corresponding adapter-primer P7 sequences EEEEE changes
For ACAGT)：Above-mentioned gained fragment extension products are taken, 10 × NEB CutSmart buffer are added according to its quality x (ug)：2x
Ul, HpyCH4III (5U/ul)：2x ul, volume is supplied with ddH2O to 20x ul, after mixing, is rotated and is incubated in 37 DEG C of insulating boxs
Educate, digest 16h.
Second of precipitation：The absolute ethyl alcohol of NaAC and 2.5 times of volume of 1/10 volume is added into above-mentioned enzymolysis product, is mixed
- 20 DEG C of 2h are placed in after even；4 DEG C, 13000g centrifugations 30min；Supernatant is removed, the rinsing of the ethanol of 10ml 70% is added and precipitates, 4 DEG C,
13000g centrifuges 30min；Supernatant is removed, room temperature dries DNA 20-30min, DNA is resuspended with 1.5ml TE low buffer, be
Final random tags joint, joint through Quality Control it is qualified after dispensed, -20 DEG C freeze it is standby.
Embodiment 3：Joint after improvement is prepared (C6spacer modifies joint)
By adapter-primer P5, adapter-primer P7-A, adapter-primer P7-B, tetra- primer (adapter-primers of adapter-primer P7-C
P5 is SEQ ID NO：1；Adapter-primer P7-A is that FFFFF (Spacer) EEEEEJJJJJNNNNNNNNNNNN is connected to SEQ ID
NO：02 5 ' end gained；Adapter-primer P7-B is that FFFFF (Spacer) EEEEEKKKKKNNNNNNNNNNNN is connected to SEQ ID
NO：02 5 ' end gained；Adapter-primer P7-C is that FFFFF (Spacer) EEEEELLLLLNNNNNNNNNNNN is connected to SEQ ID
NO：02 5 ' end gained；Synthesize producer：Sangon Biotech (Shanghai) Co., Ltd.) it is dilute with ddH2O (or TE buffer solutions)
Release to 100uM；
Wherein FFFFF (Spacer) sequence is TCTTCTTCT (C6spacer) TC (C6spacer) TTCTTCT
Spacer decorating sites select C6spacer, EEEEE to be restriction enzyme site, and JJJJJ/KKKKK/LLLLL marks for positioning
Sequence is signed, NNNNNNNNNNNN is random molecular sequence label.
FFFFF/EEEEE/JJJJJ/KKKKK/LLLLL includes but is not limited to 5 identical bases simultaneously；NNNNNNNNNNNN
For 4 to 12 randomized bases, and without four consecutive identical bases.
The preparation method step of random tags joint is as follows：
Annealing：Following system is prepared in 15ml centrifuge tubes：Adapter-primer P5：1ml, adapter-primer P7 (A)：334ul, connects
Head primer P7 (B)：334ul, adapter-primer P7 (C)：334ul,NEB buffer2:300ul, ddH2O：700ul；Common 3ml.Will
This system is reacted as follows after mixing：95 DEG C, 5min in water-bath；Then it is put at once in the beaker equipped with 95 DEG C of hot water,
Slow cooling is to 24-27 DEG C under room temperature condition；
Fragment of annealing extension：Added in former 15ml centrifuge tubes：10×NEB buffer：200ul, 25mM dNTP mix：
200ul, 500mM DTT：6ul, Klenow exo- (5U/ul)：100ul, volume is supplied with ddH2O to 5ml, after mixing, 37 DEG C
Rotate and mix in insulating box, be incubated 1h.
Precipitate for the first time：The NaAC (3M) of 1/10 volume and the anhydrous second of 2.5 times of volumes are added into above-mentioned products therefrom
- 20 DEG C of 2h are placed in after alcohol, mixing；13000g centrifuges 30min；Supernatant is removed, the rinsing of the ethanol of 5ml 70% is added and precipitates, 4 DEG C,
13000g centrifuges 30min；Supernatant is removed, room temperature dries DNA 20-30min, DNA is resuspended with 3ml ddH2O, and surveyed with Quantus
Concentration.
Enzymolysis (by taking HpyCH4III restriction endonucleases as an example, restriction enzyme site：ACNGT, corresponding adapter-primer P7 sequences EEEEE changes
For ACAGT)：Above-mentioned gained fragment extension products are taken, 10 × NEB CutSmart buffer are added according to its quality x (ug)：2x
Ul, HpyCH4III (5U/ul)：2x ul, volume is supplied with ddH2O to 20x ul, after mixing, is rotated and is incubated in 37 DEG C of insulating boxs
Educate, digest 16h.
Second of precipitation：The absolute ethyl alcohol of NaAC and 2.5 times of volume of 1/10 volume is added into above-mentioned enzymolysis product, is mixed
- 20 DEG C of 2h are placed in after even；4 DEG C, 13000g centrifugations 30min；Supernatant is removed, the rinsing of the ethanol of 10ml 70% is added and precipitates, 4 DEG C,
13000g centrifuges 30min；Supernatant is removed, room temperature dries DNA 20-30min, DNA is resuspended with 1.5ml TE low buffer, be
Final random tags joint, joint through Quality Control it is qualified after dispensed, -20 DEG C freeze it is standby.
Embodiment 4：Joint sequencing PF value improvement situations (sequencing quality) after optimization
Rear leucocyte DNA (average length 220bp) is interrupted with 30ng and acts storehouse of establishing, experiment divides 4 groups, and first group using normal
Random tags joint carry out library construction, its excess-three group is respectively using dSpacer, the spacer18 prepared in embodiment 1-3
The random tags joint made with the optimize technique that C6spacer is modified builds storehouse, builds storehouse kit and uses NEBNext Ultra II
DNA Library Prep Kit, library construction step is as follows：
(1) take 30ng interrupt DNA add 7ul NEBNext Ultra II End Prep Reaction Buffer and
3ul NEBNext Ultra II End Prep Enzyme Mix, volume is supplied with deionized water to 60ul, 20 in PCR instrument
DEG C, 30min → 65 DEG C, 30min → 4 DEG C are maintained；
(2) above-mentioned system adds 1ul joints, then adds 30ul NEBNext Ultra II Ligation Master
Mix and 1ul NEBNext Ligation Enhancer, 20 DEG C of reaction 15min after mixing.Connection product using 0.9 ×
Ampure magnetic beads are purified, and purify water elution using 23ul after purification；
(3) the above-mentioned connection products of 23ul, each 1ul of I5, I7index primer (25uM), and 25ul are added in PCR pipe
NEBNext Ultra IIQ5Master Mix, are reacted as follows after mixing in PCR instrument：
98 DEG C, 30s；
98 DEG C, 10s → 65 DEG C, 75s (8 circulations)；
65 DEG C, 5min；
4 DEG C of maintenances
Purified after the completion of PCR using 0.9 × magnetic bead, then using Quantus (Promega) and
2100bioanalyzer (Agilent) carries out Quality Control
It is sequenced after the library Quality Control of structure is qualified using Illumina NextSeq500 platforms, sequencing reagent is Mid
Output kit (300cycles), Phix mixed ratios are 1%, and each literature data amount is 1Gb, and sequencing result is as follows：
Joint residue sequence can be substantially reduced using optimize technique joint, wherein, three kinds of joint spacer modifications,
DSpacer effects are best.
The foregoing is only a preferred embodiment of the present invention, therefore can not limit the scope that the present invention is implemented according to this, i.e.,
The equivalent changes and modifications made according to the scope of the claims of the present invention and description, all should still belong in the range of the present invention covers.
<110>Xiamen Ai De biological medicines Science and Technology Co., Ltd.
<120>A kind of improved two generations sequencing random tags joint preparation method
<160>2
210>1
<211>33
<212>DNA
<213>Artificial sequence
<400>1
ACACTCTTTC CCTACACGAC GCTCTTCCGA TCT 33
<210>2
<211>34
<212>DNA
<213>Artificial sequence
<400>2
AGATCGGAAG AGCACACGTC TGAACTCCAG TCAC 34
Claims (7)
1. a kind of improved two generations sequencing random tags joint preparation method, it is characterised in that：The joint made in joint draws
The Spacer modifications that polymerase can be prevented to extend are introduced on thing P7；Extend step in joint, polymerase runs into P7 template strands
Extension is terminated behind Spacer sites, one section of 5 ' protruding terminus is left；Ensuing digestion step, restriction endonuclease, which is removed, to be included
Protruding terminus including Spacer sites, produces complete joint；And the insufficient residual of digestion is not digested joint, still protect
Stay longer 5 ' protruding terminus.
2. a kind of improved two generations sequencing random tags joint preparation method as claimed in claim 1, it is characterised in that：
The species of Spacer modifications includes dSpacer, C3Spacer, C6Spacer, C12Spacer, Spacer 9 or Spacer 18
Deng.
3. a kind of improved two generations sequencing random tags joint preparation method as claimed in claim 1, it is characterised in that：
The number of Spacer modifications is 1-5；The Spacer of more than 2 connects with first Spacer, or is distributed in protruding terminus
In sequence.
4. a kind of improved two generations sequencing random tags joint preparation method as claimed in claim 1, it is characterised in that：With
Machine label joint includes random tags joint A, random tags joint B and random tags joint C, random tags joint A, random mark
Sign adapter-primer P7-A, adapter-primer that joint B and random tags joint C is modified with Spacer respectively by adapter-primer P5 respectively
P7-B and adapter-primer P7-C are synthesized into, wherein：
Adapter-primer P5 is SEQ ID NO：01：ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Adapter-primer P7-A is that FFFFF (Spacer) EEEEEJJJJJNNNNNNNNNNNN is connected to SEQ ID NO：02 5 ' ends
Gained；SEQ ID NO：02：AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC
Adapter-primer P7-B is that FFFFF (Spacer) EEEEEKKKKKNNNNNNNNNNNN is connected to SEQ ID NO：02 5 ' ends
Gained；
Adapter-primer P7-C is that FFFFF (Spacer) EEEEELLLLLNNNNNNNNNNNN is connected to SEQ ID NO：02 5 ' ends
Gained；
The FFFFF is protruding terminus sequence, and Spacer is Spacer decorating sites, and EEEEE is restriction enzyme site, JJJJJ,
KKKKK and LLLLL is positioning sequence label and JJJJJ, KKKKK and LLLLL are different, and NNNNNNNNNNNN is random molecular
Sequence label；
Wherein：
FFFFF sequences are other base sequences of inhuman source genome, and length need to be more than 5 bases, preferably 5-20 base；
Spacer decorating sites are the Spacer chains between the base or base that Spacer is modified；The number of Spacer modifications can be with
For 1-5；The Spacer of more than 2 connects with first Spacer, or is distributed in protruding terminus sequence FFFFF, Spacer points
It is distributed in the pollution that the sequence can be avoided to bring in protruding terminus sequence；
JJJJJ, KKKKK, LLLLL include but is not limited to 5 identical bases with EEEEE, and NNNNNNNNNNNN is 4 to 12 random
Base, and without four consecutive identical bases.
5. a kind of improved two generation, which is sequenced, as claimed in claim 4 uses random tags joint preparation method, it is characterised in that：It is described
NNNNNNNNNNNN is expressed as BDHVBDHV, and wherein B represents that the position is the base in addition to A, and the D positional representations position is to remove C
Outer base, H represents that the position is the base in addition to G, and V represents that the position is the base in addition to T.
6. such as a kind of improved two generations sequencing random tags joint preparation method of claim 4, it is characterised in that：It is described
JJJJJ, KKKKK and LLLLL sequence can be overlapping or completely overlapped with the Sequence of the EEEEE, when partially or completely
When overlapping, the base of lap only occurs once.
7. more position random tags joint group preparation method, comprise the following steps：
(1) anneal：By adapter-primer P5, adapter-primer P7-A, adapter-primer P7-B, adapter-primer P7-C and buffer solution and in right amount
After deionized water mixing, progress, which makes annealing treatment, obtains annealing joint A/B/C；
(2) extension annealing joint：Polymerase extension is carried out to gained annealing joint A/B/C and obtains extension joint A/B/C；
(3) precipitate for the first time：Ethanol is carried out respectively to gained extension joint A/B/C or isopropanol precipitating purifying is obtained after purification
Extend joint A/B/C；
(4) digestion：Extension joint A/B/C after purification, which is separately added into, can produce the restriction enzyme of 3 ' T protruding terminuses
Carry out digestion and obtain digestion joint A/B/C；
(5) second of precipitation：Gained digestion joint A/B/C is subjected to ethanol or isopropanol precipitating and obtains described how fixed after purification
Position random tags joint group.
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