CN107475352A - A kind of universal PCR amplification fusion primers of the sequenator for being used to be sequenced in synthesis - Google Patents
A kind of universal PCR amplification fusion primers of the sequenator for being used to be sequenced in synthesis Download PDFInfo
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Abstract
The invention discloses a kind of universal PCR amplification fusion primers of sequenator for being used to be sequenced in synthesis, the fusion primer is that a set of primer being made up of a plurality of primer combines, wherein every primer is included successively by 5 ' ends to 3 ' ends：For fixing the fusion primer in the catenation sequence fragment of solid dielectric（101）, for distinguishing the sequence label fragment of different samples（102）, for the sequencing sequence fragment matched with sequencing primer（103）, for forming base difference in the amplified fragments between different samples to balance the diversity sequence fragment of base（104）, and for capturing the amplimer sequence fragment of target area（105）, wherein the diversity sequence fragment between a plurality of primer（104）It is different.Amplification step can be reduced using the fusion primer of the present invention, realizes that base balances, makes the PCR primer sequencing quality of amplification higher.
Description
Technical field
The present invention relates to sequencing technologies field, more particularly to a kind of sequenator for being used to be sequenced in synthesis leads to
Fusion primer is expanded with type PCR.
Background technology
Illumina microarray datasets are that it passes through reversible block technology based on the sequenator that principle is sequenced in synthesis
Realize and only synthesize a base every time, recycle corresponding LASER Excited Fluorescence group, camera capture excites
Light, so as to read base information.However, the shortcomings that technology is when the reading in same circulation all sites
When section (reads) is all same base, an all light or complete dark photo will be obtained when taking pictures, this
Sample is highly detrimental to the positioning of read site, and base identification (base calling).On such case is due to
Caused by the library base balance difference of machine, that is, between each bar read sequence similitude it is too high, cause
The same position base of different reads is all same base.
PCR primer is the unbalanced library of most typical base, because the sequencing experiment of this kind of PCR primer is often used
The specificity and diversity of the same area between more different samples.Often drawn during amplification using same amplification
Thing, different samples are entered with performing PCR amplification, obtained product similitude is higher, often simply a part of wherein
There is base difference in position, and most of base is completely the same.
In sequencing, because this kind of library sequence is present, sequence polymorphism is low, base imbalance problem, causes
Sequencing quality is relatively low, it is necessary to which adding substantial amounts of phix DNA is balanced base, and even so, some are sequenced
Quality is difficult still satisfactory, and available data are relatively low after being sequenced and crossing filter data.Illumina officials
The bacterium rDNA amplicons sequencing program of offer can not solve the problems, such as sequencing quality well, and need logical
Cross two-step PCR to carry out building storehouse, operation is comparatively laborious.And the proposition such as James J.Kozich uses new fusion
Primer banking process, and sequencing primer is revised as in amplimer position, to reduce base to a certain degree not
Balance, but this method is relatively specific for 16s rDNA V4 areas after tested, and be sequenced and imitate for other regions
Fruit is still very poor, while there is also the sequencing primer problem that Illumina officials can not be used to provide, is unfavorable for big
Large-scale production.
The content of the invention
The present invention provides a kind of universal PCR amplification fusion primers of sequenator for being used to be sequenced in synthesis,
Amplification step can be reduced using the fusion primer, realizes that base balances, makes the PCR primer of amplification that matter be sequenced
Amount is higher.
The present invention is achieved through the following technical solutions：
A kind of universal PCR amplification fusion primers of sequenator for being used to be sequenced in synthesis are a set of by more
The primer combination of bar primer composition, wherein every primer is included successively by 5 ' ends to 3 ' ends：It is above-mentioned for fixing
Primer is merged in the catenation sequence fragment of solid dielectric, for distinguishing the sequence label fragment of different samples, is used
In the sequencing sequence fragment matched with sequencing primer, for forming alkali in the amplified fragments between different samples
Basis is different to balance the diversity sequence fragment of base, and for capturing the amplimer tract of target area
Section, wherein the above-mentioned diversity sequence fragment between above-mentioned a plurality of primer is different.
Further, above-mentioned diversity sequence fragment is by 0-7 base composition.
Further, above-mentioned sequenator is Illumina microarray datasets.
Further, above-mentioned catenation sequence fragment includes upstream catenation sequence and downstream connection sequence, its sequence
It is respectively：
5’-AATGATACGGCGACCACCGAGATCTACAC-3’(SEQ ID NO：1)；
5’-CAAGCAGAAGACGGCATACGAGAT-3’(SEQ ID NO：2).
Further, above-mentioned solid dielectric is chip, is combined with thereon and the pairing of above-mentioned catenation sequence fragment
Sequence, for cluster reaction of formation.
Further, above-mentioned sequencing sequence fragment includes upstream sequencing sequence and downstream sequencing sequence, its sequence
It is respectively：
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’(SEQ ID NO：3)；
5’-AGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’(SEQ ID NO：
4)。
Further, above-mentioned sequence label fragment is random sequence of the length for 6-10 base, in different samples
Between it is different from each other.
Further, above-mentioned sequence label fragment is random sequence of the length for 8 bases.
Further, the length of above-mentioned amplimer sequence fragment is 15-25 base.
Further, degeneracy base is included in above-mentioned amplimer sequence fragment.
The fusion primer of the present invention, on the one hand it is integrated with the various sequence fragments needed for the sequencing of downstream, it is only necessary to
One_step PCR is expanded and purifying can complete Library development flow, and follow-up progress quality inspection can be sequenced；On the other hand
Change the relative position of same target area base sequence between each PCR primer by diversity sequence, make same
Base distribution is sequenced in different sequencing circulations, so that base balance is effectively improved,
Finally sequencing quality is set to have qualitative leap.
Brief description of the drawings
Fig. 1 is the structural representation of the different fragments part of the fusion primer of one embodiment of the present invention；
Fig. 2 is the design principle schematic diagram for merging diversity sequence in primer of one embodiment of the present invention；
Fig. 3 is that the fusion primer of one embodiment of the present invention enters the principle schematic of performing PCR amplification.
Description of reference numerals：
101：Catenation sequence fragment；
102：Sequence label fragment；
103：Sequencing sequence fragment；
104：Diversity sequence fragment；
105：Amplimer sequence fragment；
201：Fused upstream primer；
202：Merge primer in downstream；
203：DNA.
Embodiment
The present invention is described in further detail below by embodiment combination accompanying drawing.
Fig. 1 shows the structural representation of the different fragments part of the fusion primer of one embodiment of the present invention.
It should be noted that the fusion primer of the present invention includes a plurality of primer, they are applied in combination, and are respectively used to expand
Increase the sequence fragment of the same area between different samples.It is middle using same amplimer compared with prior art, it is right
Different samples enter performing PCR amplification, and the present invention uses different primers using between fusion primer and different samples
Combine and expanded into performing PCR, therefore avoid the problem of product similitude is higher, caused PCR expands piece
There is notable difference in section, obtain obvious base counterbalance effect between different samples.
The structure that fusion primer shown in Fig. 1 can represent any primer in so-called a plurality of primer is shown
It is intended to, wherein every primer is included successively by 5 ' ends to 3 ' ends：For fixing the fusion primer in solid dielectric
Catenation sequence fragment 101, for distinguishing the sequence label fragment 102 of different samples, be used for and sequencing primer
The sequencing sequence fragment 103 of pairing, for forming base difference in the amplified fragments between different samples with flat
The diversity sequence fragment 104 for the base that weighs, and for capturing the amplimer sequence fragment 105 of target area,
Diversity sequence fragment 104 between wherein above-mentioned a plurality of primer is different.
It should be noted that the so-called fusion primer of the present invention, in fact including sense primer and anti-sense primer,
Because either sense primer or anti-sense primer have common architectural feature, therefore can use same retouch
State expression.Sense primer and anti-sense primer draw in catenation sequence fragment 101, sequencing sequence fragment 103 and amplification
In 105 3 fragments of thing sequence fragment, difference can use respective sequence fragment.Wherein, catenation sequence
Fragment 101 and sequencing sequence fragment 103 are corresponding with the particular sequence in the microarray dataset or sequenator used,
Used microarray dataset or sequenator are different, corresponding catenation sequence fragment 101 and sequencing sequence fragment 103
Also it is different；And amplimer sequence fragment 105 correspond to need sequence on the target area that expands, it is necessary to
The target area of amplification is different, and corresponding amplimer sequence fragment 105 is also different.
It is of the invention to be primarily directed to the sequenator being sequenced while in synthesis, the Typical Representative of such sequenator
Illumina microarray datasets.Using Illumina microarray datasets when, corresponding catenation sequence fragment 101 be with
The cBot catenation sequences for the cBot primers pairing that the supporting cBot equipment of Illumina microarray datasets uses, the company
Connecing sequence includes upstream catenation sequence and downstream connection sequence, and its sequence is respectively：
5’-AATGATACGGCGACCACCGAGATCTACAC-3’(SEQ ID NO：1)；
5’-CAAGCAGAAGACGGCATACGAGAT-3’(SEQ ID NO：2).
It should be noted that cBot equipment is a kind of fasciation released by Illumina into (Cluster Generator)
System, by the way that cBot primers are fixed on solid dielectric (i.e. chip), DNA cloning is guided, is realized
DNA doubles with being similar to " tufted ".The fusion primer of the present invention includes catenation sequence fragment 101, is using
During Illumina microarray datasets, catenation sequence fragment 101 is the cBot equipment supporting with Illumina microarray datasets
The cBot catenation sequences of the cBot primers pairing of use, therefore pass through cBot primers and catenation sequence fragment 101
Between pairing, fusion primer is fixed on solid dielectric (i.e. chip), and then guide DNA cloning, reality
Existing DNA doubles with being similar to " tufted ".
Meanwhile when using Illumina microarray datasets, corresponding sequencing sequence fragment 103 is sequenced including upstream
Sequence and downstream sequencing sequence, its sequence are respectively：
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’(SEQ ID NO：3)；
5’-AGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’(SEQ ID NO：
4)。
The present invention inserts the different sequence of a segment difference between sequencing sequence fragment 103 and amplimer sequence fragment 105
Column-slice section 104 is most important for realizing for the object of the invention.Because diversity sequence fragment 104 is drawn in difference
It is mutually different between thing, the length of the diversity sequence fragment 104 especially between different primers is different,
Performing PCR amplification is entered using different primers without same sample, therefore different samples obtain after PCR amplifications
Amplified fragments can be variant, it is different to be particularly due to the length of diversity sequence fragment 104, can be not same
The effect of " base dislocation " is produced between this amplified fragments, efficiently solves the unbalanced problem of base.
In the present invention, the length of diversity sequence fragment 104 can be not particularly limited, as long as diversity sequence piece
The difference in length between the fusion primer expanded for different samples be present in section 104.Certainly, need
To be illustrated, because the fusion primer expanded for each sample actually includes sense primer with
Primer is swum, as long as between different samples, an at least difference in sense primer and anti-sense primer,
Can be that different sense primer and anti-sense primer or sense primer or anti-sense primer are different.Phase
Ying Di, so-called " there is length in diversity sequence fragment 104 between the fusion primer expanded for different samples
Difference ", as long as referring to the diversity sequence fragment of at least one in sense primer and anti-sense primer not same
The difference in length between this be present.
For each specific primer, the length of diversity sequence fragment 104 for example can be several alkali
Base, even 0 base, such as can be 0-10 base, preferably 0-9 base, more preferably 0-7
Individual base.The sample size that can specifically expand as needed determines, if necessary to the sample size of amplification
Less, the length of diversity sequence fragment 104 can be shorter, you can the difference between different samples is realized,
Sample size if necessary to amplification is more, and the length needs of diversity sequence fragment 104 are longer, could realize
Difference between different samples.In a preferred embodiment of the invention, diversity sequence fragment 104 by
0-7 base composition, it is intended that for each specific primer, the base group of diversity sequence fragment 104
Into being 0-7, certainly for different samples, the base composition number of diversity sequence fragment 104 be it is different,
I.e. sense primer is different with the base composition number of the diversity sequence fragment 104 of at least one in anti-sense primer.
Fig. 2 is the design principle schematic diagram for merging diversity sequence in primer of one embodiment of the present invention, its
In, grey filling for expand purpose region primer sequence ATGCCTATCG (assuming that), it is packless
It is then diversity sequence fragment, by 0-7 base composition, after the diversity sequence fragment makes amplification, overall sequence is backward
0-7 base is shifted, it is all same base at utmost to eliminate the PCR primers of different samples in same position
Situation, be finally reached base balance.Although it should be noted that and it is nonessential, design diversity sequence when
It is too high that G/C content is avoided the occurrence of as far as possible, or continuously repeats and situations such as same base occur.
Sequence label fragment 102 is for distinguishing different samples, and its length can also not limited strictly,
But general control is in several bases or the length of more than ten of base.Label sequence can be specifically determined as needed
The length of column-slice section 102, it is however generally that, if necessary to amplification sample size it is larger, then just need compared with
Long sequence label fragment 102 distinguishes different samples, and the sample size if necessary to amplification is smaller, only needs
Sequence label fragment 102 that will be shorter can distinguish different samples.Sequence label fragment 102 can be random
Sequence, in the case, the sequence label fragment 102 of N number of base can be used in differentiation 4 in theoryNIt is individual not
Same sample, because each site can select any one in tetra- kinds of bases of A, T, G and C,
Certainly consecutive identical base is avoided as sequence label fragment 102 as far as possible in actual applications.The present invention's
In one preferred embodiment, the length of sequence label fragment 102 is 6-10 base, is random sequence, excellent
Random sequence of the length for 8 bases is selected, it is different from each other between different samples.Table 1 shows some marks
The example of (Barcode) sequence is signed, they are the random sequence of 8 bases, different.
Table 1
Amplimer sequence fragment 105 is used to capture target area, thus its length can according to capture purpose,
And the base feature in different plant species determines, general 15-25 base of length be suitable, too short expansion
Increasing primer sequence fragment 105 is unfavorable for effective target area and captured, and long amplimer sequence fragment
105 the problem of may also having regions pair mistake.As needed, degeneracy base can be included, i.e., same
Individual site uses different bases, such as selectable degeneracy base in different amplimer sequence fragments
There is V to represent A+C+G, D represents A+T+G, and B represents T+C+G, and H represents A+T+C, and W is represented
A+T, S represent C+G, and K represents T+G, and M represents A+C, and Y represents C+T, and R represents A+G, N
Represent A+G+C+T.The use of degeneracy base, it is meant that amplimer sequence fragment 105 need not be with target area
Domain capture matching completely, the efficiency of target area capture is improved to a certain extent.
Fig. 3 is that the fusion primer of one embodiment of the present invention enters the principle schematic of performing PCR amplification, upstream
Fusion primer 2 01 and downstream fusion primer 2 02 are attached to mesh by their amplimer sequence fragment respectively
The upstream and downstream of the DNA 203 in region is marked, target fragment can be amplified by PCT amplifications come two
End is respectively provided with fused upstream primer 2 01 and downstream fusion primer 2 02.
To sum up analysis is understood, fusion primer of the invention, is on the one hand integrated with the various sequences needed for the sequencing of downstream
Column-slice section, it is only necessary to which One_step PCR expands and purifying can complete Library development flow, and follow-up progress quality inspection can enter
Row sequencing；On the other hand same target area base sequence between each PCR primer is changed by diversity sequence
Relative position, same base distribution is sequenced in different sequencing circulations, so that base balances
Property is effectively improved, and finally sequencing quality is had qualitative leap.
It the experiment proved that, enter the library that performing PCR expands to obtain, matter after sequencing using the fusion primer of the present invention
Value distribution map is close with normal DNA library sequencing quality.Table 2 is the fusion primer using the present invention
Corresponding sequencing quality value is sequenced in microorganism ITS regions, and microarray dataset is Illumina Miseq.
Table 2
Note：1. the Miseq sequencing quality quality control standards that Illumina is provided are as follows：It is outstanding for PE250：Q30
Average value >=80, it is qualified：Q30 average value >=70；It is outstanding for PE300：Q30 average value >=80, it is qualified：
Q30 average value >=65.2. ITS1-2 and ITS3-4, refer to and expand eucaryote ITS1 (endogenous transcription intervals respectively
Area) and two regions of ITS2 primer；PE250, refers to the sequencing strategy using Pair-End 250bp, R1Q20,
Refer to Read1 base mass values in Pair-End sequencings and reach the Q20 (degrees of accuracy>99%) ratio；R2Q20,
Refer to Read2 base mass values in Pair-End sequencings and reach the Q20 (degrees of accuracy>99%) ratio；R1Q30,
Refer to Read1 base mass values in Pair-End sequencings and reach the Q30 (degrees of accuracy>99.9%) ratio；R2Q30,
Refer to Read2 base mass values in Pair-End sequencings and reach the Q30 (degrees of accuracy>99.9%) ratio.
Describe technical scheme and technique effect in detail by the following examples, it should be understood that following real
It is only exemplary to apply example, it is impossible to is interpreted as limiting the scope of the invention.
Embodiment
Design of primers：The present embodiment is to carry out microbial DNA extraction for 12 soil samples, and is used
Pair of primers expands to bacterium rDNA V1-V3 regions, is identified with carrying out grand genome species composition.
By Fig. 1 order by catenation sequence fragment 101 (cBot catenation sequences), sequence label fragment 102 during design
(i.e. barcode sequences), sequencing sequence fragment 103, diversity sequence fragment 104, and amplimer sequence
Fragment 105 (i.e. extension increasing sequence) combines, and upstream and downstream 90bp or so fusion primer is formed, wherein expanding
Increasing is classified as：
Positive extension increasing sequence：AGAGTTTGATYMTGGCTCAG(SEQ ID NO：5)；
Reverse extension increasing sequence：ATTACCGCGGCTGCTGG(SEQ ID NO：6).
Final positive fusion primer see the table below 3：
Table 3
Ultimately reverse fusion primer see the table below 4：
Table 4
Note：In table 3 and table 4, underscore part is catenation sequence fragment, and Blocked portion is sequence label fragment,
Italicized item is sequencing sequence fragment, and underscore thickened portion is diversity sequence fragment, and thickened portion is amplification
Primer sequence fragment.
Build storehouse：Using the Phusion enzymes of NEB companies, PCR system is as shown in table 5, wherein for each soil
The microbial DNA of sample, forward direction fusion primer are respectively selected from any bar primer sequence in table 3, and reversely fusion is drawn
Thing is respectively selected from any bar primer sequence in table 4；And for each sample, forward direction merges primer and reversely melted
Close at least one in primer differ.
Table 5
| Reagent | Dosage |
| 2×Phusion Master Mix | 25uL |
| Forward direction fusion primer | 2uL |
| Reversely fusion primer | 2uL |
| DNA profiling | 30ng |
| H2O | Mend to overall volume 50uL |
| Total amount | 50uL |
PCR conditions are 94 DEG C of 5min；94 DEG C of 30s, 50 DEG C of 1min, 72 DEG C of 1min, 35 circulations；72℃
7min；4℃∞.
It is sequenced after completing amplification using machine on Miseq sequenators progress PE300, final analysis obtains the quality of data
Situation is as shown in table 6.
Table 6
Note：Two data represent Read1 and Read2 Q20 values respectively in Q20 row；Note：Two in Q30 row
Data represent Read1 and Read2 Q30 values respectively.
The outstanding standard that Q30 reached Illumina and provided is can be seen that from the data shown in table 6.As
Control, using common fusion primer (not including the diversity sequence in the present invention) amplification method, whole service
Sequencing quality value is then unqualified, and concrete outcome is as shown in table 7.
Table 7
| Project | Numerical value |
| Read1%Q20 | 85.54 |
| Read1%Q30 | 72.66 |
| Read2%Q20 | 58.85 |
| Read2%Q30 | 40.35 |
Above content is to combine specific embodiment further description made for the present invention, it is impossible to is recognized
The specific implementation of the fixed present invention is confined to these explanations.For the ordinary skill of the technical field of the invention
For personnel, without departing from the inventive concept of the premise, some simple deduction or replace can also be made,
Protection scope of the present invention should be all considered as belonging to.
Claims (10)
1. the universal PCR amplification fusion primers of a kind of sequenator for being used to be sequenced in synthesis, it is characterised in that the fusion primer is that a set of primer being made up of a plurality of primer combines, wherein every primer is included successively by 5 ' ends to 3 ' ends：For fixing the fusion primer in the catenation sequence fragment of solid dielectric（101）, for distinguishing the sequence label fragment of different samples（102）, for the sequencing sequence fragment matched with sequencing primer（103）, for forming base difference in the amplified fragments between different samples to balance the diversity sequence fragment of base（104）, and for capturing the amplimer sequence fragment of target area（105）, wherein the diversity sequence fragment between a plurality of primer（104）It is different.
2. fusion primer according to claim 1, it is characterised in that the diversity sequence fragment（104）By 0-7 base composition.
3. fusion primer according to claim 1, it is characterised in that the sequenator is Illumina microarray datasets.
4. fusion primer according to claim 3, it is characterised in that the catenation sequence fragment（101）Including upstream catenation sequence and downstream connection sequence, its sequence is respectively：
5’-AATGATACGGCGACCACCGAGATCTACAC-3’（SEQ ID NO：1）；
5’-CAAGCAGAAGACGGCATACGAGAT-3’（SEQ ID NO：2）.
5. fusion primer according to claim 4, it is characterised in that the solid dielectric is chip, is combined with thereon and the catenation sequence fragment（101）The sequence of pairing, for cluster reaction of formation.
6. fusion primer according to claim 3, it is characterised in that the sequencing sequence fragment（103）Including upstream sequencing sequence and downstream sequencing sequence, its sequence is respectively：
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’（SEQ ID NO：3）；
5’-AGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’（SEQ ID NO：4）.
7. according to the fusion primer described in claim any one of 1-6, it is characterised in that the sequence label fragment（102）It is random sequence of the length for 6-10 base, it is different from each other between different samples.
8. fusion primer according to claim 7, it is characterised in that the sequence label fragment（102）It is random sequence of the length for 8 bases.
9. according to the fusion primer described in claim any one of 1-6, it is characterised in that the amplimer sequence fragment（105）Length be 15-25 base.
10. fusion primer according to claim 9, it is characterised in that the amplimer sequence fragment（105）In include degeneracy base.
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| CN114317528A (en) * | 2020-09-30 | 2022-04-12 | 北京吉因加科技有限公司 | Specific molecular label UMI group, mixed specific molecular label joint and application |
| WO2022141366A1 (en) * | 2020-12-31 | 2022-07-07 | 北京寻因生物科技有限公司 | Cell barcode microbead containing frameshift base, preparation method therefor and application thereof |
| CN114846152A (en) * | 2020-12-31 | 2022-08-02 | 北京寻因生物科技有限公司 | Cell label microbead containing frameshift base and preparation method and application thereof |
| CN114846152B (en) * | 2020-12-31 | 2024-02-20 | 北京寻因生物科技有限公司 | Cell label microbead containing frameshift base, and preparation method and application thereof |
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