CN109056077A - A kind of amplicon sample mixing sequencing library construction method suitable for PacBio microarray dataset - Google Patents
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Abstract
The present invention relates to a kind of amplicon sample mixing sequencing library construction methods suitable for PacBio microarray dataset, it is applied in the library construction of amplicon amplification for existing PacBio nucleic acid sequencing platform, there are reagent cost height, the technical problems such as step complexity, amplification principle of this method based on amplification subsample, in conjunction with PacBio sequencing technologies feature, the sequence label of reverse complemental is introduced in first step amplification for each sample to be tested, make all to be marked with sequence label with the amplicon once expanded, amplicon with different labels is mixed, sequence label retains always in follow-up library building, therefore this method can realize that multiple amplification subsamples only need a library construction, simplify experimental implementation process, it improves and builds library efficiency, reduce sequencing cost.The present invention makes two primers 5 ' of every kind of sample hold the sequence label reverse complemental of addition in tag design sequence, and both ends may recognize that sequence label when sequencing, and the sequence label recognition efficiency of amplification subsample is greatly improved.
Description
Technical field
The present invention relates to library construction techniques fields, more particularly, to a kind of expansion suitable for PacBio microarray dataset
Increase sub- sample mixing sequencing library construction method.
Background technique
(Single Molecule is sequenced using unimolecule in PacBio platform (PacBio nucleic acid sequencing platform) in real time
Real-Time).The sequencing of PacBio platform is based on two key technologies, the invention of first, Pacific Biosciences company
A kind of diameter there was only tens nanometers of nano-pore, can only allow monomolecular nucleic acid under archaeal dna polymerase effect in the nano-pore
It is reacted, detects the deoxynucleotide of these four fluorescent markers of A, T, C, G of synthesis process in nano-pore, nucleic acid can be realized
Sequencing；Second, using Laser Scanning Confocal Microscope in real time, rapidly to the countless nano-pores that are integrated on SMRT cell simultaneously into
Row record.PacBio nucleic acid sequencing platform, it is average to read long reachable 10-15k, and sequencing is not influenced by AT and CG content, energy
It is enough that the region of high GC content or low G/C content is sequenced, there is greater advantage compared to the sequencing of two generations.
But there are still some shortcomings for PacBio nucleic acid sequencing platform, such as when existing amplicon amplification at present, can't
Add Tag primer；It is typically also to be realized by the connector of two-wheeled PCR or the specific sequence label of addition even if introducing label,
These steps are relatively cumbersome, and cost is also higher.And in Illumina microarray dataset, label is had generally by addition
The connector of sequence, or single-ended sequence label is added by PCR in the final step of library construction, these operations equally exist
Relative complex, the at high cost problem of process.
Summary of the invention
For the technical problems in the prior art, the invention proposes a kind of expansions suitable for PacBio microarray dataset
Increase sub- sample mixing sequencing library construction method, this method mixes the amplicon with different labels, it can be achieved that multiple amplification increments
Product only need a library construction and realize sequencing, simplify experimental implementation process, improve and build library efficiency, reduce sequencing at
This.
To achieve the above object, the present invention is achieved by the following technical solutions:
A kind of amplicon sample mixing sequencing library construction method suitable for PacBio microarray dataset, includes the following steps:
(1) design the amplimer with synthesis tape label: each sample to be tested designs and synthesizes the amplification of pair for amplification
Primer, wherein 5 ' ends of each pair of primer have sequence label, the 5 ' of each pair of primer hold sequence label reverse complementals and uniquely without weights
It is multiple；3 ' ends of each pair of primer are the specific primer of amplicon；
(2) amplification of subsample is expanded: respectively using each sample DNA as template, with the amplimer of corresponding tape label
PCR amplification is carried out for primer, the size distribution of purified pcr product and determining segment after amplification；
(3) by the DNA cloning purified product equimolar amounts sample mixing of each sample, total amount is greater than 2 μ g after sample mixing；
(4) sample mixing amplified production is subjected to end reparation and purified；
(5) purified product in step (4) is attached with flat end fitting and is reacted；
(6) circumscribed enzymic digestion and purifying are carried out to the connection product that step (5) obtain, obtained flat suitable for PacBio sequencing
The amplicon sample mixing sequencing library of platform.
Further, PCR amplification system in the step (2) are as follows: 5 μ L of sample DNA of 50ng/ μ L, 10 μM of tape labels
3 μ L of amplimer F, the 3 μ L of amplimer R of 10 μM of tape labels, 10 μ L of 5X HiFi high-fidelity buffer, 10mM dNTP mixing
1.5 μ L of liquid, 1 μ L of 1U/ μ L HiFi enzyme, 26.5 μ L of nuclease-free water.
Further, amplification program in the step (2) are as follows: PCR response procedures are as follows: 98 DEG C of 2min, 20-25 circulation ×
(98 DEG C of 20s, 65 DEG C of 30s, 72 DEG C of 1kb/min), 72 DEG C of 5min.
Further, AMPure PB magnetic beads for purifying pcr amplification product is used in the step (2).
Further, reaction system is repaired in end in the step (4) are as follows: 36.5 μ L of sample mixing amplified production, 10X DNA damage
Wound repairs 5 μ L of buffer, 100X NAD+0.5 μ L, 5 μ L of 10mM ATP Hi, 0.5 μ L of 10mM dNTP, end repair enzyme premix
2.5 μ L of liquid.
Further, the step (4) uses AMPure PB magnetic beads for purifying pcr amplification product.
Further, step (5) coupled reaction system are as follows: repair 31 μ L of product, 20 μM of flat ends in the end of purifying
2 μ L of connector, 4 μ L of 10X template ready-made buffers, 2 μ L of 10mM ATP Low, 1 μ L of 30U/ μ L ligase.
Further, the excision enzyme used in the step (6) is ExoIII and ExoVII excision enzyme.
Further, step (6) the excision enzyme digestion reaction system are as follows: outside connection 40 μ L of reaction product, 100U/ μ L
1 μ L of enzyme cutting III, 1 μ L of 10U/ μ L excision enzyme VII.
Further, in step (6) to connection product carry out circumscribed enzymic digestion and after purification also to library fragments size into
Row detection.
Compared with prior art, the beneficial effects of the present invention are:
(1) the amplicon sample mixing sequencing library construction method proposed by the present invention suitable for PacBio microarray dataset, for
Sequence label is directly added separately to 5 ' ends of target primer in the first step that experiment is opened by multiple samples, each sample
It is introduced directly into sequence label when replicating first time, and makes all to be marked with sequence label with the amplicon once expanded, these marks
Label sequence retains always in follow-up library building process, and the amplicon with different labels is mixed, it can be achieved that multiple amplifications
Subsample only needs a library construction and realizes sequencing, simplifies experimental implementation process, improves and build library efficiency, reduces sequencing
Cost.
(2) the amplicon sample mixing sequencing library construction method suitable for PacBio microarray dataset of the invention is in tag design
When sequence, make the 5 ' of two primers of the every kind of sample sequence label reverse complementals for holding addition, thus the both ends in sequencing
It can recognize that sequence label, can greatly improve the sequence label recognition efficiency of amplification subsample.
Detailed description of the invention
Fig. 1 is that the amplicon sample mixing sequencing library suitable for PacBio microarray dataset of the invention constructs flow chart.
Fig. 2 is amplicon Tag primer structural schematic diagram of the invention.
Specific embodiment
It shows that example illustrates certain embodiments of the present invention, and should not be construed as limiting model of the invention
It encloses.Present disclosure can be improved from material, method and reaction condition simultaneously, all these improvement should all
It falls within spirit and scope of the invention.
Examination used amplicon sample mixing sequencing library construction method provided by the invention suitable for PacBio microarray dataset
Agent is available on the market；Wherein, AMPure PB magnetic bead, HiFi high-fidelity buffer, dNTP mixed liquor, HiFi enzyme are purchased from
KAPA company, DNA damage repair buffer, NAD+, ATP Hi, ATP Hi, flat end fitting, template ready-made buffers, ATP
Low, ligase, exonucleaseⅢ, excision enzyme VII are purchased from PacBio company, and nuclease-free water is purchased from NEB company.
The present invention is applied in the library construction of amplicon amplification for existing PacBio nucleic acid sequencing platform, there is examination
Agent cost is excessively high, builds the technical problems such as library step complexity, based on the amplification principle of amplification subsample, in conjunction with PacBio sequencing technologies
Feature, experiment start the amplification of the first step-amplification subsample in introduce reverse complemental sequence label, visualize this hair
The bright amplicon sample mixing sequencing library construction method suitable for PacBio microarray dataset.
Specifically as shown in Figure 1, this method through the following steps that realize:
1. the amplimer of design and synthesis tape label: the amplimer of each sample to be tested design pair for amplification,
In each pair of primer 5 ' end have sequence label, each pair of primer 5 ' end sequence label reverse complementals and uniquely without repeat；It is each pair of
3 ' ends of primer are the specific primer of amplicon.Amplicon Tag primer structure is as shown in Fig. 2, wherein FFFFFFFFFFFFFF
Indicate that F end-specificity primer, RRRRRRRRRRRRRRRRRRRR indicate R end-specificity primer.
Designed primer is synthesized in such as Shanghai Sheng Gongdeng company of primer Synesis Company.
2. expanding the amplification of subsample: PCR reaction system is prepared, respectively using each sample DNA as template, with corresponding band
The amplimer of label is that primer carries out PCR amplification, and AMPure PB magnetic beads for purifying PCR product is used after amplification and determines piece
The size distribution of section.
Wherein PCR amplification system are as follows: 5 μ L of sample DNA of 50ng/ μ L, 3 μ L of amplimer F of 10 μM of tape labels, 10 μM
3 μ L of amplimer R, 10 μ L of 5X HiFi high-fidelity buffer, the 1.5 μ L of 10mM dNTP mixed liquor, 1U/ μ L HiFi of tape label
1 μ L of enzyme, 26.5 μ L of nuclease-free water.Amplification program are as follows: PCR response procedures are as follows: 98 DEG C of 2min, 20-25 circulation × (98 DEG C
20s, 65 DEG C of 30s, 72 DEG C of 1kb/min), 72 DEG C of 5min.
3. total amount is greater than 2 μ g after sample mixing by the DNA cloning purified product equimolar amounts sample mixing of each sample.
4. sample mixing amplified production carries out end reparation and with AMPure PB magnetic beads for purifying.
Wherein end repair reaction system are as follows: 36.5 μ L of sample mixing amplified production, 10X DNA damage repair 5 μ L of buffer,
100X NAD+0.5 μ L, 5 μ L of 10mM ATP Hi, 0.5 μ L of 10mM dNTP, 2.5 μ L of end repair enzyme premixed liquid.
It is reacted 5. the end of pair purifying is repaired product and is attached with flat end fitting.
Wherein coupled reaction system are as follows: end reparation 31 μ L of product of purifying, 20 μM of flat 2 μ L of end fitting, 10X templates are pre-
4 μ L of buffer processed, 2 μ L of 10mM ATP Low, 1 μ L of 30U/ μ L ligase.
6. pair connection product carries out circumscribed enzymic digestion and purifying, and carries out library quality inspection to purified product, determine that library is big
It is small, to obtain the amplicon sample mixing sequencing library suitable for PacBio microarray dataset.Wherein excision enzyme digestion reaction system are as follows:
Connect 40 μ L of reaction product, 1 μ L of 100U/ μ L exonucleaseⅢ, 1 μ L of 10U/ μ L excision enzyme VII.
Embodiment 1:
The primer amplification that 9 different soil samples are extracted with progress tape label sequence after DNA respectively, carried out after amplification etc.
1 cell is sequenced using PacBio Sequel in molal quantity sample mixing, library construction, and specific experiment process is as follows:
1. the design and synthesis of the amplicon primer of tape label
The target of the project is to analyze the diversity of microorganism in soil sample, using the side of amplification microorganism 16S overall length
Method carries out edaphon constituent analysis.The universal primer of 16S overall length is 27F (AGRGTTYGATYMTGGCTCAG) and 1492R
(RGYTACCTTGTTACGACTT), according to the sequence label primer of universal primer design and corresponding sample such as table 1, nucleosides
Acid sequence is sent as shown in SEQ ID NO:1-18, and by the sequence in table 1 to the raw work synthesis in Shanghai.
Table 1
2. expanding the 16S amplification of subsample
(1) reaction system as shown in Table 2 is prepared
Table 2
(2) it mixes well, brief centrifugation, carries out PCR amplification according to following PCR condition.
(3) after the reaction was completed, magnetic beads for purifying is carried out according to the operation instruction of AMPure PB magnetic bead, is finally washed using 11 μ L
De- buffer elution, purifying are completed.
(4) 1 μ L purified product is taken, dilutes 10 times using nuclease-free water, takes 2 μ L dilutions to carry out Qubit and quantifies, determine
The concentration and total amount of PCR product；It takes 1 μ L dilution to carry out Agilent 2100 to detect, determines the size distribution of segment, it is every to calculate
The molal quantity of a PCR product realizes equimolar amounts sample mixing.
3. sample mixing
(1) according to Qubit quantitative result and 2100 testing result of Agilent, equimolar number sample mixing, molar concentration meter are carried out
It is as follows to calculate formula:
(2) 2 μ g sample mixing amplified productions are taken, using nuclease-free water by volume dilution to 36.5 μ L.
(3) it is vortexed and mixes, brief centrifugation.
4. end is repaired
(1) reaction system:
Table 3
(2) it mixes well, brief centrifugation, 25 DEG C of incubation 5min are placed on ice；
(3) it is purified using AMPure PB magnetic bead, finally uses 31 μ L nuclease free water elutions.5. jointing
(1) it is reacted by following system configurations:
Table 4
(2) it mixes well, brief centrifugation, 25 DEG C of incubations 15min, 65 DEG C of incubation 10min inactivate ligase.
6. exonuclease digestion
(1) it is reacted by following system configurations:
Table 5
(2) it mixes well, brief centrifugation, 37 DEG C of incubation 1h；
(3) it is purified using AMPure PB magnetic bead, finally uses 11 μ L nuclease free water elutions, 11 μ L eluents are
For the library built.
7. library quality inspection
It takes 1 library μ L to dilute 5 times, takes 2 μ L dilutions to carry out Qubit and quantify, calculate library concentration；Take 1 μ L dilution into
Row 2100 detects, and determines library size.
8. machine is sequenced on
(1) according to library quality inspection as a result, according to PacBio Sequel be sequenced operating instruction carry out upper machine sequencing, survey
1 Cell of sequence, output 3.97G data.
(2) sequencing data result is analyzed, data split result is as follows:
Table 6
| Sample | Sequence label | The ccs of single-ended identification BC | The ccs of both-end identification BC |
| Sample 1 | BC01 | 12,669 | 12,386 |
| Sample 2 | BC02 | 9,279 | 8,975 |
| Sample 3 | BC03 | 14,563 | 14,149 |
| Sample 4 | BC04 | 12,201 | 11,771 |
| Sample 5 | BC05 | 21,111 | 20,491 |
| Sample 6 | BC06 | 16,915 | 16,268 |
| Sample 7 | BC07 | 22,989 | 22,087 |
| Sample 8 | BC08 | 20,454 | 19,605 |
| Sample 9 | BC09 | 17,892 | 17,133 |
| total valid ccs | total BC ccs | 148,073 | 142,865 |
| raw_ccs | raw_ccs | 164,344 | 164,344 |
| Discrimination | ratio | 90.10% | 86.93% |
The CCS that can be seen that single-ended identification sequence label from analysis result can achieve 90.1%, and both-end can identify mark
The CCS of label sequence can achieve 86.93%, illustrate that the addition sequence label sample mixing builds the data of the method in only loss 10% in library
Under ratio situation, sample mixing may be implemented and build library sequencing.
Sequencing library construction method of the invention is using PCR product as library object is built, compared to original genomic fragment
Speech, PCR product is more perfect, the defects of without base deletion and single-strand break, therefore does not need progress base reparation；The present invention
Method can also realize that multiple amplification subsamples only need a library construction and realize sequencing, therefore, reduce the reagent for building library
Cost, reduction build the library period, improve and build library efficiency, reduce sequencing cost.Meanwhile method of the invention is widely applicable for
Overall length 16S rDNA builds library and library etc. is set up in overall length transcription.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
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Claims (10)
1. a kind of amplicon sample mixing sequencing library construction method suitable for PacBio microarray dataset, which is characterized in that including such as
Lower step:
(1) design the amplimer with synthesis tape label: each sample to be tested designs and synthesizes the amplimer of pair for amplification,
Wherein 5 ' ends of each pair of primer have sequence label, and the 5 ' of each pair of primer hold sequence label reverse complementals and uniquely without repetition；Often
3 ' the ends to primer are the specific primer of amplicon；
(2) it expands the amplification of subsample: being respectively to draw using each sample DNA as template, with the amplimer of corresponding tape label
Object carries out PCR amplification, the size distribution of purified pcr product and determining segment after amplification；
(3) by the DNA cloning purified product equimolar amounts sample mixing of each sample, total amount is greater than 2 μ g after sample mixing；
(4) sample mixing amplified production is subjected to end reparation and purified；
(5) purified product in step (4) is attached with flat end fitting and is reacted；
(6) circumscribed enzymic digestion and purifying are carried out to the connection product that step (5) obtain, obtained suitable for PacBio microarray dataset
Amplicon sample mixing sequencing library.
2. a kind of amplicon sample mixing sequencing library building side suitable for PacBio microarray dataset according to claim 1
Method, which is characterized in that PCR amplification system in the step (2) are as follows: the expansion of 5 μ L of sample DNA of 50ng/ μ L, 10 μM of tape labels
Increase 3 μ L of amplimer R, the 10 μ L of 5X HiFi high-fidelity buffer, 10mM dNTP mixed liquor of 3 μ L of primers F, 10 μM of tape labels
1.5 μ L, 1 μ L of 1U/ μ L HiFi enzyme, 26.5 μ L of nuclease-free water.
3. a kind of amplicon sample mixing sequencing library building side suitable for PacBio microarray dataset according to claim 1
Method, which is characterized in that amplification program in the step (2) are as follows: PCR response procedures are as follows: 98 DEG C of 2min, 20-25 circulation × (98
DEG C 20s, 65 DEG C of 30s, 72 DEG C of 1kb/min), 72 DEG C of 5min.
4. a kind of amplicon sample mixing sequencing library building side suitable for PacBio microarray dataset according to claim 1
Method, which is characterized in that AMPure PB magnetic beads for purifying pcr amplification product is used in the step (2).
5. a kind of amplicon sample mixing sequencing library building side suitable for PacBio microarray dataset according to claim 1
Method, which is characterized in that reaction system is repaired in end in the step (4) are as follows: 36.5 μ L of sample mixing amplified production, 10X DNA damage
Repair 5 μ L of buffer, 100X NAD+0.5 μ L, 5 μ L of 10mM ATP Hi, 0.5 μ L of 10mM dNTP, end repair enzyme premixed liquid
2.5μL。
6. a kind of amplicon sample mixing sequencing library building side suitable for PacBio microarray dataset according to claim 1
Method, which is characterized in that the step (4) uses AMPure PB magnetic beads for purifying pcr amplification product.
7. a kind of amplicon sample mixing sequencing library building side suitable for PacBio microarray dataset according to claim 1
Method, which is characterized in that step (5) coupled reaction system are as follows: repair 31 μ L of product, 20 μM of flat end fittings in the end of purifying
2 μ L, 4 μ L of 10X template ready-made buffers, 2 μ L of 10mM ATP Low, 1 μ L of 30U/ μ L ligase.
8. a kind of amplicon sample mixing sequencing library building side suitable for PacBio microarray dataset according to claim 1
Method, which is characterized in that the excision enzyme used in the step (6) is ExoIII and ExoVII excision enzyme.
9. a kind of amplicon sample mixing sequencing library building side suitable for PacBio microarray dataset according to claim 8
Method, which is characterized in that step (6) the excision enzyme digestion reaction system are as follows: connection 40 μ L of reaction product, 100U/ μ L excision enzyme
1 μ L of III, 1 μ L of 10U/ μ L excision enzyme VII.
10. a kind of amplicon sample mixing sequencing library building side suitable for PacBio microarray dataset according to claim 1
Method, which is characterized in that circumscribed enzymic digestion is carried out to connection product in step (6) and also library fragments size is examined after purification
It surveys.
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