CN105506063A - Primer composition and uses thereof - Google Patents
Primer composition and uses thereof Download PDFInfo
- Publication number
- CN105506063A CN105506063A CN201410485821.9A CN201410485821A CN105506063A CN 105506063 A CN105506063 A CN 105506063A CN 201410485821 A CN201410485821 A CN 201410485821A CN 105506063 A CN105506063 A CN 105506063A
- Authority
- CN
- China
- Prior art keywords
- sequencing
- nucleic acid
- linker
- target region
- acid sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 30
- 238000012163 sequencing technique Methods 0.000 claims abstract description 356
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 205
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 102
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 84
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 84
- 238000012408 PCR amplification Methods 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims description 73
- 238000006243 chemical reaction Methods 0.000 claims description 46
- 238000012360 testing method Methods 0.000 claims description 29
- 238000010276 construction Methods 0.000 claims description 25
- 108020004414 DNA Proteins 0.000 claims description 19
- 238000002156 mixing Methods 0.000 claims description 19
- 230000003321 amplification Effects 0.000 claims description 18
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 18
- 241000264288 mixed libraries Species 0.000 claims description 13
- 239000000523 sample Substances 0.000 description 102
- 239000000047 product Substances 0.000 description 26
- 238000001514 detection method Methods 0.000 description 23
- 239000003153 chemical reaction reagent Substances 0.000 description 14
- 238000005516 engineering process Methods 0.000 description 10
- 238000000746 purification Methods 0.000 description 6
- 238000013461 design Methods 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 238000012070 whole genome sequencing analysis Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012807 PCR reagent Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 238000007403 mPCR Methods 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 208000021596 pentasomy X Diseases 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a primer composition and uses thereof. The primer composition comprises: a first primer set, wherein the first primer set comprises a first forward primer and a first reverse primer, the first forward primer comprises a target region-specific forward primer and a first nucleic acid sequence, and the first reverse primer comprises a target region-specific reverse primer and a second nucleic acid sequence; and a second primer set, wherein the second primer set comprises a second forward primer and a second reverse primer, the second forward primer comprises the first nucleic acid sequence and a first sequencing linker, and the second reverse primer comprises the second nucleic acid sequence and a second sequencing linker. According to the present invention, with the application of the primer composition to carry out PCR amplification on the nucleic acid sample containing the target region of the sample to be detected, the enrichment of the nucleic acid in the target region can be achieved in one step, and the PCR amplification products directly form the sequencing library.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a primer composition and application thereof, and more particularly relates to the primer composition, a method for constructing a target region nucleic acid sequencing library of a sample to be detected, a method for determining a target region nucleic acid sequence of the sample to be detected, a method for simultaneously determining target region nucleic acid sequences of a plurality of samples to be detected, a system for determining the target region nucleic acid sequences of the samples to be detected and a system for simultaneously determining the target region nucleic acid sequences of the samples to be detected.
Background
The currently used techniques for enrichment sequencing of target regions mainly employ two strategies: probe hybridization capture and PCR amplification enrichment. In the probe hybridization capture, the whole process takes about 4 to 7 days, the cost of one target area usually varies from hundreds to thousands of yuan, and the period of probe design synthesis is usually 6 to 8 weeks or more. The target region enrichment technology based on the PCR principle can be used for machine sequencing only by carrying out PCR and joint adding operation, the whole process generally only needs 1-2 days, the early-stage primer design and synthesis period is generally 2-4 weeks, the primer design is relatively flexible, dozens to thousands of pairs of primers can be adopted, the cost of one sample can be generally as low as dozens of yuan to hundreds of yuan, and the probe hybridization capture technology has certain advantages compared with the probe hybridization capture technology.
However, the current methods for amplifying the enriched target region by PCR still remain to be improved.
Disclosure of Invention
The present invention has been completed based on the following findings of the inventors:
the current method for amplifying the enrichment target region by PCR is to add a linker on the product after the amplification is finished so as to carry out the subsequent sequencing, two PCR reactions are needed, and a purification step is needed in the middle. Therefore, the number of steps is large, the process is still complex, and the PCR amplification enrichment of a large sample size and a large target area is not facilitated.
The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a method for enriching a target region by PCR amplification, which has fewer steps, simpler operation, less time required, and more flexibility in selecting the amount of samples and the number of primers than the conventional method, by continuously optimizing primer design, PCR reaction system, PCR thermal cycle program, etc., and which can easily realize simultaneous target region enrichment of multiple sample regions by implementing the method in combination with a high-throughput chip.
Thus, according to one aspect of the invention, there is provided, in a first aspect, a primer composition. According to an embodiment of the invention, the primer composition comprises: a first primer set comprising a first forward primer and a first reverse primer, wherein the first forward primer consists of a target region-specific forward primer and a first nucleic acid sequence, the first nucleic acid sequence is 5 'of the target region-specific forward primer, the first reverse primer consists of a target region-specific reverse primer and a second nucleic acid sequence, the second nucleic acid sequence is 5' of the target region-specific reverse primer, the first nucleic acid sequence and the second nucleic acid sequence are both common sequences, and the first nucleic acid sequence and the second nucleic acid sequence are different; a second primer set comprising a second forward primer and a second reverse primer, wherein the second forward primer comprises a first nucleic acid sequence and a first sequencing linker, the first sequencing linker is located 5 'to the first nucleic acid sequence, the second reverse primer comprises a second nucleic acid sequence and a second sequencing linker, the second sequencing linker is located 5' to the second nucleic acid sequence. The inventor surprisingly finds that the primer composition provided by the invention can be used for carrying out PCR amplification on a nucleic acid sample containing a target region of a sample to be detected, the enrichment of nucleic acid in the target region can be realized in one step, and the PCR amplification product, namely the enriched nucleic acid in the target region, directly forms a sequencing library, so that the steps of connecting a sequencing joint and purifying the connecting product are not needed, the sequencing can be directly carried out, the cost is low, the efficiency is high, the sequencing result is accurate and reliable, and the repeatability is good.
According to still another aspect of the present invention, the present invention also provides a method for constructing a nucleic acid sequencing library of a target region of a test sample. According to an embodiment of the invention, the method comprises: and carrying out PCR amplification on a nucleic acid sample containing the target region of the sample to be detected by using the primer composition so as to obtain an amplification product, wherein the amplification product forms the nucleic acid sequencing library of the target region of the sample to be detected. According to the embodiment of the invention, the method for constructing the target region nucleic acid sequencing library of the sample to be detected can realize the enrichment of the target region nucleic acid of the sample to be detected and the construction of the target region nucleic acid sequencing library in one step, has the advantages of simple operation, less time requirement and more flexible selection of sample amount and primer amount, and can easily realize the simultaneous target region enrichment of multiple samples and multiple regions when being combined with a high-throughput chip to implement the method. In addition, according to the embodiment of the invention, the method is used for constructing the target region nucleic acid sequencing library of the sample to be detected, the operation is simple, the time requirement is less, the cost is low, the efficiency is high, the obtained target region nucleic acid sequencing library can be directly sequenced, the sequencing result is accurate and reliable, and the repeatability is good. In addition, the method of the invention is suitable for the enrichment of target region nucleic acids and library construction of trace DNA samples, especially nanoliter samples.
According to another aspect of the present invention, the present invention also provides a method for determining the nucleic acid sequence of the target region of a test sample. According to an embodiment of the invention, the method comprises the steps of: constructing a target region nucleic acid sequencing library of a sample to be detected according to the method for constructing the target region nucleic acid sequencing library of the sample to be detected; sequencing the target region nucleic acid sequencing library of the sample to be tested so as to obtain a sequencing result; and determining the sequence of the target region nucleic acid of the sample to be detected based on the sequencing result. The inventor finds that the method can conveniently determine the nucleic acid sequence of the target region of the sample to be detected, and has the advantages of simple operation, less time requirement, flexible selection of sample size and primer quantity, accurate and reliable sequencing result and good repeatability.
According to yet another aspect of the present invention, there is provided a method for simultaneously determining the nucleic acid sequences of target regions of a plurality of samples to be tested. According to an embodiment of the invention, the method comprises the steps of: for each of the multiple samples to be tested, independently constructing a target region nucleic acid sequencing library of the sample to be tested according to the method for constructing the target region nucleic acid sequencing library of the sample to be tested, wherein the tag sequences are further included between the first nucleic acid sequence of the second forward primer and the first sequencing adaptor and/or between the second nucleic acid sequence of the second reverse primer and the second sequencing adaptor, and the tag sequences of the multiple samples to be tested are different from each other, and the multiple number is at least 2; mixing the target region nucleic acid sequencing libraries of the plurality of test samples to obtain a mixed library; sequencing the mixed library to obtain a sequencing result, wherein the sequencing result comprises the sequence of the target region nucleic acid sequencing library and the tag sequence of the plurality of samples to be tested; and distinguishing sequences of the target region nucleic acid sequencing libraries of the plurality of test samples based on the tag sequences, and determining the target region nucleic acid sequence of each of the plurality of test samples. The inventor finds that the method can simultaneously determine the target region nucleic acid sequences of a plurality of samples to be detected, has simple operation, needs less time, is flexible in selecting the sample amount and the primer quantity, and has accurate and reliable sequencing result and good repeatability.
According to yet another aspect of the present invention, there is provided a system for determining the nucleic acid sequence of a target region of a test sample. According to an embodiment of the invention, the system comprises: the library construction device is provided with the primer composition and is used for constructing a target area nucleic acid sequencing library of a sample to be detected according to the method for constructing the target area nucleic acid sequencing library of the sample to be detected; the first sequencing device is connected with the library construction device and is used for sequencing the target region nucleic acid sequencing library of the sample to be tested so as to obtain a sequencing result; and the first nucleic acid sequence determination device is connected with the first sequencing device and is used for determining the sequence of the target region nucleic acid of the sample to be tested based on the sequencing result. According to the embodiment of the invention, the system can conveniently and quickly determine the nucleic acid sequence of the target region of the sample to be detected, and has the advantages of flexible selection of the sample size and the primer quantity, accurate and reliable sequencing result and good repeatability.
According to another aspect of the present invention, the present invention also provides a system for simultaneously determining the nucleic acid sequences of target regions of a plurality of samples to be tested. According to an embodiment of the invention, the system comprises: a library constructing and mixing device, configured to construct a target region nucleic acid sequencing library of the sample to be tested according to the method for constructing a target region nucleic acid sequencing library of the sample to be tested, and mix the target region nucleic acid sequencing libraries of the samples to be tested, so as to obtain a mixed library, wherein the tag sequences of the samples to be tested are different from each other, and the number of the samples to be tested is at least 2; a second sequencing device, connected to the library construction and mixing device, for sequencing the mixed library to obtain a sequencing result, wherein the sequencing result comprises the sequence of the target region nucleic acid sequencing library and the tag sequence of the plurality of samples to be tested; and a second nucleic acid sequence determination device, connected to the second sequencing device, for distinguishing the nucleic acid sequences of the target region sequencing libraries of the plurality of test samples based on the tag sequences and determining the target region nucleic acid sequence of each of the plurality of test samples. According to the embodiment of the invention, the method can be used for simultaneously determining the target region nucleic acid sequences of a plurality of samples to be detected, the selection of the sample size and the primer quantity is flexible, the operation is simple, the time requirement is less, the sequencing result is accurate and reliable, and the repeatability is good.
In addition, it should be noted that, by optimizing primer design, a PCR reaction system and a PCR thermal cycle program, the invention adopts a one-time PCR mode, can realize target region enrichment on a single tube and a high-throughput chip, does not need additional joint operation, also omits purification in the middle of two-step PCR steps, more importantly, can capture multiple samples in multiple regions, and can perform high-throughput sequencing only by further purification after capture.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic structural diagram of a first forward primer, a first reverse primer, a second forward primer and a second reverse primer of a primer composition according to one embodiment of the invention;
FIG. 2 is a schematic flow chart showing a method for determining a nucleic acid sequence of a target region of a test sample according to one embodiment of the present invention;
FIG. 3 is a schematic flow chart of a method for simultaneously determining target region nucleic acid sequences of a plurality of samples to be tested according to one embodiment of the present invention;
FIG. 4 is a schematic diagram showing the structure of a system for determining a nucleic acid sequence in a target region of a sample to be tested according to one embodiment of the present invention;
FIG. 5 is a schematic diagram showing the structure of a system for simultaneously determining target region nucleic acid sequences of a plurality of samples to be tested, according to one embodiment of the present invention;
FIG. 6 shows the results of electrophoresis detection using the inner and outer primers for 63 SNP sites simultaneously, according to one embodiment of the present invention.
FIG. 7 shows the coverage of the detection of 41 SNP sites in 24 samples according to one embodiment of the present invention.
FIG. 8 shows the capture specificity results of the detection of 41 SNP sites of 24 samples according to one embodiment of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
Primer composition
According to one aspect of the invention, the invention provides a primer composition. According to an embodiment of the invention, the primer composition comprises: a first primer set and a second primer set. Specifically, referring to fig. 1, the first primer set comprises a first forward primer and a first reverse primer, wherein the first forward primer is composed of a target region-specific forward primer and a first nucleic acid sequence, the first nucleic acid sequence is located at the 5 'end of the target region-specific forward primer, the first reverse primer is composed of a target region-specific reverse primer and a second nucleic acid sequence, the second nucleic acid sequence is located at the 5' end of the target region-specific reverse primer, the first nucleic acid sequence and the second nucleic acid sequence are both common sequences, and the first nucleic acid sequence and the second nucleic acid sequence are different. The second primer set comprises a second forward primer and a second reverse primer, wherein the second forward primer comprises a first nucleic acid sequence and a first sequencing linker, the first sequencing linker is located at the 5 'end of the first nucleic acid sequence, the second reverse primer comprises a second nucleic acid sequence and a second sequencing linker, and the second sequencing linker is located at the 5' end of the second nucleic acid sequence. According to the embodiment of the invention, the primer composition is utilized to carry out PCR amplification on a nucleic acid sample containing a target region of a sample to be detected, the enrichment of nucleic acid in the target region can be realized in one step, and the PCR amplification product, namely the enriched nucleic acid in the target region, directly forms a sequencing library, so that additional steps of sequencing joint connection and connection product purification are not needed, the sequencing can be directly carried out, the cost is low, the efficiency is high, the sequencing result is accurate and reliable, and the repeatability is good.
According to an embodiment of the invention, the target region-specific forward primer and the target region-specific reverse primer are each 18-25nt in length. Therefore, the primer has high specificity and good amplification effect.
According to an embodiment of the invention, the first nucleic acid sequence is: 5'-ACACTGACGACATGGTTCTACA-3' (SEQ ID NO:1), the second nucleic acid sequence being: 5'-TACGGTAGCAGAGACTTGGTCT-3' (SEQ ID NO: 2).
According to an embodiment of the invention, the first nucleic acid sequence of the first forward primer and the first sequencing adaptor, and/or the second nucleic acid sequence of the first reverse primer and the second sequencing adaptor, further comprise a tag sequence. Thus, after PCR amplification, PCR products of a plurality of samples can be subjected to mixed sequencing, and the sample source of each sequence can be distinguished based on the difference of tag sequences.
According to some embodiments of the invention, the tag sequence is 6-11nt in length.
According to some embodiments of the invention, the first sequencing linker and the second sequencing linker are a P5 sequencing linker and a P7 sequencing linker, respectively, of the Illumina sequencing platform. Wherein, according to some specific examples of the present invention, when the first sequencing linker and the second sequencing linker are a P5 sequencing linker and a P7 sequencing linker of Illumina sequencing platform, respectively, a third nucleic acid sequence with a length of 4nt is further comprised between the tag sequence and the first sequencing linker P5, preferably the third nucleic acid sequence is: 5 '-ACAC-3'. Therefore, the sequencing success rate can be obviously improved, the sequencing result is accurate and reliable, and the sequencing efficiency is high.
According to further embodiments of the invention, the first sequencing linker and the second sequencing linker are an a sequencing linker and a P sequencing linker, respectively, of an lontorrent sequencing platform.
According to a specific example of the present invention, the target region and the target region specific forward/reverse primer sequence corresponding to the target region are shown in the following table:
use of
According to still another aspect of the present invention, the present invention also provides a method for constructing a nucleic acid sequencing library of a target region of a test sample. According to an embodiment of the invention, the method comprises: and carrying out PCR amplification on a nucleic acid sample containing the target region of the sample to be detected by using the primer composition so as to obtain an amplification product, wherein the amplification product forms the nucleic acid sequencing library of the target region of the sample to be detected.
According to an embodiment of the invention, the final concentration ratio of the first primer set to the second primer set is 1: 6-10, preferably 1: 8. therefore, the PCR amplification effect is good, and after the amplification product is directly used for sequencing, the sequencing efficiency is high and the result is accurate.
According to an embodiment of the present invention, the reaction system for PCR amplification comprises, in terms of 100nl reaction system: 10nl of 8 ng/. mu.l to 20ng/ul of template DNA; 50nl2 XPCRmastermix; 10nl 4. mu.M of a second forward primer; 10nl 4. mu.M of a second reverse primer; 10nl500nM first forward primer; 10nl500nM first reverse primer. Therefore, the PCR amplification effect is good, and after the amplification product is directly used for sequencing, the sequencing efficiency is high and the result is accurate.
According to the embodiment of the invention, the reaction procedure of the PCR amplification is as follows:
therefore, the PCR amplification effect is good, the efficiency is high, and the amplification product can be directly used for sequencing.
According to some embodiments of the present invention, the method for constructing a nucleic acid sequencing library of a target region of a test sample according to the present invention may further include the following steps:
(1) aiming at each target region, target region specific forward and reverse primers suitable for the nucleic acid amplification of the target region are designed, and a common sequence is added at the 5' end of each primer: a first nucleic acid sequence and a second nucleic acid sequence, synthesized so as to obtain a first forward primer and a first reverse primer, i.e., a first primer set (also sometimes referred to herein as "inner primers").
(2) A second forward primer comprising the first nucleic acid sequence and the first sequencing linker, and a second reverse primer comprising the second nucleic acid sequence and the second sequencing linker, are synthesized to obtain a second primer set (also sometimes referred to herein as "outer primers").
(3) And performing PCR amplification and gel electrophoresis detection by using the first primer group to verify whether the first primer group meets the requirement, wherein the judgment standard is whether a single band meeting the designed size can be amplified. After the first primer group and the second primer group are verified to be qualified, the ratio of the final concentration of the first primer group to the final concentration of the second primer group is 1: 6-10, preferably 1: 8, mixing, carrying out pre-PCR reaction and gel electrophoresis detection to determine whether the target area can be effectively and specifically amplified.
(4) The test genomic DNA sample is diluted (typically to 10 ng/. mu.L).
(5) And adding the qualified first primer group and second primer group, the sample and the PCR reaction reagent into the chip through a trace sample adding instrument matched with the chip, and carrying out corresponding PCR reaction. Before sample adding, mixing the first primer group and a part of PCR reaction reagent together, and calling the mixture as a detection site reagent premix; and mixing the sample, a second primer group with a label sequence uniquely corresponding to the sample and another part of PCR reaction reagent together, and obtaining a sample premix. When adding sample, firstly adding the sample premix liquid into each micropore on the chip, dividing the chip into a plurality of areas, and adding the corresponding detection site reagent premix liquid into each micropore with the sample premix liquid. Therefore, each micropore is internally provided with a pair of inner primers, a pair of outer primers with label sequences corresponding to the samples, the samples and PCR reagents, namely, a complete PCR reaction system is formed, and after PCR is finished, PCR products can be easily mixed, centrifuged and recycled into a centrifuge tube by inverting the chip.
(6) And then, purifying the PCR product mixed with the recovered samples by using AmpureXP magnetic beads in a certain proportion (0.8-1 time), wherein the purified product is the nucleic acid library.
(7) The constructed nucleic acid library is subjected to library quality detection, and mass concentration, fragment size distribution and molarity can be detected by using Agilent2100Bioanalyzer or Caliper Bioanalyzer, and ABISTEPONERPlusReal-TimePCRSystem, for example. And (4) performing machine sequencing after the detection is qualified.
According to another aspect of the present invention, the present invention also provides a method for determining the nucleic acid sequence of the target region of a test sample. According to an embodiment of the invention, referring to fig. 2, the method comprises the steps of:
s101: constructing a target region nucleic acid sequencing library of a sample to be tested
According to the method for constructing the nucleic acid sequencing library of the target area of the sample to be detected, the nucleic acid sequencing library of the target area of the sample to be detected is constructed.
S102: sequencing a nucleic acid sequencing library of a region of interest
And sequencing the target region nucleic acid sequencing library of the sample to be tested so as to obtain a sequencing result.
Among them, according to the embodiments of the present invention, the sequencing linker should be selected according to the sequencing platform to be used. According to some embodiments of the invention, the sequencing is performed using an Illumina sequencing platform when the first sequencing linker and the second sequencing linker are a P5 sequencing linker and a P7 sequencing linker, respectively, of the Illumina sequencing platform. According to further embodiments of the invention, when the first sequencing linker and the second sequencing linker are an a sequencing linker and a P sequencing linker, respectively, of an IonTorrent sequencing platform, the sequencing is performed using the IonTorrent sequencing platform. Therefore, the obtained PCR product, namely the nucleic acid library can be directly used for sequencing, and the sequencing result is accurate and reliable and has good repeatability.
S103: determination of the nucleic acid sequence of the target region of a sample to be tested
And determining the sequence of the target region nucleic acid of the sample to be detected based on the sequencing result.
The inventor surprisingly finds that the method for determining the target region nucleic acid sequence of the sample to be detected is simple to operate, requires less time, has flexible selection of sample amount and primer quantity, can easily realize simultaneous target region enrichment of multiple samples and multiple regions, and has accurate and reliable sequencing result and good repeatability.
According to yet another aspect of the present invention, there is provided a method for simultaneously determining the nucleic acid sequences of target regions of a plurality of samples to be tested. The inventor finds that the method can be used for easily realizing the enrichment and sequencing of the target region of multiple samples and multiple regions at the same time, and has the advantages of simple operation, less time consumption, accurate and reliable sequencing result and good repeatability.
According to some specific examples of the present invention, referring to fig. 3, the method for simultaneously determining target region nucleic acid sequences of a plurality of samples to be tested according to the present invention may comprise the following steps:
s201: separately constructing a nucleic acid sequencing library of a target region for each of a plurality of test samples
And for each of the multiple samples to be tested, independently constructing a target region nucleic acid sequencing library of the sample to be tested according to the method for constructing the target region nucleic acid sequencing library of the sample to be tested, wherein the tag sequences are further included between the first nucleic acid sequence of the second forward primer and the first sequencing adaptor and/or between the second nucleic acid sequence of the second reverse primer and the second sequencing adaptor, and the tag sequences of the multiple samples to be tested are different from each other, and the multiple number is at least 2.
According to an embodiment of the present invention, the sequencing library construction is performed on the plurality of samples to be tested simultaneously in a multi-well plate, wherein each well in the multi-well plate independently becomes a reaction system. According to some specific examples of the present invention, the multi-well plate is a chip having 5184 wells, and the reaction volume of each well of the chip is 100 nanoliters.
S202: mixing the target region nucleic acid sequencing libraries of a plurality of samples to be tested
And mixing the target region nucleic acid sequencing libraries of the plurality of samples to be tested so as to obtain a mixed library.
S203: sequencing of the Mixed library
Sequencing the mixed library to obtain a sequencing result, wherein the sequencing result comprises the sequence of the target region nucleic acid sequencing library and the tag sequence of the plurality of samples to be tested.
According to an embodiment of the invention, when said sequencing is performed using the Illumina sequencing platform, said first sequencing linker and said second sequencing linker are the P5 sequencing linker and the P7 sequencing linker, respectively, of the Illumina sequencing platform. Wherein, according to some specific examples of the present invention, when the sequencing is performed using Illumina sequencing platform, a third nucleic acid sequence with a length of 4nt is further comprised between the tag sequence and the first sequencing linker P5, preferably the third nucleic acid sequence is: 5 '-ACAC-3'. Therefore, the sequencing success rate can be obviously improved, the sequencing result is accurate and reliable, and the sequencing efficiency is high.
According to an embodiment of the invention, when the sequencing is performed by using an IonTorrent sequencing platform, the first sequencing linker and the second sequencing linker are respectively an A sequencing linker and a P sequencing linker of the IonTorrent sequencing platform.
S204: determining the target region nucleic acid sequence for each of a plurality of test samples
And distinguishing sequences of the target region nucleic acid sequencing libraries of the plurality of samples to be tested based on the tag sequences, and determining the target region nucleic acid sequence of each of the plurality of samples to be tested.
According to another aspect of the present invention, the present invention also provides a system for determining the nucleic acid sequence of a target region of a test sample. Referring to fig. 4, the system 100 includes, according to an embodiment of the present invention: a library construction device 101, a first sequencing device 102 and a first nucleic acid sequence determination device 103. According to the embodiment of the invention, the system is used for determining the target region nucleic acid sequence of the sample to be detected, the operation is simple, the time is less, the sequencing result is accurate and reliable, and the repeatability is good.
Specifically, the library constructing apparatus 101 is provided with the primer composition described above, and is configured to construct a target region nucleic acid sequencing library of a sample to be tested according to the method for constructing a target region nucleic acid sequencing library of a sample to be tested described above; the first sequencing device 102 is connected to the library constructing device 101, and is configured to sequence a target region nucleic acid sequencing library of the sample to be tested, so as to obtain a sequencing result; the first nucleic acid sequence determination device 103 is connected to the first sequencing device 102, and is configured to determine a sequence of a target region nucleic acid of a sample to be tested based on the sequencing result.
According to some embodiments of the invention, the first sequencing device 102 is an Illumina sequencing platform and the first sequencing linker and the second sequencing linker are a P5 sequencing linker and a P7 sequencing linker, respectively, of the Illumina sequencing platform.
According to other embodiments of the invention, the first sequencing device 102 is an iontornt sequencing platform, and the first sequencing linker and the second sequencing linker are an a sequencing linker and a P sequencing linker, respectively, of the iontornt sequencing platform.
According to yet another aspect of the present invention, there is provided a system for simultaneously determining the nucleic acid sequences of target regions of a plurality of test samples. Referring to fig. 5, the system 200 includes, according to an embodiment of the present invention: a library construction and mixing device 201, a second sequencing device 202 and a second nucleic acid sequence determination device 203. According to the embodiment of the invention, the system can be used for easily realizing the enrichment and sequencing of the target region of multiple sample regions simultaneously, and has the advantages of simple structure, convenient operation, less time, low cost, accurate and reliable sequencing result and good repeatability.
Specifically, the library constructing and mixing apparatus 201 is configured to construct a target region nucleic acid sequencing library of the sample to be tested according to the method for constructing a target region nucleic acid sequencing library of the sample to be tested, and mix the target region nucleic acid sequencing libraries of the samples to be tested, so as to obtain a mixed library, wherein the tag sequences of the samples to be tested are different from each other, and the number of the samples to be tested is at least 2. The second sequencing device 202 is connected to the library constructing and mixing device 201, and is configured to sequence the mixed library so as to obtain a sequencing result, where the sequencing result includes sequences of the target region nucleic acid sequencing libraries of the multiple samples to be tested and the tag sequence. The second nucleic acid sequence determination device 203 is connected to the second sequencing device 202, and is configured to distinguish the nucleic acid sequences of the target region sequencing libraries of the plurality of samples to be tested based on the tag sequences, and determine the target region nucleic acid sequence of each of the plurality of samples to be tested.
According to some embodiments of the present invention, a multi-well plate is disposed in the library constructing and mixing apparatus 201, so that the multi-well plate can be used to simultaneously perform sequencing library construction on the plurality of samples to be tested, wherein each well in the multi-well plate independently becomes a reaction system. According to some specific examples of the present invention, the multi-well plate is a chip having 5184 wells, and the reaction volume of each well of the chip is 100 nanoliters.
According to some embodiments of the invention, the second sequencing device 202 is an Illumina sequencing platform and the first sequencing linker and the second sequencing linker are a P5 sequencing linker and a P7 sequencing linker, respectively, of the Illumina sequencing platform. Wherein, according to some specific examples of the present invention, when the second sequencing device 202 is Illumina sequencing platform, a third nucleic acid sequence with a length of 4nt is further included between the tag sequence and the first sequencing linker P5, preferably the third nucleic acid sequence is: 5 '-ACAC-3'. Therefore, the sequencing success rate can be obviously improved, the sequencing result is accurate and reliable, and the sequencing efficiency is high.
According to other embodiments of the present invention, the second sequencing device 202 is an iontornt sequencing platform, and the first sequencing linker and the second sequencing linker are an a sequencing linker and a P sequencing linker, respectively, of the iontornt sequencing platform.
In addition, according to some embodiments of the present invention, the enrichment and sequencing of multiple regions of multiple samples can be conveniently and rapidly achieved by combining the method of the present invention with high throughput chip technology. Specifically, the size of the chip that can be used may be 72 × 72 — 5184 (holes), but is not limited to this size, and may also be 36 × 36 holes, 48 × 48 holes, and 60 × 60 holes, for example. According to one embodiment of the present invention, the enrichment of the target region is performed using a 72 x 72 (5184) well chip from WaferGen, usa, which is made of aluminum with good thermal conductivity and is surrounded by an inert material, and the volume of each well is equal to nano-scale, so that each well can perform a single PCR reaction.
Furthermore, in practical experimental operations, a plurality of samples and a plurality of target areas can be combined according to any scientific combination, for example, the combination form of 24 samples and 216 target areas can be: dividing the chip into 24 regions according to the number of samples, each region using one sample and one tag (as described above, the tags are located on the second primer set), the samples and tags used in each of the 24 regions being different from each other, so that 24 samples and 24 tag sequences exist in the 24 regions; each of the 24 regions contains 216 microwells, and the samples and tags used in the 216 microwells are the same for the same region, while the target region-specific primers are different. Furthermore, each of the 24 regions is capable of amplifying and enriching 216 target region nucleic acid sequences for one sample, so that the whole chip can amplify and enrich 216 target region nucleic acid sequences for 24 samples, that is, a total of 24 × 216 — 5184 target regions are enriched. Based on that each micropore is an independent PCR reaction system, namely each amplification enrichment reaction is independent, the specificity and the efficiency of nucleic acid enrichment capture can be ensured. Furthermore, based on the fact that the amplification products of 24 samples all carry mutually different tag sequences, all 5184 amplification products can be subjected to mixed sequencing, and finally, the tag sequences are used for distinguishing and identifying different samples.
It should also be noted that the method and system of the present invention can be combined with a high throughput chip, and multiple (e.g. 5184) target fragment regions of interest of multiple samples can be enriched from genomic DNA by one-step PCR reaction, and the enriched amplified products can directly constitute a sequenced DNA library (since it is a one-step PCR reaction, and no purification step is needed in the middle, the whole library construction process can be completed in a microwell chip). Compared with the existing PCR enrichment amplification technology, the method shortens the experimental process and time, and is more convenient and faster. Compared with the traditional target area capturing technology, the method has the following advantages: the traditional DNA library captured by the target region generally takes 4-7 days (probe hybridization kit of agent and NimbleGen), and the invention only needs 3-4 hours; the traditional DNA library construction captured by the target region needs a plurality of steps including target region enrichment, library construction and the like, the technology only needs one-step PCR reaction, can operate on a trace DNA sample as low as 50ng, and can capture a DNA fragment of about 200bp by a 100nL system, for example, the 100nL reaction system comprises: 10nl of template DNA (10 ng/. mu.l), 10nl of 2XPCRmastermix50nl, 10nl of second forward primer (4. mu.M), 10nl of second reverse primer (4. mu.M) and 20nl of first primer group (250nM each), namely, the reagent cost is greatly reduced. Compared with single-tube multiplex PCR amplification enrichment technology (Ampli-seq, Life technologies), the method uses a reaction system with high integration and a PCR amplification method, and effectively solves the problem of low efficiency caused by mutual interference among multiplex PCR primers while ensuring flux. Compared with the traditional method, the library construction method can be carried out on a high-throughput chip, the volume of each PCR reaction is nanoliter-level (such as a 100nl reaction system), so that the consumption of samples and reagents is greatly reduced, and the method is particularly suitable for nucleic acid enrichment and sequencing of trace samples. In conclusion, the method and the system of the invention have the advantages of rapidness, simple operation, low cost, high flux, high enrichment efficiency and the like, and have the advantages that other technologies in the technical field of small-area target region enrichment sequencing library construction cannot be compared favorably.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1:
referring to fig. 2 and fig. 3, according to the method for constructing a target region nucleic acid sequencing library and sequencing of a sample to be tested, an Illumina sequencing platform (Miseq sequencer) is utilized to perform variation detection of 63 SNP sites and target region sequencing on 24 human genome DNA samples with known whole genome sequencing results, which are specifically as follows:
designing multiple pairs of inner primers aiming at 63 personal SNP loci, and further testing genomic DNA samples of multiple samples, wherein the specific operations are as follows:
(1) respectively designing target region specific forward and reverse primers aiming at the region where each SNP locus is located, ensuring that the SNP locus is located in the middle of the region where the forward and reverse primers cover the initial position and the sequencing read length, the length of the primers is 18-25nt, the number of Poly-X is less than or equal to 3, and the Tm value of the primers is within the range of 58-60 ℃;
(2) adding common sequences to the 5' ends of the designed target region specific forward and reverse primers respectively and synthesizing so as to obtain inner primers (i.e., a first primer group: a first forward primer and a first reverse primer):
first forward primer: 5'-ACACTGACGACATGGTTCTACA-3' + 5-NNNNNNNNNNNNNNNN NNNN-3 ', wherein 5'-ACACTGACGACATGGTTCTACA-3' is the first nucleic acid sequence and the target region specific forward primer sequence is underlined;
a first reverse primer: 5'-TACGGTAGCAGAGACTTGGTCT-3' + 5-NNNNNNNNNNNNNNNN NNNN-3 ', wherein "5'-TACGGTAGCAGAGACTTGGTCT-3'" is the second nucleic acid sequence, the target region specific reverse primer sequence is underlined,
the specific sequences of the first forward primer and the first reverse primer for each SNP site are shown in Table 1.
TABLE 1
(3) Primers with sequencing adaptors and common sequences were synthesized so as to obtain outer primers (i.e.second primer set: second forward primer and second reverse primer), the sequence of the outer primers of each SNP site being identical:
a second forward primer: 5'-AATGATACGGCGACCACCGAGATCT-3' +5 '-ACAC-3' +NNNNNNN N+ 5'-ACACTGACGACATGGTTCTACA-3', wherein "5'-AATGATACGGCGACCACCGAGATCT-3'" is the first sequencing linker (seq id no: 266, P5 sequencing linker of Illumina sequencing platform), underlined is a tag sequence with a base length of 8nt, and further comprising between the tag sequence and the first sequencing linker (P5 sequencing linker) a third nucleic acid sequence with a length of 4 nt: 5 '-ACAC-3';
a second reverse primer: 5'-CAAGCAGAAGACGGCATACGAGAT-3' +NNNNNNNN+ 5'-TACGGTAGCAGAGACTTGGTCT-3', wherein "5'-CAAGCAGAAGACGGCATACGAGAT-3'" is the second sequencing linker (SEQ ID NO: 267, P7 sequencing linker of Illumina sequencing platform), and underlined is a tag sequence with a base length of 8 nt.
In this embodiment, 8 × 3-24 tag combinations are formed by combining 8 second forward primer tag sequences (seq id no: 255-. Furthermore, after subsequent library construction and sequencing is completed, 24 samples can be easily distinguished based on the tag combinations (i.e., the second forward primer tag sequence and the second reverse primer tag sequence).
The specific sequences of 8 second forward primer tag sequences (SEQ ID NO: 255-262) are shown below:
the specific sequence of the 3 second reverse primer tag sequences (SEQ ID NO: 263-265) is shown below:
(4) and (3) carrying out PCR amplification and gel electrophoresis detection by using a human genome DNA standard as a template and using the first primer group to verify whether the first primer group meets the requirement, wherein the judgment standard is that whether a single band meeting the designed size can be amplified.
Wherein, the total volume of the PCR system is 10 mul, and the system comprises:
template DNA (10 ng/. mu.l) 1. mu.l, 2XPCRmastermix 5. mu.l, first forward primer (10. mu.M) 0.5. mu.l, first reverse primer (10. mu.M) 0.5. mu.l, ddH2O3μl。
The PCR reaction procedure is shown in table 2 below:
TABLE 2PCR procedure
Then, the first primer group and the second primer group which are qualified are mixed according to the ratio of the final concentration of 1: 8, mixing, and carrying out pre-PCR reaction and gel electrophoresis detection by using a human genome DNA standard product as a template so as to determine whether the target region can be effectively and specifically amplified. Wherein, the total volume of the PCR reaction system is 10 mul, and the method comprises the following steps: template DNA (10 ng/. mu.l) 1. mu.l, 2XPCRMastermix 5. mu.l, second forward primer (10. mu.M) 0.4. mu.l, second reverse primer (10. mu.M) 0.4. mu.l, first primer set (pair of inner primers) (250nM each) 2. mu.l, ddH2O1.2. mu.l. The PCR reaction procedure is shown in Table 2.
In which, FIG. 6 shows the results of electrophoresis detection using the inner and outer primers of each SNP site simultaneously. As shown in FIG. 6, the correspondence between each lane band and the first primer set for the target region to be used (i.e., the lane number and the first primer set for the corresponding target region to be used for detection) is shown in the following table:
the results show that the first primer sets (i.e., the inner primers of the 41 target regions) corresponding to the lanes 133, 134, 145, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 155, 156, 157, 158, 159, 161, 167, 170, 171, 173, 175, 176, 177, 178, 180, 181, 182, 187, 188, 189, 190, 193 are qualified and can be used for subsequent library construction. The first primer set corresponding to the remaining lanes is not qualified and is no longer used for library construction.
The genomic DNA of the sample to be tested was diluted to 10 ng/. mu.l for use.
(5) Then, using a 72 × 72 ═ 5184 well chip from WaferGen corporation, usa, PCR reaction was performed according to the following 100nl reaction system: template DNA (10 ng/. mu.l) 10nl, 2XPCRmastermix50nl, second forward primer (4. mu.M) 10nl, second reverse primer (4. mu.M) 10nl, and first primer set (pair of inner primers) (250nM each) 20 nl. Wherein, sample adding and PCR reaction are carried out by a microscale sample adding instrument (MultisampleNanodispenser, provided by WaferGen) and a PCR instrument (Bio-Rad, provided by model T-100) which are matched with the chip. Specifically, the method comprises the following steps:
and adding the first primer group and the second primer group which are qualified through pre-PCR verification, the sample and the PCR reaction reagent into the chip, and carrying out corresponding PCR reaction. Before sample adding, mixing the first primer group and a part of PCR reaction reagent together, and calling the mixture as a detection site reagent premix; and mixing the sample, a second primer group with a label sequence uniquely corresponding to the sample and another part of PCR reaction reagent together, and obtaining a sample premix. Before sample adding, 24 areas are selected on the chip, each area is provided with 41 micropores (corresponding to 41 target areas, namely 41 SNP sites), when sample adding is carried out, firstly, the sample premix is added into each micropore of the 24 areas on the chip, and then, the corresponding detection site reagent premix is added into the micropore of the sample premix existing in each area. Therefore, each micropore is internally provided with a pair of inner primers, a pair of outer primers with label sequences corresponding to the samples, the samples and PCR reagents, namely, a complete PCR reaction system is formed, and after PCR is finished, PCR products can be easily recovered into a centrifuge tube by inverting the chip. The PCR procedure was as in Table 2.
(6) After the PCR is finished, the chip is subjected to membrane removal and then inverted centrifugation, PCR products in different holes are mixed and centrifuged at the moment, the PCR products are recovered in a centrifuge tube, then, 1-time volume of AmpureXP magnetic beads are used for purification to remove PCR reaction reagents and primers which are not reacted, and the purified products are the nucleic acid library obtained by construction.
(7) And (3) detecting the quality of the library: the constructed library was tested for quality and yield using an Agilent2100Bioanalyzer and ABISTEPOnerPlusReal-TimePCRSystem.
(8) After the quality of the library is qualified, sequencing is carried out on a Miseq sequencer, the length is read by 150bp, the sequencing depth is required to reach 800 times, and the data volume of an amplification product is 0.24 Mb.
(9) And (3) information analysis flow: firstly, the software fqclean is used to filter out the base sequences of non-target regions, such as linkers and low-quality reads, from the raw data (all data) obtained by sequencing, resulting in clean data. Cleardata was aligned onto the reference genome hg19 using base sequence alignment software Bwa, statistical data quality control information (where sequencing data for each sample was distinguished based on the tag sequence of each sample). Meanwhile, cleardata is input into base variation detection software Samtools to carry out SNPfilling (searching for base sites with variation), finally 41 SNPs (base variation sites) position information concerned in the embodiment is introduced to carry out SNP filtration to obtain final Genotype, and then the obtained Genotype is compared with whole genome sequencing data of a corresponding sample to analyze the accuracy of SNP detection.
(10) The coverage and capture specificity of 41 SNP sites of 24 samples were counted, and the results are shown in FIG. 7 and FIG. 8. The final data results for the 800X data showed that the average coverage was 95% and the capture specificity was greater than 90% for each sample (as shown in figures 7 and 8). Specifically, in fig. 7, coverage is the number of sites detected/the number of sites of interest. As can be seen from FIG. 7, the coverage of 41 SNP sites of 24 samples was 0.91 or more, indicating that the detection sites were effective; the coverage of more than 10 times of detection is more than 0.94, and the coverage of more than 50 times of detection is more than 0.91, which shows that the obtained results are uniform. In fig. 8, the capture specificity is the number of sequences identical to the sequence of interest/the number of all sequences sequenced, and a higher value indicates a higher availability of data sequenced. As can be seen from fig. 8, the lowest capture specificity of 24 samples was also close to 0.9, indicating that the availability of sequencing data was very high.
The results of variation detection of 41 SNP sites in 24 samples were compared with the known whole genome sequencing results, and the results are shown in Table 3 below. The result of the variation detection of 41 SNP sites in 24 samples is accurate and reliable.
TABLE 3 consistency of SNP detection results with partial sample whole genome sequencing results
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. A primer composition, comprising:
a first primer set comprising a first forward primer and a first reverse primer,
wherein,
the first forward primer is composed of a target region-specific forward primer and a first nucleic acid sequence located 5' of the target region-specific forward primer,
the first reverse primer is composed of a target region-specific reverse primer and a second nucleic acid sequence located 5' to the target region-specific reverse primer,
the first nucleic acid sequence and the second nucleic acid sequence are both a common sequence, and the first nucleic acid sequence and the second nucleic acid sequence are different;
a second primer set comprising a second forward primer and a second reverse primer,
wherein,
the second forward primer comprises a first nucleic acid sequence and a first sequencing linker, the first sequencing linker is located 5' to the first nucleic acid sequence,
the second reverse primer comprises a second nucleic acid sequence and a second sequencing linker, the second sequencing linker being located 5' to the second nucleic acid sequence.
2. The primer composition of claim 1, wherein the first nucleic acid sequence of the second forward primer and the first sequencing linker, and/or the second nucleic acid sequence of the second reverse primer and the second sequencing linker, further comprise a tag sequence,
optionally, the tag sequence is 6-11nt in length,
optionally, the target region-specific forward primer and the target region-specific reverse primer are each 18-25nt in length,
optionally, the first nucleic acid sequence is: 5'-ACACTGACGACATGGTTCTACA-3' (SEQ ID NO:1), the second nucleic acid sequence being: 5'-TACGGTAGCAGAGACTTGGTCT-3' (SEQ ID NO:2),
optionally, the first sequencing linker and the second sequencing linker are a P5 sequencing linker and a P7 sequencing linker, respectively, of an Illumina sequencing platform,
preferably, a third nucleic acid sequence with a length of 4nt is further comprised between the tag sequence and the first sequencing linker P5, more preferably the third nucleic acid sequence is: 5 '-ACAC-3'.
3. The primer composition of claim 1, wherein the first sequencing linker and the second sequencing linker are an a sequencing linker and a P sequencing linker, respectively, of an IonTorrent sequencing platform.
4. The primer composition of claim 1, wherein the target region and the target region-specific forward/reverse primer sequence corresponding to the target region are as shown in the following table:
5. a method for constructing a nucleic acid sequencing library of a target region of a sample to be tested, which is characterized by comprising the following steps:
performing PCR amplification on a nucleic acid sample containing a target region in a test sample by using the primer composition of any one of claims 1 to 4, so as to obtain an amplification product, wherein the amplification product constitutes a nucleic acid sequencing library of the target region in the test sample.
6. The method of claim 5, wherein the final concentration ratio of the first primer set to the second primer set is 1: 6-10, preferably 1: 8,
optionally, the reaction system for PCR amplification comprises, calculated on a 100nl reaction system:
10nl of 8 ng/. mu.l to 20ng/ul of template DNA;
50nl2XPCRmastermix；
10nl 4. mu.M of a second forward primer;
10nl 4. mu.M of a second reverse primer;
10nl500nM first forward primer;
10nl500nM first reverse primer,
optionally, the reaction procedure of the PCR amplification is:
7. a method for determining a nucleic acid sequence of a target region of a test sample, comprising the steps of:
constructing a nucleic acid sequencing library of a target region of a test sample according to the method of claim 5 or 6;
sequencing the target region nucleic acid sequencing library of the sample to be tested so as to obtain a sequencing result; and
determining the sequence of the nucleic acid of the target region of the sample to be tested based on the sequencing result,
optionally, the sequencing is performed using an Illumina sequencing platform when the first sequencing linker and the second sequencing linker are the P5 sequencing linker and the P7 sequencing linker, respectively, of the Illumina sequencing platform,
optionally, the sequencing is performed using an iontornt sequencing platform when the first sequencing linker and the second sequencing linker are the a sequencing linker and the P sequencing linker, respectively, of the iontornt sequencing platform.
8. A method for simultaneously determining the nucleic acid sequences of target regions of a plurality of samples to be tested, comprising the steps of:
for each of the plurality of samples to be tested, independently constructing a target region nucleic acid sequencing library of the sample to be tested according to the method of claim 5 or 6, wherein the sequence between the first nucleic acid sequence of the second forward primer and the first sequencing adaptor and/or the sequence between the second nucleic acid sequence of the second reverse primer and the second sequencing adaptor further comprises a tag sequence, and the tag sequences of the plurality of samples to be tested are different from each other, and the plurality is at least 2;
mixing the target region nucleic acid sequencing libraries of the plurality of test samples to obtain a mixed library;
sequencing the mixed library to obtain a sequencing result, wherein the sequencing result comprises the sequence of the target region nucleic acid sequencing library and the tag sequence of the plurality of samples to be tested; and
differentiating the sequences of the target region nucleic acid sequencing libraries of the plurality of test samples based on the tag sequences and determining the target region nucleic acid sequence of each of the plurality of test samples,
optionally, simultaneously performing sequencing library construction on the plurality of samples to be tested in a multi-well plate, wherein each well in the multi-well plate independently becomes a reaction system,
optionally, the multi-well plate is a chip having 5184 wells, each well of the chip having a reaction volume of 100 nanoliters,
optionally, when the sequencing is performed using the Illumina sequencing platform, the first sequencing linker and the second sequencing linker are the P5 sequencing linker and the P7 sequencing linker, respectively, of the Illumina sequencing platform, preferably further comprising a third nucleic acid sequence of 4nt in length between the tag sequence and the first sequencing linker P5, more preferably the third nucleic acid sequence is: 5 '-ACAC-3',
optionally, when the sequencing is performed using an iontornt sequencing platform, the first sequencing linker and the second sequencing linker are an a sequencing linker and a P sequencing linker, respectively, of the iontornt sequencing platform.
9. A system for determining the nucleic acid sequence of a target region of a test sample, comprising:
a library construction device, wherein the primer composition of any one of claims 1-4 is arranged in the library construction device, and is used for constructing a target region nucleic acid sequencing library of a sample to be tested according to the method of claim 5 or 6;
the first sequencing device is connected with the library construction device and is used for sequencing the target region nucleic acid sequencing library of the sample to be tested so as to obtain a sequencing result; and
a first nucleic acid sequence determination device connected with the first sequencing device and used for determining the sequence of the target area nucleic acid of the sample to be tested based on the sequencing result,
optionally, the first sequencing device is an Illumina sequencing platform and the first sequencing linker and the second sequencing linker are a P5 sequencing linker and a P7 sequencing linker, respectively, of the Illumina sequencing platform,
optionally, the first sequencing device is an iontornt sequencing platform, and the first sequencing linker and the second sequencing linker are an a sequencing linker and a P sequencing linker, respectively, of the iontornt sequencing platform.
10. A system for simultaneously determining target region nucleic acid sequences of a plurality of test samples, comprising:
a library constructing and mixing device, configured to construct a target region nucleic acid sequencing library of the sample to be tested according to the method of claim 5 or 6 independently for each of the plurality of samples to be tested, and mix the target region nucleic acid sequencing libraries of the plurality of samples to be tested, so as to obtain a mixed library, wherein tag sequences are further included between the first nucleic acid sequence of the second forward primer and the first sequencing adaptor, and/or between the second nucleic acid sequence of the second reverse primer and the second sequencing adaptor, and the tag sequences of the plurality of samples to be tested are different from each other, and the number is at least 2;
a second sequencing device, connected to the library construction and mixing device, for sequencing the mixed library to obtain a sequencing result, wherein the sequencing result comprises the sequence of the target region nucleic acid sequencing library and the tag sequence of the plurality of samples to be tested; and
a second nucleic acid sequence determination device, connected to the second sequencing device, for distinguishing the nucleic acid sequences of the target region sequencing libraries of the plurality of test samples based on the tag sequences and determining the target region nucleic acid sequence of each of the plurality of test samples,
optionally, a multi-well plate is arranged in the library construction and mixing device so as to simultaneously perform sequencing library construction on the plurality of samples to be tested by using the multi-well plate, wherein each well in the multi-well plate independently becomes a reaction system,
optionally, the multi-well plate is a chip having 5184 wells, each well of the chip having a reaction volume of 100 nanoliters,
optionally, the second sequencing device is an Illumina sequencing platform, the first sequencing linker and the second sequencing linker are respectively a P5 sequencing linker and a P7 sequencing linker of the Illumina sequencing platform, preferably, a third nucleic acid sequence with a length of 4nt is further comprised between the tag sequence and the first sequencing linker, more preferably, the third nucleic acid sequence is: 5 '-ACAC-3',
optionally, the second sequencing device is an iontorent sequencing platform, and the first sequencing linker and the second sequencing linker are an a sequencing linker and a P sequencing linker of the iontorent sequencing platform, respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410485821.9A CN105506063A (en) | 2014-09-22 | 2014-09-22 | Primer composition and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410485821.9A CN105506063A (en) | 2014-09-22 | 2014-09-22 | Primer composition and uses thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105506063A true CN105506063A (en) | 2016-04-20 |
Family
ID=55714353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410485821.9A Pending CN105506063A (en) | 2014-09-22 | 2014-09-22 | Primer composition and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105506063A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861724A (en) * | 2016-06-03 | 2016-08-17 | 人和未来生物科技（长沙）有限公司 | KRAS gene ultralow frequency mutation detection kit |
| CN106011230A (en) * | 2016-05-10 | 2016-10-12 | 人和未来生物科技（长沙）有限公司 | Primer composition for detecting fragmentized DNA target area and application thereof |
| CN106048008A (en) * | 2016-06-03 | 2016-10-26 | 人和未来生物科技（长沙）有限公司 | EGFR gene ultralow frequency mutation detection kit |
| CN106048009A (en) * | 2016-06-03 | 2016-10-26 | 人和未来生物科技（长沙）有限公司 | Label joint for detection of ultra-low-frequency gene mutation and application of label joint |
| CN106636063A (en) * | 2016-09-27 | 2017-05-10 | 广州精科医学检验所有限公司 | Primer compound, application thereof and method for establishing library and confirming nucleotide sequence |
| CN106755349A (en) * | 2016-12-02 | 2017-05-31 | 上海爱她基因科技有限公司 | A kind of mastocarcinoma gene detection kit |
| CN107365867A (en) * | 2017-09-04 | 2017-11-21 | 天津华大医学检验所有限公司 | A kind of Primer composition and its application for being used to detect genome target region |
| CN107868837A (en) * | 2017-12-12 | 2018-04-03 | 苏州普瑞森基因科技有限公司 | A kind of Primer composition and its application for being used to analyze enteric microorganism |
| CN108998508A (en) * | 2018-08-28 | 2018-12-14 | 深圳联合医学科技有限公司 | The construction method and primer sets and kit of amplicon sequencing library |
| WO2020113569A1 (en) * | 2018-12-07 | 2020-06-11 | 深圳华大生命科学研究院 | Method for sequencing long fragment nucleic acid |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102758026A (en) * | 2012-06-29 | 2012-10-31 | 深圳华大基因科技有限公司 | HiSeq sequencing technology-based method for detecting hepatitis B virus type and drug resistance gene |
| CN103045726A (en) * | 2012-11-20 | 2013-04-17 | 南方科技大学 | Method and apparatus for gene sequencing of multiple mixed DNA or RNA sequences |
| CN103060924A (en) * | 2011-10-18 | 2013-04-24 | 深圳华大基因科技有限公司 | Library preparation method of trace nucleic acid sample and application thereof |
| CN103397089A (en) * | 2013-07-24 | 2013-11-20 | 安诺优达基因科技(北京)有限公司 | Semi-specific amplification primer group, method and kit for quickly detecting chromosome number abnormality |
-
2014
- 2014-09-22 CN CN201410485821.9A patent/CN105506063A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060924A (en) * | 2011-10-18 | 2013-04-24 | 深圳华大基因科技有限公司 | Library preparation method of trace nucleic acid sample and application thereof |
| CN102758026A (en) * | 2012-06-29 | 2012-10-31 | 深圳华大基因科技有限公司 | HiSeq sequencing technology-based method for detecting hepatitis B virus type and drug resistance gene |
| CN103045726A (en) * | 2012-11-20 | 2013-04-17 | 南方科技大学 | Method and apparatus for gene sequencing of multiple mixed DNA or RNA sequences |
| CN103397089A (en) * | 2013-07-24 | 2013-11-20 | 安诺优达基因科技(北京)有限公司 | Semi-specific amplification primer group, method and kit for quickly detecting chromosome number abnormality |
Non-Patent Citations (9)
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011230A (en) * | 2016-05-10 | 2016-10-12 | 人和未来生物科技（长沙）有限公司 | Primer composition for detecting fragmentized DNA target area and application thereof |
| CN105861724B (en) * | 2016-06-03 | 2019-07-16 | 人和未来生物科技（长沙）有限公司 | A kind of KRAS gene ultralow frequency mutation detection kit |
| CN106048008A (en) * | 2016-06-03 | 2016-10-26 | 人和未来生物科技（长沙）有限公司 | EGFR gene ultralow frequency mutation detection kit |
| CN106048009A (en) * | 2016-06-03 | 2016-10-26 | 人和未来生物科技（长沙）有限公司 | Label joint for detection of ultra-low-frequency gene mutation and application of label joint |
| CN105861724A (en) * | 2016-06-03 | 2016-08-17 | 人和未来生物科技（长沙）有限公司 | KRAS gene ultralow frequency mutation detection kit |
| CN106048009B (en) * | 2016-06-03 | 2020-02-18 | 人和未来生物科技（长沙）有限公司 | Label joint for ultralow frequency gene mutation detection and application thereof |
| CN106636063A (en) * | 2016-09-27 | 2017-05-10 | 广州精科医学检验所有限公司 | Primer compound, application thereof and method for establishing library and confirming nucleotide sequence |
| WO2018095108A1 (en) * | 2016-09-27 | 2018-05-31 | 广州精科医学检验所有限公司 | Primer composition, use thereof, and methods for constructing library and for determining nucleic acid sequence |
| CN106755349A (en) * | 2016-12-02 | 2017-05-31 | 上海爱她基因科技有限公司 | A kind of mastocarcinoma gene detection kit |
| CN107365867A (en) * | 2017-09-04 | 2017-11-21 | 天津华大医学检验所有限公司 | A kind of Primer composition and its application for being used to detect genome target region |
| CN107868837A (en) * | 2017-12-12 | 2018-04-03 | 苏州普瑞森基因科技有限公司 | A kind of Primer composition and its application for being used to analyze enteric microorganism |
| WO2019114049A1 (en) * | 2017-12-12 | 2019-06-20 | 苏州普瑞森基因科技有限公司 | Primer composition for analyzing intestinal flora and application thereof |
| US11401561B2 (en) | 2017-12-12 | 2022-08-02 | Suzhou Precision Gene Co. Ltd. | Primer composition for analyzing intestinal flora and application thereof |
| CN108998508A (en) * | 2018-08-28 | 2018-12-14 | 深圳联合医学科技有限公司 | The construction method and primer sets and kit of amplicon sequencing library |
| CN108998508B (en) * | 2018-08-28 | 2022-09-16 | 深圳联合医学科技有限公司 | Construction method of amplicon sequencing library, primer group and kit |
| WO2020113569A1 (en) * | 2018-12-07 | 2020-06-11 | 深圳华大生命科学研究院 | Method for sequencing long fragment nucleic acid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105506063A (en) | Primer composition and uses thereof | |
| JP6483249B2 (en) | Isolated oligonucleotides and their use in sequencing nucleic acids | |
| US9133513B2 (en) | High throughput methylation detection method | |
| CN105886608B (en) | ApoE gene primer group, detection kit and detection method | |
| CN110129415B (en) | NGS library-building molecular joint and preparation method and application thereof | |
| CN103602726B (en) | Method for simultaneously sequencing a plurality of nucleic acid samples | |
| CN108998508B (en) | Construction method of amplicon sequencing library, primer group and kit | |
| TW201321518A (en) | Method of micro-scale nucleic acid library construction and application thereof | |
| CN109593757B (en) | Probe and method for enriching target region by using same and applicable to high-throughput sequencing | |
| CN105734048A (en) | PCR-free sequencing library preparation method for genome DNA | |
| WO2016095736A1 (en) | Target region enrichment method based on multiplex pcr, and reagent | |
| CN113811618B (en) | Sequencing library construction based on methylated DNA target region, system and application | |
| CN105986015A (en) | Method and kit for detecting one or more target sequence of multiple samples based on high-throughput sequencing | |
| KR20170133270A (en) | Method for preparing libraries for massively parallel sequencing using molecular barcoding and the use thereof | |
| WO2018113799A1 (en) | Method and test kit for constructing simplified genomic library | |
| CN106795650B (en) | PF quick database building method and application thereof | |
| WO2024104129A1 (en) | Method for enriching part of genome and related use | |
| CN108866155B (en) | Preparation method of next generation sequencing library | |
| WO2024104130A1 (en) | Whole genome molecular marker development method utilizing degenerate primer amplification | |
| CN105400864B (en) | Method for constructing sequencing libraries based on blood samples and use thereof in determining fetal genetic abnormalities | |
| CN109280696B (en) | Method for splitting mixed sample by SNP detection technology | |
| CN108103177A (en) | High-throughput exploitation SNP site and the method for InDel in a kind of genome from pteria martensii | |
| CN116083529B (en) | Method for targeted enrichment of DNA of genome target region and application thereof | |
| CN107904297B (en) | Primer group, joint group and sequencing method for microbial diversity research | |
| CN112251491A (en) | cDNA library construction method of capillary 96-well plate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wang Ou Inventor after: Cheng Xiaofang Inventor after: Zhang Wenwei Inventor after: Guo Fengting Inventor after: Chang Cankun Inventor after: Jiang Hui Inventor before: Wang Ou Inventor before: Cheng Xiaofang Inventor before: Guo Fengting Inventor before: Chang Cankun Inventor before: Jiang Hui Inventor before: Zhang Wenwei |
|
| CB03 | Change of inventor or designer information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180104 Address after: 518083 comprehensive building of Beishan industrial zone and 11 Building 2, Yantian District, Guangdong, Shenzhen Applicant after: Shenzhen Hua made Dazhi Technology Co. Ltd. Address before: North Road No. 146, building 11F-3 Industrial Zone in Yantian District of Shenzhen city of Guangdong Province in 518083 Applicant before: BGI-Shenzhen Co., Ltd. |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160420 |
|
| RJ01 | Rejection of invention patent application after publication |