CN117683867A - Hybridization capture universal joint sequence blocking reagent for target region targeted sequencing and application thereof - Google Patents
Hybridization capture universal joint sequence blocking reagent for target region targeted sequencing and application thereof Download PDFInfo
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- 230000000903 blocking effect Effects 0.000 title claims abstract description 94
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 69
- 238000009396 hybridization Methods 0.000 title claims abstract description 45
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- 238000012986 modification Methods 0.000 claims abstract description 22
- 230000004048 modification Effects 0.000 claims abstract description 22
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000007789 sealing Methods 0.000 claims abstract description 11
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 125000001921 locked nucleotide group Chemical group 0.000 claims abstract description 5
- 239000011324 bead Substances 0.000 claims description 31
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- 239000011534 wash buffer Substances 0.000 claims description 14
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- 238000004140 cleaning Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 108091081062 Repeated sequence (DNA) Proteins 0.000 claims description 5
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims description 4
- 239000002853 nucleic acid probe Substances 0.000 claims description 4
- 108020004414 DNA Proteins 0.000 claims description 3
- 108020003215 DNA Probes Proteins 0.000 claims description 3
- 239000003298 DNA probe Substances 0.000 claims description 3
- 230000003252 repetitive effect Effects 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 101100384865 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cot-1 gene Proteins 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
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- 101000641221 Homo sapiens Chromaffin granule amine transporter Proteins 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
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Abstract
The invention relates to the field of gene sequencing, in particular to a hybridization capture universal joint sequence blocking reagent for target region targeted sequencing and application thereof. The general connector sequence blocking reagent provided by the invention consists of a P5 blocking sequence and a P7 blocking sequence, wherein the middle of the sequence is modified by using a locked nucleotide and hypoxanthine, and the 3' end of the sequence is modified by using an MGB. The invention improves the sequence structure of the sealing reagent, optimizes the types and the number of the modification groups, can be compatible with various joints under an Illumina TruSeq system, is not influenced by the sequence change of the joint label, effectively improves the sealing effect, improves the capturing efficiency, reduces the synthesis cost and reduces the production and experimental cost.
Description
Technical Field
The invention relates to the field of gene sequencing, in particular to a hybridization capture universal joint sequence blocking reagent for target region targeted sequencing and application thereof.
Background
The target region targeted sequencing technology is one of the core technical means in the current medical inspection and biological research process, and compared with whole genome sequencing, the target region targeted sequencing only sequences the detected or researched target region, so that the sequencing data volume can be reduced, the sequencing cost can be reduced, the analysis period can be shortened, the sequencing depth, accuracy and sensitivity can be improved, and the method can be better applied to a plurality of fields such as clinical diagnosis, disease screening, reproduction assistance and the like.
The liquid phase hybridization capture is the mainstream target sequencing technology at present, and the scheme is that the repeated sequence and the joint sequence on the whole genome library fragment are blocked by a blocking reagent, then the DNA probe with biotin mark is hybridized and combined with the target area library fragment, then the target area library fragment is captured by utilizing the affinity effect of streptavidin magnetic beads and biotin, and then the sequencing can be performed after a plurality of times of washing and PCR amplification. The blocking reagent can be divided into a genome repeated sequence blocking reagent and a linker sequence blocking reagent, and has the functions of blocking a large number of repeated sequences and linker sequences existing in genome library fragments, blocking base complementary pairing combination among genome repeated sequences and base complementary pairing combination among linker sequences, avoiding serial introduction of non-target region fragments, and preventing interference with hybridization between probes and target region fragments, thereby improving capture efficiency and data utilization rate.
Currently, there are two types of blocking methods, one-to-one and one-to-many, for the tag sequence portion in the linker sequence, one-to-one meaning that each tag sequence corresponds to a blocking agent that is perfectly complementary to the sequence, one-to-many meaning that a universal blocking agent with a fixed sequence can block all tag sequences.
However, in the existing hybridization capture sequencing technology and the joint sealing reagent adopted by the commercialized kit, one-to-one joint sealing reagent needs to synthesize tens of thousands of sealing reagents with different sequences, so that the production cost is high, the actual application operation is complicated, and the error probability is high; the one-to-many linker blocking reagent requires modification of the tag sequence portion to 8-10 hypoxanthines, which is costly to modify. Meanwhile, in order to enhance the binding strength, ensure the blocking effect, prevent the blocking reagent sequence from being used as a primer for amplification in the subsequent PCR reaction, and further need to make a lock nucleic acid modification on tens of cytosine nucleotides in the linker sequence part of the blocking reagent, and add AMN or MGB or Spacer and other blocking modifications at the 3' end of the blocking sequence. The cost of the modification is generally high, the synthesis cost of the joint sealing reagent is greatly increased, the overall production cost of the kit is increased, and the experimental cost of hybridization capture is increased.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the invention provides a hybridization capture universal joint sequence blocking reagent for target region targeted sequencing and application thereof, and aims to solve a part of problems in the prior art or at least relieve a part of problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a hybridization capture universal joint sequence blocking reagent for target sequencing of target region is prepared from P5 blocking sequence and P7 blocking sequence through mixing.
Further, the P5 blocking sequence and the P7 blocking sequence are modified with locked nucleotides and hypoxanthine in the middle of the sequences.
Further, the P5 blocking sequence and the P7 blocking sequence are modified at the 3' end of the sequence by MGB.
Further, the sequence structure of the P5 blocking sequence and the P7 blocking sequence is:
p5 blocking sequence:
AATGATACGG+CGACCA+CCGAGAT+CTACACIIIIIIIIACACTCTTT+CCCTA+CA+C GACGCTCTTCCGATCT-MGB；
p7 blocking sequence:
CAAG+CAGAAGA+CGGCATA+CGAGATIIIIIIIIGTGA+CTGGAGTT+CAGACGTGTG+CTCTTCCGATC-MGB；
wherein +C represents LNA-C modification, I represents hypoxanthine, and-MGB represents MGB modification.
Furthermore, the universal joint sequence blocking reagent can be compatible with various joints under an Illumina TruSeq system and is not influenced by the change of the joint tag sequence.
The invention also provides a hybridization capture kit for target region targeted sequencing, which comprises the universal joint sequence blocking reagent.
Further, the hybridization capture kit also comprises genome repetitive sequence blocking reagent, hybridization reagent, nucleic acid probe, capture magnetic beads, magnetic bead cleaning reagent, washing reagent and PCR amplification reagent.
Further, the genome repetitive sequence blocking agent is Human Cot-1 DNA; the hybridization reagents include Hybridization Buffer, hybridization Enhancer; the nucleic acid probe is a DNA probe; the capture magnetic beads are Streptavidin Beads; the magnetic bead cleaning reagent is 2X Beads Wash Buffer; the washing reagent comprises 10X Wash Buffer I, 10X Wash Buffer II, 10X Wash Buffer III and 10X Wash Buffer S; the PCR amplification reagent comprises 2X POST-PCR Mix and POST-PCR Primers.
The invention also provides an application of the hybridization capture universal joint sequence blocking reagent or the hybridization capture kit in target region targeted sequencing.
After the technical scheme is adopted, the invention has the beneficial effects that:
(1) The invention provides a universal hybridization capture joint sealing reagent which can be compatible with various joints under an Illumina TruSeq system and is not influenced by the sequence change of a joint label.
(2) The invention improves the structure of the sealing reagent sequence, optimizes the types of the modification groups, can effectively improve the sealing effect and improves the capturing efficiency.
(3) The invention properly reduces the modification quantity of the base sequence on the premise of not affecting the sealing effect and the capturing efficiency, can effectively reduce the synthesis cost and reduces the production and experimental cost.
Detailed Description
The present invention is further described below with reference to specific examples, but the present invention is not limited to the following examples.
Example 1
The purpose of this example was to test the effect of different numbers of locked nucleotide LNA-C modifications in the P5 and P7 blocking sequences on blocking effect.
The P5 blocking sequence and the P7 blocking sequence of the synthetic hybrid capture universal linker sequence blocking reagent are designed, and two experimental groups Lib1 and Lib2 are set.
For Lib1, all C in the P5 and P7 blocking sequences were modified with LNA-C, the sequence structure is as follows:
p5 blocking sequence:
AATGATA+CGG+CGA+C+CA+C+CGAGAT+CTA+CA+CIIIIIIIIA+CA+CT+CTTT+C+C+CTA+CA+CGA+CG+CT+CTT+C+CGAT+CT-MGB；
p7 blocking sequence:
+CAAG+CAGAAGA+CGG+CATA+CGAGATIIIIIIIIGTGA+CTGGAGTT+CAGA+CGTG TG+CT+CTT+C+CGAT+C-MGB；
wherein +C represents LNA-C modification, I represents hypoxanthine, and-MGB represents MGB modification.
For Lib2, part C of the P5 and P7 blocking sequences was modified with LNA-C, the sequence structure was as follows:
p5 blocking sequence:
AATGATACGG+CGACCA+CCGAGAT+CTACACIIIIIIIIACACTCTTT+CCCTA+CA+C GACGCTCTTCCGATCT-MGB；
p7 blocking sequence:
CAAG+CAGAAGA+CGGCATA+CGAGATIIIIIIIIGTGA+CTGGAGTT+CAGACGTGTG+CTCTTCCGATC-MGB；
wherein +C represents LNA-C modification, I represents hypoxanthine, and-MGB represents MGB modification.
The general linker sequence blocking reagent can be obtained by mixing the amounts of substances such as P5 blocking sequence and P7 blocking sequence.
The same genomic library was used for both experimental groups Lib1 and Lib2 by performing a hybridization capture experiment on the genomic library using a hybridization capture kit comprising the universal linker sequence blocking reagent described above.
The hybrid capture assay comprises the steps of:
s1, library concentration:
the following system was prepared in a clean PCR tube:
wherein Universal Blockers is the english name of the hybrid capture universal linker sequence blocking reagent of the invention;
vortex vibration mixing, short centrifuging to collect liquid to the bottom of the tube, uncapping the PCR tube, placing in a vacuum rotary evaporator, evaporating at 60deg.C, and concentrating.
S2, hybridization of a probe:
the following reaction components were added to a PCR tube containing the S1 library concentrated product:
vortex vibration mixing, standing at room temperature for 5min, vortex vibration mixing again, centrifuging briefly, collecting the reaction solution to the bottom of the tube, placing the PCR tube in a preheated 95 ℃ PCR instrument, and heating the cover to 105 ℃ for denaturation at 95 ℃ for 5min;
mixing by rapid vortex oscillation, collecting the reaction liquid to the bottom of the tube by short centrifugation, immediately placing the reaction liquid in a preheated PCR instrument at 65 ℃, setting a heat cover at 75 ℃, and hybridizing at 65 ℃ for 16 hours.
S3, preparing a washing reagent:
1 Xbuffer was prepared as follows:
vortex vibration mixing, placing at corresponding temperature for standby according to the following table:
s4, preparing to capture magnetic beads:
placing Streptavidin Beads on a magnetic rack, balancing at room temperature for at least 10min, stirring by vortex, mixing thoroughly, placing 50 mu L Streptavidin Beads in a PCR tube, placing the PCR tube on the magnetic rack, and carefully removing the supernatant after the solution is clarified;
taking the PCR tube off the magnetic frame, adding 100 mu L of 1X Beads Wash Buffer, uniformly mixing by vortex oscillation, briefly centrifuging the PCR tube, placing the PCR tube on the magnetic frame, carefully removing the supernatant after the solution is clarified, and repeatedly cleaning once again by 100 mu L of 1X Beads Wash Buffer;
the PCR tube was removed from the magnetic rack, 50. Mu.L of 1X Beads Wash Buffer was added, vortexed and mixed well, and the beads were collected to the bottom of the tube by brief centrifugation.
S5, capturing magnetic beads:
placing the Streptavidin Beads suspension prepared in the step S4 on a magnetic rack, and carefully removing the supernatant after the solution is clarified;
taking out the PCR tube filled with the S2 probe hybridization product from the PCR instrument, collecting the reaction liquid to the bottom of the tube by short centrifugation, transferring all 16 mu L of probe hybridization reaction product into the PCR tube filled with Streptavidin Beads, and uniformly mixing by vortex oscillation;
incubation was continued in a PCR instrument at 65 ℃ for 40min, during which time the PCR instrument was immediately placed into the PCR instrument after shaking every 10min for 5s until the total incubation period was met.
S6, cleaning:
adding 120 mu L of 1 XWash Buffer I preheated at 65 ℃ into the S5 magnetic bead capturing product, blowing and mixing uniformly by using a pipettor, incubating for 10-20S at 65 ℃, briefly centrifuging a PCR tube, placing the PCR tube on a magnetic rack, and immediately removing the supernatant after the solution is clarified;
putting the PCR tube back into a PCR instrument at 65 ℃, adding 150 mu L of 1 XWash Buffer S preheated at 65 ℃, blowing and mixing uniformly by using a pipettor, incubating for 5min at 65 ℃, briefly centrifuging the PCR tube, placing the PCR tube on a magnetic rack, immediately removing the supernatant after the solution is clarified, and repeatedly cleaning for two times;
adding 150 mu L of 1 XWash Buffer I into the PCR tube, uniformly mixing by vortex oscillation for 30s, uniformly mixing for 1 time at intervals of 5-10s, uniformly mixing for 4 times, fully suspending magnetic beads, briefly centrifuging the PCR tube, placing the PCR tube on a magnetic rack, and carefully removing the supernatant after the solution is clarified;
adding 150 mu L of 1 XWash Buffer II into the PCR tube, uniformly mixing by vortex oscillation for 30s, uniformly mixing for 1 time at intervals of 5-10s, uniformly mixing for 2 times, fully suspending magnetic beads, briefly centrifuging the PCR tube, placing the PCR tube on a magnetic rack, and carefully removing the supernatant after the solution is clarified;
adding 150 mu L of 1X Wash Buffer III into the PCR tube, blowing and mixing uniformly by using a liquid transfer device, transferring all the magnetic bead suspension into a new PCR tube, placing the PCR tube on a magnetic rack, and carefully removing the supernatant after the solution is clarified;
the PCR tube was removed from the magnetic rack, 23. Mu.L of enzyme-free water was added, and the beads were resuspended using pipetting or vortexing.
S7, amplification after capture:
the following reaction system was prepared in a PCR tube containing S6 wash product:
the reaction solution is collected to the bottom of the tube by short centrifugation, vortex oscillation and uniform mixing are carried out, the PCR tube is placed in a PCR instrument, a thermal cover is set at 105 ℃, and the following reaction procedure is operated:
s8, purifying after amplification:
adding 50 mu L of DNA purification magnetic beads into a PCR tube filled with the S7 PCR amplification product, uniformly mixing by vortex oscillation, incubating for 5min at room temperature, placing the PCR tube on a magnetic rack, and removing the supernatant after the solution is clarified; keeping the PCR tube on a magnetic frame, adding 100 mu L of freshly prepared 80% ethanol, transferring the PCR tube on the magnetic frame, enabling the magnetic beads to be adsorbed on the opposite side tube wall, fully washing the magnetic beads, and removing the supernatant; keeping the PCR tube on a magnetic rack, and uncovering and drying for 5min; and taking the PCR tube off the magnetic rack, adding 25 mu L of enzyme-free water, uniformly mixing by vortex oscillation, incubating for 5min at room temperature, placing the PCR tube on the magnetic rack, and sucking 23 mu L of supernatant into a clean centrifuge tube after the solution is clarified to obtain the capture library.
Sequencing the capture library.
Analysis of sequencing results, data of indexes such as capturing efficiency, uniformity, repetition rate, sequencing depth and the like are shown in the following table:
the capturing efficiency and other performance indexes of Lib2 and Lib1 are basically consistent, but the number of modified locked nucleotides LNA-C in the P5 closed sequence and the P7 closed sequence used by Lib2 is small, so that the synthesis cost can be effectively reduced, and the production and experiment cost can be reduced.
Based on the above results, the sequence structures of the P5 blocking sequence and the P7 blocking sequence in Lib2 are preferred.
Example 2
The purpose of this example was to test the effect of different types of modifying groups at the 3' end of the P5 blocking sequence and the P7 blocking sequence on blocking effect.
Designing a P5 blocking sequence and a P7 blocking sequence of a synthetic hybrid capture universal joint sequence blocking reagent, and setting four experimental groups: lib1, lib2, lib3. The sequence structure of Lib1 adopts the sequence structure selected in example 1, the sequence structures of Lib2 and Lib3 are different from Lib1 only in 3 'end modification group, and the types of 3' end modification groups are shown in the following table:
| experimental grouping | 3' -terminal modifying group |
| Lib1 | MGB |
| Lib2 | AMN |
| Lib3 | Spacer C3 |
The general linker sequence blocking reagent can be obtained by mixing the amounts of substances such as P5 blocking sequence and P7 blocking sequence.
The hybridization capture assay was performed on the genomic library using a hybridization capture kit comprising the universal linker sequence blocking reagent described above, and the same genomic library was used for different assay groups.
The steps of the hybrid capture assay are described in reference to example 1 and are not set forth in detail herein.
Analysis of sequencing results, data of indexes such as capturing efficiency, uniformity, repetition rate, sequencing depth and the like are shown in the following table:
the capture efficiency of Lib1 is high, and MGB modification is preferably used as a 3' -end modification group of the P5 blocking sequence and the P7 blocking sequence in the universal joint sequence blocking reagent.
Example 3
The purpose of this example is to compare the difference in performance of the universal linker sequence blocking reagent provided by the present invention with that of existing linker sequence blocking reagents in a hybridization capture experiment. The P5 blocking sequence and the P7 blocking sequence of the universal linker sequence blocking reagent of the invention adopt the sequence structures and modification groups selected in the preferred embodiments of example 1 and example 2. The existing reagent product is QuarAcces Universal Blocker (NF 3005, dynegene).
The universal joint sequence blocking reagent and the existing joint sequence blocking reagent are respectively used to match the hybridization capture kit of the invention, so that the hybridization capture experiment is carried out on the same genome library. The steps of the hybridization capture assay are described in detail in reference to example 1 and are not discussed in detail herein. Genomic libraries used for testing included FFPE sample libraries and cfDNA sample libraries, with the experimental groupings shown in the table below:
| experimental grouping | Genomic library types | Linker sequence blocking reagents |
| Lib1 | FFPE | Existing reagents |
| Lib2 | FFPE | The invention is that |
| Lib3 | cfDNA | Existing reagents |
| Lib4 | cfDNA | The invention is that |
Analysis of sequencing results, data of indexes such as capturing efficiency, uniformity, repetition rate, sequencing depth and the like are shown in the following table:
in a combined view, the invention can obtain higher capturing efficiency for FFPE samples and cfDNA samples, and other performance indexes are also good.
The foregoing is merely illustrative of the present invention and not restrictive, and other modifications and equivalents thereof may occur to those skilled in the art without departing from the spirit and scope of the present invention.
Claims (9)
1. A hybridization capture universal adaptor sequence blocking reagent for targeted sequencing of a target region, characterized in that: it is formed by mixing the amounts of substances such as P5 blocking sequence, P7 blocking sequence and the like.
2. A hybridization capture universal adaptor sequence blocking reagent for targeted sequencing of a target region according to claim 1, wherein: the P5 blocking sequence and the P7 blocking sequence are modified by adopting a locked nucleotide and hypoxanthine in the middle of the sequences.
3. A hybridization capture universal adaptor sequence blocking reagent for targeted sequencing of a target region according to claim 1, wherein: the P5 blocking sequence and the P7 blocking sequence are modified by MGB at the 3' end of the sequence.
4. A hybridization capture universal adaptor sequence blocking reagent for targeted sequencing of a target region according to claim 1, wherein: the sequence structure of the P5 blocking sequence and the P7 blocking sequence is as follows:
p5 blocking sequence:
AATGATACGG+CGACCA+CCGAGAT+CTACACIIIIIIIIACACTCTTT+CCCTA+CA+CGACGCTCTTCCGATCT-MGB；
p7 blocking sequence:
CAAG+CAGAAGA+CGGCATA+CGAGATIIIIIIIIGTGA+CTGGAGTT+CAGACGTGTG+CTCTTCCGATC-MGB；
wherein +C represents LNA-C modification, I represents hypoxanthine, and-MGB represents MGB modification.
5. A hybridization capture universal adaptor sequence blocking reagent for targeted sequencing of a target region according to claim 1, wherein: the universal joint sequence sealing reagent can be compatible with various joints under an Illumina TruSeq system and is not influenced by the sequence change of the joint label.
6. A hybridization capture kit for targeted sequencing of a target region, characterized in that: a universal linker sequence blocking reagent comprising the universal linker sequence of any one of claims 1 to 5.
7. A hybridization capture kit for targeted sequencing of a target region according to claim 6, wherein: the hybridization capture kit also comprises a genome repeated sequence blocking reagent, a hybridization reagent, a nucleic acid probe, a capture magnetic bead, a magnetic bead cleaning reagent, a washing reagent and a PCR amplification reagent.
8. A hybridization capture kit for targeted sequencing of a target region according to claim 7, wherein: the genome repetitive sequence blocking reagent is Human Cot-1 DNA; the hybridization reagents include Hybridization Buffer, hybridization Enhancer; the nucleic acid probe is a DNA probe; the capture magnetic beads are Streptavidin Beads; the magnetic bead cleaning reagent is 2X Beads Wash Buffer; the washing reagent comprises 10X Wash Buffer I, 10X Wash Buffer II, 10X Wash Buffer III and 10X Wash Buffer S; the PCR amplification reagent comprises 2X POST-PCR Mix and POST-PCR Primers.
9. Use of a hybridization capture universal adaptor sequence blocking reagent according to any one of claims 1 to 5, or a hybridization capture kit according to any one of claims 6 to 8, for targeted sequencing of a target region.
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