CN117487897A - Second generation sequencing method with unequal depths of capture areas - Google Patents
Second generation sequencing method with unequal depths of capture areas Download PDFInfo
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- CN117487897A CN117487897A CN202311850933.5A CN202311850933A CN117487897A CN 117487897 A CN117487897 A CN 117487897A CN 202311850933 A CN202311850933 A CN 202311850933A CN 117487897 A CN117487897 A CN 117487897A
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- 238000012163 sequencing technique Methods 0.000 title claims abstract description 36
- 239000000523 sample Substances 0.000 claims abstract description 48
- 230000006287 biotinylation Effects 0.000 claims abstract description 12
- 238000007413 biotinylation Methods 0.000 claims abstract description 12
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 12
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 12
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 12
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 11
- 238000013467 fragmentation Methods 0.000 claims abstract description 6
- 238000006062 fragmentation reaction Methods 0.000 claims abstract description 6
- 238000009396 hybridization Methods 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 16
- 238000002474 experimental method Methods 0.000 claims description 12
- 238000000746 purification Methods 0.000 claims description 10
- 230000000295 complement effect Effects 0.000 claims description 7
- 238000011002 quantification Methods 0.000 claims description 5
- 230000000903 blocking effect Effects 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 3
- 238000003752 polymerase chain reaction Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 12
- 239000012634 fragment Substances 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 4
- 239000007984 Tris EDTA buffer Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention relates to a second generation sequencing method with unequal depths of capture areas, which comprises the following steps: extracting DNA double-stranded nucleic acid, performing fragmentation treatment after extracting the DNA double-stranded nucleic acid, adding protruding A base at the tail end, connecting truncated linker, and forming a pre-library by using PCR with index primer. The invention utilizes the streptavidin to partially seal the biotinylation probe which needs to reduce the depth of the region, and then mixes the biotinylation probe with the common biotinylation probe which needs to deepen the region in a certain proportion, thereby achieving the purpose of unequal depths of the regions, not only forming the difference between the original depths of the two regions, but also forming the great difference between the effective depths, reducing the reagent cost and the personnel operation cost, and being convenient and quick to operate.
Description
Technical Field
The invention relates to the technical field of accurate medical treatment, in particular to a second-generation sequencing method with unequal depths of capture areas in the aspect of detecting tumor variation by second-generation sequencing.
Background
Capture sequencing is a common means of tumor detection by hybridization of biotinylated probes to target molecules, followed by a series of washing processes to finally enrich the region of interest. In some applications, different coverage depths are required for different target areas, such as hot spot deepening or low depth SNP detection. Traditional methods are accomplished by adjusting the ratio of regional probe concentrations or by separately hybridizing the different regional probes individually. And because probes are far excessive for target molecules, the ratio of the probes can be adjusted to influence the depth of a region only by a small amplitude, and the effect is not ideal. Separate hybridization adds to the cost of reagents and time costs.
Region unequal depth hybridization is currently mainly performed in two ways:
1. mixing probes targeting different areas in a certain proportion, wherein the ratio of the low-depth area to the high-depth area is low;
2. and respectively carrying out hybridization reaction on probes targeting different areas, then respectively measuring different data amounts, and carrying out low-depth less measurement and high-depth more measurement. The two methods have the defects that the probe is far excessive for the target, the probe of the targeted low-depth region is reduced, the depth of the region cannot be effectively reduced, the depth of the high-depth region cannot be effectively improved relatively, the maximum depth difference can only reach about 1.5-2 times, and the effect is limited in the practical application process. For the second method, the separate hybridization is effective in making the depth difference, but it doubles the reagent cost and the personnel operation cost, which makes the practical operation difficult.
Disclosure of Invention
(one) solving the technical problems
The invention uses streptavidin to seal the biotinylation probe with a certain proportion which needs to reduce the depth of the region, then mixes with the common biotinylation probe with a certain proportion which needs to deepen the region, thus achieving the purpose of unequal depths of the region.
(II) technical scheme
Based on the above, the invention provides the following technical scheme: a second generation sequencing method with unequal depths of capture areas comprises the following specific steps:
step S1: extracting and fragmenting nucleic acid, namely extracting DNA double-stranded nucleic acid, and performing fragmenting treatment after extracting the DNA double-stranded nucleic acid:
step S2: repairing the tail end, adding an A tail, specifically adding a protruding A base at the tail end, and generating a sticky tail end;
step S3: adaptor ligation, adaptor purification, specifically, adaptor purification with complementary pairing to the adaptor containing the T end, adaptor purification with the primary purpose of adding adaptors to add a tag of the library and an oligonucleotide sequence that can be complementary to the sequencing platform at the end of the fragment DNA;
step S4: the method comprises the steps of Index PCR, purification and quantification, specifically, amplifying the DNA by using a primer complementary to the linker, and then inserting purification and quantification steps in the middle so as to complete the construction of a library;
step S5: hybridization incubation, specifically: the hybridization experiments were performed in duplicate with the pre-library, and the blocked probe ratios were found. In the practical use process, the pre-library is not required to be equally divided, the blocked probes are directly hybridized, the hybridization experiment is carried out, the streptavidin blocked probes are firstly carried out in an experiment group before the hybridization experiment, and the used probes are double-chain 120bp biotinylation hybridization capture probes;
diluting streptavidin by 100 times with TE buffer solution, uniformly mixing with a targeted low-depth region probe-A, incubating, then respectively adding a targeted high-depth region probe-B, uniformly mixing to be used as an experimental group hybridization capture probe, wherein the sealing process is only needed once, the once-sealed probe can be used for hundreds of times, and then can be used all the time;
step S6: rinsing and eluting;
step S7: performing a polymerase chain reaction;
step S8: and (5) quantifying and sequencing on a machine.
(III) beneficial effects
Compared with the prior art, the invention provides a second generation sequencing method with unequal depths of capture areas, which has the following beneficial effects:
according to the second generation sequencing method for the unequal depths of the capturing areas, the biotinylation probe which needs to be reduced in the depth of the areas is sealed by the streptavidin, and then the biotinylation probe and the common biotinylation probe which needs to be deepened in the areas are mixed in a certain proportion, so that the purpose of unequal depths of the areas is achieved, the original depths of the two areas can be different, the effective depths can be greatly different, meanwhile, the reagent cost and the personnel operation cost are reduced, and the operation is convenient and quick.
Drawings
FIG. 1 is a flow chart of a sequencing method of the present invention;
FIG. 2 is a schematic diagram of the comparison of the original sequencing depth according to the present invention;
FIG. 3 is a schematic representation of an effective sequencing depth comparison according to the present invention;
FIG. 4 is a comparative schematic diagram of capture efficiency according to the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Referring to fig. 1, a second generation sequencing method with unequal capture areas comprises the following specific steps:
firstly, extracting DNA double-stranded nucleic acid, and then carrying out fragmentation treatment after extracting the DNA double-stranded nucleic acid, wherein the fragmentation treatment method can be as follows: ultrasonic disruption or enzymatic cutting
During the last step of the fragmentation, the DNA fragments may be blunt-ended because they are randomly broken, and may be uneven-ended. Therefore, the end of the DNA fragment generated in the previous step is supplemented and a protruding A base is added at the end, and the purpose of adding the A base is mainly to generate a sticky end, so that the adapter primer can be added in the next step;
a DNA fragment with the A-terminal added thereto has an overhanging A-terminal, and thus can be complementarily paired with a linker having a T-terminal. The main purpose of adding the linker is to add a tag of a library and an oligonucleotide sequence which can be complementary to a sequencing platform at the tail end of fragment DNA, wherein the linker is an important component in the library, and an Illumina platform Y-type linker commonly used in sequencing comprises P5/P7, index and Rd1/Rd2 SP sequences, wherein the P5/P7 sequences are used for pairing with sequences on a sequencing chip, and the fragment to be detected is fixed on a Flowcell to complete bridge amplification; index was used to distinguish between different samples in the on-machine sequencing mix library, while Rd1/Rd2 SP was the Read1 and Read2 sequencing primer binding region. The adaptor ligation is typically performed using T4 DNA ligase (KAPA Cat#KK 8504) which restores the single-stranded nick on double-stranded DNA to allow two adjacent nucleotides to be re-ligated together, and in the adaptor ligation, the adaptor with the "T" cohesive end and the DNA fragment with the "A" cohesive end are ligated together to form a complete double-strand;
because the adaptor is added before, primers complementary to the adaptor can be directly used for amplifying the adaptor at the rear;
the experimental group is then subjected to streptavidin blocking probe manipulation prior to hybridization experiments, using a double-stranded 120bp biotinylated hybrid capture probe (which may also be used for single-stranded or other length biotinylated probes). 5. 5 mg/mL streptavidin was diluted 100-fold with TE buffer, 0.5/1/2.5/5/10. Mu.L of each was mixed with 4. Mu.L of the targeted low depth region probe-A (about 2 ten thousand probes) at 25℃and incubated for 1 h, and then 4. Mu.L of the targeted high depth region probe-B (about 1000 probes) were added, respectively, and mixed uniformly to obtain a capture probe for hybridization in the experimental group. Diluting the targeted low-depth region probe-A by using a TEbuffer for 20 times, and taking 4 mu L of the targeted high-depth region probe-B as a control group probe by mixing the targeted low-depth region probe-A with 4 mu L of the targeted high-depth region probe-B (the mixing ratio is 1:20);
rinsing and eluting;
finally, the polymerase chain reaction can be carried out; and (5) quantifying and sequencing on a machine.
Example two
Referring to fig. 2-3, a second generation sequencing method with unequal depths of capture areas is disclosed, wherein cfDNA is subjected to sequencing library construction according to a KAPA mechanical disruption method kit specification, and a pre-library is divided into a plurality of parts for hybridization experiments;
the experiment group is firstly subjected to streptavidin blocking probe operation before hybridization experiment, the used probes are double-chain 120bp biotinylation hybridization capture probes (which can also be used for single-chain or biotinylation probes with other lengths), 5. 5 mg/mL streptavidin is diluted 100 times by TE buffer solution, 0.5/1/2.5/5/10 mu L of the streptavidin is respectively mixed with 4 mu L of targeted low-depth region probes-A (about 2 ten thousand probes) at 25 ℃ for incubation at 1 h, and then 4 mu L of targeted high-depth region probes-B (about 1000 probes) are respectively added to be uniformly mixed to obtain the hybridization capture probes of the experiment group. Diluting the targeted low-depth region probe-A by using a TEbuffer for 20 times, and taking 4 mu L of the targeted high-depth region probe-B as a control group probe by mixing the targeted low-depth region probe-A with 4 mu L of the targeted high-depth region probe-B (the mixing ratio is 1:20);
then as shown in figure 1, after the nucleic acid is fragmented by using the conventional pre-library construction process of NGS, sequentially performing end repair, end A adding, joint connection, joint purification, index PCR, purification and quantification steps, respectively taking 500 ng pre-libraries, and respectively performing hybridization with probes of a control group and an experimental group at 60 ℃ and 16 h; 68 ℃, 48 ℃ after 2 times of rinsing and 3 times of rinsing. Then carrying out hybridization post amplification, wherein the hybridization product is 22.5 mu L, hifi hotstart ready mix mu L, the general primer is 2.5 mu L, and the temperature is 98 ℃ for 1 min; 15s at 98 ℃, 30s at 60 ℃, 30s at 72 ℃ and 16 cycles; and 3 min at 72 ℃. Purifying by 1 time of magnetic beads to generate a sequencing library. Performing second-generation sequencing on-line, uniformly intercepting 20G data according to 25G data quantity in PE100 mode, and performing biological control analysis, wherein the capturing efficiency, the original sequencing depth and the effective depth of two areas are focused on;
as shown in FIG. 2, the ratio of the original sequencing depth of the A probe region to the B probe region can be adjusted according to the probe blocking condition of the low depth region, and the ratio is 1:3.5 to 1:560, which can be adjusted at will; control group depth ratio was about 1:2;
the ratio of effective sequencing depth A probe region to B probe region was arbitrarily adjustable between 1:2 to 1:35, while the control group depth ratio was about 1: about 1.7, as shown in fig. 3.
The method can not only make the original depth of two areas different, but also make the effective depth greatly different. And the experimental method does not affect the overall capturing efficiency, as shown in fig. 4.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A second generation sequencing method with unequal depths of capture areas, which is characterized in that: the sequencing method comprises the following specific steps:
step S1: nucleic acid extraction and fragmentation:
step S2: repairing the tail end, and adding an A tail;
step S3: connecting joints and purifying the joints;
step S4: index PCR, purification, quantification;
step S5: hybridization and incubation;
step S6: rinsing and eluting;
step S7: performing a polymerase chain reaction;
step S8: and (5) quantifying and sequencing on a machine.
2. A method of second generation sequencing of capture areas of unequal depth according to claim 1, wherein: specifically, in step S1, a DNA double-stranded nucleic acid is extracted, and then a fragmentation treatment is performed after the DNA double-stranded nucleic acid is extracted.
3. A method of second generation sequencing of capture areas of unequal depth according to claim 1, wherein: as described above, step S2 is specifically to add a protruding A base to the end, which can result in a cohesive end.
4. A method of second generation sequencing of capture areas of unequal depth according to claim 1, wherein: specifically, the step S3 is complementary pairing with a T-terminal-containing linker and purification of the linker is performed as described above.
5. A method of second generation sequencing of capture areas of unequal depth according to claim 1, wherein: specifically, in step S4, the library is constructed by amplifying the nucleic acid using a primer complementary to the adaptor, followed by a purification and quantification step.
6. A method of second generation sequencing of capture areas of unequal depth according to claim 1, wherein: the step S5 specifically includes: the pre-library is divided into a plurality of parts for hybridization experiments, and streptavidin blocking probe operation is carried out on the experimental group before the hybridization experiments, wherein the used probes are double-chain 120bp biotinylation hybridization capture probes.
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