Splint sequence and application thereof in nucleic acid capture

Abstract

The invention discloses a Splint sequence and application of the Splint sequence in nucleic acid capture. A first basic group at the 5'end of the Splint sequence is U. When nucleic acid is captured, by using the splint sequence provided by the invention, the effect of effectively reducing the number of linkers generated by the splint sequence in a sequencing library can be achieved, the comparison rate of sequencing data can be improved, and the sequencing cost can be reduced.

Description

A splint sequence and its application in nucleic acid capture Technical Field

[0001] The present invention belongs to the field of genes, and in particular relates to a splint sequence and an application thereof in nucleic acid capture. Background Art

[0002] Based on the spatiotemporal transcriptome technology of random probe capture of RNA, the current existing technology generally uses random probes (such as 6N) to capture RNA in slices and perform reverse transcription to obtain full transcriptome information.

[0003] The process of the existing technology basically involves fixing and permeabilizing the tissue slices attached to the surface of the chip, then adding a 5' random probe with a fixed nucleic acid sequence for hybridization, and then performing a reverse transcription reaction (hereinafter referred to as RT). After the reaction is completed, a ligation reaction is performed. The gap caused by the 3' end of the oligonucleotide sequence fixed on the chip and the 5' end of the random probe can be filled by T4 ligase, and the spatial position information of the RNA in the slice can be obtained by subsequent high-throughput sequencing. However, this method of RT first and then T4 ligation will result in the loss of the captured cDNA information; in addition, the RT and T4 ligation are divided into two steps. RT needs to react at 42°C for at least 3 hours, and T4 ligation needs to react at 16°C overnight. Two constant temperature equipment are required, and it takes a long time.

[0004] As shown in Figure 1A and Figure 1B, whether the T4 ligase fails to fill the nick gap in time or there is a splint that fails to hybridize successfully with the random probe (including 6N), it will first cause the probe sequence fixed on the chip to change. The oligonucleotide sequence fixed on the random probe cannot hybridize with the extended capture probe through the splint sequence, and thus cannot be connected, and the original function of the extended oligonucleotide chain is lost.

[0005] As shown in Figure 1C, this is a common method for adding sequencing adapters during high-throughput sequencing library construction. This method involves adding a known, fixed oligonucleotide sequence to the 3' end of the cDNA, and then amplifying the library using primers. Ultimately, under the action of the RT enzyme, the fixed oligonucleotide chains on the capture chip undergo changes, and sequencing adapters are added, resulting in the final sequencing library containing a large number of nucleotide chains containing only the splint sequence.

[0006] On the one hand, the above method causes the oligonucleotide chains on the capture chip to be unable to connect to the captured cDNA, becoming useless nucleic acid chains. This leads to a decrease in the cDNA that can ultimately be fixed to the chip, the loss of many gene transcripts, and the inability to obtain the true expression of slice RNA. On the other hand, the obtained sequencing data that can be mapped to the genome is relatively small (less than 10% of the effective sequencing data). This problem is particularly prominent when using the direct circularization library construction method. Taking the MGISEQ-2000RS sequencer as an example, the library obtained using the above method and sequencing one lane generates approximately 220MB of effective sequencing data, of which only about 10MB can be mapped to the genome, greatly increasing the sequencing cost of this technical method.

[0007] Summary of the Invention

[0008] To address the drawback of existing technologies that generate a large number of splints during nucleic acid capture, the present invention provides a splint sequence and its application in nucleic acid capture. Specifically, the 5' end of the splint sequence is uracil (U) (5'- / rU / SEQ ID NO:5-3'). When the immobilized oligonucleotide sequence on the chip is extended using this splint as a template, it cannot hybridize to TSO at the 5' end of the splint, thereby preventing amplification and sequencing.

[0009] The first base at the 5' end of the splint sequence (5'-SEQ ID NO:3-3') is G. The terminal transferase activity of the RT enzyme will tend to continue polymerizing base C at the 3' end of extended sequence 1 (Figure 1-C). However, 5'- / rU / SEQ ID NO:5-3' prevents the sequence fixed on the chip from extending beyond the sequencing adapter, preventing this sequence from appearing in the sequencing library.

[0010] To address the deficiencies of the prior art, the present invention provides a splint sequence in a first aspect, wherein the first base at the 5' end of the splint sequence is U; preferably, the nucleotide sequence after the first base at the 5' end of the splint sequence is as shown in SEQ ID NO:5.

[0011] A second aspect of the present invention provides a nucleic acid capture kit comprising the splint sequence described in the first aspect of the present invention. The nucleic acid capture kit further comprises at least one reagent used in conventional nucleic acid capture kits in the art, such as random probes, T4 ligase, reverse transcriptase, and a buffer for ligation / reverse transcription.

[0012] The third aspect of the present invention provides a use of the splint sequence according to the first aspect of the present invention in nucleic acid capture.

[0013] The fourth aspect of the present invention provides a method for capturing nucleic acids, wherein the RNA in the sample captured by the random probe is reverse transcribed into cDNA by hybridizing a splint sequence random probe and a fixed oligonucleotide sequence as described in the first aspect of the present invention, and the cDNA is ligated to the random probe to form a long single-stranded nucleotide having, from 5' to 3', an oligonucleotide sequence, a random probe, and cDNA, in sequence, wherein the reverse transcription and the ligation are performed in the same reaction system.

[0014] In certain embodiments, it comprises the following steps:

[0015] (1) hybridizing the random probe and the splint sequence; wherein the 5' end of the random probe is partially complementary to the 5' end of the splint sequence;

[0016] (2) the random probe hybridizes with RNA in the sample; wherein the 3' end of the random probe is partially complementary to the 3' end of the RNA;

[0017] (3) The splint sequence hybridizes to the fixed oligonucleotide sequence.

[0018] Among them, (1), (2) and (3) have no order and can occur simultaneously in the same reaction system.

[0019] Preferably, the oligonucleotide sequence is immobilized on a capture chip;

[0020] (4) In the same reaction system, cDNA is synthesized using the RNA in the complex formed by the simultaneous hybridization in (1), (2) and (3) as a template, and the fixed oligonucleotide sequence and the random probe on the complex are connected, so that the fixed oligonucleotide sequence, the random probe and the cDNA form a long single-stranded nucleotide containing cDNA in the 5' to 3' direction.

[0021] In certain embodiments, the capture chip is placed at 30-45° C., such as 37° C., for reaction for 3-24 hours, such as 3-5 hours.

[0022] Preferably, in step (1), the random probe comprises 6-20N.

[0023] In certain embodiments, the method further comprises:

[0024] (i) mixing the random probe and the splint sequence with 5×SSC, hybridizing in an incubator at 50-60° C., for example, 55° C., for 5-30 minutes, for example, 10 minutes, to form a primer hybridization mixture, cooling the mixture, and adding 2-15% by volume, for example, 5% by volume, of an RNase inhibitor; or

[0025] (i') The random probe and the splint sequence are mixed in a solution, and the temperature is gradually reduced to room temperature, and then further reduced to 0-4°C to obtain a primer hybridization mixture. The primer hybridization mixture is mixed evenly with 5×SSC and 2-15% (e.g., 5% by volume) RNase inhibitor.

[0026] In certain embodiments, after step (i) or step (i'), the method further comprises:

[0027] (ii) The surface liquid of the capture chip was removed by aspiration, and the capture chip was washed with mixed solution 2. The primer hybridization mixed solution was added, hybridized at room temperature for 15-60 minutes, and then washed again with mixed solution 2; the mixed solution 2 contained 5×SSC and RNase inhibitor.

[0028] In certain embodiments, in step (i'), the gradient cooling is to cool the temperature from 90-100°C at a rate of 1-5°C per minute to 20-28°C.

[0029] In step (i) or (i'), the random probe is 6N;

[0030] In step (i) or (i'), the molar ratio of the random probe to the splint sequence is (1-10):(1-10); and / or,

[0031] In step (ii), the volume ratio of 5×SSC to RNase inhibitor in the mixed solution 2 is (5-30):1.

[0032] In certain embodiments, in step (i'), the temperature is lowered from 95°C to 25°C at 2°C per minute;

[0033] In step (i) or (i'), the molar ratio of the random probe to the splint is 1:1; and / or,

[0034] In step (ii), the volume ratio of 5×SSC to RNase inhibitor in the mixed solution 2 is 19:1.

[0035] In certain embodiments, the capture chip includes a capture probe, wherein the capture probe includes spatial barcode position information and a fixed oligonucleotide sequence; the fixed oligonucleotide sequence hybridizes to the 3' end of the splint. Furthermore, the fixed oligonucleotide sequence can be connected to the 5' end of the random probe.

[0036] In certain embodiments, the spatial barcode position information is Nm, the fixed oligonucleotide sequence is SEQ ID NO: 1, the capture probe is 5'Nm-SEQ ID NO: 1-3', and m is 8-30.

[0037] In certain embodiments, said m≥25.

[0038] The capture chip also includes an immobilized oligonucleotide sequence', which is used for PCR amplification of the cDNA library and sequencing of one strand of the library.

[0039] In certain embodiments, the method further comprises the steps of:

[0040] (a) After completing step (ii), the liquid on the surface of the capture chip is aspirated, a mixture of reverse transcriptase and T4 ligase 1 is added, and the capture chip is placed in an incubator for reaction for 3-24 hours;

[0041] (b) removing the sample tissue on the capture chip; and / or,

[0042] (c) recovering cDNA from the capture chip; optionally, further comprising steps of cDNA amplification and purification.

[0043] Preferably, the mixed solution 1 comprises reverse transcription reagent, 3-25 mM MgCl2, 1-50 mM DTT, and ≥1 mM ATP;

[0044] The volume ratio of the reverse transcriptase to the T4 ligase is 2:1 or 1:1; and / or,

[0045] The temperature of the incubator is 35-42°C.

[0046] A fifth aspect of the present invention provides a high-throughput sequencing method, comprising the following steps:

[0047] (I) obtaining the long single-stranded nucleotide sequence containing cDNA to be sequenced according to the method according to the third aspect of the present invention;

[0048] (II) Construction of cDNA library;

[0049] (III) Perform sequencing and analyze the obtained data.

[0050] In certain embodiments, (I) further comprises the steps of cDNA shearing, amplification and purification; and / or, in (II), the cDNA library is circularized to obtain a circularized DNB library.

[0051] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0052] The reagents and raw materials used in the present invention are commercially available.

[0053] The positive progress effect of the present invention is:

[0054] Using the splint sequence (5'- / rU / SEQ ID NO:5-3') provided by the present invention can not only effectively reduce the number of splint-generated adapters in the sequencing library, but also improve the alignment rate of sequencing data and reduce sequencing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 shows the principle of a splint-induced junction (the pointed end represents the 3' end of the nucleic acid chain).

[0056] Figure 2 shows the principle of reducing splint-induced junctions (the pointed end represents the 3' end of the nucleic acid chain).

[0057] FIG3 is a comparison of the linker data of splint (5′-SEQ ID NO: 3-3′) and splint (5′- / rU / SEQ ID NO: 5-3′). DETAILED DESCRIPTION

[0058] Example 1

[0059] 1.1 Sample Slicing

[0060] (1) Slicing frozen OCT samples: Pre-cool the freezing microtome cabinet (-20°C) and the sample head (-10°C to -15°C, adjusted according to the actual operation process), and pre-cool the brushes, blades and other experimental tools in the -20°C cabinet in advance. Take the frozen OCT tissue block out of the -80°C freezer and place it in the freezing microtome for equilibrium. Use liquid OCT at room temperature to fix the tissue block to the sample holder and freeze it until the OCT solidifies. If needed, use the pre-cooled blade to trim the tissue block and then slice it.

[0061] (2) Paraffin sample sectioning: Use a Leica paraffin slicer, set the paraffin section thickness to 5 μm, and trim the paraffin-embedded blocks appropriately before sectioning.

[0062] 1.2 RNA quality control

[0063] (1) Frozen OCT samples: Cut 10-20 tissue sections with a thickness of 10 μm and place them in 1.5 ml EP tubes pre-cooled at -20°C. Use the RNeasy Mini Kit (QIAGEN) to extract total RNA, and use the Agilent RNA 6000 Pico Kit (Agilent) to test RNA quality. Samples with RIN ≥ 7 can be used for subsequent experiments.

[0064] (2) Paraffin samples: Cut 2-4 5 μm sections and place the sections in RNase-free 1.5 ml centrifuge tubes. Use the RNeasy FFPE kit to extract RNA and use the Agilent RNA 6000 Pico Kit to test RNA quality. Samples with a DV200 ≥ 50% can be used for subsequent experiments.

[0065] 1.3 Chip processing and tissue patching

[0066] (1) Frozen OCT samples: Carefully pick up the capture chip with tweezers and place it in a new 24-well plate. Record the capture chip number. The capture chip contains a capture probe, which includes a spatial barcode and a fixed oligonucleotide sequence: 5'-N 25 -TTGTCTTCCTAAGACCGCTTGG-3' (the underlined part is SEQ ID NO: 1, N 25 (N represents 25 consecutive Ns). Wash the chip twice with 400 μl of 0.1× SSC, rinse thoroughly with nuclease-free water (NF-H2O), and bake the chip at 37°C to dry. Cut frozen OCT tissue into 10 μm sections, flatten the sections, and use forceps to hold the capture chip. Quickly approach the frozen section, and the tissue section will automatically adhere to the chip. Then bake the attached tissue chip at 37°C for 3-5 minutes.

[0067] (2) Paraffin samples: Chip processing is the same as above. Paraffin samples are cut into 5-8 μm slices, spread in a 42°C water bath, picked up with a capture chip, and baked at 60°C. The chip with the tissue attached is then placed in a dewaxing reagent for 30 minutes, repeated once; removed, blotted dry on absorbent paper, and placed in 100% anhydrous ethanol for 5 minutes, repeated once; removed, and placed in 96% ethanol for 5 minutes, repeated once.

[0068] 1.4 Tissue section fixation (decrosslinking) and permeabilization

[0069] (1) Frozen OCT samples: After the above chip is dried, place it in a pre-cooled methanol solution at -20°C for 30 minutes. After taking it out and drying the methanol, add permeabilization reagent (permeabilization enzyme mixed with 10μl 0.1N HCl + 90μl NF-H2O) and react in a 37°C incubator for 10-13 minutes.

[0070] (2) Paraffin sample: After the chip is dried, place it in a 24-well cell plate, add cross-linking buffer (380 μl of pH 9.0 TE buffer + 20 μl of RNase Inhibitor (RI) and mix well), and react in a 70°C incubator for 1 hour. After the reaction is completed, remove the chip, wait for the chip to return to room temperature, and place it in a -20°C pre-cooled methanol solution for 20 minutes. After removing it, dry the methanol, add permeabilization reagent (same as above), and react in a 37°C incubator for 20 minutes.

[0071] 1.5 Random probe (6N) hybridization with splint

[0072] While the tissue sections are being fixed, prepare the primer hybridization mix. Add 5 μM random probe 6N (5'pho-SEQ ID NO:4-3') and 5 μM splint (5'- / rU / SEQ ID NO:5-3') to 5× SSC, mix thoroughly, and hybridize in a 55°C incubator for 10 minutes. Remove from the incubator and place on ice. Add 5% by volume of RNase Inhibitor (RI), mix thoroughly, and return to ice.

[0073] Table 1. Random probe 6N and splint sequences and hybridization methods used in this application

[0074]

[0075] (pho- represents phosphorylation modification; rU represents U is ribonucleotide)

[0076] After the permeabilization reaction is complete, remove the chip, aspirate the surface liquid, and wash the chip once with 190 μl of 5×SSC + 10 μl of RI. Add the hybridized primer hybridization mixture and hybridize for 15 minutes at room temperature. Then wash once with 5×SSC + RI. This yields the primer hybridization mixture, as shown in Set 3 in Figure 3.

[0077] 1.6 Reverse transcription (RT) and T4 connection

[0078] The liquid on the chip surface was aspirated, and a mixture of RT and T4 ligase (containing RT reagent, 3 mM MgCl2, 10 mM DTT, 1 mM ATP, pH 8.3 @ 25°C; RT enzyme 2.5 μl / 100 μl and T4 ligase 5 μl / 100 μl) was added. The chip was placed in a 37°C incubator for 5 hours.

[0079] 1.7 Tissue Removal

[0080] After the reaction, remove the chip, blot the surface liquid, and rinse once with NF-H2O. Add tissue removal solution and place the chip in a 55°C incubator for 20 minutes. Remove the chip and aspirate the chip surface, then aspirate twice with NF-H2O to remove all tissue.

[0081] 1.8 cDNA recovery, amplification, and purification

[0082] (1) cDNA recovery: Add 400 μl / well cDNA release solution to the reaction wells of the above chip, cover the reaction wells with the chip with sealing film to seal, cover the plate cover and seal the outer ring to prevent volatilization, and react in a 55℃ incubator for 3 hours to release cDNA; recover the liquid in the reaction well into a new 1.5ml centrifuge tube, add 350 μl / well NF-H2O to clean the chip, and recover the cleaning solution into the same centrifuge tube. Add VAHTSTM DNA Clean Beads (VAZYME) to the recovery solution in a ratio of 0.8:1 to the 1.5ml centrifuge tube, shake and mix, and incubate at room temperature for 10 minutes. After instant centrifugation, place the EP tube on a magnetic stand and let it stand for 3 minutes. After the liquid is clear, remove the supernatant, add 1ml of freshly prepared 80% ethanol, shake and mix, then centrifuge for instantaneous centrifugation and let it stand for 30 seconds; discard the supernatant and repeat the washing with 80% ethanol. Discard the supernatant and air-dry at room temperature until the surface of the magnetic beads is non-reflective and crack-free; add 42 μl NF-H2O to dissolve, shake and mix, and let it stand at room temperature for 5 minutes. After instant centrifugation, let it stand on the magnetic stand for 3-5 minutes. After the liquid is clear, recover the supernatant into a new PCR tube.

[0083] (2) cDNA amplification: Add 58 μl of PCR mixture (including 50 μl of cDNA HIFI Master Mix and 8 μl of cDNA Primers) to the above PCR tube, totaling 100 μl, and perform PCR reaction. The PCR reaction steps are as follows: ① 95℃ for 5 minutes, ② 98℃ for 20 seconds, ③ 58℃ for 20 seconds, ④ 72℃ for 3 minutes, ②-④ for 15 cycles, then 72℃ for 5 minutes, remove and place on ice. Use Qubit dsDNA Mix to detect the concentration of the amplified cDNA: InvitrogenTM Qubit dsDNA HS Buffer 198 μl, Qubit dsDNA HS Reagent 200×1 μl, cDNA product 1 μl.

[0084] (3) cDNA purification: Transfer the PCR product from the previous step to a new 1.5ml centrifuge tube and mix it with VAHTSTM DNA Clean Beads (VAZYME) equilibrated at room temperature in a volume ratio of 1:1, shake and mix, and incubate at room temperature for 10 minutes. After instant centrifugation, place the EP tube on a magnetic stand and let it stand for 3 minutes. After the liquid is clear, remove the supernatant. Add 1ml of freshly prepared 80% ethanol, let it stand for 30 seconds, discard the supernatant, repeat the washing with 80% ethanol, discard the supernatant, and dry at room temperature until the surface of the magnetic beads is non-reflective and cracked. Add 40μl NF-H2O to dissolve it back, shake and mix, and let it stand at room temperature for 5 minutes. After instant centrifugation, let it stand on a magnetic stand for 3-5 minutes. After the liquid is clear, recover the supernatant and transfer it to a new 1.5ml centrifuge tube. Take 1μl of cDNA sample and use Qubit dsDNA HS Kit to detect the concentration. Use Agilent 2100 High Sensitivity DNA kit to detect the distribution of cDNA fragments. (QC standard: fragment size is mainly distributed between 1000-1500bp)

[0085] 1.9 cDNA library circularization, sequencing, and data analysis

[0086] Transfer 50 ng of the purified product to a new PCR tube, add NF-H2O to make up to 20 μl, then add 20 μl of 2× Make DNB buffer. Incubate in a PCR instrument at 95°C for 3 minutes and then at 40°C for 3 minutes. Place the reaction mixture on ice, then add 39 μl of RCA buffer, 1 μl of 10 mM ATP, and 4 μl of One Step Enzyme. Incubate in a PCR instrument at 30°C for 30 minutes. Remove the mixture and add 20 μl of DNB stop buffer to create the circularized DNB library. The library was sequenced using the MGISEQ-2000RS sequencer. The data was automatically analyzed at https: / / uat.stomics.tech / sap / to generate a heatmap.

[0087] Example 2

[0088] Steps 2.1-2.4 are the same as steps 1.1-1.4 in Example 1.

[0089] 2.5 Random probe (6N) and splint hybridization

[0090] While the tissue sections are being fixed, prepare the primer and hybridization mixture.

[0091] 10 μM random probe 6N (5'pho-SEQ ID NO:4-3') and 10 μM splint (5'- / rU / SEQ ID NO:5-3') were mixed in a PCR tube. The temperature was set to a gradient in a PCR instrument, starting from 95°C and decreasing by 2°C per minute to 25°C. The tube was then removed and placed on ice. 1 μl of the primer hybridization mixture was added to 5×SSC, followed by the addition of 5% by volume of an RNase inhibitor. The mixture was mixed and placed on ice.

[0092] After the permeabilization reaction is complete, remove the chip, aspirate the surface liquid, and wash the chip once with 190 μl of 5×SSC + 10 μl of RI. Add the hybridized primer hybridization mixture and hybridize for 15 minutes at room temperature. Then, wash once with 5×SSC + RI. This yields the primer hybridization mixture, as shown in Set 4 in Figure 3.

[0093] Steps 2.6-2.9 are the same as steps 1.6-1.9 in Example 1.

[0094] Comparative Example 1

[0095] Steps 1.1-1.4 are the same as steps 1.1-1.4 in Example 1.

[0096] 1.5 Random probe (6N) hybridization with splint

[0097] While the tissue sections are being fixed, prepare the primer hybridization mixture. Add 5 μM random probe 6N (5'pho-SEQ ID NO:2-3') and 5 μM splint (5'-SEQ ID NO:3-3') to 5× SSC, mix thoroughly, and hybridize in a 55°C incubator for 10 minutes. Remove from the oven and place on ice. Add 5% by volume of RNase Inhibitor (RI), mix thoroughly, and return to ice.

[0098] After the permeabilization reaction is complete, remove the chip, aspirate the surface liquid, and wash the chip once with 190 μl of 5×SSC + 10 μl of RI. Add the hybridized primer hybridization mixture and hybridize for 15 minutes at room temperature. Then wash once with 5×SSC + RI. This yields the primer hybridization mixture, as shown in Set 1 in Figure 3.

[0099] Steps 1.6-1.9 are the same as steps 1.6-1.9 in Example 1.

[0100] Comparative Example 2

[0101] Steps 2.1-2.4 are the same as steps 1.1-1.4 in Example 1.

[0102] 2.5 Random probe (6N) and splint hybridization

[0103] While the tissue sections are being fixed, prepare the primer and hybridization mixture.

[0104] 10 μM random probe 6N (5'pho-SEQ ID NO:2-3') and 10 μM splint (5'-SEQ ID NO:3-3') were mixed in a PCR tube. The temperature was set to a gradient in a PCR instrument, starting from 95°C and decreasing by 2°C per minute to 25°C. The tube was then removed and placed on ice. 1 μl of the primer hybridization mixture was added to 5×SSC, followed by the addition of 5% by volume of an RNase inhibitor. The mixture was mixed and placed on ice.

[0105] After the permeabilization reaction is complete, remove the chip, aspirate the surface liquid, and wash the chip once with 190 μl of 5×SSC + 10 μl of RI. Add the hybridized primer hybridization mixture and hybridize for 15 minutes at room temperature. Then, wash once with 5×SSC + RI. This yields the primer hybridization mixture, as shown in Set 2 in Figure 3.

[0106] Steps 2.6-2.9 are the same as steps 1.6-1.9 in Example 1.

[0107] in conclusion

[0108] As shown in Figure 3, spatiotemporal transcriptome capture was tested on fresh mouse hemi-brain sections embedded in OCT (A) and paraffin-embedded mouse hemi-brain sections (B), and the number of junctions generated by splint for each method was analyzed.

[0109] Valid reads: After sequencing, through algorithm analysis, it is determined that the nucleic acid sequence is captured by random probes, reverse transcribed and connected to the chip.

[0110] Adapter generated by splint: As described in the principle, the sequence generated due to the chain displacement function of the RT enzyme is called the adapter sequence.

[0111] Unique mapping: After the sequences in the valid reads are aligned with the reference genome, they can be mapped to the sequences at the unique position in the genome.

[0112] Figure A shows test data from fresh OCT-embedded mouse brain. Data analysis revealed that, with approximately identical valid read counts (both approximately 210M), the use of 5'pho-SEQ ID NO:4-3' and 5'- / rU / SEQ ID NO:5-3' reduced the proportion of splint-induced junctions from 45% to 20%, a reduction of approximately 25%. Consequently, the proportion of unique mappings increased from approximately 2.5% to approximately 8.5%, a 3.4-fold increase. These results demonstrate that 5'pho-SEQ ID NO:4-3' and 5'- / rU / SEQ ID NO:5-3' can reduce splint-induced junctions and increase the proportion of unique mappings, regardless of hybridization method.

[0113] Figure B shows test data from paraffin-embedded (FFPE) mouse brain. Data analysis revealed that, while valid reads were largely identical, the use of 5'pho-SEQ ID NO:4-3' and 5'- / rU / SEQ ID NO:5-3' reduced the proportion of splint-induced junctions from 50% to 22%, a reduction of approximately 28%. Consequently, the proportion of unique mappings increased from approximately 1% to approximately 2%, a two-fold increase. The conclusions were similar to those for fresh OCT mouse brain.

[0114] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A splint sequence, characterized in that: The first base at the 5' end of the splint sequence is U; preferably, the nucleotide sequence after the first base at the 5' end of the splint sequence is as shown in SEQ ID NO:

5.

2. A nucleic acid capture kit, characterized in that: Comprising the splint sequence as claimed in claim 1; preferably, the nucleic acid capture kit further comprises at least one of a random probe, a T4 ligase, a reverse transcriptase and a buffer for ligation / reverse transcription.

3. Use of the splint sequence as claimed in claim 1 in nucleic acid capture.

4. A method for capturing nucleic acid, characterized in that: By hybridizing the random probe and the fixed oligonucleotide sequence with the splint sequence as claimed in claim 1, the RNA in the sample captured by the random probe is reverse transcribed into cDNA, and the cDNA is connected to the random probe to form a long single-stranded nucleotide having, from 5' to 3', the oligonucleotide sequence, the random probe and the cDNA in sequence, wherein the reverse transcription and the connection are carried out in the same reaction system.

5. The method according to claim 4, characterized in that It includes the following steps: (1) the random probe hybridizes with the splint sequence; wherein the 5' end of the random probe is partially complementary to the 5' end of the splint sequence; (2) the random probe hybridizes with RNA in the sample; wherein the 3' end of the random probe is partially complementary to the 3' end of the RNA; (3) the splint sequence is hybridized with an immobilized oligonucleotide sequence; preferably, the oligonucleotide sequence is immobilized on a capture chip; (4) In the same reaction system, cDNA is synthesized using the RNA in the complex formed by the simultaneous hybridization in (1), (2) and (3) as a template, and the fixed oligonucleotide sequence and the random probe on the complex are connected to form a long single-stranded nucleotide containing cDNA in the 5' to 3' direction.

6. The method according to claim 5, characterized in that The capture chip is placed at 30-45° C., such as 37° C., for reaction for 3-24 hours, such as 3-5 hours; Preferably, in step (1), the random probe comprises 6-20N.

7. The method according to claim 4 or 5, characterized in that The method further comprises: (i) mixing the random probe and the splint sequence with 5×SSC, placing in a 50-60° C., for example, 55° C. incubator for hybridization for 5-30 minutes, for example, 10 minutes, to form a primer hybridization mixture, taking it out and cooling it, and adding 2-15% by volume, for example, 5% by volume of an RNase inhibitor; or (i') mixing the random probe and the splint sequence in a solution, setting a gradient to cool to room temperature, and then cooling to 0-4°C to obtain a primer hybridization mixture, and mixing the primer hybridization mixture with 5×SSC and 2-15% (eg, 5% by volume) RNase inhibitor.

8. The method according to claim 7, characterized in that After step (i) or step (i'), the method further comprises: (ii) The surface liquid of the capture chip is removed by aspiration, and the capture chip is washed with mixed solution 2, and the primer hybridization mixed solution is added, hybridized at room temperature for 15-60 minutes, and then washed again with mixed solution 2; the mixed solution 2 is 5×SSC and RNase inhibitor.

9. The method according to claim 8, characterized in that In step (i'), the gradient cooling is to cool the temperature from 90-100°C by 1-5°C per minute to 20-28°C; In step (i) or (i'), the random probe is 6N; In step (i) or (i'), the molar ratio of the random probe to the splint sequence is (1-10):(1-10); and / or, In step (ii), the volume ratio of 5×SSC to RNase inhibitor in the mixed solution 2 is (5-30):

1.

10. The method according to claim 9, characterized in that In step (i'), the temperature is lowered from 95°C by 2°C per minute to 25°C; In step (i) or (i'), the molar ratio of the random probe to the splint is 1:1; and / or, In step (ii), the volume ratio of 5×SSC to RNase inhibitor in the mixed solution 2 is 19:

1.

11. The method according to any one of claims 4 to 10, characterized in that: The capture chip includes a capture probe, wherein the capture probe includes spatial barcode position information and a fixed oligonucleotide sequence; the fixed oligonucleotide sequence hybridizes with the 3' end of the splint.

12. The method according to claim 11, characterized in that The capture probe is 5'Nm-SEQ ID NO: 1-3', and m is 8-30.

13. The method according to claim 12, characterized in that Said m≥25.

14. A high-throughput sequencing method, characterized in that: It includes the following steps: (I) obtaining the long single-stranded nucleotide containing cDNA to be sequenced according to the method described in any one of claims 3 to 13; (II) constructing a cDNA library; (III) Perform sequencing and analyze the acquired data.

15. The method according to claim 14, characterized in that (I) further comprises the steps of cDNA shearing, amplification and purification; and / or, in (II), the cDNA library is circularized to obtain a circularized DNB library.

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