Combined coding Tn5 reagent, library construction kit and library construction method

Abstract

The invention relates to the technical field of library construction for next-generation sequencing, in particular to a combined coding Tn5 reagent, a kit for library construction and a library construction method. Wherein the combination coding Tn5 reagent comprises different types of Tn5-Ax transposase which is used for marking double-stranded cDNA captured on a substrate in a first direction; the Tn5-By transposase is used for marking the double-stranded cDNA captured on the substrate in a second direction, and the Tn5-By transposase is different from the double-stranded cDNA captured on the substrate; wherein the first direction and the second direction are not overlapped or parallel; wherein the Tn5-Ax transposase contains space coding information Ax, and the Tn5-By transposase contains space coding information By. The kit contains the combined coding Tn5 reagent. The library building method comprises the step of using the combined coding Tn5 reagent. According to the method, the full-length transcriptome library containing spatial information can be efficiently obtained.

Description

Technical Field

[0001] The present application relates to the technical field of library construction for next-generation sequencing, and in particular to a combination encoding Tn5 reagent, a kit for library construction, and a library construction method. Background Art

[0002] Next-generation sequencing (NGS), also known as high-throughput sequencing, is a DNA sequencing technology developed based on PCR and gene chips, which can quickly sequence the base pairs of DNA or RNA samples. NGS supports a wide range of applications, including gene expression profiling, chromosome counting, epigenetic change detection and molecular analysis, and is promoting the development and realization of the future of personalized medicine. It determines the gene sequence by sequencing multiple small DNA fragments in parallel.

[0003] Spatial transcriptomics is a technology that analyzes and describes the expression profiles of specific cell types in a spatial dimension to understand the expression differences between organs, tissues and pathological states. It can parse the transcriptome of tissues in different spatial locations.

[0004] However, the existing methods based on second-generation sequencing either only capture the 3' end information of mRNA or only capture the 5' end information, and cannot capture the full-length information of the entire mRNA. The technology that is similar to this method is mainly full-length transcriptome sequencing based on third-generation sequencing. Although it can capture the full-length information of mRNA, due to the short length of mRNA (generally around 1.5kb), it is not very consistent with the fragment length required by conventional third-generation sequencers (more than 10-20kb). Therefore, there are often problems such as low detection efficiency and low accuracy, and its sequencing price is also relatively expensive.

[0005] However, the existing technology cannot capture the full-length information of mRNA, so it is impossible to build a library of spatial full-length transcriptome based on second-generation sequencing. Therefore, how to obtain the complete mRNA spatial full-length transcriptome using second-generation sequencing has become an urgent problem to be solved. Summary of the invention

[0006] The present application provides a combination of Tn5 encoding reagents, a library construction kit and a library construction method.

[0007] Tn5 transposase is a sequence-specific DNA binding protein expressed by the DNA transposon sequence. The transposition of Tn5 transposon has two processes: cutting and pasting. At the beginning of transposition, two transposases (Tnp) recognize the external end sequence (OE) sequences at both ends of the transposon to form a Tnp OE complex. Then the two Tnp OE complexes bind to each other and form a dimer synaptic complex with the ability to cut DNA. In genomic and transcriptomic applications, Tn5 transposase randomly inserts into the DNA double strand and inserts the two adapters it carries into the gene sequence to complete the adapter ligation reaction and perform the subsequent library construction process. In a single sample, the full-length transcriptome sequencing analysis of mRNA can be achieved using Smart-seq2 technology and its interruption of Tn5 transposase, adapter ligation reaction and other steps.

[0008] In the scheme of the present application, a tissue or RNA solution sample is captured by a polyT probe modified on the surface of a substrate (e.g., a glass slide), and a double-stranded cDNA is obtained after reverse transcription and two-strand synthesis. For example, 50 different TN5-Ax are introduced into the cDNA region through parallel PDMS microfluidic lanes for reaction and incubation; for example, 50 different TN5-By are introduced into the cDNA region through vertically arranged PDMS microfluidic lanes for reaction and incubation. The incubation products are interrupted and collected for purification. The interrupted products are used to construct a library to obtain a full-length transcriptome library containing spatial information.

[0009] The present application discloses a combination encoding Tn5 reagent, the scheme of which is as follows:

[0010] A combination of Tn5-encoded reagents comprising different species of Tn5-A for labeling double-stranded cDNA captured on a substrate in a first direction. x transposase; and,

[0011] Different species of Tn5-B are used to label the double-stranded cDNA captured on the substrate in the second direction. y Transposase;

[0012] wherein the first direction and the second direction are non-overlapping or parallel;

[0013] Wherein, the Tn5-A x Transposase contains spatially encoded information A x , the Tn5-B y Transposase contains spatially encoded information B y ;

[0014] Among them, x refers to Tn5-A x Transposase number; y refers to Tn5-B y The transposase number, x and y are each independently selected from positive integers.

[0015] Preferably, x and y are each independently selected from a positive integer of 1-100; more preferably, x and y are each independently selected from a positive integer of 1-50.

[0016] Optionally, the spatial coding information A x Contains or is any one or more of the sequences SEQ ID NO.2-51, or contains or is a sequence having at least 80% identity with any one or more of the sequences SEQ ID NO.2-51;

[0017] and / or,

[0018] Among them, the spatial coding information B y It comprises or is any one or more of the sequences SEQ ID NO.52-101, or it comprises or is a sequence having at least 80% identity with any one or more of the sequences SEQ ID NO.52-101.

[0019] Optionally, the spatial coding information A x Contains or is any one or more of the sequences SEQ ID NO.2-51;

[0020] and / or,

[0021] Among them, the spatial coding information B y Contains or is any one or more of the sequences SEQ ID NO.52-101.

[0022] More preferably, the spatial coding information A x Containing or being any eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or more of the sequences SEQ ID NO.2-51;

[0023] and / or,

[0024] Among them, the spatial coding information B y It comprises or is any eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or more of the sequences SEQ ID NO.52-101.

[0025] It should be noted that, here, the specific selection of the spatial coding information is not important, and it is mainly necessary to select different spatial coding information for distinction.

[0026] Optionally, the spatial coding information A x It also contains the MErev sequence;

[0027] And / or, the spatial coding information By It also contains the MErev sequence;

[0028] Wherein, the MErev sequence comprises or is the sequence shown in SEQ ID NO.1, or the MErev sequence comprises or is a sequence having at least 80% identity with the sequence shown in SEQ ID NO.1.

[0029] Preferably, the Tn5-A x The preparation method of the transposase comprises the following steps:

[0030] So that the spatial coding information A x Mixed with MErev sequence and amplified to obtain Mix-A x sequence;

[0031] Make Mix-A x The sequence and Tn5 transposase were mixed and incubated in a buffer system to obtain Tn5-A x Transposase.

[0032] Preferably, the Tn5-B y The preparation method of the transposase comprises the following steps:

[0033] So that the spatial encoding information B y Mixed with MErev sequence and amplified to obtain Mix-B y sequence;

[0034] Mix-B y The sequence and Tn5 transposase were mixed and incubated in a buffer system to obtain Tn5-B y Transposase.

[0035] Preferably, Tn5-A is prepared x When transposase is used, the mixed incubation time is 45-75 minutes, and the mixed incubation temperature is 22-37°C.

[0036] Preferably, Tn5-B is prepared y When transposase is used, the mixed incubation time is 45-75 minutes, and the mixed incubation temperature is 22-37°C.

[0037] The present application also provides a kit for library construction, the scheme of which is as follows:

[0038] A kit for library construction, comprising the above-mentioned combination encoding Tn5 reagent.

[0039] Preferably, the library construction kit further comprises a substrate, wherein the substrate comprises a polyT sequence with an amino group modification at one end for capturing RNA;

[0040] Preferably, the 5' end of the polyT sequence is amino-modified;

[0041] More preferably, the length of the polyT sequence is 20-30 nt;

[0042] More preferably, the polyT sequence comprises or is the sequence shown in SEQ ID NO.102, or the polyT sequence comprises or is a sequence having at least 80% identity with the sequence shown in SEQ ID NO.102.

[0043] Preferably, the library construction kit further comprises a reverse transcription reagent and a PCR reagent.

[0044] The present application also provides a library construction method based on the spatial full-length transcriptome of combined encoding Tn5, and the scheme is as follows:

[0045] A library construction method based on the spatial full-length transcriptome encoding Tn5, wherein the library construction method comprises the following steps:

[0046] The double-stranded cDNA captured on the substrate is labeled with Tn5 transposase twice in different directions using the combination encoding Tn5 reagent according to any one of claims 1 to 4 to obtain Tn5 transposase-labeled double-stranded cDNA;

[0047] The double-stranded cDNA labeled with Tn5 transposase was sheared and the fragmented cDNA was collected;

[0048] After the fragmented cDNA is amplified, adapters are connected to construct a library; the library can be used for second-generation sequencing.

[0049] Optionally, the angle between the first direction and the second direction is 10-90°, including endpoint values.

[0050] Preferably, the first direction and the second direction are perpendicular to each other.

[0051] Further preferably, when the double-stranded cDNA is labeled with Tn5 transposase twice in different directions, the following steps are included:

[0052] The double-stranded cDNA captured on the substrate was labeled with different Tn5-A species using parallel PDMS microfluidic lanes. x Transposase;

[0053] The double-stranded cDNA captured on the substrate was labeled with different Tn5-B species using vertically arranged PDMS microfluidic lanes. y Transposase;

[0054] or,

[0055] Further preferably, when the double-stranded cDNA is labeled with Tn5 transposase twice in different directions, the following steps are included:

[0056] The double-stranded cDNA captured on the substrate was labeled with different types of Tn5-A in parallel using a spotter. x Transposase;

[0057] The double-stranded cDNA captured on the substrate was vertically spotted with different types of Tn5-B using a spotter. y Transposase.

[0058] Optionally, when the double-stranded cDNA captured on the substrate is labeled with Tn5 transposase twice in different directions, the labeling temperature is 22°C-37°C;

[0059] and / or,

[0060] The labeling time is 20-40 minutes.

[0061] Optionally, the size of any single-stranded cDNA in the double-stranded cDNA is 50-5000 bp.

[0062] Preferably, when the double-stranded cDNA labeled with Tn5 transposase is sheared, the shearing reagent is selected from any one or more of SDS, EDTA and commercial shearing reagents.

[0063] Preferably, the disrupting reagent is 0.1-0.3% SDS.

[0064] Optionally, the method for obtaining double-stranded cDNA captured on a substrate comprises the following steps:

[0065] mixing the tissue or RNA with the substrate to capture the RNA;

[0066] The RNA captured on the substrate is reverse transcribed and double-stranded synthesized to obtain double-stranded cDNA captured on the substrate.

[0067] Preferably, after the tissue or RNA is mixed with the substrate, the capture time is 5-20 min; and / or,

[0068] The capture temperature is 22-37°C.

[0069] Preferably, the reverse transcription temperature is 37-46° C.; and / or the reaction time is 100-140 min.

[0070] Preferably, the temperature during the second-chain synthesis is 22-37° C.; and / or the reaction time is 100-140 min.

[0071] Effects of the Invention

[0072] 1. The present application adds spatial coding information to both ends of cDNA by performing Tn5 transposase labeling multiple times in different directions, and can obtain spatial transcriptome full-length information. Of course, it is understandable that those skilled in the art can also design different groups of Tn5 transposase labels for at least two directions (e.g., two directions, three directions, and more directions) of double-stranded cDNA as needed, so as to more comprehensively grasp its spatial information.

[0073] 2. The present application directly labels cDNA (eg, double-stranded cDNA), which saves pre-amplification time and reduces reagent and time costs compared to the conventional slide spatial transcription library construction method. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 . Flowchart of library construction based on the spatial full-length transcriptome encoding Tn5.

[0075] Figure 2 .The result of conventional PE150 second-generation high-throughput sequencing of the library constructed in Example 1.

[0076] Figure 3 .In Example 1, the distribution results of reads on genes in the second-generation sequencing results of the library are shown.

[0077] Figure 4 .Example: Distribution ratio diagram of sequencing fragments of the library constructed in the embodiment on the genome. DETAILED DESCRIPTION

[0078] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in components' functions as the criterion for distinction.

[0079] The specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0080] Example 1

[0081] The reagents involved in the following embodiments are all commercially available unless otherwise specified.

[0082] 1. Preparation of the combination encoding Tn5 transposase

[0083] (1) Sequence annealing

[0084] The related sequences are from bioengineering biosynthesis, and the sequences are shown in Table 1.

[0085] Table 1

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] In Table 1, MEB y Each N in NNNNNN is independently selected from the bases A, T, C, and G. It should be noted that NNNNNN is randomly synthesized and is called the UMI value, which is used to mark each unique DNA or RNA molecule. The main function of these UMIs is to identify and correct the deviations in the PCR amplification process in high-throughput sequencing. In the experiment, each DNA or RNA molecule will be added with a randomly generated short sequence (UMI), which remains unchanged during the PCR amplification process; it should also be understood that the short sequence UMI added to different molecules is not exactly the same, that is, the UMI in each MEB is different. When the sequencing results are analyzed, sequences with the same UMI will be classified as the same molecule, thereby avoiding repeated reading errors.

[0094] Take 4 μL of 20 μM MErev and 4 μL of 20 μM ME-A respectively. x (x = 1-50, respectively, sequences SEQ ID NO. 2-51), 2 μL annealing buffer (Annealing buffer, Ibotek, RM20821), a total of 10 μL, shake and mix. Take 4 μL 20 μM MErev (sequence SEQ ID NO. 1), 4 μL 20 μM ME-B y (y=1-50, respectively, sequences SEQ ID NO.52-101), 2 μL Annealing buffer, a total of 10 μL, shake and mix. According to the system shown in Table 2 below, the reaction procedure is carried out to obtain Mix-A x Sequence, Mix-B y sequence.

[0095] Table 2

[0096] temperature time Temperature change speed Number of cycles 95℃ 2min 1℃ / s 1 22℃ 5min 0.1℃ / s 1 4℃ hold 1℃ / s 1

[0097] (2) Tn5 transposase embedding

[0098] Take 2 μL Max-A x , 1 μL Tn5 transposase (purchased from Ibotek, RM21303), 5 μL Assemblebuffer (purchased from Ibotek, RM20187), 17 μL enzyme-free water, a total of 25 μL, oscillate to mix, and incubate at 37°C for 1 hour. x .

[0099] Take 2 μL Max-B y , 1μL Tn5 transposase (purchased from Ibotek, RM21303), 5μL 5×Assemblebuffer (purchased from Ibotek, RM20187), 17μL enzyme-free water, a total of 25μL, shake to mix, incubate at 37℃ for 1h. y .

[0100] 2. Preparation of capture chip and synthesis of cDNA

[0101] (1) Preparation of capture chip

[0102] The capture chip (also referred to as the amino glass slide below) was prepared using a polyT sequence with an amino group at the 5' end synthesized by Bio-Engineering (5'-NH2-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN, SEQ ID NO.102; wherein V refers to base A, C or G; N refers to base A, T, C or G; any one of them can be selected; for example, V is selected as A and N is selected as C). 3.7 mg of disuccinimidyl suberate (DSS) was dissolved in dimethyl sulfoxide (DMSO) and mixed by oscillation. Added to a centrifuge tube according to the system shown in Table 3 below, oscillated and mixed to obtain a mixed solution. 50 μL of the mixed solution was added to the reaction area of ​​the amino glass slide and incubated at room temperature for 1 hour. The reaction area was washed twice with a 2×SSC (Saline-Sodium Citrate, a salt solution) + 0.1% SDS (a surfactant) solution, washed once with ultrapure water, and dried with nitrogen. The blocking buffer was prepared according to the system shown in Table 4 below. Immerse the glass slide in the blocking solution and incubate at room temperature for 1 hour. Wash with ultrapure water and blow dry with nitrogen to complete the preparation of the capture chip.

[0103] Table 3

[0104] Reagents Brand and item number Volume (μL) PBS Bioengineering, E607008- 42.5 DSS SIGMA, S1885 2.5 polyT Biosynthesis 5 Total / 50

[0105] Table 4

[0106]

[0107] (2) cDNA synthesis

[0108] Take 10 μL of 200 μg / μL mouse reference RNA solution (TAKARA, 636657) and add it to the slide reaction area, incubate at 37°C for 10 min. Add the reverse transcription reaction solution according to the system shown in Table 5 below, seal with tape, and react at 42°C for 2 h. Discard the solution, wash the reaction area twice with 2×SSC+0.1% SDS solution, add 80 mM KOH and incubate at room temperature for 5 min, and wash twice with EB solution (purchased from QIAGEN, product number 19086).

[0109] Table 5

[0110]

[0111] Add the double-strand synthesis reaction solution according to the system shown in Table 6 below, seal with tape, and react at 37°C for 2 hours. Discard the solution, wash twice with EB solution, and complete the synthesis of double-stranded cDNA.

[0112] Table 6

[0113] Reagents Brand and item number Volume (μL) PEG8000 Blue Sky, R0056 9 Enzyme-free water Tiangen, RT121-01 13.5 TSOprimer Biosynthesis 3.75 dNTP Bioengineering, A610056-0500 3.75 Klenow Fragment (3'→5'exo-) NEB,M0212L 3.75 Klenowbuffer NEB,M0212L 3.75 Total / 37.5

[0114] 3. Tn5 tagging of the combination encoding double-stranded cDNA

[0115] Align the parallel array of PDMS microfluidic channels with the glass slide ( Figure 1 ), 10 kinds of Tn5-A were introduced into parallel channels x The solution was fixed with a clamp, sealed with tape, and incubated at 37°C for 30 min to allow Tn5-A x Bind to cDNA, remove the reaction liquid, and uncover the microfluidic channel. Then align the vertically arranged PDMS microfluidic channel with the glass slide, and introduce 10 kinds of Tn5-B into the vertically arranged microfluidic channel. y The solution was fixed with a clamp, sealed with tape, and incubated at 37°C for 30 min to allow Tn5-B y Bind to cDNA, remove the reaction liquid, and uncover the microfluidic channel. Complete the cDNA combination encoding Tn5 tag. Among them, Tn5-A x MEA modified on transposase x Select MEA1 to MEA 10 The sequences of Tn5-B y MEB modified on transposase yThe selected is MEB1 to MEB 10 sequences (i.e., sequences SEQ ID NO.52 to SEQ ID NO.61).

[0116] 4. DNA collection, library amplification and construction ( Figure 1 )

[0117] Combination Figure 1 , add 50μL 0.2% SDS to the reaction area and incubate at room temperature for 5min. Collect the liquid into a PCR tube, add 75μL purified magnetic beads, mix thoroughly and incubate at room temperature for 8min. Then place it on a magnetic rack for 5min, remove the magnetic beads and retain the supernatant after the liquid is clarified, add 200μL 80% alcohol to wash the magnetic beads twice, dry them at room temperature for 2min, add 21μL ddH2O to elute, place it on a magnetic rack, and carefully pipette 20μL of liquid into a new PCR tube after the liquid is clarified. Add the PCR amplification solution according to the system shown in Table 7 below. The above system was amplified, and the amplification program was: 72℃3min, 98℃30s, 12 cycles (98℃15s, 60℃30s, 72℃3min), 72℃5min, and keep at 4℃.

[0118] Table 7

[0119] Reagents Brand and item number Volume (μL) product The above purified product 20 Enzyme-free water Tiangen, RT121-01 3 PCRMIX Ibotek, RM20242 25 Indexp7 Biosynthesis 1 Indexp5 Biosynthesis 1 Total / 50

[0120] Purify the amplified product, add 37.5μL purified magnetic beads to the PCR tube, mix thoroughly and incubate at room temperature for 8min. Then place it on the magnetic stand for 5min, remove the magnetic beads and retain the supernatant after the liquid is clear, add 200μL 80% alcohol to wash the magnetic beads twice, dry them at room temperature for 2min, add 22μL ddH2O to elute, place it on the magnetic stand, and carefully draw 20μL after the liquid is clear and send it to sequencing. Take 1μL for library concentration detection.

[0121] 5. Second-generation high-throughput sequencing

[0122] The library obtained above was subjected to conventional PE150 second-generation high-throughput sequencing, and the library results were as follows: Figure 2 As shown, the reads obtained after sequencing are aligned back to the corresponding reference genome. The distribution results of the reads on the gene after alignment are as follows Figure 3 As shown, the reads obtained by conventional 3' end sequencing are mainly distributed at the 3' end of the gene. The combined encoding Tn5 transposase used in the present invention can achieve balanced coverage of the sample genome and also has spatial encoding information. Figure 4 It is a distribution ratio diagram of library sequencing fragments on the genome.

[0123] In summary, the method of the present application can perform spatial sequencing of complete mRNA, which makes up for the defect that the second-generation sequencing cannot be used to perform spatial sequencing of complete mRNA in the prior art. This method has the characteristics of high detection efficiency and high accuracy.

Claims

1. A combined Tn5-encoding reagent comprising different species of Tn5-A for labeling double-stranded cDNA captured on a substrate in a first direction. x transposase; and, Different species of Tn5-B are used to label the double-stranded cDNA captured on the substrate in the second direction. y Transposase; in, The first direction and the second direction are non-overlapping or parallel; Wherein, the Tn5-A x Transposase contains spatially encoded information A x , the Tn5-B y Transposase contains spatially encoded information B y ; Among them, x refers to Tn5-A x Transposase number; y refers to Tn5-B y The transposase number, x and y are each independently selected from positive integers.

2. The combined encoded Tn5 reagent according to claim 1, wherein The spatial coding information A x Contains or is any one or more of the sequences SEQ ID NO.2-51, or contains or is a sequence having at least 80% identity with any one or more of the sequences SEQ ID NO.2-51; and / or, Among them, the spatial coding information B y It comprises or is any one or more of the sequences SEQ ID NO.52-101, or it comprises or is a sequence having at least 80% identity with any one or more of the sequences SEQ ID NO.52-101.

3. The combined coded Tn5 reagent according to claim 1, wherein the spatially coded information A x Contains or is any one or more of the sequences SEQ ID NO.2-51; and / or, in, The spatial coding information B y Contains or is any one or more of the sequences SEQ ID NO.52-101.

4. The combined encoding Tn5 reagent according to claim 2 or 3, wherein The spatial coding information A x It also contains the MErev sequence; and / or, Among them, the spatial coding information B y It also contains the MErev sequence; Wherein, the MErev sequence comprises or is the sequence shown in SEQ ID NO.1, or the MErev sequence comprises or is a sequence having at least 80% identity with the sequence shown in SEQ ID NO.

1.

5. A kit for library construction, comprising the combination encoding Tn5 reagent according to any one of claims 1 to 4.

6. A library construction method based on the spatial full-length transcriptome encoding Tn5, wherein the library construction method comprises the following steps: The double-stranded cDNA captured on the substrate is labeled with Tn5 transposase twice in different directions using the combination encoding Tn5 reagent according to any one of claims 1 to 4 to obtain Tn5 transposase-labeled double-stranded cDNA; The double-stranded cDNA labeled with Tn5 transposase was sheared and the fragmented cDNA was collected; After the fragmented cDNA is amplified, adapters are connected to construct a library; the library can be used for second-generation sequencing.

7. The library construction method according to claim 6, wherein the angle between the first direction and the second direction is 10-90°, including the endpoint values.

8. The library construction method according to claim 6, wherein the double-stranded cDNA captured on the substrate is labeled with Tn5 transposase twice in different directions at a labeling temperature of 22°C-37°C; and / or, The labeling time is 20-40 minutes.

9. The library construction method according to claim 6, wherein the size of any single-stranded cDNA in the double-stranded cDNA is 50-5000 bp.

10. The library construction method according to claim 6, wherein the method for obtaining the double-stranded cDNA captured on the substrate comprises the following steps: mixing the tissue or RNA with the substrate to capture the RNA; The RNA captured on the substrate is reverse transcribed and double-stranded synthesized to obtain double-stranded cDNA captured on the substrate.

Images