Method for single cell or low quantity sample full-length transcriptome library construction for nanopore sequencing

Abstract

The present application relates to the technical field of single cell sequencing, and particularly relates to a method for constructing a full-length transcriptome library of a single cell or a trace sample for nanopore sequencing. The method for constructing a library provided by the present application comprises: after lysing a single cell or a trace sample, mRNA is reversely transcribed to prepare cDNA by using a Smart-seq2 method, PCR amplification is performed by taking the cDNA as a template, the PCR amplification product is subjected to FFPE and end repair, and then a nanopore sequencing adaptor is connected. The method can realize true single cell sequencing, and has the advantages of high transcript coverage, high resolution and strong flexibility, and can be applied to differential gene expression analysis of a single cell, transcript structure analysis, full-length isoform identification and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of single cell sequencing, in particular to a method for constructing a full-length transcriptome library of single cells or trace samples for nanopore sequencing. BACKGROUND

[0002] For multicellular organisms, although from a fertilized egg, as the individual develops, the cells gradually differentiate into different subgroups, each expressing different genetic information and performing different physiological functions. For the same tissue, there is also heterogeneity between adjacent cells, such as brain tissue and heart tissue. In particular, a tumor is a collection of mutant cells, and bulk phase sequencing of tumor tissue and microenvironment cells can mask some key information. Single cell sequencing can perfectly solve this problem, greatly improving the resolution of sequencing analysis, and providing strong help for individual development and disease research. In recent years, single cell sequencing technology is leading a new round of technological revolution in the field of biomedicine.

[0003] Current commercial single cell processing platforms include Fluidigm C1, 10X genomics, etc. The advantage of these methods is high throughput, but the current experimental process is limited to sequencing analysis of the 3' end of the transcript. Therefore, the overall information of the transcript is ignored. SMART-seq is a landmark technology that can cover the entire transcript, thereby further realizing in-depth research such as variable splicing and SNV detection on the basis of expression analysis.

[0004] Nanopore platform is a new real-time single-molecule nanopore sequencing technology platform: by means of the potential difference on both sides of the nanopore caused by a single molecule passing through the nanopore to realize signal detection, the diameter of the nanopore only allows a single nucleotide polymer to pass through, and the electric properties of A, T, C and G four bases are different, so the type of base passing through the nanopore can be detected by the difference in electrical signal characteristics, thereby realizing sequencing. The read length of nanopore sequencing is longer, the average read length can reach 20kb, and the maximum can reach Mb level, and there is no GC bias caused by PCR reaction in the sequencing process, and the coverage is more uniform. SUMMARY

[0005] The purpose of the present application is to provide a method for constructing a full-length transcriptome library of single cells or trace samples for nanopore sequencing.

[0006] To achieve the above object, the application combines SMART technology and ONT nanopore sequencing technology to develop a single cell / trace sample full-length transcriptome library construction method.

[0007] Specifically, the application provides the following technical solutions:

[0008] The application provides a single cell or trace sample full-length transcriptome library construction method for nanopore sequencing, which comprises the following steps: after lysing a single cell or trace sample, mRNA is reversely transcribed into cDNA by using a Smart-seq2 method, PCR amplification is performed by taking the cDNA as a template, and the PCR amplification product is subjected to FFPE and end repair, and then connected to a nanopore sequencing adapter.

[0009] Specifically, a single cell sample or trace RNA sample obtained by a cell sorting method or a fiber cutting method is lysed in a single tube, and subsequent operations can be performed in the tube.

[0010] After lysing the single cell or trace sample, cDNA synthesis is performed by using a reverse transcription primer of Smart-seq2, a linker sequence is added to the 5' end of the cDNA by using the template-switching activity of reverse transcriptase, then chain conversion primer is combined and extended to synthesize double-stranded cDNA by PCR amplification, full-length transcript sequence is obtained, the double-stranded cDNA is subjected to FFPE repair and end repair, and then A is added, and then a sequencing adapter of ONT is added by using a TA connection method, and finally nanopore sequencing technology is used to realize end-to-end sequencing of the full-length transcript.

[0011] The application finds that the connection efficiency of the full-length transcriptome obtained by using SMART technology and the ONT adapter is low, and it is difficult to meet the high efficiency requirement of nanopore sequencing. In order to improve the connection efficiency of the full-length transcriptome obtained by using SMART technology and the ONT adapter, the application screens and optimizes the factors affecting the connection efficiency of the adapter, and finds that by simultaneously controlling the reaction conditions of FFPE and end repair and the reaction conditions of nanopore sequencing adapter connection, the connection efficiency of the full-length transcriptome obtained by using SMART technology and the ONT adapter can be significantly improved.

[0012] Preferably, the reaction conditions of FFPE and end repair are: reacting the reaction system at 20℃ for 30-35min; then reacting at 65℃ for 10min; or, first adding FFPE Master Mix and FFPE buffer in the PCR purified product, reacting at 20℃ for 10-15min, then adding Endprep Master Mix, Endprep buffer and water, reacting at 20℃ for 30-35min, and then reacting at 65℃ for 10min.

[0013] The reaction conditions of connecting the nanopore sequencing adapter are: reacting at 22-25℃ for 1h-1.5h.

[0014] The present application finds that, by simultaneously controlling the reaction conditions of FFPE and end repair and the reaction conditions of connecting the nanopore sequencing adapter in the above conditions, the efficiency of connecting the full-length transcriptome obtained by SMART technology with the ONT adapter can be significantly improved, and the subsequent ONT sequencing efficiency can be improved.

[0015] Preferably, the 60μL reaction system of FFPE and end repair includes the following components: 200ng-1500ng of PCR purified product, 3.5μL of FFPE buffer, 2μL of FFPE Master Mix, 3.5μL of End prep buffer, 2-3μL of End prep Master Mix, and water to make up to a total volume of 60μL.

[0016] Further, the present application also finds that, compared with other sequencing adapters recommended by ONT, the AMX sequencing adapter can better match the full-length transcriptome obtained by SMART technology, improve the adapter connection efficiency and ONT sequencing efficiency.

[0017] Based on this, the nanopore sequencing adapter is preferably an ONT Adapter Mix (AMX) sequencing adapter.

[0018] Further preferably, the 60μL reaction system of connecting the nanopore sequencing adapter includes the following components: 100-1300ng of FFPE and end repair purified product, 5μL of ONT Adapter Mix (AMX) sequencing adapter, 25μL of ONT LNB, 10μL of T4 DNA ligase, and water to make up to a total volume of 60μL.

[0019] In the library construction method described above, the reverse transcription by the Smart-seq2 method preferably comprises the following steps: first, mix the mRNA, reaction buffer and water, and then place them at low temperature, then mix with 3'SMART-Seq CDS Primer II A and treat at 72°C for 2-3 min to obtain a first premix; mix 5xUltra Low First-Strand Buffer, SMART-Seq v4 Oligonucleotide and RNase inhibitor, and then mix with SMARTScribe reverse transcriptase to obtain a second premix; mix the first premix and the second premix for reverse transcription reaction.

[0020] Preferably, the conditions of the reverse transcription reaction are: 42°C, 85-95 min; 70°C, 8-12 min. More preferably, 42°C, 90 min; 70°C, 10 min.

[0021] In the reverse transcription reaction described above, the reverse transcription primer is combined with the polyA tail of the mRNA through annealing, which is a modified Oligo dT primer that can hybridize with any polyA RNA and add a tail at the primer site for subsequent amplification; then the RT reverse transcriptase is added to synthesize the first strand (the characteristic of the RT reverse transcriptase is to automatically add three C bases when reverse transcribing to the end of the mRNA template), and the strand conversion primer is combined with it, and the reverse transcriptase continues to extend to obtain a sequence complementary to the strand conversion primer.

[0022] After the reverse transcription reaction described above, the double-stranded cDNA is synthesized by PCR amplification, and the cDNA is the full-length transcript.

[0023] In the library construction method described above, the reaction conditions of the PCR amplification are: 95°C, 1-1.5 min; 98°C, 10-15 sec, 65°C, 25-35 sec, 68°C, 3-4 min, 15-20 cycles; 72°C, 8-12 min. Preferably: 95°C, 1 min; 98°C, 10 sec, 65°C, 30 sec, 68°C, 3 min, 15-20 cycles; 72°C, 10 min.

[0024] Preferably, the 30μL reaction system of the PCR amplification comprises the following components: 2xSeqAmp PCR Buffer (or 2xSeqAmp CB PCR Buffer) 25μL, PCR Primer II A (12μM) 1μL, SeqAmp DNA polymerase 1μL, water 3μL.

[0025] In the above-mentioned method, a purification step can be set after PCR amplification, after FFPE and end repair, and after adapter ligation. The purification can use a purification method commonly used in the art, including but not limited to magnetic bead purification, etc.

[0026] Further, the present application also provides a single cell or trace sample full-length transcriptome sequencing method, which comprises: constructing a single cell or trace sample full-length transcriptome library by using the single cell or trace sample full-length transcriptome library construction method for nanopore sequencing, and sequencing the single cell or trace sample full-length transcriptome library.

[0027] Preferably, the sequencing is performed by using a nanopore sequencing platform. Further preferably, the nanopore sequencing platform is an ONT sequencing platform.

[0028] The present application has the following advantages:

[0029] (1) Low template starting amount: a single cell or 10 pg of total RNA can be used as a starting template for efficient amplification;

[0030] (2) High transcript coverage: full transcriptome information is obtained by double-end primer amplification of full-length cDNA, avoiding 5' and 3' bias, and the product integrity is good;

[0031] (3) High base resolution: single-base resolution can be achieved, and specific transcripts of specific genes can be located;

[0032] (4) High operation success rate: the processing of RNA samples is greatly reduced, thereby minimizing the risk of sample loss;

[0033] (5) High amplification sensitivity: the detection amount of low expression is greatly increased;

[0034] (6) Full transcript analysis: can be used for single cell level differential gene expression analysis, transcript structure analysis, and full-length isoform identification.

[0035] The present application optimizes the library construction method, combines the SMART technology and the ONT nanopore sequencing technology well, and effectively improves the connection efficiency of the ONT nanopore sequencing adapter and the data effectiveness of the ONT sequencing. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The figure is a whole flow chart of the single cell library construction method of Example 1 of the present application.

[0037] Figure 2Agilent 2100 Bioanalyzer quality check of cDNA after single cell amplification in Example 1 of the present application.

[0038] Figure 3 Library sequencing quality plot of Example 1 in Experimental Example of the present application.

[0039] Figure 4 Library sequencing pore speed detection result of Example 1 in Experimental Example of the present application.

[0040] Figure 5 Library sequencing quality plot of Example 2 in Experimental Example of the present application.

[0041] Figure 6 Library sequencing pore speed detection result of Example 2 in Experimental Example of the present application.

[0042] Figure 7 Library sequencing quality plot of Comparative Example in Experimental Example of the present application.

[0043] Figure 8 Library sequencing pore speed detection result of Comparative Example in Experimental Example of the present application. DETAILED DESCRIPTION

[0044] The following examples are intended to illustrate the present application but not to limit the scope of the present application.

[0045] Example 1

[0046] The present embodiment provides a method for constructing a full-length transcriptome library of single cell or micro sample for nanopore sequencing (basic flow as shown in Figure 1 The method comprises the following steps:

[0047] 1. Single cell lysis

[0048] Prepare 10x Reaction Buffer according to Table 1, add into single cell tube, mix gently, and centrifuge (lysis buffer contains detergent, and air bubbles must be avoided when mixing gently), and lyse at room temperature for 5 min.

[0049] Table 1. Single cell lysis reaction buffer formula

[0050] Component Amount 10x lysis buffer 19 μL RNase inhibitor 1 μL Total volume 20 μL

[0051] 2. Reverse transcription

[0052] (1) Take 0.2 mL centrifuge tube, and prepare reverse transcription reagent according to Table 2.

[0053] Table 2. Reverse transcription reaction system formula (1)

[0054]

[0055]

[0056] (2) Place the sample on ice and add 2 μL of 3' SMART-Seq CDS Primer II A (12 μM), flick to mix, and spin down. The resulting reaction is shown in Table 3.

[0057] Table 3. Reverse transcription reaction formulation (2)

[0058] Component Amount Reaction from Table 2 10.5 μL 3' SMART-Seq CDS Primer II A (12 μM) 2 μL Total volume 12.5 μL

[0059] Place the reaction in a pre-heated PCR machine at 72°C for 3 min.

[0060] (3) Prepare the reverse transcription Mix according to Table 4 at the time of incubation.

[0061] Table 4. Reverse transcription reaction formulation (3)

[0062] Component Amount 5x Ultra Low First-Strand Buffer 4 μL SMART-Seq v4 Oligonucleotide (48 μM) 1 μL RNase inhibitor (40 U / μL) 0.5 μL Total volume 5.5 μL

[0063] (4) After the 72°C incubation for 3 min, place the sample on ice for 2 min.

[0064] (5) Add 2 μL of SMARTScribe Reverse Transcriptase to the reverse transcription Mix from step (4). Note: Mix the master mix by flicking, and make sure to mix gently after adding the reverse transcriptase without vortexing.

[0065] (6) Add 7.5 μL of the reverse transcription Mix to each reaction tube (total volume 20 μL). Flick to mix and spin down.

[0066] (7) Pre-heat the PCR machine to 42°C and perform the reverse transcription reaction according to the following conditions: 42°C for 90 min; 70°C for 10 min; 4°C for storage.

[0067] 3. PCR amplification

[0068] (1) Take out all the reagents needed for PCR (except the enzyme) and thaw on ice. Gently vortex each reagent and spin down, keeping on ice.

[0069] (2) Prepare the PCR Mix according to Table 5.

[0070] Table 5. PCR reaction formulation

[0071] Component Amount 2x SeqAmp PCR Buffer OR 2X SeqAmp CB PCR Buffer 25 μL PCR Primer II A (12 μM) 1 μL SeqAmp DNA Polymerase 1 μL Nuclease-free water 3 μL Total volume 30 μL

[0072] Note: SeqAmp DNA Polymerase is added last.

[0073] (3) Add 30 μL PCR Mix to the reverse transcription product (20 μL) obtained in step 2, and mix by flicking, and elute.

[0074] (4) Preheat the PCR instrument to 95°C, and perform PCR reaction under the conditions shown in Table 6.

[0075] Table 6 PCR reaction conditions

[0076]

[0077] (5) Take 1 μL of the PCR product, and measure the concentration using Qubit.

[0078] 4. Magnetic bead purification and quality detection

[0079] (1) Before use, mix the AMPure XP magnetic beads at room temperature for 30 min to improve the nucleic acid binding efficiency.

[0080] (2) Add 1 μL of 10X Lysis Buffer to the PCR product.

[0081] (3) Transfer the PCR product to a 1.5 ml centrifuge tube, add 50 μL of magnetic beads, mix by flicking, and then stand at room temperature for 8 min.

[0082] (4) Place the sample on a magnetic stand for about 5 min until the liquid becomes clear, and remove the supernatant.

[0083] (5) Add 200 μL of freshly prepared 80% ethanol to clean the magnetic beads, stand on the magnetic stand for 30 s, and remove the supernatant.

[0084] (6) Repeat the above step once, and after the second cleaning, elute by removing the supernatant using 10 μL of a pipette tip.

[0085] (7) Place the centrifuge tube on the magnetic stand, open the tube cap, and dry at room temperature for 2-2.5 min. The drying standard is that the surface of the magnetic beads is not smooth, but no cracks appear.

[0086] (8) Take down the centrifuge tube, add 17 μL of Elution Buffer to cover the magnetic beads, mix by flicking, and stand at room temperature for 2 min.

[0087] (9) Place the sample back on the magnetic stand for 1 min or more until the solution is completely clear.

[0088] (10) Transfer the supernatant to a nuclease-free, low-adhesion centrifuge tube.

[0089] (11) Take 1 μL of the purified product and measure the concentration using Qubit. Take 1 μL of the amplified cDNA and verify using Agilent 2100 Bioanalyzer and Agilent High Sensitivity DNA Kit (Agilent, Cat# 5067-4626). The results are shown in FIG. 6. Figure 2

[0090] 5. End repair

[0091] (1) Prepare the reaction system according to Table 7.

[0092] Table 7 End repair reaction system

[0093] Component Amount Nuclease-free water (60-X) μL PCR purified product X μL (500 ng) FFPE buffer 3.5 μL FFPE Master Mix 2 μL Endprep buffer 3.5 μL Endprep Master Mix 3 μL

[0094] (2) Gently mix, centrifuge for a moment, 20°C for 35 min, 65°C for 10 min.

[0095] 6. Magnetic bead purification of end repair product

[0096] (1) Before using Agencourt AMPure XP magnetic beads, rotate and mix at room temperature for 30 min to improve nucleic acid binding efficiency.

[0097] (2) Transfer the end repair product into a clean 1.5 ml centrifuge tube, add 48 μL of magnetic beads, gently mix and stand at room temperature for 5 min. Place the sample tube on the magnetic stand and stand for 5 min to allow the magnetic beads to be completely absorbed to the magnet side, and discard the supernatant.

[0098] (3) Place the centrifuge tube on the magnetic stand, wash the magnetic beads six times by blowing and sucking 200 μL of freshly prepared 70% ethanol (require the pipette to be perpendicular to the central hole, do not blow and suck against the magnetic beads), stand on the magnetic stand for 30 s, discard the supernatant. Repeat the washing once, and after the second discarding, centrifuge for a moment, place on the magnetic stand and remove the supernatant with 10 μL of the gun head.

[0099] (4) Place the centrifuge tube on the magnetic stand, open the tube cap, and air dry for about 5 min to completely volatilize the residual ethanol.

[0100] (5) Add 61 μL of Nuclease-free water, gently mix, and stand at room temperature for 5 min; then stand on the magnetic stand for 3 min, and transfer 61 μL of the eluate into a new 1.5 ml centrifuge tube (try not to suck the magnetic beads).

[0101] 7. Connection of sequencing adapter

[0102] (1) Prepare the adapter connection reaction system according to Table 8.

[0103] Table 8 Adapter connection reaction system ​

[0104] Nuclease-free water (60-X) μL End-repaired purified product X μL (420 ng) LNB (ONT) 25 μL AMX (ONT) 5 μL NEBNext Quick T4 DNA Ligase 10 μL

[0105] (2) Lightly mix, instantaneous centrifugation, 25℃ reaction for 1h.

[0106] 8. Magnetic bead purification of ligation product

[0107] (1) Before using Agencourt AMPure XP magnetic beads, rotate and mix at room temperature for 30 min to improve nucleic acid binding efficiency.

[0108] (2) Add 40 μL magnetic beads to the ligation product, lightly mix, and then let stand at room temperature for 5 min.

[0109] (3) Place the sample tube in the magnetic stand and let stand for 5 min to allow the magnetic beads to be completely adsorbed to the magnet side, and then discard the supernatant.

[0110] (4) Add 250 μL SFB (ONT) to wash the magnetic beads, lightly mix, and then let stand in the magnetic stand for 3 min after instantaneous centrifugation, and then discard the supernatant. Repeat the washing once, and then remove the supernatant after the second washing, and then remove the supernatant by 10 μL gun tip after instantaneous centrifugation.

[0111] (5) Place the centrifuge tube in the magnetic stand, open the tube cap, and air dry for about 1 min.

[0112] (6) Add 25 μL EB, lightly mix, and let stand at room temperature for 5 min; and then let stand in the magnetic stand for 3 min.

[0113] 9. Sequencing

[0114] Sequencing is performed using the ONT sequencing platform.

[0115] Example 2

[0116] The present example provides a method for constructing a full-length transcriptome library of a single cell or a micro-sample for nanopore sequencing (the basic process is shown in Figure 1 The difference between the method of the present example and the method of Example 1 is only that the end repair reaction conditions in step 5 are to first add FFPE Master Mix 2 μL, FFPE buffer 3.5 μL to the PCR purified product, and then react at 20℃ for 15 min, and then add Endprep Master Mix 2 μL, Endprep buffer 3.5 μL and water to the reaction system, and then react at 20℃ for 30 min, 65℃ for 10 min, and the ligation reaction conditions in step 7 are to react at 22℃ for 1.5 h.

[0117] Comparative Example

[0118] The comparative example provides a single cell or trace sample full-length transcriptome library construction method for nanopore sequencing, which is only different from the method of Example 1 in that the end repair reaction conditions in step 5 are 20℃, 5min, 65℃, 5min (recommended by commercial kit), and the linker ligation reaction conditions in step 7 are 25℃ for 10min.

[0119] Experimental example

[0120] The single cell sample was subjected to full-length transcriptome library construction using the single cell or trace sample full-length transcriptome library construction method for nanopore sequencing of Examples 1 and 2 and Comparative Example, and the detection results of library sequencing quality and library sequencing pore speed are shown in Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 and Figure 8 The actual measured data of the two samples are shown in Table 9, and the actual measured transcript full-length sequence statistics are shown in Table 10. The results show that the sequencing effect of Examples 1 (N01, N02) and 2 (N03, N04) is equivalent, compared with Comparative Example (N05, N06), the library quality of Examples 1 and 2 is obviously improved Figure 3 、 Figure 5 and Figure 7 ), the pore speed is relatively stable in a short time Figure 4 、 Figure 6 and Figure 8 ), the connection efficiency of the linker is higher, and the amount of data obtained by sequencing is more.

[0121] Table 9 Actual measured data off-line results

[0122]

[0123] Note: In Table 9, MeanLength represents average length, MaxLength represents maximum fragment length, and MeanQscore represents average Q value.

[0124] Table 10 Actual measured transcript full-length sequence statistics

[0125]

[0126] Note: In Table 10, Number of clean reads (except rRNA) represents the number of reads after removing rRNA, Number of full-length reads represents the number of full-length reads, and Full-Length Percentage (FL%) represents the proportion of full-length reads.

[0127] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. It is therefore intended that whatever lies within the scope of the application be covered by the appended claims.

Claims

1. A method for constructing full-length transcriptome libraries from single cells or trace samples for nanopore sequencing, characterized in that, include: After lysing single cells or trace samples, mRNA was reverse transcribed into cDNA using the Smart-seq2 method. The cDNA was then used as a template for PCR amplification. The PCR amplification products were subjected to FFPE and end repair, and then ligated into nanopore sequencing adapters. The reaction conditions for FFPE and end repair are as follows: react the reaction system at 20℃ for 30-35 min, and then at 65℃ for 10 min; or, first add FFPE Master Mix and FFPE buffer to the PCR purified product, react at 20℃ for 10-15 min, then add Endprep Master Mix, Endprep buffer and water, react at 20℃ for 30-35 min, and then at 65℃ for 10 min. The reaction conditions for connecting the nanopore sequencing adapter are: 22-25℃ for 1-1.5h. The 60 μL reaction system of FFPE and end-repair includes the following components: 200-1500 ng of PCR purified product, 3.5 μL of FFPE buffer, 2 μL of FFPE Master Mix, 3.5 μL of Endprep buffer, 2-3 μL of Endprep Master Mix, and water to a total volume of 60 μL. The 60 μL reaction system for connecting nanopore sequencing adapters comprises the following components: 100-1300 ng of FFPE and end-repair purification product, 25 μL of ONT LBN, 5 μL of ONT Adapter Mix, 10 μL of T4 DNA ligase, and water to a total volume of 60 μL.

2. The method for constructing a full-length transcriptome library from a single cell or trace sample for nanopore sequencing according to claim 1, characterized in that, The reverse transcription process includes: first, mixing mRNA, reaction buffer, and water and placing the mixture at low temperature; then mixing it with 3'SMART-Seq CDS Primer II A and treating it at 72°C for 2-3 min to obtain a first premix; then mixing 5×Ultra LowFirst-Strand Buffer, SMART-Seq v4 Oligonucleotide, and RNase inhibitor, and then mixing them with SMARTScribe reverse transcriptase to obtain a second premix; finally, mixing the first and second premixes to perform the reverse transcription reaction.

3. The method for constructing a full-length transcriptome library from a single cell or trace sample for nanopore sequencing according to claim 2, characterized in that, The conditions for the reverse transcription reaction are: 42℃, 85-95 min; 70℃, 8-12 min.

4. The method for constructing a full-length transcriptome library from a single cell or a small sample for nanopore sequencing according to any one of claims 1 to 3, characterized in that, The PCR amplification reaction conditions were as follows: 95℃, 1-1.5 min; 98℃, 10-15 sec, 65℃, 25-35 sec, 68℃, 3-4 min, 15-20 cycles; 72℃, 8-12 min.

5. A method for full-length transcriptome sequencing of single-cell or trace samples, characterized in that, It includes: constructing a full-length transcriptome library of a single cell or a trace sample using the method for constructing a full-length transcriptome library of a single cell or a trace sample for nanopore sequencing as described in any one of claims 1 to 4, and performing sequencing on the full-length transcriptome library of the single cell or a trace sample.

6. The method for full-length transcriptome sequencing of single cells or trace samples according to claim 5, characterized in that, The sequencing was performed using a nanopore sequencing platform.

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