Reverse transcription primer set, primer combination, sequencing library construction method and kit

Abstract

A reverse transcription primer set, a primer combination, a method for constructing a sequencing library and a kit. The reverse transcription primer set comprises a reverse transcription primer and a template switching primer, wherein the reverse transcription primer has a structure sequentially comprising, from the 5' end to the 3' end: a universal sequence, a random sequence, an Oligo(dT) and an anchor sequence; and the template switching primer has a structure sequentially comprising, from the 5' end to the 3' end: the universal sequence and rGrG+G, wherein rG is riboguanosine (RNA), and +G is LNA-modified guanosine. In the present invention, the library yield is effectively improved, the accuracy and sensitivity of sequencing are improved, the sequencing data output is increased, the splitting rate of the full-length sequence is increased, and the length of the full-length sequence is also increased.

Description

Reverse transcription primer set, primer combination, sequencing library construction method and kit Technical Field

[0001] The present invention relates to the technical field of gene sequencing, and in particular to a reverse transcription primer set, a primer combination, a method for constructing a sequencing library, and a kit. Background Art

[0002] The full-length transcriptome is based on a third-generation sequencing platform. Without interruption and splicing, the full-length mRNA sequence and complete structural information including 5'UTR, 3'UTR, and polyA tail can be directly obtained. This allows for accurate analysis of structural information such as alternative splicing and fusion genes in species with reference genomes, overcoming the problem of short transcript splicing and incomplete information in species without reference genomes.

[0003] ONT (Oxford nanopore technology) full-length transcriptome sequencing, a newly emerging third-generation sequencing technology, preserves full-length sequence information while offering throughput advantages. With typical data volumes, it can saturate the majority of transcripts in a sample and quantify them. Currently, a single ONT sequencer cell can produce up to 100GB of data, while a single library typically requires only 6-8GB of data. This requires pooling multiple libraries before sequencing. However, ONT's official library preparation kits exhibit low sequencing yields.

[0004] Summary of the Invention

[0005] According to the first aspect, in one embodiment, there is provided a reverse transcription primer set, comprising: a reverse transcription primer and a template switching primer;

[0006] The reverse transcription primer comprises a structure including, from the 5' end to the 3' end, a universal sequence, a random sequence, an Oligo (dT) and an anchor sequence;

[0007] The structure of the template switching primer includes the universal sequence, rGrG+G in sequence from the 5' end to the 3' end, where rG is riboguanosine (RNA) and +G is LNA-modified guanosine.

[0008] According to the second aspect, in one embodiment, a primer combination is provided, comprising: the reverse transcription primer set and a PCR primer according to the first aspect;

[0009] The structure of the PCR primer includes, from the 5' end to the 3' end, an adapter connection sequence, a sample tag sequence, and a target fragment complementary sequence;

[0010] The 5' end of the PCR primer is modified with a phosphate group;

[0011] The adapter connection sequence is used for splitting the sample tags;

[0012] The sample tag sequences of different samples are different;

[0013] The target fragment is the product obtained by extending the reverse transcription primer set.

[0014] According to the third aspect, in one embodiment, a method for constructing a sequencing library is provided, comprising the following steps:

[0015] a reverse transcription step, comprising reverse transcribing the RNA sample using a reverse transcription primer set to obtain a reverse transcription product;

[0016] The amplification step includes performing PCR amplification on the reverse transcription product to obtain a PCR amplification product.

[0017] According to the fourth aspect, in one embodiment, a sequencing library construction kit is provided, comprising the reverse transcription primer set described in the first aspect or the primer combination described in the second aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 is a schematic diagram of the experimental principle of full-length transcriptase library construction according to an embodiment of the present invention;

[0019] Figure 2.1 is an amplification detection diagram of the ONT official kit provided in Example 1 of the present invention;

[0020] Figure 2.2 is an amplification detection diagram of the full-length transcriptome library construction provided in embodiment 2 of the present invention. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can readily appreciate that some of the features may be omitted in different circumstances, or may be replaced by other materials or methods. In some cases, some operations related to the present application are not shown or described in the specification. This is to avoid the core of the present application being overwhelmed by excessive descriptions, and for those skilled in the art, it is not necessary to describe these related operations in detail. They can fully understand the related operations based on the description in the specification and the general technical knowledge in the art.

[0022] In addition, the features, operations, or characteristics described in the specification may be combined in any appropriate manner to form various embodiments. Furthermore, the steps or actions in the method description may be reordered or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various sequences in the specification and drawings are provided solely for the purpose of clearly describing a particular embodiment and are not intended to be mandatory, unless otherwise specified.

[0023] The serial numbers assigned to the components in this document, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0024] ONT sequencing suffers from low sequencing accuracy. The official ONT kit suffers from low adapter ligation efficiency when constructing full-length transcriptome libraries. This low adapter ligation efficiency is primarily manifested in the following ways: 1. Low sequencing yield, less than half of the chip yield; 2. Short fragments of reverse transcription products, with a high proportion of small fragments; and 3. Changing the existing library construction method and continuing to use the original primers will result in a low full-length sequence resolution rate after pooling.

[0025] Therefore, new strategies need to be developed to improve sequencing yield and the efficiency of full-length sequence resolution.

[0026] According to the first aspect, in one embodiment, a reverse transcription primer set is provided, comprising:

[0027] Reverse transcription primers and template switching primers;

[0028] The reverse transcription primer comprises a structure including, from the 5' end to the 3' end, a universal sequence, a random sequence, an Oligo (dT) and an anchor sequence;

[0029] The structure of the template switching primer includes the universal sequence, rGrG+G in sequence from the 5' end to the 3' end, where rG is riboguanosine (RNA) and +G is LNA-modified guanosine.

[0030] From the experimental data of the present invention, it can be found that the reverse transcription primer set provided in this embodiment can increase the sequencing data output and increase the full-length sequence length.

[0031] In one embodiment, a marker base sequence is provided between the universal sequence and the random sequence in the reverse transcription primer;

[0032] The landmark base sequence consists of at least one base.

[0033] In one embodiment, the marker base sequence consists of 1 to 20 bases.

[0034] This example unexpectedly discovered that adding a landmark base sequence between the universal sequence and the random sequence can improve the sequencing data resolution rate without significantly reducing the reverse transcription efficiency or causing significant nonspecific amplification.

[0035] In one embodiment, the landmark base sequence consists of n landmark base units, where n is at least 1 and is an integer; the sequence of the landmark base unit consists of at least 1 base, and the landmark base is identical to the 3' end sequence of the universal sequence.

[0036] In one embodiment, n is 1 or 2.

[0037] For example, the universal sequence of the reverse transcription primer from 5' to 3' is: AAGCAGTGGTATCAACGCAGAGTAC. When the 3' end sequence is GTAC and n is 1 or 2, the landmark base sequence from 5' to 3' is: GCAC or GTACGTAC.

[0038] In one embodiment, the random sequence consists of 6 to 12 random bases N, specifically 6, 7, 8, 9, 10, 11, or 12, where N is any base.

[0039] In one embodiment, the random sequence consists of 8 random bases N, where N is any base.

[0040] Exemplarily, the random sequence is: NNNNNNNN.

[0041] It should be noted that the difference in bases in the random sequence will not affect the experimental results.

[0042] In one embodiment, the Oligo(dT) consists of 15 to 40 thymines T.

[0043] In one embodiment, Oligo(dT) consists of 30 thymines T.

[0044] Illustratively, Oligo(dT) is TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT.

[0045] In one embodiment, the anchor sequence is VN, where V is adenine A, guanine G, or cytosine C, and N is any base.

[0046] In one embodiment, the reverse transcription primer comprises the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 9;

[0047] SEQ ID NO: 1: 5'-AAGCAGTGGTATCAACGCAGAGTACNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3'

[0048] SEQ ID NO: 2: 5'-AAGCAGTGGTATCAACGCAGAGTACGTACNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3';

[0049] SEQ ID NO: 9: 5'-AAGCAGTGGTATCAACGCAGAGTACGTACGTACNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3'.

[0050] In one embodiment, the template switching primer comprises the nucleotide sequence shown in SEQ ID NO: 3;

[0051] SEQ ID NO: 3: 5'-AAGCAGTGGTATCAACGCAGAGTACrGrG+G-3'.

[0052] The reverse transcription primer set of the present invention operates as follows: during reverse transcription, the universal sequence of the reverse transcription primer serves as the primer recognition sequence for PCR amplification, the oligo(dT) recognizes the poly(A) sequence at the end of the RNA, and the anchor sequence specifically anchors to the junction of the 3' end of the RNA and the poly(A). During transcription to the 5' end of the RNA, the reverse transcriptase adds several C bases to the 3' end of the cDNA strand to synthesize the first strand of cDNA. The G base at the 3' end of the template-switching primer, TSO, is complementary to the aforementioned C bases, and the primer is extended to synthesize double-stranded cDNA.

[0053] Furthermore, the RNA is reverse transcribed using a combination of reverse transcription primers to synthesize double-stranded cDNA, and the PCR primers are used to amplify the double-stranded cDNA as a template.

[0054] According to the second aspect, in one embodiment, there is provided a primer combination comprising: the reverse transcription primer set and PCR primers described in the first aspect;

[0055] The structure of the PCR primer includes, from the 5' end to the 3' end, an adapter connection sequence, a sample tag sequence, and a target fragment complementary sequence;

[0056] The 5' end of the PCR primer is modified with a phosphate group;

[0057] The adapter connection sequence is used for splitting the sample tags;

[0058] The sample tag sequences of different samples are different;

[0059] The target fragment is the product obtained by extending the reverse transcription primer set.

[0060] In this example, the adapter sequence is used to connect to the adapter during the sequencing library construction process. In addition, the adapter sequence is also used to detect the accuracy of the barcode. Due to the low accuracy of ONT, when the detected sequence is consistent with the known sequence at both ends of the barcode, it can be confirmed that the detected barcode sequence is accurate and the barcode can be directly split.

[0061] The linker sequence in this embodiment preferably adopts the sequence officially provided by ONT: 5'-ATCGCCTACCGTGAC-3'.

[0062] The target fragment is specifically a double-stranded cDNA obtained by extension of the template switching primer. Therefore, the complementary sequence of the target fragment is partially identical or completely identical to the universal sequence of the reverse transcription primer.

[0063] The sample tag sequence is a barcode, which is used to distinguish different RNA samples. Therefore, different samples have different barcodes. The barcodes can be purchased commercially.

[0064] In one embodiment, the PCR primer comprises the nucleotide sequence shown in SEQ ID NO: 4;

[0065] SEQ ID NO: 4: 5'-phos / ATCGCCTACCGTGAC-barcode-AAGCAGTGGTATCAACGCAGAGT-3'.

[0066] The upstream primer and downstream primer of the PCR primer of the present invention are the same, that is, the two ends of the single-stranded cDNA are complementary. Sequences less than 500 bp are easily connected to each other and cannot be amplified by the primer, thereby increasing the length of the cDNA library.

[0067] According to the third aspect, in one embodiment, a method for constructing a sequencing library is provided, comprising the following steps:

[0068] A reverse transcription step, comprising reverse transcribing the RNA sample using the reverse transcription primer set provided in the first aspect to obtain a reverse transcription product;

[0069] The amplification step includes PCR amplification of the reverse transcription product to obtain the PCR amplification product of the sequencing library.

[0070] The reverse transcription step specifically includes the following steps:

[0071] 1) Mix the RNA sample with the reverse transcription primer for reaction.

[0072] In this step, the RNA sample is total RNA; the reverse transcription primer includes a structure including, from the 5' end to the 3' end, a universal sequence, a random sequence, an Oligo(dT) and an anchor sequence. The reverse transcription primer is the same as the reverse transcription primer described in the first aspect and will not be repeated here.

[0073] The RNA sample is mixed with the reverse transcription primer and reacted at 72°C for 3-5 minutes, followed by 42°C for 1-5 minutes. The preferred reaction schedule is 72°C for 3 minutes, followed by 42°C for 2 minutes, with a cooling rate of -0.1°C / s from 72°C to 42°C. The RNA sample is first mixed with the reverse transcription primer to initiate the reaction, which is intended to open up the RNA's secondary structure.

[0074] 2) Mixing the reverse transcription system containing the template switching primer with the reaction product of step 1) to perform a reverse transcription reaction.

[0075] In this step, the template switching primer is the same as the reverse transcription primer described in the first aspect and will not be described in detail here.

[0076] In this step, before mixing the reverse transcription system containing the template switching primer with the reaction product of step 1), the step further includes: incubating the prepared reverse transcription system on a PCR instrument, preferably at 42° C. for 1 min.

[0077] After mixing the reverse transcription system containing the template switching primer with the reaction system in step 1), set the reaction temperature to 42°C and the reverse transcription reaction time to 60-90 minutes. After the reaction is completed, cool to 4°C. Preferably, after mixing the reaction product in step 1) with the reverse transcription system containing the template primer, preheat the PCR instrument to 42°C and perform the reverse transcription reaction according to the following program: 42°C for 90 minutes, 4°C for ∞.

[0078] The reverse transcription system includes: template switching primer, reverse transcriptase, dNTP, dithiothreitol (DTT), template strand, and buffer, wherein the reverse transcriptase is preferably Superscript II RT.

[0079] 3) Purifying the product of the reverse transcription reaction in step 2) to obtain a reverse transcription product.

[0080] In this step, the purification aims, firstly, to remove excess template-switching primer (TSO) to prevent it from participating in the amplification reaction; secondly, to remove the reverse transcriptase in the reaction to prevent it from interfering with the amplification reaction. Preferably, the purification is performed using magnetic beads. The reverse transcription product obtained after magnetic bead purification is double-stranded cDNA.

[0081] The magnetic bead purification specifically comprises the following steps:

[0082] Add nuclease-free water to the product of the reverse transcription reaction in step (2), and then add magnetic beads and mix well.

[0083] In this step, the magnetic beads are preferably 1 volume of AMPure XP magnetic beads; after adding the magnetic beads and mixing, the mixture is preferably left at room temperature for 5 minutes.

[0084] (1) Place the reaction tube on a magnetic rack and let it stand until the liquid becomes clear, then remove the supernatant.

[0085] In this step, the standing time is preferably 2-5 minutes.

[0086] (3) Keep the reaction tube on the magnetic stand at all times, add ethanol to rinse the magnetic beads, and remove the supernatant after standing.

[0087] In this step, the mass concentration of ethanol is 80%; step (3) is preferably repeated twice, and the washing is performed three times.

[0088] (4) Open the lid and let it dry, remove the reaction tube from the magnetic stand, add the eluent and place it aside.

[0089] In this step, the elution buffer is preferably Elution Buffer; and the storage condition is preferably room temperature for 4 minutes.

[0090] (5) Centrifuge the reaction tube and place it on a magnetic rack. After the solution is clarified, take the supernatant and save it.

[0091] In this step, the centrifugation is preferably short centrifugation, and the present invention has no particular limitation on the centrifugation conditions; the supernatant is preferably stored at -20°C.

[0092] The PCR amplification step specifically includes the following steps:

[0093] 1) Mix the reverse transcription product with the PCR reaction system for amplification reaction.

[0094] In this step, the PCR reaction system includes: nuclease-free water, amplification enzyme and PCR primers.

[0095] The PCR amplification enzymes used included 1) KAPA HiFi HotStart Uracil+ReadyMix or 2) KAPA HiFi HotStart ReadyMix. Both enzymes demonstrated superior fidelity to ONT's official 2x LongAmp Taq Master Mix. Experimental results indicate that using KAPA HiFi HotStart Uracil+ReadyMix resulted in higher library yields.

[0096] The PCR primers in this step are the same as those described in the second aspect and will not be described again here.

[0097] In this step, the amplification reaction is preferably performed at 95-98°C for 15-30 seconds, 62-65°C for 15-30 seconds, and 72°C for 4-6 minutes. Preferably, the amplification reaction is performed at 98°C for 20 seconds, 65°C for 15 seconds, and 72°C for 4.5 minutes. The number of cycles is preferably 16-19, including but not limited to 16, 17, 18, and 19.

[0098] After the cycle is completed, keep it in a 72℃ environment for 5 to 10 minutes and then cool it to 12℃.

[0099] 2) Purifying the reaction product of step 1) to obtain a PCR amplification product.

[0100] In this step, purification is preferably performed by magnetic bead purification.

[0101] The magnetic bead purification preferably comprises the following steps:

[0102] (1) Add magnetic beads to the reaction product and mix well.

[0103] In this step, the magnetic beads are preferably 1 volume of AMPure XP magnetic beads; after adding the magnetic beads and mixing, it is preferably allowed to stand at room temperature for 10 minutes.

[0104] (2) Place the reaction tube on a magnetic rack, add ethanol to rinse the magnetic beads, and remove the supernatant after standing.

[0105] In this step, the mass concentration of ethanol is 80%; the standing time is preferably 30 seconds; and step (2) is preferably repeated once more, and the rinsing is performed twice.

[0106] (3) Remove the ethanol from the reaction tube, open the lid and let it dry, remove the reaction tube from the magnetic stand, add the eluent and place it aside.

[0107] In this step, the elution buffer is preferably Elution Buffer; and the storage condition is preferably room temperature for 4 minutes.

[0108] (4) Centrifuge the reaction tube and place it on a magnetic rack. After the solution is clarified, collect the supernatant.

[0109] The purpose of this step is to remove dimers and recover most of the fragments.

[0110] In this step, the centrifugation is preferably short centrifugation, and the present invention has no particular limitation on the centrifugation conditions.

[0111] (5) Take 1 / 3 of the purified product obtained in step (4), mix it with water and magnetic beads, and set aside.

[0112] In this step, the magnetic beads are preferably 0.4 times the volume of AMPure XP magnetic beads.

[0113] (6) Place the reaction tube from step (5) on a magnetic rack and collect the supernatant after the solution is clarified.

[0114] In this step, the solution is allowed to stand until it becomes clear; the standing time is preferably 2-5 minutes.

[0115] (7) Keep the reaction tube in step (6) always on the magnetic rack, add ethanol to rinse the magnetic beads, and remove the supernatant after standing.

[0116] In this step, the mass concentration of ethanol is 80%; the standing time is preferably 30 seconds; and step (7) is preferably repeated once more, and the rinsing is performed twice.

[0117] (8) Remove the ethanol from the reaction tube in step (7), open the lid and dry it, then remove it from the magnetic rack, add the remaining 2 / 3 of the purified product in step (4), mix well and set aside.

[0118] In this step, it is preferably placed at room temperature for 4 minutes.

[0119] (9) Centrifuge the reaction tube in step (8) and place it on a magnetic rack. After the solution is clarified, take the supernatant to obtain the purified PCR amplification product.

[0120] The purpose of this purification step is to remove small fragments and recover large fragments.

[0121] Furthermore, after obtaining the PCR amplification products, the sequencing library construction method further includes: linker ligating the PCR products to obtain a sequencing library.

[0122] After the PCR amplification step is completed, the full-length transcript sequence is obtained. Then, after pooling the libraries of different samples and adding sequencing adapters, sequencing can be performed on the machine.

[0123] In this step, the adapter is preferably an ONT sequencing adapter, which is commercially available.

[0124] According to the fourth aspect, in one embodiment, a sequencing library construction kit is provided, comprising the reverse transcription primer set described in the first aspect or the primer combination described in the second aspect.

[0125] In one embodiment, the sequencing library construction kit further includes: a sequencing adapter, and the sequencing adapter is preferably an ONT sequencing adapter.

[0126] In one embodiment, the sequencing library construction kit further includes: a reverse transcriptase and an amplification enzyme, wherein the reverse transcriptase is preferably Super script II RT, and the amplification enzyme is preferably 1) KAPA HiFi HotStart Uracil and ReadyMix, or 2) KAPA HiFi HotStart ReadyMix.

[0127] The reverse transcription primer set provided by the present invention improves the accuracy and sensitivity of sequencing, increases sequencing data output, increases the full-length sequence length, improves data splitting rate, and improves the utilization of effective data; the F end of the PCR primer is identical to the R end, which increases the cDNA library length; and KAPA HiFi HotStart Uracil+ReadyMix (2x) is used to improve library output. Therefore, the reverse transcription primer set, primer combination, sequencing library construction method and sequencing library construction kit provided by the present invention can effectively improve sequencing library output and fragment size, to obtain more offline data volume, and make the splitting rate of the full-length sequence higher, and obtain more effective data that can be used for analysis.

[0128] Example 1

[0129] This example uses four test samples (total RNA samples from humans, rice, mice, and chrysanthemum) to construct an ONT full-length transcriptome library. The official ONT full-length transcriptome library construction process is as follows:

[0130] a) Transfer 1 μg of total RNA to a 0.2 mL PCR tube, add water to 9 μL, then add 1 μL of 2 μM VN Primers (VNP) and 1 μL of 10 mM dNTPs, for a total of 11 μL. Mix by pipetting and centrifuge.

[0131] b) Incubate at 65°C for 5 minutes and then cool rapidly on a pre-chilled ice box;

[0132] c) In a new tube, prepare a mixture of the following reagents: 4 μL 5x RT Buffer, 1 μL RNaseOUT, 1 μL Nuclease-free water, and 2 μL Strand-Switching Primer (SSP, at 10 μM), totaling 8 μL. Mix by pipetting and centrifuge.

[0133] d) Add the mix prepared in the previous step to the rapidly cooled and annealed mRNA in step b), mix thoroughly by pipetting, centrifuge, and incubate at 42°C in a PCR instrument for 2 minutes;

[0134] e) Add 1 μL of Maxima H Minus Reverse Transcriptase to a total of 20 μL, mix thoroughly by pipetting, and centrifuge. Place the reaction in a PCR instrument and perform the reverse transcription reaction according to the following protocol: 42°C, 90 min; 85°C, 5 min; 4°C, ∞;

[0135] f) Divide the reaction from step e) into four equal portions. Transfer 5 μL of each aliquot to a new 0.2 mL PCR tube and add 45 μL of PCR amplification solution. The PCR amplification solution includes: 1.5 μL Barcode Primers (BP01-BP12), 18.5 μL Nuclease-free water, and 25 μL 2x LongAmp Taq Master Mix, for a total PCR reaction volume of 50 μL.

[0136] Each PCR reaction uses 5 μL of reverse-transcribed RNA sample (20 μL total). Therefore, each sample has enough material to perform up to 4 PCR reactions. The same sample is added with the same barcode number.

[0137] g) Mix thoroughly by pipetting, centrifuge, and perform amplification in a PCR instrument according to the following protocol: 95°C for 30 seconds, followed by 11-18 cycles of: 95°C for 15 seconds, 62°C for 15 seconds, and 65°C for 50 seconds. After the end of the cycle, incubate at 65°C for 6 minutes, then hold at 4°C.

[0138] h) Add 1 μL of NEB Exonuclease 1 to the completed PCR product, pipette to mix thoroughly, and centrifuge. Place the product in a PCR instrument and perform the digestion reaction according to the following schedule: 37°C for 15 minutes, 80°C for 15 minutes, and 4°C for ∞.

[0139] i) Pool the PCR reactions containing samples with different barcodes into new 1.5 mL centrifuge tubes;

[0140] j) Resuspend the AMPure XP beads by vortexing, add 0.8 times the amount of AMPure XP beads resuspended in step i), mix thoroughly by pipetting, and place on a room temperature mixer for 5 minutes;

[0141] k) Briefly centrifuge the reaction tube and place it on a magnetic rack. Once the solution is clear, carefully remove the supernatant.

[0142] l) Keeping the centrifuge tube on the magnetic stand, add 200 μL of freshly prepared 70% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds and carefully remove the supernatant.

[0143] m) Repeat the previous step once, rinsing twice in total;

[0144] n) Remove any residual 70% ethanol by aspirating, open the lid and briefly air dry, remove the tube from the magnetic stand, add 12 μL of Elution Buffer (EB), mix gently by pipetting, and place on a room temperature mixer for 10 minutes.

[0145] o) Briefly centrifuge the reaction tube and place it on a magnetic rack. Once the solution is clear, carefully pipette the entire supernatant into a new 1.5 mL EP tube.

[0146] p) Take 1 μL of the purified product to detect the fragment size and concentration of DNA;

[0147] q) Transfer 100 fmol of the purified product to a new 1.5 mL EP tube and fill with Elution Buffer (EB) to a final volume of 11 μL. Add 1 μL of Rapid Adapter (RAP), pipette to mix thoroughly, and centrifuge. Incubate at room temperature for 5 minutes. After a brief centrifugation, place the tube at -20°C and wait for sequencing.

[0148] r) The primer nucleotide sequences and modifications used in the above experimental procedures are as follows:

[0149] VNP: 5′-phos / ACTTGCCTGTCGCTCTATCTTCTTTTTTTTTTTTTTTTTTVN-3′ (SEQ ID NO: 5)

[0150] V is A, C or G, and N is any base.

[0151] SSP: 5′-TTTCTGTTGGTGCTGATATTGCTmGmGmG-3′ (SEQ ID NO: 6)

[0152] In the nucleotide sequence shown in SEQ ID NO: 6, "m" indicates methylation.

[0153] Barcode Primer:

[0154] F-terminal: 5′-ATCGCCTACCGTGAC-barcode-ACTTGCCTGTCGCTCTATCTTC-3′ (SEQ ID NO: 7)

[0155] R-terminal: 5′-ATCGCCTACCGTGAC-barcode-TTTCTGTTGGTGCTGATATTGC-3′ (SEQ ID NO: 8)

[0156] The reagents mentioned in the above method are all provided in the PCR-cDNA Barcoding (SQK-PCB109) kit.

[0157] Example 2

[0158] This example uses 4 test samples (the same 4 test samples as in Example 1) to construct an ONT full-length transcriptome library. The experimental process for constructing the full-length transcriptome library is as follows:

[0159] 1. Reverse Transcription

[0160] a) Transfer 1 μg of total RNA to a 0.2 mL PCR tube, add water to 3 μL, and then add 1 μL of 12 μM dT Primer, for a total of 4 μL. Mix by pipetting and centrifuge.

[0161] SMART technology reverse transcription primer dT primer:

[0162] 5'-AAGCAGTGGTATCAACGCAGAGTACNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' (SEQ ID NO: 1)

[0163] b) V is A, C, or G, and N is any base. React on a PCR instrument: 72°C for 3 min; 42°C for 2 min (cool from 72°C to 42°C at a rate of -0.1°C / s);

[0164] c) Prepare the reverse transcription mix in a new 0.2 mL PCR tube using the following reagents:

[0165] Table 1

[0166] TSO: 5'-AAGCAGTGGTATCAACGCAGAGTACrGrG+G-3' (SEQ ID NO: 3)

[0167] d) Mix the reverse transcription mix prepared in the previous step by pipetting and incubate in a PCR instrument at 42°C for 1 minute. Then add it to the PCR tube containing the reaction in step b), mix it by pipetting and centrifuge.

[0168] e) Preheat a PCR machine to 42°C and perform the reverse transcription reaction on the PCR machine according to the following protocol: 42°C for 90 min, 4°C for ∞;

[0169] f) Add 40 μL of Nuclease-Free Water to the reaction in step e), then add 1 volume (50 μL) of Ampure XP Magnetic Beads, mix thoroughly by pipetting, and let stand at room temperature for 5 minutes.

[0170] AMPure XP magnetic beads were rotated and mixed at room temperature for 30 minutes before use to improve nucleic acid binding efficiency.

[0171] g) Place the reaction tube on a magnetic stand and let it stand for 2-5 minutes until the liquid becomes clear. Pipette out and discard the supernatant.

[0172] h) Keeping the reaction tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads. Let it stand at room temperature for 30 seconds, then carefully discard the supernatant.

[0173] i) Repeat the previous step twice, rinsing three times in total, and remove the supernatant with a 10 μL pipette tip;

[0174] j) Open the lid and let it dry briefly. The standard of dryness is that the surface of the magnetic beads is free of reflection and cracks. Remove the reaction tube from the magnetic stand, add 51 μL Elution Buffer, pipette gently to mix, and let it stand at room temperature for 4 minutes.

[0175] k) Briefly centrifuge the reaction tube and place it on a magnetic rack. Once the solution has clarified, carefully pipette the entire supernatant into a new 1.5 mL EP tube and store at -20°C for long-term storage.

[0176] 2. PCR Amplification

[0177] a) Remove all reagents needed for PCR, thaw on ice, gently vortex each reagent, centrifuge briefly, and store on ice;

[0178] b) Prepare the PCR reaction mix using the following reagents:

[0179] Table 2

[0180] The F and R ends of the PCR primer in Table 2 are both: 5'-phos / ATCGCCTACCGTGAC-barcode-AAGCAG TGGTATCAACGCAGAGT-3' (SEQ ID NO: 4)

[0181] c) Transfer 20 μL of the purified product from the previous reverse transcription step to a new 0.2 mL PCR tube, add 80 μL of the prepared PCR reaction mix, mix by pipetting, and centrifuge briefly.

[0182] d) Place the sample on a PCR instrument and perform the PCR amplification reaction according to the following procedure:

[0183] Table 4

[0184] e) Take 1 μL of PCR product and measure the concentration using Qubit. A concentration between 20-40 ng / μL is acceptable. If the concentration is lower than 20 ng / μL, perform PCR amplification on the backup sample and combine them after amplification.

[0185] 3. PCR Product Purification

[0186] a) Remove the Ampure XP Beads 30 minutes in advance and place them at room temperature. Vortex and mix thoroughly before use.

[0187] b) Add 1 volume (100 μL) of Ampure XP beads to the PCR product, pipette to mix thoroughly, and let stand at room temperature for 10 minutes.

[0188] c) Place the reaction tube on a magnetic stand and let it stand for 2-5 minutes until the liquid becomes clear. Pipette out and discard the supernatant.

[0189] d) Keeping the reaction tube on the magnetic stand, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads. Let it stand at room temperature for 30 seconds and then carefully discard the supernatant.

[0190] e) Repeat the previous step once, rinsing twice in total;

[0191] f) Remove any residual 80% ethanol by aspirating, open the lid and briefly air dry, remove the reaction tube from the magnetic stand, add 51 μL of Elution Buffer (EB), mix gently by pipetting, and let it stand at room temperature for 4 minutes;

[0192] g) Briefly centrifuge the reaction tube and place it on a magnetic rack. Once the solution is clear, carefully pipette the entire supernatant into a new 1.5 mL EP tube.

[0193] h) Transfer 1 / 3 of the purified product (17 μL) from the previous step to a new 1.5 mL centrifuge tube, add water to 50 μL, add 0.4 times the volume (20 μL) of Ampure XP beads, mix thoroughly by pipetting, and let stand at room temperature for 10 minutes.

[0194] i) Place the reaction tube on a magnetic stand and let it stand for 2-5 minutes until the liquid becomes clear. Pipette out and discard the supernatant.

[0195] j) Keeping the reaction tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads. Let it stand at room temperature for 30 seconds and then carefully discard the supernatant.

[0196] k) Repeat the previous step once, rinsing twice in total;

[0197] l) Remove any remaining 80% ethanol by aspirating, briefly open the lid to air dry, remove the reaction tube from the magnetic rack, add the remaining volume from step g), gently pipette to mix, and let stand at room temperature for 4 minutes;

[0198] m) Briefly centrifuge the reaction tube and place it on a magnetic rack. Once the solution is clear, carefully pipette the entire supernatant into a new 1.5 mL EP tube.

[0199] n) Take 1 μL of the purified product to detect the DNA fragment size and concentration.

[0200] 4. Add ONT connector

[0201] Pool the libraries with different barcodes, add 1 μL Rapid Adapter (RAP), mix thoroughly by pipetting, centrifuge, and incubate at room temperature for 5 minutes.

[0202] 5. Sequencing on Nanopore

[0203] Figure 2.1 is an amplification detection diagram of the library construction using the ONT official kit in Example 1, and Figure 2.2 is an amplification detection diagram of the full-length transcriptome library construction provided in this example.

[0204] Comparison of Figures 2.1 and 2.2 shows the effect of using the PCR amplification primers of Example 1 (SEQ ID NO: 7 and SEQ ID NO: 8) and the PCR amplification primer of Example 2 (SEQ ID NO: 4) on the size of the amplified fragment. Compared with the PCR amplification primers of Example 1 with different F ends and R ends, the PCR amplification primers of Example 2 with the same F end and R end greatly increase the length of the cDNA library.

[0205] Example 3

[0206] In this example, a full-length transcriptome library construction method was used to construct an ONT full-length transcript library.

[0207] The experimental process of the full-length transcriptome library construction method used in this example is the same as that in Example 2, except that: 1) the RNA sample is: Universal Human Reference RNA (UHRR, purchased from Agilent, catalog number 740000) sample; 2) PCR amplification enzymes are KAPA HiFi HotStart Uracil+ReadyMix (2x) and KAPA HiFi HotStart ReadyMix (2x) respectively for PCR amplification.

[0208] Table 3 shows the effects of two amplification enzymes tested using UHRR. The results show that KAPA HiFi HotStart Uracil+ReadyMix (2x) has better amplification effect, and the library yield is about doubled compared to the other enzyme.

[0209] Table 3: Amplification results of different amplification enzymes

[0210] Example 4

[0211] In this example, a full-length transcriptome library construction method was used to construct an ONT full-length transcript library.

[0212] The experimental procedures of this embodiment and the method for constructing a full-length transcriptome library are the same as those of Example 2, with the only difference being that the reverse transcription primers used in this embodiment are: SEQ ID NO: 2, SEQ ID NO: 9 are:

[0213] SEQ ID NO: 2: 5'-AAGCAGTGGTATCAACGCAGAGTACGTACNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3'

[0214] V is A, C or G, and N is any base.

[0215] SEQ ID NO: 9: 5'-AAGCAGTGGTATCAACGCAGAGTACGTACGTACNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3'

[0216] V is A, C or G, and N is any base.

[0217] Since the sequence before the random primer (universal sequence) of the reverse transcription primer in Example 2 is the same as the sequence before the template-switching primer TSO rGrG+G (universal sequence), the sequences at both ends of the target fragment of the sequencing library in Example 2 are identical. Since the sequence before the random primer (universal primer + landmark base sequence) of the reverse transcription primer in Example 4 is different from the sequence before TSO rGrG+G (universal sequence), the sequences at both ends of the target fragment of the library in Example 4 are different. Using Example 2 and Example 4, the effects of using the reverse transcription primers in Example 2 and Example 4 on the resolution of the full-length sequence are compared. The results of the comparison of the full-length sequence resolution are shown in Table 5.

[0218] Table 5 Full-length sequence splitting comparison results

[0219] Table 5 shows the splitting rates of the full-length sequences using the ONT official library construction kit, the full-length transcription library construction method used in Example 1, and the methods used in Example 2 and Example 4. As can be seen from Table 5, the full-length sequence splitting rate of Example 2 is on the low side, while the full-length sequence splitting rate of Example 4 is at the same level as that of the ONT official kit in Example 1. The full-length sequence splitting rates of the two reverse transcription primers used in Example 4 are not much different. The experimental results of Example 2 and Example 4 in Table 5 indicate that when splitting the full-length sequence, the sequence before the random primer of the reverse transcription primer differs from the sequence before TSO rGrG+G, which can reduce judgment errors and thus improve the splitting rate.

[0220] Regarding the splitting rate of full-length sequences, full-length sequences refer to sequences containing complete cDNA structures (containing 5′ and 3′ primers at both ends and correctly paired). The full-length sequence splitting rate refers to the ratio of the number of full-length sequences to the number of sequenced sequences.

[0221] Table 6 Comparison of sequencing results

[0222] Table 6 shows the data output and full-length sequence lengths for Example 1 using the official ONT library construction kit, and for Examples 2 and 4 using the full-length transcription library construction method. As can be seen from Table 6, compared to Example 1, the library output for Examples 2 and 4 increased by over 130%, and the full-length sequence length increased by an average of approximately 36%. The full-length sequence lengths of the two reverse transcription primers used in Example 4 differed little.

[0223] The above examples are used to illustrate the present invention, which are only used to help understand the present invention and are not intended to limit the present invention. Those skilled in the art can make several simple deductions, modifications or substitutions based on the concept of the present invention.

Claims

1. A reverse transcription primer set, characterized in that: include: Reverse transcription primers and template switching primers; The structure of the reverse transcription primer includes, from the 5' end to the 3' end, a universal sequence, a random sequence, Oligo (dT) and an anchor sequence; The structure of the template switching primer includes the universal sequence, rGrG+G in sequence from the 5' end to the 3' end, where rG is riboguanosine (RNA) and +G is LNA-modified guanosine.

2. The reverse transcription primer set according to claim 1, characterized in that A landmark base sequence is arranged between the universal sequence and the random sequence of the reverse transcription primer; The iconic base sequence consists of at least one base; Preferably, the landmark base sequence consists of 1 to 20 bases.

3. The reverse transcription primer set according to claim 1 or 2, characterized in that: The landmark base sequence consists of n landmark base units, where n is at least 1 and is an integer; The sequence of the marker base unit consists of at least one base, and the sequence of the marker base unit is identical to the 3' terminal sequence of the universal sequence.

4. The reverse transcription primer set according to any one of claims 1 to 3, characterized in that: The universal sequence is 5'-AAGCAGTGGTATCAACGCAGAGTAC-3'; Preferably, the random sequence consists of 6 to 12 random bases N, where N is any base; Preferably, the Oligo(dT) consists of 15 to 40 thymine Ts; Preferably, the anchor sequence is VN, V is adenine A, guanine G or cytosine C, and N is any base.

5. The reverse transcription primer set according to any one of claims 1 to 4, characterized in that: The landmark base is GTAC or GTACGTAC.

6. The reverse transcription primer set according to any one of claims 1 to 5, characterized in that: The reverse transcription primer includes the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 9; The template switching primer includes the nucleotide sequence shown in SEQ ID NO:

3.

7. A primer combination, characterized in that: include: The reverse transcription primer set and PCR primer according to any one of claims 1 to 6; The structure of the PCR primer includes, from the 5' end to the 3' end, a linker connection sequence, a sample tag sequence, and a complementary sequence of the target fragment; The 5' end of the PCR primer is modified with a phosphate group; The adapter connection sequence is used for splitting sample tags; The sample label sequences of different samples are different; The target fragment is the product obtained by extending the reverse transcription primer set according to claim 1.

8. The primer combination according to claim 7, characterized in that: The PCR primer includes the nucleotide sequence shown in SEQ ID NO:

4.

9. A method for constructing a sequencing library, characterized in that: The following steps are involved: A reverse transcription step, comprising reverse transcribing the RNA sample using the reverse transcription primer set according to any one of claims 1 to 6 to obtain a reverse transcription product; The amplification step includes performing PCR amplification on the reverse transcription product to obtain a PCR amplification product.

10. The construction method according to claim 9, characterized in that: In the reverse transcription step, the RNA sample is first mixed with a reverse transcription primer for reaction, and then the reaction product is mixed with a reverse transcription system containing a template switching primer for reverse transcription reaction to obtain double-stranded cDNA.

11. The construction method according to claim 9 or 10, characterized in that: In the amplification step, the reverse transcription product is PCR amplified using PCR primers; The structure of the PCR primer includes, from the 5' end to the 3' end, a linker connection sequence, a sample tag sequence and a target fragment complementary sequence, and the 5' end of the PCR primer is modified with a phosphate group; The sample label sequences of different samples are different; The target fragment is the reverse transcription product according to claim 9; Preferably, the PCR primer comprises the nucleotide sequence shown in SEQ ID NO:

4.

12. The construction method according to any one of claims 9 to 11, characterized in that: In the amplification step, the amplification enzyme used for PCR amplification includes 1) KAPA HiFi HotStart Uracil and ReadyMix, or 2) KAPA HiFi HotStart ReadyMix.

13. The construction method according to any one of claims 9 to 12, characterized in that: In the reverse transcription step, the RNA sample is mixed with the reverse transcription primer for reaction at the following procedures: 72°C, 3 to 5 min; 42°C, 1 to 5 min; Preferably, after the reaction product is mixed with the reverse transcription system containing the template switching primer, the reaction temperature is set to 42° C., the reverse transcription reaction time is 60 to 90 min, and after the reaction is completed, the temperature is lowered to 4° C.

14. The construction method according to any one of claims 9 to 13, characterized in that: In the amplification step, the program of each cycle was as follows: 95-98°C, 15-30s; 62-65°C, 15-30s; 72°C, 4-6min; Preferably, in the amplification step, the number of cycles is 16 to 19; Preferably, in the amplification step, after the cycle is completed, the environment is maintained at 72°C for 5 to 10 minutes and then cooled to 12°C; Preferably, the method further comprises a linker connection step, including linking the PCR products to obtain a sequencing library.

15. A sequencing library construction kit, characterized in that: Comprising the reverse transcription primer set according to any one of claims 1 to 6 or the primer combination according to claim 7 or 8; Preferably, it also includes: a sequencing adapter.

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