CN114763546B - dU5' adapter and application thereof, and cDNA library constructed by same - Google Patents

Abstract

The invention provides dU5' adapter, application thereof and a constructed cDNA library. The dU5 'adapter has a structure of 5' -p-S1-S2-S3-S4-SpC3-3', S1, S2, S3 and S4 are gene fragments connected by chemical bonds in sequence, and p represents phosphorylation modification of the 5' end; s1 is a first stem; s2 is a ring part; s3 is a second stem containing 1 deoxyuracil dU; the 5 '. Fwdarw.3' sequence of S1 is complementary to the 3'. Fwdarw.5' sequence of S3; s4 is a guide (N) _m Wherein N is selected from any one of A, T, G and C bases, and m is 10; spC 3C 3 spacer modification at the 3' end. The dU5' adapter can reduce the formation of a linker-primer dimer, improve the PCR amplification efficiency, reduce the PCR period and improve the library quality; provides a new important platform for constructing cDNA library with wide biological application.

Description

A dU5' adapter and its application and cDNA library constructed

Technical Field

The invention belongs to the field of biotechnology and relates to a dU5' adapter and its application and constructed cDNA library.

Background technique

cDNA library construction is crucial for many high-throughput sequencing applications. In recent years, a variety of cDNA library construction methods have been developed to explore and study new properties of RNA, for example, in-depth research in the field of RNA structure.

In recent years, our research team has developed RNA G-quadruplex structure sequencing (rG4-seq) technology (Kwok, Marsico et al. 2016), which revealed the existence of RNA G-quadruplex structure in the human body from the transcriptome level, providing an effective basis for further research on rG4 structure and function. The 5' adapter used in library construction can reduce non-specific binding during cDNA ligation, thereby improving ligation efficiency. This design has been patented (No. 9,816,120, USA, authorized). However, the experimental process of rG4-seq sequencing technology requires a large amount of RNA starting material and a long library construction time. Therefore, we have systematically evaluated and optimized some key experimental steps in the entire cDNA library construction process, hoping to improve the efficiency and quality of library construction. This research result has been published in 2019 (Yeung, Zhao et al. 2019) and a patent has been applied for (No. 15/957,037, USA, pending).

Based on the above research, we found that the PCR efficiency was low during library construction, and the number of reaction cycles required to complete the library construction was large, which affected the quality of the library and resulted in less valid data obtained from subsequent second-generation sequencing analysis. Therefore, it is urgent to design a new 5' adapter to improve PCR amplification efficiency, reduce PCR cycles, improve library quality, and provide a new and important platform for the construction of cDNA libraries for a wide range of biological applications.

Summary of the invention

Based on the problems existing in the prior art, the first purpose of the present invention is to provide a dU5’ adaptor, which is a deoxyuracil-containing structure specially designed by the inventor, which can reduce the formation of linker-primer dimers and improve PCR amplification efficiency; the second purpose of the present invention is to provide the use of the dU5’ adaptor in constructing a cDNA library; the third purpose of the present invention is to provide a method for constructing a cDNA library, in which the deoxyuracil-containing dU5’ adaptor specially designed by the present invention is used; the fourth purpose of the present invention is to provide a cDNA library constructed by the above-mentioned construction method.

The purpose of the present invention is achieved by the following technical means:

In one aspect, the present invention provides a dU5' adaptor having a structure as shown in the following formula (I):

5'-p-S1-S2-S3-S4-SpC3-3' (I)

Among them, S1, S2, S3 and S4 are gene fragments connected in sequence by chemical bonds, p represents phosphorylation modification at the 5'end; S1 is the first stem; S2 is the loop; S3 is the second stem, which contains 1 deoxyuracil dU; the 5'→3' sequence of S1 is complementary to the 3'→5' sequence of S3; S4 is the guide part (N) _m , wherein N is selected from any one of A, T, G and C bases, and N in (N) _m is the same or different, and m is 10; SpC3 represents C3 interval modification at the 3' end.

The design of the guide (N) _m at the 3' end of the dU5' adapter and the C3 spacer modification can reduce non-specific binding, prevent and avoid self-aggregation, and improve the connection efficiency during the connection process of the dU5' adapter with the cDNA nucleic acid sample. The C3 spacer modification at the 3' end and the phosphorylation modification at the 5' end are both conventional modification methods in the art.

In the above-mentioned dU5' adaptor, preferably, the S1 has the following nucleotide sequence:

5'-AGATCGGAAGAGC-3' (SEQ ID NO: 1).

In the above-mentioned dU5' adapter, preferably, the S2 has the following nucleotide sequence:

5’-GTCGTGTA-3’.

In the above-mentioned dU5' adaptor, preferably, the S3 has the following nucleotide sequence:

5'-GC/ dU /CTTCCGATCT-3' (SEQ ID NO: 2).

The dU5' adaptor of the present invention has the nucleotide sequence shown in SEQ ID NO:3.

SEQ ID NO: 3 is as follows:

5′-/5Phos/-AGATCGGAAGAGCGTCGTGTAGC/ dU /CTTCCGATCTNNNNNNNNNN-/3SpC3/-3′.

During the design of the dU5' adapter, the applicant analyzed that the original 5' adapter had a special stem-loop structure, which might hinder the efficiency of the forward primer binding in the subsequent PCR amplification process, thereby reducing the PCR amplification efficiency. Therefore, the applicant creatively replaced the adjacent position with T-dU while ensuring the maximum binding of the forward primer, so that the insertion position of dU was the 24th position from the 5' end. This insertion position was more conducive to the pairing of the subsequent PCR forward primer, and its complementary pairing part was kept intact, which was conducive to the binding of the forward primer to the template, and could effectively reduce the formation of adapter-primer dimers and reduce the number of cycles required for the reaction, thereby improving the PCR amplification efficiency and thus improving the quality of the library.

On the other hand, the present invention also provides the use of the above-mentioned dU5' adaptor in constructing a cDNA library.

Since the 5’ adapter ligation step is widely used in the library construction process of other RNA high-throughput sequencing, this study is not only applicable to rG4-seq technology, but also to other DNA and RNA research based on second-generation sequencing, providing a new and important platform for constructing cDNA libraries for a wide range of biological applications.

In another aspect, the present invention further provides a method for constructing a cDNA library based on an improved method for constructing a RNA-G quadruplex sequencing library, wherein the improved method comprises the following steps:

The cDNA nucleic acid sample with the 3' adapter is mixed with the dU5' adapter and then subjected to a ligation reaction to obtain a dU5' adapter-cDNA nucleic acid complex, which is then purified by a column;

The deoxyuracil in the purified dU5' adapter-cDNA nucleic acid complex is cleaved to form an adapter-nucleic acid complex with complementary end portions; residual adapter fragments after cleavage are removed by column purification;

The adapter-nucleic acid complex with complementary end portions is used as a template, and a PCR reaction is performed using a specifically bound forward primer, a reverse primer and a polymerase to amplify the product and construct a cDNA library.

In the above construction method, preferably, the ligation reaction is carried out using the rapid ligation kit NEB M2200L.

In the above construction method, preferably, the specific process of cleavage includes: adding USER II enzyme (NEB M5508S) and buffer to the purified dU5' adapter-cDNA nucleic acid complex to cleave deoxyuracil.

The buffer may include CutSmart buffer (CS, NEB B7204S), Quick ligation buffer (QL), etc.; the polyethylene glycol may include polyethylene glycol PEG 6000, etc.

In the above construction method, preferably, during the PCR reaction, the polymerase includes KAPA HiFi hot start high-fidelity polymerase (Roche KK2601).

In the above construction method, preferably, the cDNA nucleic acid sample with a 3' adaptor has a nucleotide sequence shown in SEQ ID NO:4.

SEQ ID NO: 4 is as follows:

5′-CAGACGTGTGCTCTTCCGATCT(N) ₄₀ -3′.

Wherein, N is selected from any one of A, T, G and C bases, and N in (N) ₄₀ are the same or different.

In the above construction method, preferably, the forward primer has the nucleotide sequence shown in SEQ ID NO: 5; and the reverse primer has the nucleotide sequence shown in SEQ ID NO: 6.

SEQ ID NO: 5 is as follows:

5’-AATGATACGGCGACCACCGAGATCTACACTACACGACGCTCTTCCGATCT-3’.

SEQ ID NO: 6 is as follows:

5’-CAAGCAGAAGACGGCATACGAGAT-(6nt index sequence)-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’.

SEQ ID NO: 6 is a universal sequencing primer sequence in the Truseq Kit of the Illumina sequencing platform; wherein, the "6nt index sequence" is a universal specific index to facilitate distinguishing different samples during the sequencing process, and the specific index sequence is well known in the art.

In the above construction method, preferably, the cleavage time of the USER II enzyme is 15 minutes.

In the above construction method, preferably, the molar ratio of the cDNA nucleic acid sample with 3’ adaptor to the dU5’ adaptor is 1:10.

In yet another aspect, the present invention also provides a cDNA library, which is constructed by the above construction method.

The dU5' adapter of the present invention can reduce the formation of linker-primer dimers, improve PCR amplification efficiency, reduce the number of PCR cycles, and improve library quality, thereby improving effective analysis data and reducing costs in the subsequent NGS data analysis process; it provides a new and important platform for building a wide range of biological application cDNA libraries. Due to differences in gene expression, some RNA samples with low expression levels cannot reach the starting amount of ordinary library construction samples, and this method can reduce the starting amount of the reaction, thereby expanding the scope of RNA research, especially for RNA research in the fields of low expression rG4 sequencing, transcriptome structure sequencing, etc., which is of great significance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart of the construction of a cDNA library using the dU5′ adapter in Example 2 of the present invention.

Figure 2 is a graph showing the experimental results of the effect of USER II enzyme on the cleavage of deoxyuracil in the dU5' linker at different cleavage times in Example 3 of the present invention.

FIG3 is a graph showing the experimental results of the effect of USER II enzyme on the cleavage efficiency of deoxyuracil in dU5' linker in different reaction buffers and different concentrations of polyethylene glycol in Example 4 of the present invention.

Figure 4 is a graph showing the experimental results of the effect of the USER II enzyme in Example 4 of the present invention on the cleavage efficiency of deoxyuracil in the dU5' linker of Comparative Examples 2 and 3 in different reaction buffers and specific proportions of polyethylene glycol.

Figure 5 is a graph showing the results of an optimization experiment on the effect of removal of the residual dU5' linker in Example 5 of the present invention.

Figure 6 is a graph showing the optimization experimental results of the influence of the connection ratio of the simulated cDNA sample and the dU5' adaptor in Example 6 of the present invention.

FIG. 7 is a graph showing the experimental results of the effect of column purification after the ligation reaction on the cleavage rate of USER II enzyme in Example 7 of the present invention.

FIG8 is a graph showing the results of a column purification experiment after USER II enzyme cleavage in Example 8 of the present invention.

FIG9 is a graph showing the comparison of the PCR amplification results of Example 2 of the present invention and Comparative Example 1.

Detailed ways

In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

Embodiment 1:

This embodiment provides a dU5' adaptor having the following nucleotide sequence: 5'-/5Phos/-AGATCGGAAGAGCGTCGTGTAGC/ dU /CTTCCGATCTNNNNNNNNNN-/3SpC3/-3' (SEQ ID NO: 3).

The dU5' adapter is a deoxyuracil-containing structure specially designed by the inventors. In the newly designed dU5' adapter, the insertion position of dU is the 24th position from the 5' end. This insertion position is more conducive to the pairing of the subsequent PCR forward primer, and its complementary pairing part is kept intact, which is conducive to the binding of the forward primer to the template, can effectively reduce the formation of adapter-primer dimers, and reduce the number of cycles required for the reaction, thereby improving the amplification efficiency of PCR, thereby improving the quality of the library.

The dU5' adaptor of this example was obtained by commercial synthesis according to its nucleotide sequence. The commercially synthesized dU5' adaptor nucleic acid sample was dissolved in ultrapure water to obtain a solution with a concentration of 100 μM, and stored at -20°C.

Embodiment 2:

This embodiment provides an improved method for constructing a cDNA library, the improved part of which is shown in FIG1 , and comprises the following steps:

The simulated cDNA nucleic acid sample (with a 3' adapter, the sequence of which is shown in SEQ ID NO: 4) was mixed with the dU5' adapter of Example 1 and then subjected to a ligation reaction using a quick ligation kit (NEB, M2200L), the molar ratio of the nucleic acid sample to the dU5' adapter being 1:10, and the reaction conditions being 37°C for 2h; the dU5' adapter-cDNA nucleic acid complex was obtained and column purified (Zymo Research, R1016);

Add CutSmart buffer (1X final) USERII enzyme (0.1U/μL final) to the purified dU5' adapter-cDNA nucleic acid complex to cleave deoxyuracil at 37°C for 15 min to form an adapter-nucleic acid complex with complementary ends; remove excess adapters by column purification (Zymo Research, R1016);

Using the terminal partially complementary adapter-nucleic acid complex as a template, a specifically bound forward primer (sequence see SEQ ID NO: 5) and a reverse primer (sequence see SEQ ID NO: 6) were used to perform a PCR reaction under the action of KAPA HiFi hot start high-fidelity DNA polymerase to amplify the product and construct a cDNA library.

Comparative Example 1:

This comparative example provides a nucleotide sequence based on a common 5' adapter as follows: 5'-/5Phos/-AGATCGGAAGAGCGTCGTGTAGCTCTTCCGATCTNNNNNNNNNN-/SpC3/-3' (SEQ ID NO: 7). The 5' adapter of the comparative example is compared with the dU5' adapter, and the experimental conditions are optimized. The difference is that the dU5' adapter used in Example 2 is replaced by the common 5' adapter of the comparative example 1.

Comparative Example 2:

This comparative example provides a 5' adaptor into which dU is inserted, wherein dU is at the 18th position (Loop18) at the 5' end.

The nucleotide sequence of the 5' adapter for inserting dU is as follows: 5'-/5Phos/-AGATCGGAAGAGCGTCG/ dU /GTAGCTCTTCCGATCTNNNNNNNNNN-/3SpC3/-3' (SEQ ID NO: 8).

Comparative Example 3:

This comparative example provides a 5' adaptor into which dU is inserted, wherein dU is at the 20th position (Loop20) at the 5' end.

The nucleotide sequence of the 5' adapter for inserting dU is as follows: 5'-/5Phos/-AGATCGGAAGAGCGTCGTG/ dU /AGCTCTTCCGATCTNNNNNNNNNN-/3SpC3/-3' (SEQ ID NO: 9).

Example 3: Optimization of cleavage time of deoxyuracil in dU5' adapter by USER II enzyme

This example investigates the effect of USER II enzyme on the cleavage of deoxyuracil in the dU5' linker at different cleavage times, and the reaction condition is set at 37° C. The experimental results are shown in FIG2 .

As can be seen from FIG. 2 , the cleavage rate increases with the increase of reaction time. In order to maximize the cleavage rate, a cleavage time of 15 min is preferably used.

Example 4: Optimization of the effects of reaction buffer and polyethylene glycol on cleavage efficiency during cleavage of deoxyuracil in dU5' linker by USER II enzyme

This example investigates the effect of USER II enzyme on the cleavage efficiency of deoxyuracil in dU5' linker in reaction buffer and polyethylene glycol at different concentrations, wherein the buffer is CutSmart buffer (CS) or Quick ligation buffer (QL), and the polyethylene glycol is PEG 6000, with mass concentrations of 7.5%, 12.5% and 17.5%, respectively, wherein QL itself contains PEG 6000 with a mass concentration of 7.5%. The experimental results are shown in Figures 3 and 4.

As shown in Figure 3, for the dU5' adaptor designed in the present invention (see SEQ ID NO: 3), there was no significant difference in the cleavage rate between the two buffers, both of which were 97%. There was also no significant difference in the cleavage rate between the three concentrations of polyethylene glycol, which were 97%, 97% and 98% respectively.

As can be seen from Figure 4: whether in CS or QL buffer, the efficiency of USER II cleaving dU Loop 18 (see SEQ ID NO: 8) and dU Loop 20 (see SEQ ID NO: 9) ranges from 37% to 68%, and the dU linker cleavage is incomplete, while the cleavage rate of the 24th dU design of the present invention (see SEQ ID NO: 3) reaches 98%, which is conducive to maximizing the binding of the forward primer. The experimental results show that the dU5' linker designed by the present invention is better than the designs of Comparative Examples 2 and 3.

Example 5: Removal of residual dU5' adapter affects optimization

In order to avoid transfer contamination and reduce the generation of adapter-primer dimers, it is necessary to remove excess adapters before PCR amplification. This example investigates the column purification removal strategy after treatment with different mass concentrations of PEG6000 and USER II in CS buffer and QL buffer, and compares it with the removal without column purification to determine whether the purification efficiency is affected by the type of buffer and the concentration of PEG 6000. The experimental results are shown in Figure 5.

As shown in Figure 5, residual dU5' linker can be effectively removed by column purification, and the reaction conditions are not affected by the two buffer types and the concentration of PEG 6000.

Example 6: Optimization of the influence of the ligation ratio of the simulated cDNA nucleic acid sample and the dU5' adapter

A cDNA nucleic acid sample (66 nt DNA) with a 3' adapter simulation was synthesized using a commercial synthesis method, including 40 random nucleotides and a 22 nt 3' adapter sequence (N40+22) to simulate the cDNA template in the real library (see SEQ ID NO: 4). The ligation yield of ssDNA (dU5' adapter-cDNA nucleic acid complex) with a molar ratio of (N40+22) cDNA template to dU5' adapter of 1:1, 1:2.5, 1:5 and 1:10 was tested, and the experimental results are shown in Figure 6.

As can be seen from Figure 6, when the molar ratio of the (N40+22) cDNA template to the dU5' adaptor is 1:10, the ssDNA connection yield is as high as 90%.

Example 7: Effect of column purification after ligation reaction on USER II enzyme cleavage rate

After the dU5' adapter and the nucleic acid sample are connected, the next step of USER II enzyme cleavage is required. In order to study the effect of the reaction environment of the connection process on the subsequent USER II enzyme cleavage, column purification is used to remove substances other than the product of the connection process before USER II enzyme cleavage, and the efficiency is compared with that of USER II enzyme cleavage directly after the connection reaction. The experimental results are shown in Figure 7.

As can be seen from Figure 7, after the ligation reaction, column purification was performed, and the USER II enzyme cleavage rate reached 88±1%, while without column purification after the ligation reaction, the USER II enzyme cleavage rate was only 24±1%. It can be seen that column purification after the ligation reaction helps to improve the USER II enzyme cleavage rate. Based on the above results, for the subsequent purification of USER II cleavage process, we finally used CS buffer without adding polyethylene glycol. This preference can save both reagents and costs.

Example 8: Column purification after USER II enzyme cleavage

After USER II enzyme cleavage, the residual adapter-primer dimer and the formation of non-specific products will affect PCR amplification, thereby reducing the generation of the target product. Therefore, column purification was performed after USER II enzyme cleavage to remove excess dU5' adapter cleavage products. The experimental results are shown in Figure 8.

As can be seen from Figure 8, column purification after USER II enzyme cleavage can remove 91±1% of the dU5' adapter cleavage products.

Example 9: Comparison of PCR amplification experiments

The experimental results of PCR amplification and construction of cDNA library were compared using the adapter-nucleic acid complexes with complementary ends of the dU5' adapter designed by the present invention (see SEQ ID NO: 3) and the common 5' adapter (see SEQ ID NO: 7) of Comparative Example 1 as templates. The experimental results are shown in FIG9 .

As can be seen from FIG9 , the relative yields of the PCR products obtained by using the dU5' adapter of the present invention in PCR cycle numbers 4, 6, 8, 10, 12 and 14 are 1.1, 1.1, 1.3, 1.4, 1.1 and 1.1, respectively, and the yields of the PCR products are all higher than those of the common 5' adapter. Specifically, when the PCR cycle numbers are 4 and 6, due to the limited initial product of PCR amplification, the yields of the PCR products of the dU5' adapter of the present invention and the common 5' adapter of Comparative Example 1 are similar; and when the PCR cycle numbers are 8 and 10, the yield of the PCR products of the present invention is 1.3 to 1.4 times that of the common 5' adapter of Comparative Example 1, and the amplification efficiency is significantly higher than that of the common 5' adapter; in addition, when the PCR cycle numbers are 12 and 14, the PCR products have begun to show over-amplification, and the product length increases, but the yield is still higher than that of the common 5' adapter of Comparative Example 1, which is 1.1 times.

This shows that under the premise of the same number of amplification cycles, the specially designed dU5' adapter containing deoxyuracil in the present invention has significantly improved PCR efficiency compared with the common 5' adapter in comparative example 1.

Sequence Listing

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Claims (10)

1. A dU5' adaptor having the structure of formula (I) below:

5’-p-S1-S2-S3-S4-SpC3-3’ （I）

wherein S1, S2, S3 and S4 are gene fragments which are connected in sequence through chemical bonds, and p represents5' -terminal phosphorylation modification; s1 is a first stem; s2 is a ring part; s3 is a second stem containing 1 deoxyuracil dU; the 5 '. Fwdarw.3' sequence of S1 is complementary to the 3'. Fwdarw.5' sequence of S3; s4 is a guide (N) _m Wherein N is selected from any one of A, T, G and C bases, and (N) _m N in (a) are the same or different, m is 10; spC 3C 3 spacer modification at the 3' end;

wherein the nucleotide sequence of S1 is as follows:

5’-AGATCGGAAGAGC-3’（SEQ ID NO：1）；

the nucleotide sequence of S2 is as follows:

5’-GTCGTGTA-3’；

the nucleotide sequence of S3 is as follows:

5’-GC/dU/CTTCCGATCT-3’（SEQ ID NO：2）。

2. use of the dU5' adaptor of claim 1 in constructing a cDNA library.

3. A method of constructing a cDNA library, comprising the steps of:

mixing a cDNA nucleic acid sample with a 3' adapter with the dU5' adapter of claim 1, performing ligation reaction to obtain a dU5' adapter-cDNA nucleic acid complex, and performing column purification;

cleaving deoxyuracil in the purified dU5' adaptor-cDNA nucleic acid complex to form an adaptor-nucleic acid complex with complementary terminal portions; removing the cleaved residual adaptor fragments by column purification;

and (3) taking the adapter-nucleic acid complex with the complementary tail end part as a template, adopting a forward primer, a reverse primer and polymerase with specific binding to perform PCR reaction, amplifying a product, and constructing to obtain a cDNA library.

4. The construction method according to claim 3, wherein the ligation reaction is performed using the rapid ligation kit NEB M2200L.

5. A construction method according to claim 3, wherein the specific process of performing the cleavage comprises: to the purified dU5' adapter-cDNA nucleic acid complex is added the USER II enzyme, buffer and cleaved deoxyuracil.

6. A method of construction according to claim 3, wherein the polymerase during the PCR reaction comprises KAPA HiFi hot start high fidelity polymerase.

7. The method of claim 3, wherein the nucleotide sequence of the cDNA nucleic acid sample with 3' adapter is as set forth in SEQ ID NO: 4.

8. A method of construction according to claim 3, wherein the forward primer has a nucleotide sequence as set forth in SEQ ID NO:5 is shown in the figure; the nucleotide sequence of the reverse primer is shown as SEQ ID NO: shown at 6.

9. The construction method according to claim 5, wherein the cleavage with the USER II enzyme is performed for 15min.

10. The method of construction of claim 3, wherein the molar ratio of the 3 'adaptor-bearing cDNA nucleic acid sample to the dU5' adaptor is 1:10.