WO2021232793A1

WO2021232793A1 - Universal closed sequence and use thereof

Info

Publication number: WO2021232793A1
Application number: PCT/CN2020/139918
Authority: WO
Inventors: 胡玉刚; 汪彪; 郑文莉; 吴强
Original assignee: 纳昂达(南京)生物科技有限公司
Priority date: 2020-05-18
Filing date: 2020-12-28
Publication date: 2021-11-25
Also published as: CN111534518A; CN111534518B

Abstract

Provided in the present invention are a universal closed sequence and a use thereof. The universal closed sequence comprises a left non-tag region closed sequence, a middle tag region closed sequence and a right non-tag region closed sequence which are sequentially connected from 5' to 3'. The left non-tag region closed sequence comprises 5-7 LAN or BNA modified bases, the middle tag region closed sequence is a universal closed base sequence, the right non-tag region closed sequence comprises 7-10 LAN or BNA modified bases, and a 3' end of the right non-tag region closed sequence is provided with a closed modification. The structure can significantly enhance the binding capacity with a sequence to be closed, thereby improving the closing effect, and meanwhile, can reduce or avoid non-specific capture of a library, thereby improving the target rate of target library capture.

Description

General closed sequence and its application

Technical field

The present invention relates to the field of high-throughput sequencing library construction, in particular to a universal closed sequence and its application.

Background technique

With the increasing importance of high-throughput sequencing in assisted diagnosis in clinical applications, how to reduce sequencing costs is a critical issue. Reducing sequencing costs has corresponding performance at different levels: MGI (MGI) continues to introduce higher Sequencers with sequencing throughput have continuously reduced sequencing costs, and successively launched MGI-200, MGI-2000 and T7 sequencers. Among them, the T7 sequencer is currently the sequencer with the highest sequencing throughput and the lowest sequencing cost on the market. In addition, targeted capture sequencing is also an effective way to achieve large-scale reduction of detection costs while detecting target sequences.

In the sequencing process, different samples are distinguished by different Index sequences, and then multiple samples are mixed and sequenced, which is also a way for high-throughput sequencing to reduce the cost of a single sample. However, if a single-ended Index is used, since Index adapters or primers will inevitably lead to contamination and/or crosstalk in each link of synthesis, library construction, experimental operations, and sequencing. Therefore, there is a need for a way to solve the low-frequency mutual crosstalk between samples. The current method to solve this problem is to use double-ended Index to distinguish different samples. The double-ended Index method can effectively remove the mutual crosstalk between samples.

The hybridization capture can effectively reduce the sequencing cost of the detection target, and at the same time, if the proportion of the captured target area can be increased during the hybridization sealing process, the sequencing cost can also be saved. However, there is no effective solution for how to efficiently capture the sample library with index on both ends.

Summary of the invention

The main purpose of the present invention is to provide a universal closed sequence and its application to solve the problem of low hybridization and capture efficiency of double-ended index libraries in the prior art.

In order to achieve the above objective, according to one aspect of the present invention, a universal closing sequence is provided, which includes a left non-tag region closing sequence and a middle tag region closing sequence sequentially connected in a direction from 5'to 3'. And the right non-tag region closed sequence, where the left non-tag region closed sequence includes 5-7 LAN or BNA modified bases, the middle label region closed sequence is a universal closed base sequence, and the right non-tag region closed sequence Including 7-10 LAN or BNA modified bases, and the 3'end of the blocking sequence in the right non-tag region has a blocking modification.

Further, the 3'end blocking modification is MGB modification, C3 spacer modification, phosphorylation modification, digoxigenin modification or biotin modification, or the 3'end base is a dideoxy base.

Further, the universal blocking base is hypoxanthine or C3 spacer.

Further, the universal blocking sequence is the blocking sequence of the P1 linker with the first tag sequence or the blocking sequence of the P2 linker with the second tag sequence of the MGI sequencing platform, where the blocking sequence of the P1 linker is SEQ ID NO: 3 :

CTCTCA+GTACG+TCA+GCA+GT+TXXXXXXXXXXCA+ACTCCT+TGGC+TCACAGA+ACGA+CATGG+CTACGATC+CGACTT/3SpC3/; where + means LAN or BNA modification, X means hypoxanthine or C3 spacer, /3SpC3/is Modification of the C3 spacer at the 3'end;

The blocking sequence of the P2 linker is SEQ ID NO: 4:

GCA+TGGC+GA+CCTT+ATCA+GXXXXXXXXXXXTTGTCTT+CCTA+AGA+CCGC+TTG+GCC+TCCGA+CTT/3SpC3/; where + means LAN or BNA modification, X means hypoxanthine or C3 spacer, /3SpC3/ is The C3 spacer at the 3'end is modified.

Further, the universal blocking sequence is the blocking sequence of the P1 linker with the first tag sequence or the blocking sequence of the P2 linker with the second tag sequence of the MGI sequencing platform;

The blocking sequence of the P1 linker is SEQ ID NO: 5:

CTC+TCA+GT+ACG+TCA+GCA+GT+TXXXXXXXXXXCA+ACTCCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CTAC+GATC+CGACTT/3SpC3/; where + means LAN or BNA modification, X means times Xanthine or C3 spacer, /3SpC3/ is the modification of the C3 spacer at the 3'end;

The blocking sequence of the P2 linker SEQ ID NO: 6:

GCA+TG+GC+GA+CC+TT+ATCA+GXXXXXXXXXXTTG+TCTT+CCTA+AGA+CC+GC+TTG+GCC+TCC+GA+CTT/3SpC3/; where + means LAN or BNA modification, X means times Xanthine or C3 spacer, /3SpC3/ is the C3 spacer modification at the 3'end.

In order to achieve the above objective, according to the second aspect of the present invention, a capture kit is provided, the capture kit includes a universal blocking sequence, and the blocking sequence is any of the above universal blocking sequences.

Further, the working concentration of the universal capture probe in the kit is based on a single library, which is 0.4-0.8 μg of the universal blocking sequence/1 μg of the library to be captured.

According to a third aspect of the present invention, there is provided a library hybridization capture method, the method includes using a capture kit to capture the library to be captured, and the capture kit uses any of the foregoing capture kits.

Further, the step of using the capture kit to capture the library to be captured includes blocking the blocking sequence and the library to be captured in a molar ratio of 10:1 to 20:1.

According to the fourth aspect of the present invention, there is provided a method for constructing a library. The method for constructing a library includes: constructing a fragmented library; hybridizing and capturing the fragmented library to obtain a captured library; performing PCR amplification on the captured library to obtain sequencing Library; use any of the above capture kits for hybrid capture, or use any of the above methods for hybrid capture.

By applying the technical solution of the present invention, 5-7 and 7-10 bases are modified by LNA or BNA respectively on the closed sequence of the left non-tag region and the closed sequence of the right non-tag region, which can enhance the closed sequence to be blocked. Binding ability, thereby enhancing the blocking effect; blocking modification at the 3'end of the blocking sequence in the right non-tag region, so that the blocking sequence of the present application can reduce or avoid the capture of non-target libraries during library capture, and improve the capture of target libraries Rate (or called the target rate of the target library).

Description of the drawings

The drawings of the specification constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and the description thereof are used to explain the present invention, and do not constitute an improper limitation of the present invention. In the attached picture:

Figure 1 shows a schematic diagram of the current MGI platform dual-end Index database construction process;

Fig. 2 shows a schematic diagram of the principle of improving the target sequence capture rate when a universal closed sequence is used for capture in the prior art;

Fig. 3 shows a statistical result diagram of crosstalk between libraries constructed by using 12 double-ended Indexes in the double-ended Index library of the MGI platform in the prior art;

4A and 4B show schematic diagrams of the normal and abnormal blocking of the hybrid library by the improved universal blocking sequence;

Figure 5 shows a schematic diagram of the effect of using different modification quantities and different concentrations of universal blocking sequences on the target sequence capture rate;

Figure 6 shows the influence of too few and too many base modification universal blocking sequences on the blocking effect;

Figure 7 shows the effect of universal blocking sequences with the same number of modified bases but different modified positions on the blocking effect;

Figure 8 shows the effect of different library input on the blocking effect.

Detailed ways

It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other if there is no conflict. The present invention will be described in detail below in conjunction with embodiments.

Term explanation:

Double-ended Index adapter: For high-throughput sequencing, a universal sequencing adapter is required to connect the end of each fragment. Each non-complementary region of the adapter has a variable sequence region. The sequence is the Index sequence, which is used to split data during sequencing.

Adaptor blocking sequence: During library capture, each library has the same and similar adaptor sequence. During hybridization, the target fragment and non-target fragment adaptor parts will bind to each other, reducing the target rate. The adaptor sealing sequence is used to specifically bind the adaptor part. Sequence, play a role in improving the target rate. The universal blocking sequence is a sequence that can be blocked for all linkers with different Inedx in the library.

C3 arm: C3 Splicer is mainly used to imitate the three-carbon gap between the 3'and 5'hydroxyl groups of ribose, or to "replace" an unknown base in a sequence. The middle of the nucleic acid sequence is mainly used for connection and cannot be complementary. The base pairing of the base pair plays a stabilizing effect, and only plays a role in connecting the front and rear bases.

It should be noted that the index length used for the MGI sequencing platform mentioned in this application is described by taking 10 bp as an example. The length of the label closure section for index in this application can be adapted to the closure of index joints of different lengths by adjusting the length of the hypoxanthine I and C3 spacer arms.

As mentioned in the background technology, current high-throughput sequencers need to reduce sequencing costs, and the throughput of single sequencing is getting higher and higher. In order to save sequencing costs, the existing MGI sequencers have also launched the highest throughput on the market. The T7 sequencer, which has the lowest sequencing cost, requires that many sequencing samples must be mixed for sequencing at a time when the sequencing throughput increases. High-throughput sequencing is to connect different samples to unused Index connectors to achieve multiple samples mixed together for sequencing. MGI first introduced a single-ended Index connector library building solution. This solution has a significant problem that there will be crosstalk between samples. In order to solve the crosstalk problem, MGI also launched a double-ended Index library building solution, which can be solved. Due to the low-frequency crosstalk problem caused by the interaction of joint synthesis, experimental operation and sequencing process, the crosstalk data is filtered out through two Index data splitting.

However, when performing capture library sequencing for its paired-end index library, there is also the defect of low capture efficiency for the library containing the target fragment. In order to further improve the capture efficiency of the paired-end Index library, this application uses Nanoda to launch the MGI sequencer. The double-ended Index database construction program (the specific double-ended Index database construction process is shown in Figure 1), and the corresponding hybrid capture improvement program is proposed.

Another effective way to reduce the cost of sequencing is to target capture sequencing. The size of the human genome is 3Gb, and the region encoding the gene accounts for less than 2% of the region. (In the case of IDT's all-exon V2 version), it covers humans. There are about 20,000 genes, about 34Mb in size, so the cost of a whole-genome sequencing can measure 10 whole-exome sequencing. Compared with whole-genome sequencing, the detection area of tumor-targeted drugs has a greater cost difference and can be more reflected. The importance of targeted sequencing. Moreover, tumor mutations are low-frequency mutations. Two main issues need to be considered. One is that low-frequency mutations cannot have crosstalk between samples, or if crosstalk cannot be avoided, there must be a way to eliminate crosstalk data. Double-ended Index means when crosstalk cannot be avoided. It is a necessary solution to eliminate crosstalk when avoiding, so this is also the greatest significance of the existence of double-ended Index. Another condition for detecting low-frequency mutations is that the sequencing depth must be guaranteed, which generally requires a sequencing depth of several thousand to tens of thousands of times.

In the process of targeted sequencing, there are two key factors that determine the target rate of targeted capture. On the one hand, the specificity of the design area, the designed probe cannot fall into the highly repetitive area, and the other is the capture time. Joint closure effect. The probe sequence is determined by the sequence of the region to be captured. Generally, highly repetitive regions are avoided when designing the probe, so what we can improve is the blocking effect during targeted capture. If the blocking sequence is not added during capture, theoretical and practical tests have shown that the target rate of (target fragment library) will not exceed 50%. As shown in Figure 2, when the blocking linker sequence is not added, the linker part will bind to the non-targeting library to reduce the target rate. Under the premise that the specificity of the capture probe region is relatively good, adding different levels of blocking sequences can make the target rate fluctuate between 45-90%. Therefore, this application takes the double-ended Index linker library of the MGI platform as an example to explain the improved design of the universal blocking sequence and the achieved blocking effect, which can greatly increase the target rate and reduce the sequencing cost.

There is a linker sequence at both ends of the double-ended Index library of the MGI platform. The index sequence carried by the Index linker of different numbers is a 10bp variable region, which is used for hybrid capture and hybrid sequencing. Different samples are distinguished. The purpose of the double-ended Index mentioned above is to remove crosstalk. As shown in Figure 3, 3-7 sequences can be filtered out through the double-ended Index for every 10,000 sequences. If the double-ended Index is not used for one-thousandth and below The mutation detection is unreliable data, and the accuracy of detection can be improved through double-ended Index. In order to improve the target rate during the hybridization capture process, the present invention develops a universal closed sequence for the double-ended Index of the MGI platform. One of the characteristics of this universal sequence is that the 10bp Index region selects universal bases (hypoxanthine or C3 spacer) to play a blocking/occupying role. In order to improve the blocking effect, the fixed sequence regions at both ends are used to increase the hybridization temperature. Base modification and substitution, some bases in the fixed sequence are modified with LNA or BNA. The specific characteristics and requirements of the double-ended Index universal probe are as follows: (1) The tag sequence region is a universal closed base, such as hypoxanthine (I), C3 spacer equidistant sequence or a combination thereof; (2) In order to enhance this The binding efficiency of the universal closed sequence is that some bases in the non-tag sequence region upstream and downstream of the closed sequence are modified by LNA or BNA, and the number of modified bases is 5-7 and 7-10, or 20%-40, respectively. %between. (3) The optimal use concentration of universal blocking sequences is inversely proportional to the number of modified bases. There are many modified bases, and the optimal concentration is relatively low. Too high a concentration will have a negative effect; on the contrary, there are fewer modified bases, and the blocking effect must be achieved. A higher blocking sequence concentration is required.

The present invention designs a universal closed modified sequence based on the linker sequence characteristics of the MGI double-ended Index. The two universal closed original sequences of MGI are as follows:

The part of N is the closed sequence of the Index sequence. Compared with the Index of 6bp and 8bp, the index of 10bp length has the advantage that it can increase the choice when designing different indexes. The disadvantage is that the design of general closed sequence increases the difficulty and Instability. When designing a universal closed sequence, the bases in the Index area are designed to be degenerate bases N, C3 spacer and hypoxanthine. The obtained universal closed sequence is more unstable, which requires a universal closure at both ends of the Index. There are more bases modified in the sequence. Therefore, this application increases the number of modified bases that improve the blocking effect from 3-6 to 5-10.

In a preferred embodiment of the present application, the P1 (5+7 modified) end blocking sequence is SEQ ID NO: 3:

CTCTCA+GTACG+TCA+GCA+GT+TXXXXXXXXXXCA+ACTCCT+TGGC+TCACAGA+ACGA+CATGG+CTACGATC+CGACTT/3SpC3/; where + means LAN or BNA modification, X means hypoxanthine or C3 spacer, /3SpC3/is Block modification at the 3'end. In a preferred embodiment, the P2 end blocking sequence is SEQ ID NO: 4:

GCA+TGGC+GA+CCTT+ATCA+GXXXXXXXXXXXTTGTCTT+CCTA+AGA+CCGC+TTG+GCC+TCCGA+CTT/3SpC3/; where + means LAN or BNA modification, X means hypoxanthine or C3 spacer, /3SpC3/ is Block modification at the 3'end.

In another preferred embodiment of the present application, the P1 end blocking sequence is SEQ ID NO: 5:

CTC+TCA+GT+ACG+TCA+GCA+GT+TXXXXXXXXXXCA+ACTCCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CTAC+GATC+CGACTT/3SpC3/; where + means LAN or BNA modification, X means times Xanthine or C3 spacer, /3SpC3/ is a blocking modification at the 3'end. In a preferred embodiment, the P2 end blocking sequence is SEQ ID NO: 6:

GCA+TG+GC+GA+CC+TT+ATCA+GXXXXXXXXXXTTG+TCTT+CCTA+AGA+CC+GC+TTG+GCC+TCC+GA+CTT/3SpC3/; where + means LAN or BNA modification, X means times Xanthine or C3 spacer, /3SpC3/ is a blocking modification at the 3'end.

Further research found that the number of modified bases is relatively large, and the concentration of the universal blocking sequence added at the same time is relatively high, but the blocking effect is not good. As shown in Figure 4A and Figure 4B, when the number of modified bases in the closed sequence is large, The local area forms a strong binding ability. Most of them will be like Figure 4A. The blocking sequence can correctly block the library sequence, but at the same time it will also form a cross-star block to capture a non-target sequence, thereby reducing the target rate in the library. As shown in Figure 4B.

It is further found that during the hybridization process, the amount of library added is also limited to the maximum amount. Calculated as the 250bp length insert library, it cannot exceed 6.5μg (25pmol/L, here refers to the single-ended sequence) in a single hybridization. The whole exome test found that each library invested 500ng during hybridization, and the hybridization effect of 12 libraries was better than that of 14-16 libraries. During hybridization, the library space distance would be shortened due to excessive library input. The two libraries and universal blocking of the library form a cross-star structure of the double library and double blocking sequence shown in FIG. 4B, resulting in a decrease in capture efficiency.

Based on the above-mentioned research and findings, the applicant proposed the technical solution of this application. In a typical embodiment, a universal closing sequence is provided. The universal closing sequence includes, in a direction from 5'to 3': a left non-tag region closing sequence, a middle tag region closing sequence, and The right non-tag region blocking sequence, where the left non-tag region blocking sequence includes 5-7 LAN (Locked nucleic acid, locked nucleic acid) or BNA (Bridged nucleic acid 2', 4'-BNA ^NC , ie 2 '-O,4'-aminoethylene bridged nucleic acid is a compound containing a six-member bridged structure with an NO bond) modified bases (mainly C bases are modified by LNA or BNA), the middle tag region blocking sequence is universal The closed base sequence, the closed sequence of the right non-tag region includes 7-10 LAN or BNA modified bases, and the 3'end of the closed sequence of the right non-tag region has a closed modification.

Modification of LNA or BNA by 5-7 bases on the closed sequence in the left non-tag region and 7-10 bases in the closed sequence in the right non-tag region can significantly enhance the binding ability to the sequence to be blocked , Thereby increasing the blocking effect; and blocking and modifying the 3'end of the blocking sequence in the right non-tag region, so that the excess adapters in the library cannot be used as primers to amplify the adapters of other libraries, thereby reducing or avoiding non-specific libraries. Heterosexual capture improves the target rate of target library capture.

The blocking modification of the 3'end of the above-mentioned right non-tag region blocking sequence can be MGB modification, C3 spacer modification, 3'phosphorylation modification, 3'digoxigenin modification, 3'biotin modification or 3'end base Dideoxy base. In this application, it is preferred to use C3 spacer modification.

The above-mentioned tag region blocking sequence adopts a universal blocking base, and a base sequence that has weak binding ability to all four bases of A, T, C, and G can be used. In a preferred embodiment of the present application, the universal blocking base is hypoguanine I and/or C3 spacer. The number of bases in the blocking sequence of the specific tag region is not limited to 10 bp, and can be set reasonably according to the number of bases in the sample tag in the library to be blocked. For example, it can also be 6bp, 7bp, 8bp, 9bp, 11bp or 12bp.

When the C3 spacer is used as the tag region blocking sequence, take the blocking sequence of the P1 and P2 linker of the aforementioned MGI platform as an example, which is 10 C3 spacers, or 10 hypoxanthines (I). The advantage of hypoxanthine is that it has weak pairing ability with all bases, while the C3 spacer only occupies one base, which has no binding ability with the paired base and cannot play a stable role.

In the above-mentioned universal closed sequence, the number of bases modified by LNA or BNA in the closed sequence of the left non-tag region and the closed sequence of the right non-tag region is generally considered to be the same as the amount of modified base data and the closed sequence of the left non-tag region or the right The sequence length of the closed sequence in the non-tag region is negatively correlated. Longer sequences require fewer bases to be modified, while shorter sequences require more bases to be modified. However, in this application, the inventors found that for the closed sequence of the non-tag region of a specific length, the number of bases modified by LNA or BNA in the closed sequence of the left non-tag region or the closed sequence of the right non-tag region is When the length is 5 to 10 bases, the blocking sequence has the strongest binding ability to the target linker. When it is less than 5 bases, the binding to the target linker is unstable, which makes the capture efficiency low.

In addition, when using the above-mentioned universal blocking sequence for library capture, the total amount of the library to be captured and the amount of the added universal blocking sequence should also match. Shape structure, which leads to non-specific capture and reduces capture efficiency. For example, when the added amount of the universal blocking sequence is 2.4 μg, calculated based on 500 ng per library, a total of 12 libraries are hybridized, that is, when the total amount is 6 μg, the hybridization capture effect is better than the capture effect of hybridization of 14-16 libraries. Of course, when each library is less than 500ng, such as 400ng, 2.4μg of universal blocking sequence and hybridization of 15 libraries at the same time have the highest capture efficiency.

Regarding the double-ended index adapter of the MGI platform, this application also provides a universal closed sequence that can inhibit the capture of the double-ended Index sequencing library of the MGI sequencing platform. In a preferred embodiment of the present application, the P1 (5+7 modified) end blocking sequence is SEQ ID NO: 3:

The universal blocking sequences provided by the above two preferred embodiments not only increase the number of modified bases to improve the binding ability with the target linker, but also the specific positions of the modified bases on the above universal blocking sequences are also modified compared to In the case of bases in other positions, the binding ability to the target linker is strong. That is to say, the above-mentioned preferred universal blocking sequence has the best blocking effect on the target linker, and the capture efficiency of the target library is the highest during hybridization and capture.

On the basis of the aforementioned various improved universal blocking sequences, in a second exemplary embodiment of the present application, a capture kit is provided, which includes any of the aforementioned universal blocking sequences. The universal blocking sequence in the capture kit has strong binding ability to the target linker, and when used for the capture library construction, it can achieve efficient capture of the target library.

In order to further improve the capture efficiency of the target library (ie, the target rate), in a preferred embodiment, the working concentration of the universal capture probe in the above kit is 0.4-0.8 μg universal blocking sequence/1 μg of the library to be captured. The capture according to the above dosage can further avoid the formation of a cross-star closure due to the excessive amount of the library, thereby reducing the target rate in the library. The above working concentration can be different according to the specific blocking scheme. For example, when the blocking schemes of SEQ ID NO: 5 and SEQ ID NO: 6 of this application are adopted, the working concentration is 0.4 μg general blocking sequence/1 μg to be captured The library is captured, and the target rate of the target library is higher. When the blocking schemes of SEQ ID NO: 3 and SEQ ID NO: 4 of the present application are adopted, the target library for capturing the target library according to the working concentration of 0.8 μg universal blocking sequence/1 μg library to be captured is relatively high.

In a third exemplary embodiment of the present application, a library hybridization capture method is also provided. The method includes using a capture kit to capture the library to be captured, and the capture kit uses the above-mentioned capture kit. The blocking sequence in the capture kit can achieve efficient capture of the target library when the capture library is constructed.

The inventors also found that when the molar ratio of the universal blocking sequence to the library to be captured is 10:1-20:1, the blocking effect is better. Therefore, in a preferred embodiment of the present application, in the step of using the capture kit to capture the library to be captured, the blocking sequence and the library to be captured are blocked in a molar ratio of 10:1-20:1.

In a fourth exemplary embodiment of the present application, a method for constructing a library is provided. The method for constructing a library includes: constructing a fragmented library; hybridizing and capturing the fragmented library to obtain a capture library; and performing PCR amplification on the capture library , To obtain a sequencing library; use the above-mentioned capture kit for hybridization capture, or use any of the above-mentioned methods for hybridization capture. In the library constructed by the library construction method of the present application, the target library accounts for a relatively high proportion, and the effective data produced by the library accounts for a high proportion.

The following will further illustrate the beneficial effects of the present application in conjunction with specific embodiments.

It should be noted that the following examples are performed using ^{the library construction process provided by the NadPrep TM} DNA Library Construction Kit (for MGI) (201909 Version 2.0) (Naonda (Nanjing) Biotechnology Co., Ltd.). It should also be noted that the following embodiments are only exemplary descriptions, and do not limit the method of the present application to only the following methods. The specific process is briefly described as follows:

DNA sample fragmentation---end repair and A addition---adapter ligation---fragment screening---PCR amplification---library purification, quantification and quality inspection---after sequencing or targeted capture using the MGI platform Sequencing.

Example 1 How much modification and the general blocking scheme of adding concentration difference

Steps: Library construction was carried out with reference to the instructions of NadPrep ^TM DNA Library Construction Kit (for MGI) (201909 Version 2.0). The steps of hybridization and capture are carried out as follows. When multi-library hybridization and hybridization are performed after vacuum concentration, the specific hybridization library mixing steps are as follows:

Table 1:

组分Component	总文库量Total library volume	数量quantity
总文库Total library	6μg6μg	1～121～12
Human Cot DNAHuman Cot DNA	5μl5μl	//
通用封闭序列(下面列出序列)Universal closed sequence (sequences listed below)	2μl2μl	//

1) The application's improved MGI connector double-ended Index universal closed sequence

1.1 The P1 end blocking sequence shown in SEQ ID NO: 3 and the P2 end closing sequence shown in SEQ ID NO: 4.

CTCTCA+GTACG+TCA+GCA+GT+T10XXXXXXXXXXCA+ACTCCT+TGGC+TCACAGA+ACGA+CATGG+CTACGATC+CGACTT/3SpC3/;

GCA+TGGC+GA+CCTT+ATCA+GXXXXXXXXXXTTGTCTT+CCTA+AGA+CCGC+TTG+GCC+TCCGA+CTT/3SpC3/

1.2 SEQ ID NO: 5: P1 end blocking sequence shown in SEQ ID NO: 6 P2 end blocking sequence

CTC+TCA+GT+ACG+TCA+GCA+GT+TXXXXXXXXXXCA+ACTCCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CTAC+GATC+CGACTT/3SpC3/;

GCA+TG+GC+GA+CC+TT+ATCA+GXXXXXXXXXXTTG+TCTT+CCTA+AGA+CC+GC+TTG+GCC+TCC+GA+CTT/3SpC3/

In the above four sequences, X represents C3 Spacer/hypoxanthine, +N represents LNA or BNA modified base (the two modification effects are equivalent, taking LNA modification as an example), and /3SpC3/ represents the 3'C3 spacer arm closure.

The general blocking sequence and concentration table put in the hybridization process are as follows:

Table 2:

2) The specific hybridization capture steps are as follows:

1. Mix the components in a 0.2/1.5ml low-adsorption centrifuge tube according to the above table, vortex to mix, and centrifuge immediately.

2. Put the centrifuge tube into a vacuum concentrator preheated to 60℃ for drying.

3. After all the liquid has evaporated and dried completely, seal the centrifuge tube for later use.

4. Take out

Exome Research Panel v2.0 melts naturally on ice, and aliquoted in small quantities as needed after use.

5. Prepare the hybridization reaction solution according to Table 3 below, mix it with a pipette and add it to the bottom of the centrifuge tube that has been vacuum concentrated and dry, use the pipette to gently pipette and mix 15-20 times, centrifuge briefly, and incubate at 25°C 5 ~10min.

table 3:

6. Vortex and mix the hybridization reaction mixture. After instant centrifugation, transfer all 17μl of the hybridization reaction mixture in the centrifuge tube to a new 0.2ml PCR tube, centrifuge briefly, put it in the PCR machine, and start the following hybridization program :

Table 4:

7. Hybrid library elution

(1) Preparation

1. Take out

The other reagents in the Hybridization and Wash Kit are naturally thawed at room temperature and mixed evenly by vortexing.

2. Dynabeads ^TM M-270 Streptavidin Beads are vortexed and mixed uniformly, and the streptavidin magnetic beads can be cleaned and captured only after 30 minutes of equilibration at room temperature.

(2) Reagent preparation

1. Preparation of elution buffer

Prepare 1X working solution of elution buffer according to the following system:

table 5:

组分名称Component name	Rnase-free水Rnase-free water	缓冲液Buffer	总计total
2X磁珠洗脱缓冲液2X magnetic bead elution buffer	160μl160μl	160μl160μl	320μl320μl
10X洗脱缓冲液I10X Elution Buffer I	252μl252μl	28μl28μl	280μl280μl
10X洗脱缓冲液II10X Elution Buffer II	144μl144μl	16μl16μl	160μl160μl
10X洗脱缓冲液III10X Elution Buffer III	144μl144μl	16μl16μl	160μl160μl
10X Stringent洗脱缓冲液10X Stringent Elution Buffer	288μl288μl	32μl32μl	320μl320μl

2. Preparation of magnetic bead suspension, as shown in Table 6.

Table 6:

(3) Avidin magnetic beads cleaning

1. ^{Vortex and mix Dynabeads TM} M-270 Streptavidin Beads for 15 seconds to ensure complete mixing. Pipette 50μl M270 magnetic beads into a 1.5ml low adsorption centrifuge tube.

2. Add 100μl 1X Bead Wash Buffer to the centrifuge tube, gently pipette and mix 10 times, centrifuge briefly, and place it on a magnetic stand for a few minutes. When the liquid is completely clear, use a pipette to remove the supernatant. Remove the centrifuge tube from the magnetic stand.

3. Repeat step 2 twice.

4. Add 17μl magnetic bead suspension to the centrifuge tube, gently pipette and mix, and transfer all the magnetic bead suspension to a new 0.2ml low-adsorption PCR tube.

(4) Streptavidin magnetic bead capture

1. After the 16h hybridization reaction, adjust the PCR instrument to enter the elution program.

2. Add the resuspended streptavidin magnetic beads to the hybridization system, and use a pipette to gently pipette or vortex to mix.

3. Incubate at 65°C for 45 minutes, vortex gently every 10-12 minutes to ensure that the magnetic beads are completely resuspended.

(5) Thermal elution (note: the thermal elution process should be operated quickly; try to avoid bubbles during the blowing and mixing process)

1. After the incubation, remove the PCR tube from the PCR machine, and add 100μl 65°C 1X Wash Buffer I to it, and mix the hybridization system containing magnetic beads by pipetting and mixing.

2. Place the PCR tube on the magnetic stand for 1 min. After the liquid is completely clarified, use a pipette to remove and discard the supernatant.

3. Remove the PCR tube from the magnetic stand, add 150μl 65°C 1X Stringent Wash Buffer, gently pipette 10 times to mix evenly, put it in the PCR machine and incubate at 65°C for 5 minutes.

4. Repeat steps 2 and 3 once.

(6) Elution at room temperature

1. Centrifuge the PCR tube briefly and place it on the magnetic stand for 1 min. After the liquid is completely clear, pipette and discard the supernatant, add 150μl 1X Wash Buffer I at room temperature, vortex to mix, incubate at room temperature for 2 min, vortex and mix for 30 seconds during this time Let stand for 30 seconds, alternately, to ensure sufficient mixing.

2. Centrifuge the PCR tube briefly and place it on the magnetic stand for 1 min. After the liquid is completely clear, pipette and discard the supernatant, add 150μl of room temperature 1X Wash Buffer II, vortex and mix, incubate at room temperature for 2 minutes, vortex and mix for 30 seconds. Let stand for 30 seconds, alternately, to ensure sufficient mixing.

3. Centrifuge the PCR tube briefly and place it on a magnetic stand for 1 min. After the liquid is completely clear, pipette and discard the supernatant, add 150μl room temperature 1X Wash Buffer III, vortex to mix, incubate at room temperature for 2 minutes, vortex and mix for 30 seconds. Let stand for 30 seconds, alternately, to ensure sufficient mixing.

4. Centrifuge the PCR tube briefly and place it on the magnetic stand for 1 min. After the liquid is completely clear, pipette and discard the supernatant, then use a 10μl tip to remove a small amount of residual Buffer.

5. Remove the PCR tube from the magnetic stand, add 22.5μl Nuclease Free Water, use a pipette to gently pipette 10 times to ensure uniform mixing, and transfer all the liquid to a new 0.2ml PCR tube.

The subsequent PCR amplification and library purification and quantification steps can be performed in accordance with the instructions of the NadPrep ^TM DNA Library Construction Kit (for MGI) (201909 Version 2.0).

The test in this example found that when universal bases are used in the Index area, the number of base modifications in the fixed regions at both ends and the use concentration of the universal blocking sequence have a greater impact on the final blocking effect, and fewer modified bases (5+ 7) To achieve the best blocking effect, you need to add 200μmol/L (the ratio of the blocking sequence to the library is 20:1); the more modified bases (7+10) are 100μmol/L to achieve the best effect, reaching 200μmol/ Inhibition has been produced at L. As shown in Figure 5, it may be that the local area has strong binding ability. When too many universal closed sequences are added, the chance of collision in the reaction system increases, thus forming an abnormal cross star structure (as shown in the figure). Shown in 4B).

Example 2

The steps of Example 2 are the same as those of Example 1, with the only difference being that the number of modified bases of the universal blocking sequence used is different. In this embodiment, the number of modified bases of the universal blocking sequence is shown in the following table:

Table 7:

封闭组合Closed combination	4+6修饰封闭(封闭方案3)4+6 modified blocking (blocking scheme 3)	8+11修饰组合(封闭方案4)8+11 modification combination (closed solution 4)
浓度(μmol/L)Concentration (μmol/L)	200200	100100

In the blocking scheme 3, the P1 sequence remains unchanged (SEQ ID NO: P1 end blocking sequence shown in SEQ ID NO: 3,

CTCTCA+GTACG+TCA+GCA+GT+T10XXXXXXXXXXCA+ACTCCT+TGGC+TCACAGA+ACGA+CATGG+CTACGATC+CGACTT/3SpC3/), the sequence of P2 is: SEQ ID NO: 7

GCA+TGGC+GA+CCTT+ATCAGXXXXXXXXXXTTGTCTT+CCTA+AGA+CCGC+TTG+GCCTCCGA+CTT/3SpC3/.

In the blocking scheme 4, the P1 blocking sequence is SEQ ID NO: 8

CTC+TCA+GT+ACG+TCA+G+CA+GT+TXXXXXXXXXXCA+ACTCCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CTAC+GATC+CGA+CTT/3SpC3/; P2 blocking sequence is SEQ ID NO :9

GCA+TG+GC+GA+CC+TT+AT+CA+GXXXXXXXXXXTTG+TCTT+CCTA+AGA+CC+GC+TTG+GCC+TC+C+GA+CTT/3SpC3/.

Scheme 3 is to leave the modification of P1 unchanged on the basis of scheme 1, and reduce the number of blocked modified bases at both ends of the P2 tag by one, and the number of modifications is 4+6; scheme 4 is in scheme 3 Basically, add a block modification base to the universal block at both ends of the tag. When there are 4 blocking modified bases on the left side of the tag sequence and 6 on the right side, the blocking effect is obviously inferior to the 5+7 combination scheme of Scheme 1. At the same time, the 8+11 modification method of Scheme 4 is also less effective than the 7+10 scheme of Scheme 2, and the result is shown in Figure 6. Therefore, the closed sequence of the left non-tag region includes 5-7 modified bases, and the closed sequence of the right non-tag region includes 7-10 modified bases is a better solution.

Example 3

The steps of Example 3 are the same as those of Example 1. The number of modified bases of the universal blocking sequence is also the same as that of Scheme 2 of Example 1. The only difference is that the positions of modified bases of the universal blocking sequence are different. The specific sequence is as follows:

Blocking scheme 5: P1 blocking sequence is SEQ ID NO: 10

CTC+T+CA+GT+ACG+TCA+GCA+GTTXXXXXXXXXXCAACTCCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CTAC+GATC+CGA+CTT/3SpC3/, the P2 blocking sequence is SEQ ID NO: 11

G+CA+TG+GC+GA+CC+TT+ATCAGXXXXXXXXXXTTGTCTT+CCTA+AGA+CC+GC+TTG+GCC+TC+C+GA+CTT/3SpC3/.

Blocking scheme 6: P1 blocking sequence is SEQ ID NO: 12

CTCTCA+GT+ACG+TCA+G+CA+GT+TXXXXXXXXXXCA+ACT+CCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CTAC+GATCCGACTT/3SpC3/; P2 blocking sequence is SEQ ID NO: 13

GCATG+GC+GA+CC+TT+AT+CA+GXXXXXXXXXXTTG+TCTT+C+CTA+AGA+CC+GC+TTG+GCC+TCC+GACTT/3SpC3/.

The closed scheme 5 changed the position of the modification based on the same number of modifications as scheme 2. The scheme 5 reduced one modification at each end and increased the modification at a position closer to the middle label; the closed scheme 6 is just the opposite. The closed modified bases at both ends are increased, and the modifications closer to the middle tag are reduced. The general blocking protocol 5, protocol 6 and protocol 2 used the blocking sequence at a concentration of 100 μmol/L, and hybridized with the same input amount of the library. As a result, it is found that the effects of Scheme 5 and Scheme 2 are close, and the effect of Scheme 6 is worse, as shown in Figure 7, which shows that not only the number of modified bases affects the blocking effect, but also the modified position has an impact on the blocking. The present invention finds balanced modification Adding modification to the region closer to the middle tag sequence will be significantly better than increasing the number of modifications at the two ends.

Example 4 Multiple library hybridization test

The experimental procedures for library construction and hybridization are the same as in Example 1. The universal blocking selection is 7+10 base modifications, and the concentration is 100 μmol/L. The difference is that multiple libraries are mixed and hybridized. The specific library input quantity and total library input are as follows:

Table 8.

投入文库数量(500ng/文库)Number of input libraries (500ng/library)	10个10	12个12	14个14	16个16
文库投入量总量Total amount of library input	5μg5μg	6μg6μg	7μg7μg	8μg8μg

When the input amount for hybridization capture is 500ng/library, the sequencing indicators perform better. If a single hybridization can allow more input, that is, more samples in a single hybridization will reduce the cost of hybridization capture for each sample. . The test of the universal closed sequence in this application found that when no more than 12 samples are hybridized together in a single time, the hybridization indicators perform well, and when the number of hybridized libraries reaches 14 and 16 samples, the hit rate will have a certain degree Decline. As shown in Figure 8, the total input of 14 libraries and 16 libraries is 7μg and 8μg, respectively. In a fixed capture system, as the number of libraries increases, some libraries have the opportunity to form a cross between two libraries and universal closure. The star structure is closed, thereby reducing the hit rate.

In summary, the present invention designs a universal blocking sequence for the hybridization of the double-ended Index library of the MGI platform, by replacing the variable region of the Index with universal bases, and adding the fixed sequence on both sides of the index to increase the annealing temperature. Base modification can improve the capture efficiency of the double-ended index library. Furthermore, this application also finds that the blocking effect of the universal blocking sequence and the number of modified bases that increase the annealing temperature are controlled at 5-7 on the left and 7-10 on the right, the blocking effect of the closed sequence is better. . In addition, it has also been found that if the specific positions of the modified bases are further optimized for the preferred positions of the examples of this application, the blocking effect is the best. Correspondingly, the concentration of the universal blocking sequence also affects the target rate of the target library. When the amount of the universal blocking sequence is 0.4-0.8 μg, the application can support 12 samples to be hybridized together at the same time, which greatly reduces the hybridization and capture cost of a single sample.

The above are only preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

A universal closed sequence, characterized in that the universal closed sequence includes a left non-tag region closed sequence, a middle label region closed sequence, and a right non-tag region closed sequence sequentially connected in a direction from 5'to 3',

Wherein, the closed sequence of the left non-tag region includes 5-7 bases modified by LAN or BNA, the closed sequence of the middle label region is a universal closed base sequence, and the closed sequence of the right non-tag region includes 7~ 10 LAN or BNA modified bases, and the 3'end of the right non-tag region closed sequence has a closed modification.
The universal blocking sequence according to claim 1, wherein the blocking modification at the 3'end is MGB modification, C3 spacer modification, phosphorylation modification, digoxigenin modification or biotin modification, or the 3' The'terminal base is a dideoxy base.
The universal blocking sequence of claim 1, wherein the universal blocking base is hypoxanthine or a C3 spacer.
The universal closed sequence according to any one of claims 1 to 3, wherein the universal closed sequence is a closed sequence of a P1 linker with a first tag sequence of an MGI sequencing platform or a sequence with a second tag The closed sequence of the P2 linker, where,

The blocking sequence of the P1 linker is SEQ ID NO: 3:

CTCTCA+GTACG+TCA+GCA+GT+TXXXXXXXXXXCA+ACTCCT+TGGC+TCACAGA+ACGA+CATGG+CTACGATC+CGACTT/3SpC3/; where + means LAN or BNA modification, X means hypoxanthine or C3 spacer, /3SpC3/is Modification of the C3 spacer at the 3'end;

The blocking sequence of the P2 linker is SEQ ID NO: 4:

GCA+TGGC+GA+CCTT+ATCA+GXXXXXXXXXXXTTGTCTT+CCTA+AGA+CCGC+TTG+GCC+TCCGA+CTT/3SpC3/; where + means LAN or BNA modification, X means hypoxanthine or C3 spacer, /3SpC3/ is The C3 spacer at the 3'end is modified.
The universal closed sequence according to any one of claims 1 to 3, wherein the universal closed sequence is a closed sequence of a P1 linker with a first tag sequence of an MGI sequencing platform or a sequence with a second tag The closed sequence of the P2 linker;

The blocking sequence of the P1 linker is SEQ ID NO: 5:

CTC+TCA+GT+ACG+TCA+GCA+GT+TXXXXXXXXXXCA+ACTCCT+TGGC+TCAC+AGA+ACGA+CAT+GG+CTAC+GATC+CGACTT/3SpC3/; where + means LAN or BNA modification, X means times Xanthine or C3 spacer, /3SpC3/ is the modification of the C3 spacer at the 3'end;

The blocking sequence of the P2 linker SEQ ID NO: 6:

GCA+TG+GC+GA+CC+TT+ATCA+GXXXXXXXXXXTTG+TCTT+CCTA+AGA+CC+GC+TTG+GCC+TCC+GA+CTT/3SpC3/; where + means LAN or BNA modification, X means times Xanthine or C3 spacer, /3SpC3/ is the C3 spacer modification at the 3'end.
A capture kit comprising a universal blocking sequence, characterized in that the universal blocking sequence is the universal blocking sequence according to any one of claims 1 to 5.
The kit according to claim 6, wherein the working concentration of the universal capture probe in the kit is 0.4-0.8 μg of the universal blocking sequence/1 μg of the library to be captured.
A method for library hybridization capture, said method comprising using a capture kit to capture the library to be captured, characterized in that said capture kit uses the capture kit of claim 6 or 7.
The method according to claim 8, wherein the universal blocking sequence and the library to be captured are blocked at a molar ratio of 10:1 to 20:1.
A method for building a database, the method for building a database includes:

Construction of fragmented library;

Hybridize and capture the fragmented library to obtain a capture library;

Performing PCR amplification on the capture library to obtain a sequencing library;

It is characterized in that the capture kit according to claim 6 or 7 is used for the hybrid capture, or the method according to claim 9 or 10 is used for the hybrid capture.