CN116121352A - Method for improving double-end sequencing quality, chip and application thereof - Google Patents

Abstract

The invention relates to a chip for double-end sequencing, wherein three primers, namely P5, P7 and P5a, are fixed on the chip, the P5 and the P7 respectively have one enzyme cutting site which is one of ideoxyU and i8 oxyG and is different from each other, and the P5a is a nucleotide sequence which does not have an enzyme cutting site and has the same composition as P5 base. When the sequencing kit containing the chip for double-end sequencing is used for sequencing PE150 and above, the initial sequencing signal intensity of double-end sequencing of Read1 and Read2 can be effectively balanced, and the Q30 of double-end sequencing can be improved, so that a method for improving the quality of double-end sequencing is obtained.

Description

Method for improving double-end sequencing quality, chip and application thereof

Technical Field

The invention belongs to the technical field of biology, relates to a high-throughput sequencing technology, and in particular relates to a method and a chip for improving double-end sequencing quality and application thereof.

Background

In recent years, high-throughput sequencing technology (Next Generation Sequencing, NGS) has gradually entered into people's daily life as an important technology for molecular diagnostics, which plays an important role in personalized medicine, genetic diseases, clinical diagnostics, and the like. Compared with the traditional nucleic acid detection means, the high-throughput sequencing technology has obvious advantages, can simultaneously identify the base sequences of millions of DNA, and has the characteristics of high speed, low cost, high accuracy and the like. Double-end sequencing is an important sequencing method for high-throughput sequencing, can increase sequencing read length and sequencing data volume, improves the accuracy of a result ratio, and plays an important role in De-novo assembly, detection of chromosome structural variation and the like. There are various double-end sequencing procedures and methods in the market at present, for example, one of the double-end sequencing methods of illumine company is to fix two primers P5 and P7 on the surface, which can be complementarily paired with the library linker, and the library is bridge amplified on the chip by the two primers P5 and P7 to obtain DNA cluster (cluster), thereby playing the role of signal amplification. Wherein, the two primers P5 and P7 respectively have two enzyme cutting sites of the oxyU and the i8 oxyG, for example, when 1 chain is sequenced, all the oxyU at the P5 end of the surface needs to be cracked by using USER enzyme, only the chain growing out of P7 is left, and SBS sequencing can be carried out after hybridization of the sequencing primer 1. After the 1-chain sequencing is completed, the 1-chain sequencing chain is denatured and eluted by formamide, the 3' -end of the surface P5 is dephosphorylated (namely, end repair) by phosphokinase, then the 2-chain is generated by taking the P5 as a primer, the i8oxodG on the P7 chain is cracked by FPG enzyme, only the P5 regrown chain is left, and the 2-chain sequencing can be performed after hybridization of the sequencing primer 2 (patent US7754429B2, US10457985B 2). The method is easy to be influenced by a plurality of links of two-chain generation, so that the signal-to-noise ratio of 2-chain signals is lower than that of 1-chain signals, and the quality of 2-chain sequencing is generally lower than that of 1-chain signals. For example, the Huada gene BGISEQ platform utilizes the characteristic that the USER enzyme can make uracil position generate single nucleotide gap, part dUTP is introduced in the 1-chain synthesis process, 1-chain is fragmented by the USER enzyme cutting, the binding capacity of 1-chain and template chain is effectively reduced, then 1-chain is eluted and digested to reduce the influence of 1-chain on two-chain sequencing quality, and DNB double-end sequencing data quality and reading length are effectively improved (patent CN 108070642B). Usually, the sequencing quality of double-end sequencing is increased along with the sequencing read length and is mainly influenced by factors such as accumulation of base lag reaction, reduction of sequencing fluorescent signal intensity and the like, the sequencing quality of double-end sequencing is gradually reduced, particularly when the read length of PE150, PE200, PE250 and the like or the insert fragment is longer, the sequencing quality of the double-end sequencing is obviously reduced, and the sequencing quality difference of both 1 chain and 2 chain is also obviously different along with the increase of the read length. Therefore, it is important to improve the quality of double-ended sequencing.

Disclosure of Invention

Based on this, it is an object of the present invention to provide a method for improving the quality of double ended sequencing.

The method comprises the following technical scheme.

In a first aspect of the present invention, there is provided a chip for double-ended sequencing, wherein three primers, namely P5, P7 and P5a, are immobilized on the chip, wherein P5 and P7 respectively carry different cleavage sites in ideoxyU and i8 oxyG, and P5a is a nucleotide sequence which does not carry cleavage sites and has the same composition as P5 base.

In a second aspect, the invention provides the use of the chip in double-ended sequencing, and a double-ended sequencing kit comprising the chip.

In a third aspect of the invention, a method for improving the quality of double ended sequencing is provided.

A method of improving the quality of double ended sequencing comprising the steps of:

preparing a chip: preparing three primers P5, P5a and P7 in a certain proportion, wherein the 5' end of each primer is provided with a-DBCO group and can react with a macromolecule with an azide group on a glass chip with silanized surface, fixing the primers on the glass chip, and cleaning to obtain a sequencing chip;

surface amplification and sequencing were performed.

In a fourth aspect of the invention, there is provided a method of double ended sequencing comprising the steps of:

s1, obtaining the sequencing chip;

s2, obtaining a library to be amplified, diluting, and carrying out denaturation on a 2pM sample loading solution;

s3, loading the sequencing chip and the 2pM library loading liquid onto a sequencing system to amplify and double-end PE sequencing.

According to the invention, when the sequencing chip is prepared, a proper proportion of P5a is doped when the conventional primers P5 and P7 are fixed, and when the chip is used for double-end sequencing, the invention discovers that the initial sequencing signal intensity of double-end sequencing of Read1 and Read2 can be effectively balanced, and the Q30 (sequencing quality) of double-end sequencing of PE150 and above can be improved.

Drawings

FIG. 1a and FIG. 1b show PE sequencing when P5a is mixed.

FIG. 2 is a schematic diagram of the results of comparison of PE150 sequencing Q30 in example 1.

FIG. 3 is a schematic representation of the results of a comparison of example 2PE200 sequencing Q30.

FIG. 4 is a schematic representation of the results of comparison of PE250 sequencing Q30 of example 3.

FIG. 5 is a schematic representation of the results of a comparison of PE200 sequencing Q30 according to embodiment 4.

FIG. 6 is a schematic representation of the results of a comparison of PE200 sequencing Q30 according to embodiment 5.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental methods, in which specific conditions are not noted in the examples below, are generally carried out according to conventional conditions, for example, green and Sambrook-s.A.fourth edition, molecular cloning, A.laboratory Manual (Molecular Cloning: A Laboratory Manual), published in 2013, or according to the conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

To facilitate an understanding of the present technology, some terms and phrases are defined below.

Double-ended sequencing: the amplified-end sequencing is performed by adding a linker to both ends of the DNA fragment, performing a first sequencing, washing off the template strand, amplifying the module in situ, and performing a second sequencing round.

PE: pair End, double ended sequencing.

Read length: the length of reads obtained by sequencing can be used to reflect the length of the sequence that can be measured.

Q30: representing the percentage of bases with a mass value of 30.

In some embodiments, the invention relates to a chip for double-ended sequencing, on which three primers P5, P7 and P5a are immobilized, wherein P5 and P7 respectively have different enzyme digestion sites in the ideoxyU and i8 oxyG, and P5a has no enzyme digestion site and has the same nucleotide sequence with the P5 base composition as the conventional chip.

In some of these embodiments, the P5a is present in a ratio of 5% to 25%, preferably 10% to 20%, of the total of P5 and P5 a.

In some of these embodiments, the ratio of the total amount of P5 and P5a to the fixed amount of P7 is 0.9-2:0.9:2, preferably: 0.9-1.1:0.9-1.1, further preferably 1:1.

in some of these embodiments, the base sequence of P5 is shown in SEQ ID NO. 1; the base sequence of P7 is shown as SEQ ID NO. 2.

The chip is applied to double-end sequencing, can effectively balance the initial sequencing signal intensity of double-end sequencing of Read1 and Read2, and can improve the Q30 of double-end sequencing of PE150 and above.

In some embodiments, the kit for double-ended sequencing comprises the chip for double-ended sequencing; conventionally, the linker sequence corresponding to the sequence of the P5 primer and/or the P7 primer is also included.

In some embodiments, the kit further involves components such as a common polymerase, a denaturing agent, and various common buffers.

In some embodiments of the present invention, there is also provided a method of improving the quality of double-ended sequencing, comprising the steps of:

preparing a chip: preparing three primers P5, P5a and P7 in a certain proportion, wherein the 5' end of each primer is provided with a-DBCO group and can react with a macromolecule with an azide group on a glass chip with silanized surface, fixing the primers on the glass chip, and cleaning to obtain a sequencing chip;

the sequencing chip is used for the next surface amplification and sequencing according to the conventional method.

The 5' ends of all 3 primers carry-DBCO groups.

In some embodiments of the invention, there is also provided a method of double-ended sequencing comprising the steps of:

the sequencing chip is prepared, namely, a proper amount of P5a is fixed on a glass chip besides the P5 and P7 primers;

obtaining a library to be amplified, diluting, and carrying out denaturation to obtain a 2pM loading liquid;

loading the sequencing chip and the 2pM library loading liquid onto a sequencing system, and performing amplification and double-end PE sequencing by the system according to the following sequence after setting parameters: amplification-1 chain template strand cleavage-1 chain sequencing-elution-surface P5 3' end dephosphorylation-2 chain amplification-2 chain template strand cleavage-2 chain sequencing.

The PE read length in the parameters is 150-300, preferably 150-250, and can be 150, 200, 250, 300, etc.

The present invention will be described in further detail with reference to specific examples.

The sequencing chip in the following examples was prepared according to the conventional method, and the preparation steps thereof were as follows: cleaning the surface of the glass raw material; 2) Surface silanization; 3) And (3) packaging a chip: packaging the silanized surface into a flow cell (flow cell), wherein the flow cell is provided with 2,3,4 or a plurality of flow channels according to experimental requirements; 4) Oligonucleotide primer immobilization: preparing three oligonucleotide primers P5, P5a and P7 in a certain proportion, wherein the 5' ends of the 3 primers are provided with-DBCO groups and are used for reacting with macromolecules with azide groups so as to fix the probe primers on a sequencing chip, and cleaning the chip for later use.

Wherein, the base composition of P5 and P7 is as follows, and when in use, the 5' is added with polyT, and the polyT can be 6-10T.

The base sequence of P5 is 5'-AATGATACGGCGACCACCGAATCTACAC-3' (SEQ ID NO. 1)

The base sequence of P7 is 5'-CAAGCAGAAGACGGCAACGAGAT-3' (SEQ ID NO. 2). Surface P5, P5a, P7 oligonucleotide probe sequences used in examples 1-4 below:

p5 sequence 5'-TTTTTTTTAATGATACGGCGACCACCGA/i8 oxadG/ATCTACC-3' P5a sequence 5'-TTTTTTTTAATGATACGGCGACCACCGAGATCTACAC-3'

P7 sequence 5 '-TTTTTTTTCAAGCAGAAGACGGCA/ideoxyU/ACGAGAGAT-3'

Surface P5, P5a, P7 oligonucleotide probe sequences used in example 5 below:

p5 sequence 5'-TTTTTTTTAATGATACGGCGACCACCGAGATC/ideoxyU/ACAC-3' P5a sequence 5'-TTTTTTTTAATGATACGGCGACCACCGAGATCTACAC-3'

P7 sequence 5'-TTTTTTTTCAAGCAGAAGACGGCATAC/i8oxodG/AGAT-3'.

The oligonucleotide primer is immobilized: the three oligonucleotide primers P5, P5a and P7 are prepared in a certain proportion, and in the experiment, the concentration of 3 primers can be carried out in various proportions (such as 1:1:1,2:1:1,2:1:2,3:1:3 and the like). The 5' ends of the 3 primers carry-DBCO.

Base composition of linker sequence in the following examples:

i5:AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T(SEQ ID NO.3)；

i7: CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC T ((SEQ ID No. 4)) represents a phosphorothioate modification.

Example 1

1) On the chip surface grafted with the polymer with azide group: two proportions of three oligonucleotide mix primers P5, P5a, and P7 were prepared with 3xssc solution. P5: p5a: the molar concentration ratio of the P7 primer is as follows: the flow channels 1-3 are experimental groups with different mixing P5 a/(P5+P5a) of 10%,20% and 40% in sequence; the flow channel 4 is a control group;

flow passage	P5 concentration (uM)	P5a concentration (uM)	P7 concentration (uM)
				1	4.5	0.5	5
2	4	1	5
				3	3	2	5
4	5	0	5

2) The prepared primer is introduced into a corresponding flow channel, and the reaction is carried out for 4 hours by heating in a 55 ℃ oven;

3) Washing each flow channel by using a 3xssc solution to obtain a sequencing chip a;

4) E.coli ATCC8739 was used as the library to be amplified with a length of 550bp, and the library was prepared by a commercially available library-building kit (e.g., nuo Wei Zan VAHTS Universal Pro DNA Library Prep Kit for Illumina, # ND608-01; VAHTS Multiplex Oligos Set 4/5for Illumina, # N321/N322-01) were obtained by library construction, and the corresponding linker sequences contained in the library were compatible with the second generation sequencing platforms of illumine, MGI, etc.), diluted to 2nM, and then alkaline denatured at room temperature for 3 min (0.1M NaOH solution as the base);

5) Preparing a 2pM loading solution by using a 3XSSC buffer solution after denaturation, and loading the prepared sequencing chip a and the 2pM library loading solution after denaturation dilution onto a salus pro sequencing system;

6) Setting parameters to perform PE150 amplification sequencing;

7) Referring to FIG. 1a, after the library has been surface amplified to double strand, to hybridize to sequencing primer 1 for read1 sequencing (1 strand), either P5 or P5a strand Shen Lian complementary to the P7 strand is excised, leaving a separate P7 strand Shen Lian with a 1 strand sequencing primer hybridization site on the P7 strand. Since the P5a primer does not have the cleavage site of i8oxodG, the P5a extension strand cannot be excised and remains, and part of the P7 extension strand will hybridize to P5a in read1 sequencing to continue to form a double strand without participating in sequencing. The sequencing result of the flow channel read1 with P5a shows that the signal is lower than that of the control (without P5 a). The results in Table 2a show that when P5 a/(P5+P5a) was 10%,20% and 40% in this order, i.e., P7 was unchanged, and the content of incorporated P5a was increased, the 1-strand sequencing signals were lower than those of the control (flow channel 4) and decreased with the increase in the ratio. With reference to FIG. 1b, after 1-strand sequencing, the excised P5 is repaired and P5 after the repair is used as a primer to regenerate P5 extension Shen Lian as 2-strand, and as P5a cannot be excised, the extension strand directly serves as 2-strand, so that the link of 2-strand regeneration is avoided. Complete excision of P7 extension Shen Lian (P7 primer with cleavage site ideoxyU) prior to 2 strand sequencing leaves only the regenerated P5 extension and the unresectable P5a extension hybridized to sequencing primer 2 for read2 sequencing. Therefore, the channel containing P5a, the 2-strand sequencing signal was higher than the control.

In the results of tables 2a, 2b and FIG. 2, when the ratio of P5 a/(P5+P5a) was 10% and 20%, the sequencing signal of the 1-strand PE150 was slightly lower than that of the control, and the effect on the 1-strand Q30 was insignificant, and the 2-strand signal was slightly higher than that of the control, so that the 2-strand Q30 was improved. When the ratio P5 a/(P5+P5a) reaches 40%, 1 chain Q30 is already affected due to the excessively low 1 chain signal. PE150 reads the sequencing Q30 of long lower runners 1,2, 4, and is slightly better mixed with low content of P5a, and runner 3 influences sequencing Q30 because high content of P5a influences 1-chain sequencing signals.

TABLE 1a example 1PE150 sequencing initiation Signal

TABLE 1b example 1PE150 sequencing Q30

Example 2

1) On the surface of the polymer chip grafted with the azide group: the mixed primers of the three oligonucleotide probes P5, P5a and P7 in two proportions are prepared by using a 3xssc solution, wherein the total concentration of P5 and P5a is 8uM, and the concentration of P7 is 8uM. P5: p5a: the molar concentration ratio of the P7 primer is as follows: the flow channels 1-3 are experimental groups with different mixing P5 a/(P5+P5a) of 10%,15% and 40% in sequence; the flow channel 4 is a control group without P5a;

flow passage	P5 concentration (uM)	P5a concentration (uM)	P7 concentration (uM)
				1	7.2	0.8	8
2	6.8	1.2	8
				3	4.8	3.2	8
4	8	0	8

2) The prepared primer is introduced into a corresponding flow channel, and the reaction is carried out for 4 hours by heating in a 55 ℃ oven;

3) Washing each flow channel by using a 3xssc solution to obtain a sequencing chip b;

4) The library to be amplified (E.coli ATCC8739 as in example 1 above, 550bp in length, library containing adaptor-compatible second generation sequencing platforms such as illumine and MGI, etc.) was diluted to 2nM and then alkaline denatured at room temperature for 3 min (0.1M NaOH solution as the base);

5) Preparing a 2pM loading solution by using a 3XSSC buffer solution after denaturation, and loading the prepared sequencing chip b and the 2pM library loading solution after denaturation dilution onto a salus pro sequencing system;

6) Setting parameters to perform PE200 amplification sequencing;

7) Referring to FIG. 1a, after the library has been surface amplified to double strand, to hybridize to sequencing primer 1 for read1 sequencing (1 strand), either P5 or P5a strand Shen Lian complementary to the P7 strand is excised, leaving a separate P7 strand Shen Lian with a 1 strand sequencing primer hybridization site on the P7 strand. Since the P5a primer does not have the cleavage site of i8oxodG, the P5a extension strand cannot be excised and remains, and part of the P7 extension strand will hybridize to P5a in read1 sequencing to continue to form a double strand without participating in sequencing. The sequencing result of the flow channel read1 with P5a shows that the signal is lower than that of the control (without P5 a). The results in Table 3a show that when P5 a/(P5+P5a) was 10%,15% and 40% in this order, i.e., P7 was unchanged, and the content of incorporated P5a was increased, the 1-strand sequencing signals were lower than those of the control (flow channel 4) and decreased with the increase in the ratio. With reference to FIG. 1b, after 1-strand sequencing, the excised P5 is repaired and P5 after the repair is used as a primer to regenerate P5 extension Shen Lian as 2-strand, and as P5a cannot be excised, the extension strand directly serves as 2-strand, so that the link of 2-strand regeneration is avoided. Complete excision of P7 extension Shen Lian (P7 primer with cleavage site ideoxyU) prior to 2 strand sequencing leaves only the regenerated P5 extension and the unresectable P5a extension hybridized to sequencing primer 2 for read2 sequencing. Therefore, the channel containing P5a, the 2-strand sequencing signal was higher than the control. From a combination of the results in tables 2a, 2b and FIG. 3, when the total concentration of P5 and P5a was increased to 8uM and the concentration of P7 was increased to 8uM, the ratio of P5 a/(P5+P5a) was 10% and 15%, the 1-strand PE200 sequencing signal was slightly lower than the control, and the effect on 1-strand Q30 was insignificant, and the 2-strand signal was slightly higher than the control, which was advantageous for increasing 2-strand Q30. When the ratio P5 a/(P5+P5a) reaches 40%, 1 chain Q30 is already affected due to the excessively low 1 chain signal. The sequencing Q30 of the PE200 read long lower flow channels 1,2 and 4 is compared, the low-content P5a is better than the control without the P5a, but the flow channel 3 influences the sequencing Q30 due to the fact that the high-content P5a influences the 1-chain sequencing signal, and the P5a content is controlled to have a remarkable promoting effect on the PE200 read long under a certain proportion.

TABLE 2a example 2PE200 sequencing initiation Signal

TABLE 2b example 2PE200 sequencing Q30

Example 3

1) On the surface of the polymer chip grafted with the azide group: two proportions of three oligonucleotide probe mix primers P5, P5a, and P7 were prepared with 3xssc solution. P5: p5a: the molar concentration ratio of the P7 primer is as follows: the flow channels 1-3 are experimental groups with different mixing P5 a/(P5+P5a) of 10%,20% and 30% in sequence; the flow channel 4 is a control group;

flow passage	P5 concentration (uM)	P5a concentration (uM)	P7 concentration (uM)
				1	4.5	0.5	5
2	4	1	5
				3	3.5	1.5	5
4	5	0	5

2) The prepared primer is introduced into a corresponding flow channel, and the reaction is carried out for 4 hours by heating in a 55 ℃ oven;

3) Washing each flow channel by using a 3xssc solution to obtain a sequencing chip c;

4) The library to be amplified (E.coli ATCC8739 as in example 1, 550bp in length, containing the corresponding linker compatible with the second generation sequencing platforms such as illumine and MGI, etc.) was diluted to 2nM with TEbuffer and then alkaline denatured at room temperature for 3 min (0.1M NaOH solution as the base);

5) Preparing a 2pM loading solution by using a 3XSSC buffer solution after denaturation, and loading the prepared sequencing chip c and the 2pM library loading solution after denaturation dilution onto a salus pro sequencing system;

6) Setting parameters to perform PE250 amplification sequencing;

8) Referring to FIG. 1a, after the library has been surface amplified to double strand, to hybridize to sequencing primer 1 for read1 sequencing (1 strand), either P5 or P5a strand Shen Lian complementary to the P7 strand is excised, leaving a separate P7 strand Shen Lian with a 1 strand sequencing primer hybridization site on the P7 strand. Since the P5a primer does not have the cleavage site of i8oxodG, the P5a extension strand cannot be excised and remains, and part of the P7 extension strand will hybridize to P5a in read1 sequencing to continue to form a double strand without participating in sequencing. The sequencing result of the flow channel read1 with P5a shows that the signal is lower than that of the control (without P5 a). The results in Table 3a show that when P5 a/(P5+P5a) was 10%,20% and 30% in this order, i.e., P7 was unchanged, and the content of incorporated P5a was increased, the 1-strand sequencing signals were lower than those of the control (flow channel 4) and decreased with the increase in the ratio. With reference to FIG. 1b, after 1-strand sequencing, the excised P5 is repaired and P5 after the repair is used as a primer to regenerate P5 extension Shen Lian as 2-strand, and as P5a cannot be excised, the extension strand directly serves as 2-strand, so that the link of 2-strand regeneration is avoided. Complete excision of P7 extension Shen Lian (P7 primer with cleavage site ideoxyU) prior to 2 strand sequencing leaves only the regenerated P5 extension and the unresectable P5a extension hybridized to sequencing primer 2 for read2 sequencing. Therefore, the channel containing P5a, the 2-strand sequencing signal was higher than the control. In the results of tables 3a, 3b and FIG. 4, when the ratio of P5 a/(P5+P5a) was 10% and 20%, the sequencing signal of the 1-strand PE250 was slightly lower than that of the control, and the effect on the 1-strand Q30 was insignificant, and the 2-strand signal was slightly higher than that of the control, so that the 2-strand Q30 was improved. Whereas when the ratio of P5 a/(P5+P5a) is 30%, 1 chain Q30 is already affected due to the excessively low 1 chain signal. The sequencing Q30 of the PE250 read long lower flow channels 1,2 and 4 is compared, the low-content P5a is mixed with the sequencing Q30 to be more remarkably superior to the control without the P5a, but the flow channel 3 influences the sequencing Q30 due to the fact that the high-content P5a influences a 1-chain sequencing signal, and the control of the P5a content has more remarkable promoting effect on the PE250 read long under a certain proportion.

TABLE 3a example 3PE250 sequencing initiation Signal

TABLE 3b example 3PE250 sequencing Q30

To sum up, in examples 1,2 and 3, when mixed with a certain proportion (e.g., 20%) of P5a primer, the primers can regulate the 1-strand and 2-strand sequencing signals, balance the 1-strand and 2-strand sequencing Q30, and the improvement effect is more remarkable compared with the control group when the read length is increased in the sequencing from PE150 to PE 250. However, the P5a ratio needs to be controlled, for example, when the content reaches 30%, the PE sequencing is not positively affected.

Example 4

1) On the surface of the polymer chip grafted with the azide group: the mixed primers of the three oligonucleotide probes P5, P5a and P7 in two proportions are prepared by using a 3xssc solution, and the total concentration of P5 and P5a is set to be 10uM, and the concentration of P7 is set to be 10uM. P5: p5a: the molar concentration ratio of the P7 primer is as follows: the flow channels 1 and 3 are respectively experimental groups with different total concentrations of P5 and P5a and 20% of P5 a/(P5 + P5 a) mixed, and the flow channels 2 and 4 are control groups with different total concentrations of P5 and P5a;

flow passage	P5 concentration (uM)	P5a concentration (uM)	P7 concentration (uM)
				1	4	1	5
2	5	0	5
				3	8	2	10
4	10	0	10

2) The prepared primer is introduced into a corresponding flow channel, and the reaction is carried out for 4 hours by heating in a 55 ℃ oven;

3) Washing each flow channel by using a 3xssc solution to obtain a sequencing chip d;

4) The library to be amplified (E.coli ATCC8739 as in example 1 above, 550bp in length, library containing adaptor-compatible illumine, MGI, etc. second generation sequencing platforms) was diluted to 2nM and then alkaline denatured at room temperature for 3 min (0.1M NaOH solution as the base);

5) Preparing a 2pM loading solution by using a 3XSSC buffer solution after denaturation, and loading the prepared sequencing chip d and the 2pM library loading solution after denaturation dilution onto a salus pro sequencing system;

6) Setting parameters to perform PE200 amplification sequencing;

7) After the library has been surface amplified into double strands, to be able to hybridize to sequencing primer 1 for read1 sequencing (1 strand), either P5 or P5a extension Shen Lian complementary to the P7 extension strand needs to be excised, leaving a separate P7 extension Shen Lian with a 1 strand sequencing primer hybridization site on the P7 extension strand. Since the P5a primer does not have the cleavage site of i8oxodG, the P5a extension strand cannot be excised and remains, and part of the P7 extension strand will hybridize to P5a in read1 sequencing to continue to form a double strand without participating in sequencing. The sequencing result of the flow channel read1 with P5a shows that the signal is lower than that of the control (without P5 a). The results in tables 4a and 4b show that the total concentration of the surface primers P5 and P5a was lower than 10. Mu.M in response to a signal of 5. Mu.M and lower than 10. Mu.M in response to a signal of 5. Mu.M in response to a signal of 10. Mu.M, but that the experimental groups each mixed with 20% of P5a were superior to the corresponding control groups not mixed with P5a (see FIG. 5). Mixing proper amount of P5a under the conditions of different total P5 and P5a concentrations and different total P7 concentrations has an effect of promoting PE200 sequencing.

TABLE 4a example 4PE200 sequencing initiation Signal

TABLE 4b example 4PE200 sequencing Q30

Example 5

1) On the surface of the polymer chip grafted with the azide group: two proportions of three oligonucleotide probe mixed primers P5, P5a and P7 are prepared by using a 3xssc solution, wherein the sequence of the used primer is that P5 is provided with an ideoxyU enzyme cutting site, and P7 is provided with an i8 oxyG enzyme cutting site. P5: p5a: the molar concentration ratio of the P7 primer is as follows: the flow channels 1-3 are experimental groups mixed with 5% of P5 a/(P5+P5a) and 10% of 20% of different groups in sequence; the flow channel 4 is a control group;

flow passage	P5 concentration (uM)	P5a concentration (uM)	P7 concentration (uM)
				1	9.5	0.5	10
2	9	1	10
				3	8	2	10
4	10	0	10

2) The prepared primer is introduced into a corresponding flow channel, and the reaction is carried out for 4 hours by heating in a 55 ℃ oven;

3) Washing each flow channel by using a 3xssc solution to obtain a sequencing chip e;

4) The library to be amplified (E.coli ATCC8739 as in example 1 above, 550bp in length, library containing adaptor-compatible illumine, MGI, etc. second generation sequencing platforms) was diluted to 2nM and then alkaline denatured at room temperature for 3 min (0.1M NaOH solution as the base);

5) Preparing a 2pM loading solution by using a 3XSSC buffer solution after denaturation, and loading the prepared sequencing chip e and the 2pM library loading solution after denaturation dilution onto a salus pro sequencing system;

6) Setting parameters to perform PE200 amplification sequencing;

7) After the library has been surface amplified into double strands, to be able to hybridize to sequencing primer 1 for read1 sequencing (1 strand), either P5 or P5a extension Shen Lian complementary to the P7 extension strand needs to be excised, leaving a separate P7 extension Shen Lian with a 1 strand sequencing primer hybridization site on the P7 extension strand. The results in Table 5a show that when P5 a/(P5+P5a) was 5%,10% and 20% in this order, i.e., P7 was unchanged, and the content of incorporated P5a was increased, the 1-strand sequencing signals were lower than those of the control (flow channel 4) and decreased with the increase in the ratio. With reference to FIG. 1b, after 1-strand sequencing, the excised P5 is repaired and P5 after the repair is used as a primer to regenerate P5 extension Shen Lian as 2-strand, and as P5a cannot be excised, the extension strand directly serves as 2-strand, so that the link of 2-strand regeneration is avoided. Complete excision of P7 extension Shen Lian (P7 primer with cleavage site i8 oxodG) prior to 2 strand sequencing leaves only the regenerated P5 extension strand and the unresectable P5a extension strand hybridized to sequencing primer 2 for read2 sequencing. Therefore, the channel containing P5a, the 2-strand sequencing signal was higher than the control. In combination with the results of tables 5a, 5b and FIG. 6, the ratio of P5 a/(P5 + P5 a) was 5%,10% and 20%, the 1-strand PE200 sequencing signal was slightly lower than the control, and had no significant effect on the 1-strand Q30, the 2-strand signal was slightly higher than the control, which was advantageous for increasing the 2-strand Q30, and the ratio of P5 a/(P5 + P5 a) was 10% and 20% was slightly better than 5%.

TABLE 5a example 5PE200 sequencing initiation Signal

TABLE 5b implementation case 5PE200 sequencing Q30

The experimental results show that the P5 has the same positive effect of mixing a proper amount of P5a when the P5 is provided with the i8 oxydG enzyme cutting site and the P7 is provided with the ideoxyU enzyme cutting site, and has the effect of promoting the sequencing under the PE200 read length.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (12)

1. The chip for double-end sequencing is characterized in that three primers, namely P5, P7 and P5a, are fixed on the chip, wherein P5 and P7 respectively have one enzyme digestion site which is different from one enzyme digestion site in ideoxyU and i8 oxyG, and P5a is a nucleotide sequence which does not have an enzyme digestion site and has the same composition with P5 base.

2. Chip according to claim 1, characterized in that P5a is present in a proportion of 5% to 25%, preferably 10% to 20%, of the total of P5 and P5 a.

3. The chip of claim 1, wherein the ratio of the total amount of P5 and P5a to the amount of P7 is 0.9 to 2:0.9:2.

4. the chip of claim 3, wherein the ratio of the total amount of P5 and P5a to the amount of P7 is 0.9 to 1.1:0.9 to 1.1.

5. The chip of claim 1, wherein the base sequence of P5 is shown in SEQ ID No. 1; the base sequence of P7 is shown as SEQ ID NO. 2.

6. A kit for double-ended sequencing comprising the chip for double-ended sequencing according to any one of claims 1 to 5.

7. A method for improving the quality of double ended sequencing comprising the steps of:

preparing a chip: preparing three primers P5, P5a and P7 in a certain proportion, wherein the 5' end of each primer is provided with a-DBCO group and can react with a macromolecule with an azide group on a glass chip with a silanized surface, the P5 and the P7 are respectively provided with one enzyme cutting site which is one of ideoxyU and i8 oxyG and is different from each other, and the P5a is a nucleotide sequence which is provided with no enzyme cutting site and has the same composition as the P5 base;

fixing the primer on a glass chip, and cleaning to obtain a sequencing chip;

surface amplification and sequencing were performed.

8. The method for improving the quality of double-ended sequencing according to claim 7, characterized in that the P5a is present in a ratio of 5-25%, preferably 10-20%, of the total of P5 and P5 a.

9. The chip of claim 7, wherein the ratio of the total amount of P5 and P5a to the amount of P7 is 0.9 to 2:0.9:2.

10. the chip of claim 9, wherein the ratio of the total amount of P5 and P5a to the amount of P7 is 0.9 to 1.1:0.9 to 1.1.

11. A method of double ended sequencing comprising the steps of:

s1, obtaining the sequencing chip according to any one of claims 1 to 5;

s2, obtaining a library to be amplified, diluting, and carrying out denaturation on a 2pM sample loading solution;

s3, loading the sequencing chip and the 2pM library loading liquid onto a sequencing system, setting parameters, and performing amplification and double-end PE sequencing.

12. The method of double ended sequencing according to claim 11, wherein in step s3, the PE read length in the parameter is 150-300.

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