Nucleic acid sequencing method and nucleic acid sequencing kit

Abstract

Provided are a nucleic acid sequencing method and a nucleic acid sequencing kit. The kit comprises a nucleic acid probe, a ligase, dNTP having a blocking group attached to a 3′ end, a polymerase, a reagent 1 for excising the blocking group attached to the 3′ end of the dNTP, and a reagent 2 for excising the remaining nucleotides on the nucleic acid probe that are not bound to a to-be-tested base group.

Claims

1. A method for sequencing nucleic acid, comprising the following steps: (1) hybridizing a sequencing primer to a nucleic acid molecule to be tested; (2-1) ligating a nucleic acid probe to the sequencing primer; (3) detecting a detectable label being bound to the nucleic acid probe of the nucleic acid molecule to be tested, and obtaining information of a base to be tested; (4-1) ligating a dNTP having a blocking group ligated to the 3′-terminal thereof to a sequencing primer to which a nucleic acid probe is not ligated; (5) detecting a detectable label being bound to the nucleic acid probe of the nucleic acid molecule to be tested, and obtaining information of a base to be tested; (6-1) excising the blocking group ligated to the 3′-terminal of the dNTP; wherein, when the step (3) exists, the step (5) does not exist; or when the step (5) exists, the step (3) does not exist; wherein the nucleic acid probe comprises a first moiety, a second moiety, a linker and a detectable label, wherein: the first moiety has a sequencing base, which is one or more selected from the group consisting of A, T, U, C and G, the second moiety has random bases and/or universal bases, and the number of the bases is at least 3, the first moiety is ligated to the second moiety via the linker, and the ligation between the first moiety and the linker is cleavable, and the detectable label is ligated to the second moiety or the linker.

2. The method for sequencing nucleic acid according to claim 1, wherein between the step (2-1) and the step (3), the following step is further comprised: (2-2) eluting nucleic acid probes that are not ligated to the nucleic acid molecule to be tested.

3. The method for sequencing nucleic acid according to claim 1, wherein between the step (4-1) and the step (5), the following step is further comprised: (4-2) eluting dNTPs that are not ligated to the nucleic acid molecule to be tested.

4. The method for sequencing nucleic acid according to claim 1, wherein after the step (6-1), the following step is further comprised: (6-2) eluting a portion that is excised in the step (6-1).

5. The method for sequencing nucleic acid according to claim 1, wherein in the step (2-1), the time for the ligating is 0.5 to 30 minutes.

6. The method for sequencing nucleic acid according to claim 1, wherein in the step (2-1), the temperature for the ligating is an appropriate temperature at which the ligase being used works.

7. The method for sequencing nucleic acid according to claim 1, wherein: between the step (2-1) and the step (3), the following step is further comprised: (2-2) eluting nucleic acid probes that are not ligated to the nucleic acid molecule to be tested; between the step (4-1) and the step (5), the following step is further comprised: (4-2) eluting dNTPs that are not ligated to the nucleic acid molecule to be tested; after the step (6-1), the following step is further comprised: (6-2) eluting a portion that is excised in the step (6-1); and the eluting conditions of any two of the steps (2-2), (4-2) and (6-2) are the same, or the eluting conditions of the three steps are the same.

8. The method for sequencing nucleic acid according to claim 1, wherein in the step (4-1), the dNTP used has no detectable label.

9. The method for sequencing nucleic acid according to claim 1, wherein in the step (4-1), the dNTP is ligated by a polymerase-catalyzed polymerization reaction to the sequencing primer that is not ligated to the nucleic acid probe.

10. The method for sequencing nucleic acid according to claim 1, wherein in the step (6-1), conditions for excising the blocking group ligated to the 3′-terminal of the dNTP and the conditions for excising the remaining nucleotides on the nucleic acid probe that are not bound to the base to be tested are the same.

11. The method for sequencing nucleic acid according to claim 1, wherein the first moiety is located at the 5′-terminal or the 3′-terminal.

12. The method for sequencing nucleic acid according to claim 1, wherein the bases of the second moiety are 3 to 15 bases.

13. The method for sequencing nucleic acid according to claim 1, wherein the detectable label is a fluorophore; and the detectable label is ligated to the second moiety.

14. The method for sequencing nucleic acid according to claim 1, wherein the linker does not contain a sulfur atom.

15. The method for sequencing nucleic acid according to claim 1, wherein in the step (4-1), the blocking group is selected from the following Formula A to Formula D: ##STR00024## wherein: in Formula A, R.sub.1 and R.sub.2 are independently selected from the group consisting of H, F, CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2R alkane, COOR and CONHR, wherein R is independently C.sub.1-C.sub.6 alkyl; in Formula B, R.sub.3 to R.sub.7 are independently selected from the group consisting of H and C.sub.1-C.sub.6 alkyl; in formula C, R.sub.8 and R.sub.9 are independently selected from the group consisting of H, F, Cl, CF.sub.3, nitro, cyano, C.sub.1-C.sub.6 alkoxy and C.sub.1-C.sub.6 carboxyl; and in Formula D, R.sub.10 is selected from C.sub.1-C.sub.6 alkyl.

16. A kit for use in the method for sequencing nucleic acid according to claim 1, comprising: a nucleic acid probe, a ligase, a dNTP having a blocking group ligated to the 3′-terminal thereof, and a polymerase; Reagent 1, which is used for excising the blocking group ligated to the 3′-terminal of dNTP; Reagent 2, which is used for excising the remaining nucleotides on the nucleic acid probe that are not bound to the base to be tested; wherein Reagent 1 and Reagent 2 are the same; and wherein the kit further comprises an appropriate buffer for dissolving the nucleic acid probe; Eluent 1 for eluting the nucleic acid probe, and Eluent 2 for eluting dNTP; wherein the nucleic acid probe comprises a first moiety, a second moiety, a linker and a detectable label, wherein: the first moiety has a sequencing base, which is one or more selected from the group consisting of A, T, U, C and G, the second moiety has random bases and/or universal bases, and the number of the bases is at least 3, the first moiety is ligated to the second moiety via the linker, and the ligation between the first moiety and the linker is cleavable, and the detectable label is ligated to the second moiety or to the linker.

17. The kit according to claim 16, wherein the first moiety is located at the 5′-terminal or the 3′-terminal.

18. The kit according to claim 16, wherein the bases of the second moiety are 3 to 15 bases.

19. The kit according to claim 16, wherein the detectable label is a fluorophore; and the detectable label is ligated to the second moiety.

20. The kit according to claim 16, wherein the linker does not contain a sulfur atom; wherein the linker is selected from the group represented by the following Formula IV to Formula IX: ##STR00025## wherein, in Formula IV, R.sup.1 is selected from the group consisting of H, OH, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl; R.sup.2 is selected from the group consisting of H, OH, F, Cl, and Br.

21. The kit according to claim 16, wherein the ligase is a DNA ligase, and the polymerase is a DNA polymerase.

22. The kit according to claim 21, which is characterized by any one or more of the following items 1) to 4): 1) The DNA ligase is one or more selected from the group consisting of T4 DNA ligase, T7 DNA ligase, and T3 DNA ligase; 2) The concentration of the nucleic acid probe is 0.1 μM to 5 μM; 3) The concentration of the DNA ligase is 0.01 μM to 2 μM; and 4) the reagents in the kit are free of silver ions.

23. The kit according to claim 16, wherein Reagent 1 or Reagent 2 is an endonuclease, an organic phosphine, or a complex of PdCl.sub.2 and sulfonated triphenylphosphine.

24. The method for sequencing nucleic acid according to claim 1, wherein the above steps (2-1) to (5) are repeated, or the above steps (2-1) to (6-1) are repeated.

25. The method for sequencing nucleic acid according to claim 1, wherein in the step (6-1), further excising remaining nucleotides on the nucleic acid probe that are not bound to the base to be tested.

26. The method for sequencing nucleic acid according to claim 4, wherein the above steps (2-1) to (6-2) are repeated.

27. The method for sequencing nucleic acid according to claim 9, wherein the polymerization reaction is performed for a time of 0.5 to 10 minutes; at a temperature of 45° C. to 60° C.

28. The method for sequencing nucleic acid according to claim 13, wherein the detectable label is at least one selected from the group consisting of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647 and AF688.

29. The method for sequencing nucleic acid according to claim 1, wherein the linker is selected from the group represented by the following Formula IV to Formula IX: ##STR00026## wherein, in Formula IV, R.sup.1 is selected from the group consisting of H, OH, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, and C.sub.2-C.sub.6 alkynyl; R.sup.2 is selected from the group consisting of H, OH, F, Cl, and Br.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Schematic structural diagram of the reversibly blocked dNTP used the in sequencing-by-synthesis method.

(2) FIG. 2: Schematic diagram of excision chemical reaction in the sequencing-by-synthesis method.

(3) FIG. 3: After the excision reaction in the sequencing-by-synthesis method, the scar atoms left by dNTP on the sequencing chain are reversibly blocked.

(4) FIG. 4: Schematic diagram of AP site-containing probes removed by endonuclease IV.

(5) FIG. 5: The fluorescence signal value of the 4th base (4 types) after 64 NNN sequences carrying scar. Wherein: the ordinate represents the fluorescence signal value, the four groups of signal peak groups in the figure, from left to right, respectively represent the set of fluorescence signal values when the 4th base is A, C, G or T, and each group of signal peak groups contains 64 fluorescence signal values; the abscissa represents the permutation and combination of 64 NNN sequences when the 4th base is A, C, G, or T, there are a total of 64×4=256 permutations and combinations, of which 64 NNN sequences are shown in Table 2 as follows.

(6) FIG. 6: The fluorescence signal value of the 4th base (4 types) after 64 NNN sequences without scar for the DNA in natural state. Wherein: the ordinate represents the fluorescence signal value, the four groups of signal peak groups in the figure, from left to right, respectively represent the set of fluorescence signal values when the 4th base is A, C, G or T, and each group of signal peak groups contains 64 fluorescence signal values; the abscissa represents the permutation and combination of 64 NNN sequences when the 4th base is A, C, G, or T, there are a total of 64×4=256 permutations and combinations, of which 64 NNN sequences are shown in Table 2 as follows.

SPECIFIC MODELS FOR CARRYING OUT THE PRESENT INVENTION

(7) The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that are commercially available.

(8) The nucleic acid probes or modified nucleotide used in the following examples can be synthesized according to the methods known in the art, and unless otherwise specified, they are synthesized by a commissioned commercial company, such as Heya Medical Technology (Shanghai) Co., Ltd. or Biotech Biotechnology (Shanghai) Co., Ltd.

Example 1: Sequencing Application of AP Site Reversible Ligation Nucleic Acid Probe (3 Random Bases+3 Universal Bases) in Combination with Phosphate-Blocked Nucleotide (1)

(9) 1. Instruments and Reagents

(10) The instrument was based on a BGISEQ-500 platform. Theoretically, other sequencing platforms (such as Illumina's Hiseq platform, etc.) could be appropriately adjusted to perform the same or similar experiments as in this example.

(11) In addition, in order to enable the application on the BGISEQ-500 platform, the selected modified dye had absorption and emission wavelengths similar to those of the dye used by the BGISEQ-500 reagent, so that it could be well detected by the BGISEQ-500 optical system.

(12) Some of the reagents used in this experiment were completely the same as those of BGISEQ-500, for example, the photographic buffer reagent and elution buffer 2 used in this experiment.

(13) Some reagents used in this experiment were different from those of BGISEQ-500, including: a ligation solution containing “the probes, enzymes and buffers of this example”, the excision buffer solution of this experiment was used to replace the excision buffer solution of BGISEQ-500; specific components were shown in the Part 2 as follows.

(14) Excision reagent buffer: including 50 mM Tris-HCl, 0.1M NaCl, 2 mM MgSO.sub.4, 2 mM DTT, 1 wt % Tween-80, 0.5 wt % tert-butanol, pH 7.5, 0.5 μM endo IV (NEB: M0304S).

(15) Polymerization buffer for the phosphate-blocked nucleotide was as follows: pH 7.8, containing 20 mM sodium chloride, 3 mM magnesium sulfate, 50 mM Tris, 5% DMSO, 0.05% Tween-20, and 0.2 μM KOD156 DNA polymerase.

(16) The experimental sample was the genomic DNA of E. coli, which was a standard sample carried by BGISEQ-500.

(17) According to the manufacturer's instructions, a library for sequencing was prepared by using MGIeasy™ DNA library preparation kit (Shenzhen Huada Zhizao Technology Co., Ltd.) to extract DNA from E. coli standard strains as raw materials, and it was loaded on the sequencing chip.

(18) 2. Synthesis of Probes and Phosphate-Blocked Nucleotide

(19) Shenggong Bioengineering (Shanghai) Co., Ltd. was commissioned to synthesize the following 4 groups of AP site reversible ligation probes (3 random bases+3 universal bases), and mychemlab was commissioned to synthesize 4 phosphate-blocked nucleotides.

(20) The first group of probes (A probes): the first moiety, i.e., the sequencing base was A, and the second moiety was 3 random bases+3 universal bases.

(21) ##STR00009##

(22) The second group of probes (T probes): the first moiety, i.e., the sequencing base was T, and the second moiety was 3 random bases+3 universal bases.

(23) ##STR00010##

(24) The third group probes (C probes): the first moiety, i.e., the sequencing base was C, and the second moiety was 3 random bases+3 universal bases.

(25) ##STR00011##

(26) The fourth group probes (G probes): the first moiety, i.e., the sequencing base was G, and the second moiety was 3 random bases+3 universal bases.

(27) ##STR00012##

(28) The 4 phosphate-blocked nucleotides were as follows:

(29) ##STR00013##

(30) The above 4 groups of probes and T4 DNA ligase were dissolved in the following buffer:

(31) 50 mM CH.sub.3COOK, 20 mM Tris, 10 mM Mg(CH.sub.3COO).sub.2, 100 μg/ml BSA, 1 mM ATP, 10% PEG6000; to obtain the ligation solution. The concentration of the probe in the ligation solution was 1 μM, in which the molar ratio of A probes:T probes:C probes:G probes was approximately 1:4:4:1. The DNA ligase concentration in the ligation solution was 0.5 μM.

(32) 3. Sequencing Steps

(33) (1) Referring to the instruction manual of BGISEQ-500, the following preliminary preparations were performed: library construction, a DNA single-stranded loop was amplified into DNA nanospheres, the DNA nanospheres were loaded on the chip carried by BGISEQ-500, and the sequencing primer was loaded on the DNA nanospheres.

(34) (2) The ligation solution containing the above four kinds of probes, T4 DNA ligase and buffer solution was added by using an instrument, and the ligation reaction was performed at 25° C. for 4 minutes;

(35) (3) The elution reagent 2 was used to elute the probes that were not ligated;

(36) (4) The polymerization reaction solution containing the phosphate-blocked nucleotide, polymerase and buffer solution was added, and the polymerization reaction was performed at 55° C. for 2 minutes;

(37) (5) The elution reagent 2 was used to elute unreacted reversibly blocked nucleotides;

(38) (6) The photographic buffer for image acquisition (photographing) was added; the base information of each DNA nanosphere site was analyzed by software;

(39) (7) The endonuclease IV (New England Biolabs, article number M0304L) and its buffer solution were added, and reaction was performed at 37° C. for 5 minutes to excise the AP site and reversibly blocked group,

(40) (8) The elution reagent 2 was added to elute the excised portion of the probe and the reversibly blocked group;

(41) The four groups of probes could be added repeatedly for sequencing in the next cycle.

(42) 4. Experimental Results

(43) The result was completely consistent with the sequence of the standard sample carried by BGISEQ-500, indicating that the sequencing method of the present invention was accurate.

Example 2: Sequencing Application of AP Site Reversible Ligation Nucleic Acid Probe (3 Random Bases+3 Universal Bases) in Combination with Phosphate-Blocked Nucleotide (2)

(44) This example was performed by following the same method as Example 1, except that the sequencing steps were as follows:

(45) (1) Referring to the instruction manual of BGISEQ-500, the following preliminary preparations were performed: library construction, a DNA single-stranded loop was amplified into DNA nanospheres, the DNA nanospheres were loaded on the chip carried by BGISEQ-500, and the sequencing primer was loaded on the DNA nanospheres.

(46) (2) The ligation solution containing the above four kinds of probes, T4 DNA ligase and buffer solution was added by using instrument, and the ligation reaction was performed at 25° C. for 4 minutes;

(47) (3) The elution reagent 2 was used to elute the probes that were not ligated;

(48) (4) The photographic buffer for image acquisition (photographing) was added; the base information of each DNA nanosphere site was analyzed by software;

(49) (5) The polymerization reaction solution containing the phosphate-blocked nucleotide, polymerase and buffer solution was added, and the polymerization reaction was performed at 55° C. for 2 minutes;

(50) (6) The elution reagent 2 was used to elute unreacted reversibly blocked nucleotides;

(51) (7) The endonuclease IV (New England Biolabs, article number M0304L) and its buffer solution were added, and reaction was performed at 37° C. for 5 minutes to excise the AP site and reversibly blocked group,

(52) (8) The elution reagent 2 was added to elute the excised portion of the probe and the reversibly blocked group;

(53) The four groups of probes could be added repeatedly to perform the sequencing in the next cycle.

(54) The result was completely consistent with the sequence of the standard sample carried by BGISEQ-500, indicating that the sequencing method of the present invention was accurate.

Example 3: Sequencing Application of AP Site Reversible Ligation Nucleic Acid Probe (3 Random Bases+3 Universal Bases) in Combination with Phosphate-Blocked Nucleotide (3)

(55) This example was performed by following the same method as Example 1, except that the operation of step (2) was as follows:

(56) The ligation solution containing the above four kinds of probes, T4 DNA ligase and buffer solution was added by using instrument, and the ligation reaction was performed at 25° C. for 1 minute.

(57) The result was completely consistent with the sequence of the standard sample carried by BGISEQ-500, indicating that the sequencing method of the present invention was accurate.

Example 4: Sequencing Application of AP Site Reversible Ligation Nucleic Acid Probe (3 Random Bases+3 Universal Bases) in Combination with Phosphate-Blocked Nucleotide (4)

(58) This example was performed by following the same method as Example 1, except that the operation of step (2) was as follows:

(59) The ligation solution containing the above four kinds of probes, T4 DNA ligase and buffer solution was added by using instrument, and the ligation reaction was performed at 25° C. for 10 minutes.

(60) The result was completely consistent with the sequence of the standard sample carried by BGISEQ-500, indicating that the sequencing method of the present invention was accurate.

Example 5: Sequencing Application of Azido-Ligated Nucleic Acid Probe in Combination with Azidomethylene Blocked Nucleotide

(61) This example was performed by following the same method as Example 1, except that the following azido-ligated nucleic acid probe and azidomethylene blocked nucleotide were used:

(62) Four groups of azido-ligated nucleic acid probes:

(63) The first group (A probes): the first moiety, i.e., the sequencing base was A, and the second moiety was 6 random bases.

(64) ##STR00014##

(65) The second group (T probes): the first moiety, i.e., the sequencing base was T, and the second moiety was 6 random bases.

(66) ##STR00015##

(67) The third group (C probes): the first moiety, i.e, the sequencing base was C, and the second moiety was 6 random bases.

(68) ##STR00016##

(69) The fourth group (G probes): the first moiety, i.e., the sequencing base was G, and the second moiety was 6 random bases.

(70) ##STR00017##

(71) Four azidomethylene blocked nucleotides:

(72) ##STR00018##

(73) The azido-blocked nucleotide polymerization buffer contained 20 mM sodium chloride, 60 mM ammonium sulfate, 3 mM magnesium sulfate, 50 mM Tris, 5% DMSO, 0.05% Tween-20, and the reaction temperature was 55° C., 0.2 μM 9° N DNA polymerase; pH was 9.0.

(74) The excision reagent buffer contained 50 mM Tris-HCl, 1 M NaCl, 10 mM THPP; pH was 9.0.

(75) The results were analyzed using the sequencing analysis software of BGISEQ-500, as shown in Table 1 below.

(76) TABLE-US-00001 TABLE 1 Analysis of sequencing results Reference Genome (Reference) E. coli Number of cycles (Cycle Number) 50 Photographing area (number of areas) 384 Total reads (Total Reads) 184.72 M Mapped reads (Mapped Reads) 160.15 M .sup.a Q30 80.84% Lagging phase (Lag) 0.34% Leading phase (Runon) 0.36% Effective reads ratio (ESR) 79.51% Mapping rate (Mapping Rate) 86.7% .sup.b Error rate 1.13%

(77) a, Q30 indicated the probability of a base being mismeasured was 0.1%, that was, the accuracy was 99.9%; Q30 was 80.84%, which means that the accuracy of 80.84% of the base call reached 99.9%.

(78) b, represented an average error rate.

(79) It could be seen from the above table that the method of the present invention had a Q30 of 80.84%, the error rate was only 1.13%, and the reads reached at least 50, which was better than the existing sequencing-by-ligation method.

Example 6: Sequencing Application of AP Site Reversible Ligation Probe (6 Random Bases) in Combination with Phosphate-Blocked Nucleotide

(80) This example was performed by following the same method as Example 1, except that the following 4 groups of nucleic acid probes were used:

(81) Four groups of AP site reversible ligation probes (x=6) were as follows:

(82) The first group (A probes): the first moiety, i.e., the sequencing base was A, and the second moiety was 6 random bases.

(83) ##STR00019##

(84) The second group (T probes): the first moiety, i.e., the sequencing base was T, and the second moiety was 6 random bases.

(85) ##STR00020##

(86) The third group (C probes): the first moiety, i.e., the sequencing base was C, and the second moiety was 6 random bases.

(87) ##STR00021##

(88) The fourth group (G probes): the first moiety, i.e., the sequencing base was G, and the second moiety was 6 random bases.

(89) ##STR00022##

(90) The result was completely consistent with the sequence of the standard sample carried by BGISEQ-500, indicating that the sequencing method of the present invention was accurate.

Example 7: Sequencing Application of AP Site Reversible Ligation Probe (7 Random Bases) in Combination with Phosphate-Blocked Nucleotide

(91) This example was performed by following the same method as Example 1, except that the 4 groups of AP site reversible ligation probes were used, wherein x=7.

(92) The result was completely consistent with the sequence of the standard sample carried by BGISEQ-500, indicating that the sequencing method of the present invention was accurate.

Example 8: Sequencing Application of AP Site Reversible Ligation Probe (8 Random Bases) in Combination with Phosphate-Blocked Nucleotide

(93) This example was performed by following the same method as Example 1, except that the 4 groups of AP site reversible ligation probes were used, wherein x=8.

(94) The result was completely consistent with the sequence of the standard sample carried by BGISEQ-500, indicating that the sequencing method of the present invention was accurate.

Example 9: Sequencing Application of AP Site Reversible Ligation Probe (9 Random Bases) in Combination with Phosphate-Blocked Nucleotide

(95) This example was performed by following the same method as Example 1, except that the 4 groups of AP site reversible ligation probes were used, wherein x=9.

(96) The result was completely consistent with the sequence of the standard sample carried by BGISEQ-500, indicating that the sequencing method of the present invention was accurate.

Experimental Example 1: Effect of Scar on Sequencing

(97) 1. Experimental Instruments and Reagents

(98) The four azidomethylene blocked nucleotides were the same as in Example 5.

(99) The four reversibly blocked non-fluorescent nucleotides were as follows (purchased from mychem):

(100) ##STR00023##

(101) The experimental sample was the genomic DNA of E. coli, which was a standard sample carried by BGISEQ-500.

(102) The azido-blocked nucleotide polymerization buffer contained 20 mM sodium chloride, 60 mM ammonium sulfate, 3 mM magnesium sulfate, 50 mM Tris, 5% DMSO, 0.05% Tween-20, and the reaction temperature was 55° C., 0.2 μM 9° N (the name of a DNA polymerase) DNA polymerase; pH was 9.0.

(103) The excision reagent buffer contained 50 mM Tris-HCl, 1 M NaCl, 10 mM THPP; pH was 9.0.

(104) 2. Experimental Method

(105) According to the manufacturer's instructions, a library for sequencing was prepared by extracting DNA from E. coli standard strain as raw material using MGIeasy™ DNA library preparation kit (Shenzhen Huada Zhizao Technology Co., Ltd.); referring to the BGISEQ-500 DNB preparation loading kit (Shenzhen Huada Zhizao Technology Co., Ltd., article number 85-05531-00), the prepared DNBs (DNA nanoballs) were loaded onto the sequencing chip of the BGISEQ-500 platform. The sequencing was performed using the BGISEQ-500 sequencing kit (SE50 V3.0, Shenzhen Huada Zhizao Technology Co., Ltd., article number PF-UM-PEV30). The specific sequencing steps were as follows:

(106) Using the BGISEQ-500 platform, the same DNBs were loaded onto both sides of the chip, 4 cycles of sequencing was performed to the DNA random sequence on both sides, and the sequence at each position and its corresponding number were numbered, and then 100% formamide was used to elute the sequencing chain, and sequencing primer was reloaded; at one side the polymerization and excision of 3 cycles of fluorescently modified, reversibly blocked nucleotides (scar was left after excision) were performed, while at the other side, the polymerization and excision of 3 cycles of non-fluorescent, reversibly blocked nucleotides (there was no DNA at natural state after excision) were performed, and then at both sides, the polymerization of one cycle of fluorescently modified, reversibly blocked nucleotides was performed.

(107) Combining the sequence information of the previous 4 cycles, the distribution of the fluorescence signal of the 4th base after different NNN sequences was analyzed: FIG. 5 and FIG. 6 showed the signal effect of the scar atom modified bases on the next base, and the effect of natural nucleic acid pairs on the next base information, wherein the 64 permutations and combinations of NNN sequences were shown in Table 2 as below.

(108) TABLE-US-00002 TABLE 2 64 permutations and combinations of 3 bases 1 AAA 2 AAC 3 AAG 4 AAT 5 ACA 6 ACC 7 ACG 8 ACT 9 AGA 10 AGC 11 AGG 12 AGT 13 ATA 14 ATC 15 ATG 16 ATT 17 CAA 18 CAC 19 CAG 20 CAT 21 CCA 22 CCC 23 CCG 24 CCT 25 CGA 26 CGC 27 CGG 28 CGT 29 CTA 30 CTC 31 CTG 32 CTT 33 GAA 34 GAC 35 GAG 36 GAT 37 GCA 38 GCC 39 GCG 40 GCT 41 GGA 42 GGC 43 GGG 44 GGT 45 GTA 46 GTC 47 GTG 48 GTT 49 TAA 50 TAC 51 TAG 52 TAT 53 TCA 54 TCC 55 TCG 56 TCT 57 TGA 58 TGC 59 TGG 60 TGT 61 TTA 62 TTC 63 TTG 64 TTT

(109) The results showed that the DNA at natural state had little relationship with the previous NNN sequences, and there was no particularly strong fluctuation; however, the signals with scar had a very strong relationship with the NNN sequences, especially for the signals when the 4th base was A base, for example the first group of signal peak groups from the left as shown in FIG. 5 and the first group of signal peak groups from the left as shown in FIG. 6, some NNN sequences could cause the loss of about 70%.

(110) The results also showed that the scar had an effect on the next base, especially when the 3′ base in the NNN sequence was a G base, the scar made the next base signal very weak, so that the base recognition of the next base was prone to making mistake, especially when the A base was after G, it was more prone to making mistakes.

(111) Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that according to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.