Application of cyanuric acid in sequencing

Abstract

The invention relates to the field of biological sequencing, in particular to application of cyanuric acid in sequencing. The invention provides an application of cyanuric acid in sequencing. The technical key point of the invention is as follows: in a DIPSEQ high-throughput sequencing cPAS biochemical reagent, by adding CA (CyanuricAcid), the stability of DNB, enzyme and azide groups can be promoted, the reaction efficiency can be improved, Lag / Runon can be reduced, and the sequencing quality can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biological sequencing, and in particular to the application of cyanuric acid in sequencing. Background Art

[0002] The second-generation DNA sequencing technology has the characteristics of large sequencing throughput, low cost, high degree of automation and single-molecule sequencing. It has been widely used in whole genome sequencing, transcriptome sequencing, metagenomic sequencing, etc. It is a powerful tool for analyzing the evolution and classification of organisms, studying genes related to diseases such as cancer and autism, and conducting in vitro diagnosis. It has promoted people's further understanding of life sciences and promoted the development of the health industry. The second-generation DNA sequencing technologies currently developed mainly involve combined probe anchor synthesis sequencing technology (Combinatorial Probe Anchor Synthesis, cPAS) including sequencing by ligation (SBL) technology, sequencing by synthesis (SBS) technology, and immersion sequencing (DIPSEQ) technology.

[0003] Immersion sequencing (DIPSEQ) technology is a second-generation sequencing technology with advantages such as high throughput, low cost, and automation. It uses cPAS and DNA nanoballs (DNB) as the core technology. DIPSEQ uses a temperature-stable water bath instead of a complex temperature control system, which is conducive to providing stability for biochemical reactions. In biochemical reactions, CPAS is still the core sequencing technology. Sequencing reagents are placed in the reagent tank according to the designed specific order. The reagent tank is fixed in the water bath. After reaching the required temperature, the chip is moved between different reagent tanks through the designed sequencing equipment to complete a complete sequencing cycle. This reaction method affects the DIPSEQ sequencing quality and read length because the reagents are always immersed in a constant high temperature, enzyme activity, azide group stability and other issues.

[0004] BGI-SEQ cPAS uses advanced DNA nanoball (DNB) and combined probe anchor polymerization (CPAS) sequencing core technology, using a special surface modified regular array chip, each modification site only fixes one DNA nanoball, the array chip modification site spacing is uniform, can ensure that the light signals of different nanoballs will not interfere with each other, thereby greatly improving the accuracy of signal processing. The sequencing reagents and fluorescent labeled probes with different luminescent wavelengths are pumped into the sequencing chip flow chamber through the instrument liquid system. Each DNA nanoball only binds to one fluorescent marker per sequencing cycle, and then the laser excites the fluorescent group to emit light. The light signals emitted by different fluorescent groups are collected by a scientific-grade CMOS camera. After that, the light signals on the regular array chip are processed and converted into digital signals by random software, and transmitted to the computer for processing, so that the base sequence information of the sample to be tested can be obtained.

[0005] In BGI-DIPSEQ technology, the immersion sequencing method is used, and a temperature-stable water bath is used instead of a complex temperature control system. On the one hand, it maintains a stable biochemical reaction environment, and on the other hand, it allows the reagents to fully contact the chip. Its biochemical reaction uses traditional BGI cPAS sequencing reagents and biochemical reaction principles and steps. A complete cycle includes the following: polymerization reaction reagents, used to extend the complementary chain of the DNA chain to be tested; elution reagents, which wash away the polymerization reaction reagents remaining in the previous step of the reaction; photographing reagents, which are used to protect DNA and fluorescent markers when taking pictures; regeneration reagents, which remove the blocking of dNTP at the end of the complementary chain so that the complementary chain can continue to extend; elution reagents: wash away the regeneration reagents remaining in the previous step of the reaction.

[0006] Based on the cPAS sequencing technology of the BGI SEQ-500 sequencing platform, the biochemical reaction part mainly adopts a flow channel solution, which pumps specific reagents from the refrigeration equipment into the corresponding flow tank at a specific position at a specific time point, and is assisted by sophisticated temperature control facilities to ensure that the biochemical reaction can be fully carried out in the shortest possible time. The reaction steps have been mentioned in the description of the DIPSEQ sequencing technology. However, due to the limitations of the optical system of the BGISEQ-500 sequencing platform, its photography time is relatively long (it takes about 14 minutes / cycle to take a full chip), which means that the photography reagents stay in the flow chamber for a long time.

[0007] The existing DIPSEQ technology and BGI500 sequencing technology, the former uses the immersion sequencing method, and the sequencing reaction reagents are always in a constant high-temperature environment. This environment brings some technical difficulties to the stability of the reagent components, and may affect the reaction efficiency of the sequencing enzyme in this environment to a certain extent. For example, how to maintain the activity of the sequencing enzyme without being affected by temperature, the stability of specially labeled dNTPs and excision reagents under high temperature conditions, etc. These factors directly manifest in a relatively high lag / Run on ratio during the sequencing process. This defect of the sequencing reaction reagent in the high-throughput sequencing reagent is not conducive to the scientific research needs and clinical applications of the DIPSEQ high-throughput universal sequencing reagent in high-read length sequencing.

[0008] Although the reagent stability of the latter is better than that of the former, the photography time is longer due to the limitations of the photography system. The length of time the photography reagents used in the photography process stay on the chip surface also has a photobleaching effect on the sequencing fluorescent group (i.e., fluorescence signal quenching), which to some extent leads to regional sequencing quality deviations.

[0009] In addition, whether in BGI-DIPSEQ technology or BGI-SEQ technology, the degree of damage to the sequencing template (DNB) and specially labeled dNTPs during the photography process varies depending on the light machine. The strength of the anti-light damage effect of the photography reagent affects the sequencing quality of the platform to a certain extent. Summary of the invention

[0010] In view of this, the present invention provides the application of cyanuric acid in sequencing. The technical key of the present invention is that by adding CA (cyanuric acid) to the DIPSEQ high-throughput sequencing cPAS biochemical reagent, the stability of DNB, enzyme, and azide group can be promoted, the reaction efficiency can be improved, the Lag / Runon can be reduced, and the sequencing quality can be improved.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The invention provides application of cyanuric acid in sequencing.

[0013] In some embodiments of the present invention, in the above applications, the sequencing includes high-throughput sequencing.

[0014] In some embodiments of the present invention, in the above applications, the sequencing includes immersion sequencing technology (DIPSEQ) or probe-anchored synthesis sequencing technology (cPAS).

[0015] In some embodiments of the present invention, in the above applications, the cyanuric acid enhances the thermal stability of DNB polymerase, dNTP and / or azide groups.

[0016] In some embodiments of the present invention, in the above application, the cyanuric acid reduces the delayed reaction and / or the advanced reaction.

[0017] In some embodiments of the present invention, in the above applications, the cyanuric acid promotes polymerization reaction and / or improves sequencing quality.

[0018] In some embodiments of the present invention, in the above application, the cyanuric acid reduces the photobleaching and / or quenching effect of the fluorescent dye.

[0019] In some embodiments of the present invention, in the above application, the cyanuric acid reduces DNB photodamage.

[0020] In some embodiments of the present invention, in the above application, the added amount of cyanuric acid is 125-500 μmol / L.

[0021] The present invention also provides the use of cyanuric acid in preparing a polymerization reaction reagent, a photographic reagent, a reagent combination or a kit.

[0022] The invention also provides a polymerization reaction reagent comprising: 125-500 μmol / L cyanuric acid.

[0023] The invention also provides a photographic reagent comprising: 125-500 μmol / L cyanuric acid.

[0024] In some embodiments of the present invention, the reagent combination of the present invention (BGISEQ500-cPAS sequencing buffer) also includes: TE buffer, DNB preparation buffer, DNB polymerase mix I, DNB polymerase mix I (LC), DNB termination buffer, DNB loading buffer I, DNB loading buffer II, dNTPs mix, dNTPs mix II and DNA polymerase mix.

[0025] The present invention also provides a reagent combination, including the above-mentioned polymerization reaction reagent and / or the above-mentioned photographic reagent.

[0026] In some embodiments of the present invention, the above reagent combination includes cPAS sequencing reagents and / or DIPSEQ reagents.

[0027] The present invention provides a kit, comprising the above-mentioned polymerization reaction reagent, the above-mentioned photographic reagent or the above-mentioned reagent combination and an acceptable auxiliary agent or carrier.

[0028] The present invention also provides a sequencing method, wherein a sample to be tested is subjected to polymerization reaction, after a first elution, photographed, regenerated, and then subjected to a second elution to obtain a sequencing result;

[0029] The polymerization reaction uses the above-mentioned polymerization reaction reagent, the above-mentioned polymerization reaction reagent in the above-mentioned reagent combination, or the above-mentioned polymerization reaction reagent in the above-mentioned kit;

[0030] The photographing uses the above-mentioned photographing reagent, the photographing reagent in the above-mentioned reagent combination or the photographing reagent in the above-mentioned kit.

[0031] In some embodiments of the present invention, in the above sequencing method, the first elution washes away the polymerization reaction reagent.

[0032] In some embodiments of the present invention, in the above sequencing method, the regeneration uses a regeneration reagent, and the regeneration removes the blockage of the dNTP at the end of the complementary chain, so that the complementary chain can continue to extend.

[0033] In some embodiments of the present invention, in the above sequencing method, the second elution washes away the regeneration reagent.

[0034] The invention provides the application of cyanuric acid in sequencing. The invention also provides a polymerization reaction reagent, a photographing reagent, a reagent set and a kit.

[0035] The beneficial effects of the present invention include:

[0036] (1) The CA provided by the present invention can enhance the stability of DNB polymerase and specially labeled dNTP in the sequencing reaction reagents in the DIPSEQ technology in a high temperature environment, effectively improve the polymerization efficiency, and reduce the Lag (Lag is a lag reaction, which refers to the delay phenomenon in the sequencing process, and the causes include insufficient reaction and incomplete excision of the excision reagent) generated during the sequencing reaction, thereby achieving the purpose of significantly improving the sequencing quality.

[0037] (2) During the photography process, it can significantly resist the photobleaching effect of the light machine on the fluorescent dyes used in the BGI series sequencing technologies including DIPSEQ technology, making the fluorescent signal more stable (from the heatmap of the signal value), and significantly weakening the quenching effect of the original photography reagents on the fluorescent dyes in this type of sequencing technology.

[0038] (3) Significantly reduce or minimize the photodamage caused by optical effects to DNBs (the downward trend of the signal value becomes slower; the quality of the second-strand recovery in PE sequencing. Generally speaking, if the DNB is photodamaged, the second-strand signal will not recover). BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0040] Figure 1 Show the test plan;

[0041] Figure 2 Indicates the signal strength of Cy-SRE and Ctrl-SRE in channel A;

[0042] Figure 3 It shows the signal strength of Cy-SRE and Ctrl-SRE in C channel;

[0043] Figure 4 Shows the signal intensity of Cy-SRE and Ctrl-SRE in G channel;

[0044] Figure 5 It shows the signal intensity of Cy-SRE and Ctrl-SRE in T channel;

[0045] Figure 6 The SNR comparison results of Cy-SRE and Ctrl-SRE are shown;

[0046] Figure 7 The comparison results of BIC, Fit and ESR between Cy-SRE and Ctrl-SRE are shown;

[0047] Figure 8 The comparison results of RHO, BIC, Fit and ESR of Cy-SRE and Ctrl-SRE in the sixth shot are shown. DETAILED DESCRIPTION

[0048] The invention discloses application of cyanuric acid in sequencing.

[0049] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0050] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0051] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0052] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0053] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0054] In Examples 1 to 5 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0055] The present invention will be further described below in conjunction with embodiments:

[0056] Example 1 Test of the effect of new reaction sequencing reagents on the DIPSEQ V8 sequencing platform

[0057] This example measures the effect of CA. 0.003% (v / v) CA reagent was added to the cPAS sequencing reagent (i.e., BGISEQ500-cPAS sequencing buffer), and Hot and Cold (Hot refers to dNTPs with fluorescence and blocking groups, and Cold refers to dNTPs with blocking groups) were prepared, and the PE100 test was performed on the DIPSEQ V8 sequencing platform. During the test, the biochemical process was a total time of 4.5 min / each cycle of the cPAS biochemical script. The sequencing Lag and Runon levels with and without CA under the reaction conditions of the biochemical script were counted. As shown in Table 1, CA significantly improves Lag1, Lag2 and Runon1, but not Runon2 (Lag is a lag reaction, which refers to the delay phenomenon in the sequencing process. The causes include insufficient reaction and incomplete excision of the excision reagent. Lag 1 (%) and Lag 2 (%) refer to the lag values ​​of the first and second chains, respectively. Runon refers to an advance reaction, which is an advance phenomenon in the sequencing reaction. The causes include incomplete cold reaction. Runon 1 (%) and Runon 2 (%) refer to the Runon values ​​of the first and second chains, respectively). It is speculated that the reduction of Lag is due to the protective effect of CA on the enzyme, and the reduction of Runon is due to the protective effect of CA on fluorescence.

[0058] Table 1 PE100 sequencing results of new sequencing reaction reagents on DIPSEQ sequencing platform

[0059]

[0060] Example 2 Testing of the Effect of CA Analogs in Sequencing Reagents

[0061] This example is to test whether antioxidants such as GSH (Glutathione), EGCG (Epigallocatechin gallate), TED (Triethylene Diamine), and Rutin can promote polymerization reactions like CA when added to sequencing reaction reagents.

[0062] 20mM GSH, 10mM TED, 1mM Rutin, 20uM EGCG and 0.003% CA were added to the cPAS sequencing reaction reagents, and the PE50 test was performed on BGISEQ-500. The experimental materials in this example all used Escherichia coli circular single-stranded DNA, and the BGISEQ-500DNB preparation kit was used, and the rolling circle replication time was 20min. The sequencing reagents all used the BGISEQ-500 reagent formula, and the selected biochemical reaction processes were all standard BGISEQ-500PE50_V3 standard processes. The test results are shown in Table 2.

[0063] Table 2 PE50 test results of CA analogs in sequencing reaction reagents

[0064]

[0065] In Table 2, ChipProductivity% is the chip utilization rate, which is the ratio of the number of FOVs that normally produce reports to the number of FOVs that are taken by the lanes. This includes FOV loss that cannot be processed normally due to various problems such as image loss and excessive offset of out-of-focus bubbles, as well as read quality problems caused by algorithm alignment accuracy that are filtered out; MappedRate(%) is the mapping rate, which is the ratio of reads in the sequencing results that can be mapped to E.Coli sequences; Q30 refers to the ratio of sequencing accuracy above 99.9%; ESR (effective spot rate, effective luminescent DNB point ratio) is to exclude DNBs that are not loaded or luminescent abnormally to ensure the accuracy of the results. The quality value of each base of each DNB is calculated to determine whether the reads will be filtered due to low quality.

[0066] The experimental results show that the addition of GSH, TED, Rutin and EGCG to the sequencing reagents has no promoting effect on the polymerization reaction, but the addition of CA can promote the polymerization reaction, reducing Lag by 0.03% and Runon by 0.01%.

[0067] Example 3: Test of the effect of novel reaction sequencing reagents on the BGISEQ-500 platform

[0068] The main purpose of this example is to further accurately confirm the effect of CA on the BGISEQ-500 platform.

[0069] 0.003% (232 μM) of CA reagent was added to the cPAS sequencing reagent, and the sequencing quality performance of the BGISEQ-500 high-throughput universal sequencing kit was compared with the sequencing quality of the novel sequencing reaction reagent sequencing reaction.

[0070] The experimental materials in this example all used E. coli circular single-stranded DNA, the main peak length of the inserted fragment was 160 bp, the BGISEQ-500 DNB preparation kit was used, and the rolling circle replication time was 20 minutes. The sequencing reagents all used the BGISEQ-500 reagent formula.

[0071] The test scheme is shown in the conditions of Table 3, and is as follows:

[0072] Test 1 is the control group: PE100 was run under the normal recipe (BGISEQ-500 standard biochemical process) without heating Hot / Cold.

[0073] Test 2 is a non-heated Hot / Cold run under the flush recipe (fast biochemical process) PE100. (The flush recipe is a fast biochemical process. Based on the BGISEQ-500 standard biochemical process, the reaction reagents Hot and Cold are changed from the standard incubation of 60s to no incubation, and the flow rate of Hot and Cold is increased from 250μL / min to 600μL / min, with a total pumping time of 25s.)

[0074] Test 3 was Hot / Cold, after heating at 55°C for 3 hours in a water bath, PE100 was run under the normal recipe (standard biochemical process). (Normal recipe is the normal biochemical process, i.e. the BGISEQ-500 standard biochemical reaction protocol.)

[0075] Test 4 is a Hot / Cold test with 0.003% CA added and heated at 55°C in a water bath for 3 hours (the purpose of heating at 55°C for 3 hours is mainly to test the stability of the accelerated reagent, because the newly prepared reagents have better stability, and heating can better demonstrate that CA can improve the stability of the reagents and extend the shelf life, while other analogs do not show functions superior to CA. Secondly, it can simulate the characteristics of DIPSEQ immersion sequencing water bath heating), and run PE100 under flush recipe.

[0076] Test 5 is Hot / Cold with 0.003% CA added, running PE100 under the flush recipe;

[0077] The test results are shown in Table 3.

[0078] Table 3 PE100 test of new reaction sequencing reagents on BGISEQ-500 platform

[0079]

[0080]

[0081] In Table 3, DNB concentration: 17 ng / μL. Reagents refers to the biochemical reagents used in the sequencing reaction, +0.003% CA refers to adding 0.003% CA to the sequencing reaction reagent ICRbuffer; 55℃heat 3h refers to placing Hot and Cold in a water bath at 55℃ for 3h; Recipe refers to the biochemical process used in the sequencing reaction, where Normal refers to using the BGISEQ-500 standard biochemical process, and Flush refers to modifying the pump Hot / Cold speed to 450μL / min based on the standard biochemical process, and the Hot / Cold incubation time is changed to zero;

[0082] The results show that:

[0083] (1) Compared with Test2 and control, the lag of flush recipe is 0.03% higher, indicating that flush recipe can amplify the impact of experimental conditions on sequencing quality and can be used in subsequent CA test conditions.

[0084] (2) Test 3 simulates the reaction characteristics of DIPSEQ in a water bath. The reagents were heated for 3 h. Compared with Test 2, Lag increased by 0.04% and Runon increased by about 0.05%.

[0085] (3) Compared with Test 3, the new reaction reagent with the addition of CA can reduce Lag by 0.05% to 0.06% and Runon by 0.05% under the condition of reagent heating.

[0086] (4) Compared with Test 2, the addition of CA to the new reaction reagent can reduce Lag by 0.02%-0.03% without heating, and there is no significant difference in Runon.

[0087] Based on the above results, the new sequencing reaction reagent can reduce both Lag and Runon under heating conditions, and can reduce Lag without significant effect on Runon under non-heating conditions.

[0088] Example 4 Optimization of titration concentration of novel reaction sequencing reagents

[0089] Examples 1 to 3 have confirmed that the addition of 0.003% CA (about 232 μM) to the new reaction sequencing reagent can reduce Lag / Runon on both the BGISEQ-500 platform and the DIPSEQ platform. Considering the multi-faceted cross-linker function and "strand displacement" of Cyanuric, it may be able to stabilize the DNB structure during sequencing, and may promote the polymerization efficiency at the secondary structure of the DNA template. Therefore, different libraries were used to test the effects of different Cyanuric Acid concentrations in the reaction sequencing reagent on sequencing quality on BGISEQ-500.

[0090] Table 4 SE45 sequencing results of different concentrations of CA in Human library

[0091]

[0092] Table 5 SE45 sequencing results of E. coli library with different concentrations of CA

[0093]

[0094] As shown in Table 4 and Table 5, they are human library and ecoli library respectively. The concentrations of Cyanuric Acid are 125μM, 250μM, and 500μM respectively. It can be clearly seen from the data in the table that when the concentration is 125μM, the Lag value increases and the polymerization efficiency decreases. However, when the CA concentration is 250μM and 500μM, its lag level is lower than control, indicating that the polymerization efficiency is enhanced, and 500μM CA is more conducive to promoting polymerization efficiency.

[0095] Example 5: Test of Photobleaching Effect of CA on Fluorescent Dyes

[0096] Since Cyanuric Acid has weak reducing properties and may replace the function of Vc in SRE, and the small molecule has a structure similar to DNA and may transfer photodamage, SRE containing only small molecules and salt ions was prepared, hereinafter referred to as Cy-SRE.

[0097] This example mainly compares the photobleaching effect of Cy-SRE and Ctrl-SRE (normal are, control) on fluorescent dyes. It includes the following three aspects: 1) the effect of SRE residence time on the fluorescent signal; 2) the effect of the number of photos on the fluorescent signal; 3) the fluorescence signal intensity of the photosensitive area and the non-photosensitive area after the same SRE residence time.

[0098] The test plan is as follows Figure 1 As shown, a total of 6 photos were taken, with an interval of 20 minutes between the first five photos, and the photographed area was expanded by 1 time each time. After the fifth photo was taken, it was rinsed with fresh SRE and immediately photographed (the photographed area was expanded horizontally from 8cols to 13cols based on the fifth photo). The control group and the experimental group were conducted independently.

[0099] The experiment found that Ctrl-SRE has a significant photobleaching effect on fluorescence. As time goes by, the degree of fluorescence quenching increases. At the same time, as the number of photos increases, the degree of fluorescence signal intensity quenching in Ctrl-SRE gradually increases. There is also a very obvious difference in signal intensity between the area where no photos are taken and the area where photos are taken.

[0100] like Figure 2 As shown in the figure, in channel A, the signal intensity of dye-A in Cy-SRE is significantly higher than that in Ctrl-SRE, and is relatively stable, without bleaching with the residence time of SRE. The number of photos does not significantly change the signal intensity like in Ctrl-SRE. This seems to indicate that Cy-SRE can maintain the stability of dye-A.

[0101] like Figure 3 As shown in the figure, in the C channel, the fluorescence signal intensity of C in Ctrl-SRE is consistent with that in the A channel, and the bleaching effect is more significant with the residence time. Cy-SRE did not show the phenomenon of promoting the luminescence intensity of the C channel to the A channel, but the bleaching effect on the fluorescence signal did not increase with the residence time of SRE. The number of photos has a significant effect on the intensity of the C signal, indicating that Cy-SRE can maintain the stability of C, but not as significant as A.

[0102] like Figure 4As shown in the figure, in the G channel, the luminescence efficiency of Cy-SRE is lower than that of Ctrl-SRE. The SRE residence time is still not significant in Cy-SRE. The effect of the number of shots is not as significant in Cy-SRE as in Ctrl-SRE.

[0103] like Figure 5 As shown in the figure, in the T channel, the signal intensity of T is slightly increased in Cy-SRE, but not as significant as in the A channel. In Cy-SRE, the effect of taking a photo on the signal intensity of T is not significant. Consistent with the other three channels mentioned above, the signal intensity of T decreases in Ctrl-SRE as the SRE residence time increases, but there is no significant decrease in Cy-SRE.

[0104] like Figure 6 and Figure 7 As shown in the figure, the effects of the two SREs on SNR are almost the same except for T. The SNR-T value is lower in Cy-SRE (the average value is about 2 to 5 lower). There is no significant difference in Bic, fit and ESR.

[0105] like Figure 8 As shown in the figure, in the sixth photo result, the rightmost area of ​​the lane belongs to the first photo area, and the signal intensity on the left remains high, which may be related to the glue. In summary, the effect of photography on photobleaching is significantly stronger than the residence time of SRE. Cy-SRE is significantly more resistant to photodamage than Ctrl-SRE.

[0106] Based on the above experimental results, we can conclude that Cy-SRE has a good protective effect on fluorescent dyes.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of cyanuric acid in sequencing.

2. The use according to claim 1, characterized in that The cyanuric acid enhances the thermal stability of DNB polymerase, dNTPs and / or azide groups.

3. The use according to claim 1 or 2, characterized in that: The applications of cyanuric acid include but are not limited to any of the following: (I) reducing lag response and / or lead response; and / or (II) promoting polymerization reaction and / or improving sequencing quality; and / or (III) reducing photobleaching and / or quenching of fluorescent dyes; and / or (IV) Reduce DNB photodamage.

4. The use according to any one of claims 1 to 3, characterized in that The added amount of cyanuric acid is 125-500 μmol / L.

5. Use of cyanuric acid in the preparation of polymerization reagents, photographic reagents, reagent combinations or kits.

6. A polymerization reagent, characterized in that include: 125~500μmol / L cyanuric acid.

7. A photographic reagent, characterized in that: include: 125~500μmol / L cyanuric acid.

8. A reagent combination, characterized in that It comprises the polymerization reagent as claimed in claim 6 and / or the photographic reagent as claimed in claim 7.

9. A kit, characterized in that It comprises the polymerization reagent according to claim 6, the photographic reagent according to claim 7 or the reagent combination according to claim 8 and an acceptable auxiliary agent or carrier.

10. A sequencing method, characterized in that Take the sample to be tested for polymerization reaction, after the first elution, take a picture, regenerate, and then undergo the second elution to obtain the sequencing result; The polymerization reaction uses the polymerization reagent as claimed in claim 6, the polymerization reagent in the reagent combination as claimed in claim 8, or the polymerization reagent in the kit as claimed in claim 9; The photographing uses the photographing reagent as described in claim 7, the photographing reagent in the reagent combination as described in claim 8, or the photographing reagent in the kit as described in claim 9.

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