Sequencing reaction stabilizer composition and application thereof

Abstract

The invention relates to the technical field of sequencing, in particular to a sequencing reaction stabilizer composition and application thereof. The invention provides an application of ammonium pyrrolidine dithiocarbamate and N, N '-dimethyl thiourea in sequencing, and also provides a stabilizer composition containing the ammonium pyrrolidine dithiocarbamate and the N, N'-dimethyl thiourea, and a preparation method of the stabilizer composition containing the ammonium pyrrolidine dithiocarbamate and the N, N '-dimethyl thiourea. The invention also discloses application of the stabilizer composition in preparation of a sequencing reaction reagent, a sequencing kit and a high-throughput sequencing reaction. The stabilizer composition provided by the invention can enhance the stability of DNB polymerase and dNTPs with a special label in a sequencing reaction reagent in a high-temperature environment, prolong the shelf life of the reagent, effectively improve the polymerization efficiency, reduce the lag generated in a sequencing reaction process and achieve the purpose of remarkably improving the sequencing quality, and the stability of the DNB polymerase in the sequencing reaction reagent and the dNTPs with the special label in the high-temperature environment is improved. Therefore, on the premise of ensuring high sequencing quality, the sequencing read length is further improved, and the application range of the high-throughput universal sequencing reagent is expanded.

Description

Technical Field

[0001] The present invention relates to the technical field of sequencing, and in particular to a sequencing reaction stabilizer composition and application thereof. 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 that have been developed so far mainly involve sequencing by ligation (SBL) technology and sequencing by synthesis (SBS) technology, as well as the combined probe anchor synthesis sequencing technology (cPAS) developed by BGI, including the immersion sequencing (DIPSEQ) technology.

[0003] The immersion sequencing (DIPSEQ) technology developed by BGI (refer to our immersion sequencing patent) is a second-generation sequencing technology with advantages such as high throughput, low cost, and automation, with 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, and 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 quality and read length of DIPSEQ sequencing because the reagents are always immersed in a constant high temperature, enzyme activity, stability of azide groups 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. At present, the goal of DIPSEQ sequencing technology is 3min rapid biochemical reaction (the standard biochemical time of the BGISEQ-500 sequencing platform is about 6min). Due to the reduction of polymerization reaction time, the polymerization efficiency is reduced, which causes the lag / runon to increase, affecting the sequencing quality.

[0007] Although the sequencing reaction reagents in the existing BGISEQ-500RS high-throughput universal sequencing reagents can perfectly meet the sequencing of SE100 or even longer, under the rapid biochemical reaction process, due to the shortened polymerization reaction time and the constant reaction rate, the reaction cannot be fully completed within the limited reaction time, resulting in a relatively high starting lag value, which affects the sequencing quality. In the sequencing process, the lag value is a linear parameter with a cumulative effect and is closely related to the read length. A high lag value is prone to produce the accuracy of sequencing reads, which directly affects the sequencing quality (such as Q30). The existing DIPSEQ technology 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, which 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 the specially labeled dNTP and excision reagents under high temperature conditions, etc. These factors directly show that a relatively high lag and Runon ratio is generated during the sequencing process. These defects of the sequencing reaction reagents in the high-throughput sequencing reagent are not conducive to the scientific research needs and clinical applications of the DIPSEQ high-throughput universal sequencing reagent in high-read length sequencing. Summary of the invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a sequencing reaction stabilizer composition and its application. The stabilizer composition provided by the present invention can effectively enhance the stabilizer of the test reaction reagent and extend the shelf life of the reagent, thereby effectively improving the polymerization reaction efficiency.

[0009] The present invention provides the use of ammonium pyrrolidine dithiocarbamate and / or N,N'-dimethylthiourea in any of the following items:

[0010] (I), maintaining the high efficiency and / or specificity of the sequencing enzyme; and / or

[0011] (II), opening the DNB special structure; and / or

[0012] (III), increasing the specific binding of primers to templates; and / or

[0013] (IV) facilitating the extension of the sequencing enzyme at the secondary structure; and / or

[0014] (V), improving polymerization efficiency; and / or

[0015] (VI), reducing the lag generated during the sequencing reaction; and / or

[0016] (VII), improving sequencing quality; and / or

[0017] (VIII), increasing sequencing read length and / or

[0018] (IX) Expand the application scope of sequencing reagents.

[0019] The present invention provides a stabilizer composition comprising ammonium pyrrolidinedithiocarbamate and N,N'-dimethylthiourea.

[0020] Compared with other compositions, the stabilizer composition provided by the present invention can effectively improve the stability of sequencing reaction reagents in the DIPSEQ technology, as well as the DNB polymerase and dNTP with special fluorescence therein in a high temperature environment, maintain the effective concentration of DNB polymerase and dNTP with special fluorescence in the sequencing reagent, reduce the lag level generated during the sequencing reaction, achieve the purpose of significantly improving the sequencing quality, and obtain more accurate technical effects.

[0021] In some specific embodiments, the stabilizer composition comprises 1 mM ammonium pyrrolidinedithiocarbamate and 1 mM N,N'-dimethylthiourea.

[0022] Experiments show that the ammonium pyrrolidine dithiocarbamate and N,N'-dimethylthiourea at the above concentrations cooperate closely, maintaining the stability of the enzyme and the special group and the best effect of effective concentration, thereby obtaining a more accurate technical effect.

[0023] The present invention also provides the use of the stabilizer composition in preparing a sequencing reaction reagent or a sequencing kit.

[0024] The present invention provides a sequencing reaction reagent, comprising the stabilizer composition and acceptable auxiliary materials or additives.

[0025] The present invention provides a sequencing kit, comprising the stabilizer composition and other reagents, wherein the other reagents include at least one of a sequencing buffer, dNTPs, DNA nanoballs, a sequencing primer, a probe and a sequencing enzyme.

[0026] In some embodiments, the dNTPs are linked to fluorescent groups and / or blocking groups.

[0027] In some embodiments, the sequencing enzyme comprises a DNB polymerase and / or a DNA polymerase.

[0028] The present invention provides use of the stabilizer composition, the sequencing reaction reagent or the sequencing kit in high-throughput sequencing.

[0029] The present invention also provides a high-throughput sequencing method, comprising taking a sample to be tested, mixing it with any of the following items, and sequencing it;

[0030] (i), ammonium pyrrolidinedithiocarbamate and / or N,N'-dimethylthiourea; and / or

[0031] (ii) a stabilizer composition as claimed in any one of claims 2 to 4; and / or

[0032] (iii) the sequencing reaction reagent according to claim 6; and / or

[0033] (iv) A reagent in a sequencing kit according to any one of claims 7 to 9.

[0034] Furthermore, the sequencing also includes a heating step.

[0035] In some embodiments, the high-throughput sequencing method comprises taking a sample to be tested,

[0036] (A), adding ammonium pyrrolidine dithiocarbamate and heating; and / or

[0037] (B), adding N,N'-dimethylthiourea and heating; and / or

[0038] (C) adding the stabilizer composition according to any one of claims 2 to 4, the sequencing reaction reagent according to claim 6, or the reagent in the sequencing kit according to any one of claims 7 to 9 without heating; and / or

[0039] (D) adding the stabilizer composition according to any one of claims 2 to 4, the sequencing reaction reagent according to claim 6, or the reagent in the sequencing kit according to any one of claims 7 to 9, and heating.

[0040] Experiments show that under heating conditions, adding APDTC and DMTU separately can reduce Lag and Runon at the same time; the new reaction reagent that adds APDTC and DMTU at the same time can reduce Lag and Runon both under heating and without heating, and the effect is better than adding APDTC or DMTU alone when the reagent is heated.

[0041] Preferably, the heating temperature is 60° C. and the heating time is 3 h.

[0042] The present invention provides the use of ammonium pyrrolidine dithiocarbamate and N,N'-dimethylthiourea in sequencing, and also provides a stabilizer composition comprising ammonium pyrrolidine dithiocarbamate and N,N'-dimethylthiourea, and the use of the stabilizer composition in preparing a sequencing reaction reagent or a kit and in a high-throughput sequencing reaction. The stabilizer composition provided by the present invention can enhance the stability of the sequencing reaction reagent in a high-temperature environment and extend the shelf life of the reagent; at the same time, it can enhance the stability of the DNB polymerase and the dNTPs with special labels in the sequencing reaction reagent in the DIPSEQ technology in a high-temperature environment, effectively improve the polymerization efficiency, reduce the lag generated during the sequencing reaction, and achieve the purpose of significantly improving the sequencing quality, thereby further improving the sequencing read length under the premise of ensuring high sequencing quality, and expanding the application range of high-throughput universal sequencing reagents. Experiments show that, under heating conditions, adding 1mM APDTC and 1mM DMTU separately can reduce both Lag and Runon by 0.01-0.02%; adding 1mM APDTC and 1mM DMTU to the new reaction reagent at the same time, under heating conditions, reduces Lag by 0.03% and Runon by about 0.04%, and the effect is better than adding APDTC or DMTU alone. Adding APDTC and DMTU simultaneously without heating the reagents can also reduce lag and runon by 0.01%. DETAILED DESCRIPTION

[0043] The present invention provides a sequencing reaction stabilizer composition and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0045] Example 1 Test of the effect of new reaction sequencing reagents on the BGISEQ-500 platform

[0046] The stabilizer composition provided by the present invention comprises reagent I and reagent II, wherein:

[0047] Reagent I is APDTC (ammonium pyrrolidinedithiocarbamate). Since the APDTC molecule contains N and S atoms with strong coordination ability, it is often used as a chelating agent for atomic absorption spectrophotometry analysis. It is also an effective environmentally friendly metal corrosion inhibitor.

[0048] Reagent II is DMTU (N,N'-dimethylthiourea), a low molecular weight, highly diffusible antioxidant that has the function of scavenging hydrogen peroxide and hydroxyl groups (Plant, Cell and Environment (2011) 34, 1031–1042). It can protect DNA and enzymes under high temperature conditions, promote high-throughput sequencing polymerization reactions, reduce Lag and Runon, and stabilize the reagents to extend their shelf life.

[0049] On the one hand, APDTC and DMTU can maintain the high efficiency and specificity of the sequencing enzyme, open the special structure of DNB, increase the specific binding of primers and templates, and facilitate the extension of the sequencing enzyme at the secondary structure. On the other hand, they can improve the polymerization efficiency, thereby effectively reducing the lag generated during the sequencing reaction and achieving the purpose of significantly improving the sequencing quality. In this way, the sequencing read length can be further improved while ensuring high sequencing quality, and the application scope of DIPSEQ and BGISEQ-500RS high-throughput universal sequencing reagents can be expanded.

[0050] The APDTC and DMTU reagents in this embodiment are mainly added to the sequencing reaction reagents. The other reagent components of this technology are the same as those of the BGI sequencing series.

[0051] The main purpose of this example is to determine the effects of APDTC and DMTU on the BGISEQ-500 platform.

[0052] 1mM APDTC and 1mM DMTU reagents were added to the cPAS sequencing reaction reagents, 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 reagents.

[0053] The experimental materials in this example all used Escherichia 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 min. The sequencing reagents all used the BGISEQ-500 reagent formula.

[0054] The test plan is shown in the conditions of experience in Table 1, as follows:

[0055] Test 1 is to run PE100 using the normal standard Hot / Cold. (Hot refers to dNTPs with fluorescent and blocking groups, Cold refers to dNTPs with blocking groups)

[0056] Test 2 was to heat the Hot / Cold in a water bath at 60°C for 3 h and then run PE100.

[0057] Test 3 was performed using Hot / Cold PE100 supplemented with 1 mM APDTC.

[0058] Test 4 was run using Hot / Cold PE100 supplemented with 1 mM DMTU;

[0059] Test 5 was run using Hot / Cold PE100 with 1 mM APDTC and 1 mM DMTU added simultaneously;

[0060] Test 6 used Hot / Cold with 1 mM APDTC added, heated at 60°C in a water bath for 3 h, and then ran PE100.

[0061] Test 7 used Hot / Cold with 1 mM DMTU added, heated at 60°C in a water bath for 3 h and then ran PE100;

[0062] Test 8 used Hot / Cold with 1 mM APDTC and 1 mM DMTU added simultaneously, heated at 60°C in a water bath for 3 h, and then ran PE100;

[0063] The biochemical processes used in the above sequencing reactions were all tested under the flush recipe (fast biochemical process). The flush recipe refers to the change of the standard incubation of 60s for the reaction reagents Hot and Cold to no incubation based on the BGISEQ-500 standard biochemical process, and the flow rate of the Hot and Cold pumps was increased from 250μL / min to 450μL / min, with a total pumping time of 25s. Among them, the purpose of heating at 60℃ for 3h is to test the stability of the accelerated reagents, because the stability of the newly prepared reagents is good, and heating can better demonstrate the role of APDTC and DMTU in improving the stability of the reagents and extending the shelf life. On the other hand, it can simulate the characteristics of DIPSEQ immersion sequencing water bath heating.

[0064] The results are shown in Table 1.

[0065] Table 1 PE100 test of new reaction sequencing reagents on BGISEQ-500 platform

[0066]

[0067] Note: DNB concentration: 15.3ng / ul. reagents refers to the biochemical reagents used in sequencing reaction, +1mM APDTC refers to adding 1mM APDTC to sequencing reaction reagent ICR buffer; +1mM DMTU refers to adding 1mM DMTU to sequencing reaction reagent ICR buffer; +1mM APDTC / DMTU refers to adding 1mMAPDTC and 1mM DMTU to sequencing reaction reagent ICR buffer at the same time; 60℃heat 3h refers to placing Hot and Cold in a water bath at 60℃ for 3h; ChipProductivity% refers to chip utilization, which is the ratio of the number of FOVs that normally generate reports to the number of FOVs that are taken by lanes. This includes FOV loss that cannot be processed normally due to various problems such as image loss, excessive offset of out-of-focus bubbles, and read quality problems caused by algorithm alignment accuracy. The reads are filtered out due to read quality problems. MappedRate (%) is the alignment rate. We use the E. coli library, that is, the ratio of reads in the sequencing results that can be aligned to the E. Coli sequence. Q30 refers to the proportion 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 is calculated for each base of each DNB to determine whether the reads will be filtered due to low quality. 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 the advance reaction, which is the 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, the same below.

[0068] The results show that:

[0069] 1. Test 2 simulates the reaction characteristics of DIPSEQ water bath heating. The reagent is heated at 60℃ for 3h. Compared with test 1, there is no difference in lag, but Runon increases by about 0.08%. This is due to the effect of high temperature on the dNTP azide group, which leads to a significant increase in Runon.

[0070] 2. Comparison between test 3 and test 1 shows that adding 1 mM APDTC to the new reaction reagent has no effect on lag and runon under hot / cold conditions.

[0071] 3. Comparison between Test 4 and Test 1 shows that adding 1 mM DMTU to the new reaction reagent has no effect on lag and runon under hot / cold conditions.

[0072] 4. Comparison of Test 5 with Test 1 shows that under hot / cold conditions, adding 1mM APDTC and 1mM DMTU simultaneously can reduce Lag1 and Lag2 by 0.01% and Runon1 and Runon2 by 0.01%.

[0073] 5. Compared with test 2, adding 1mM APDTC to the new reaction reagent can reduce Lag1 by 0.02% and Lag2 by 0.01% under the condition of reagent heating. It can also reduce Runon1 and Runon2 by 0.02%.

[0074] 6. Compared with test 2, adding 1mM DMTU to the new reaction reagent in test 7 can reduce Lag1 by 0.02% and Lag2 by 0.01% under the condition of reagent heating. It can also reduce Runon1 and Runon2 by 0.02%.

[0075] 7. Test 8 was compared with test 2. When 1 mM APDTC and 1 mM DMTU were added to the new reaction reagent, under the reagent heating condition, Lag1 and Lag2 decreased by 0.03%, and Runon1 and Runon2 both decreased by 0.04%.

[0076] The above results show that:

[0077] 1. The new sequencing reaction reagent with only 1mM APDTC can reduce both Lag and Runon by 0.01-0.02% when heated. The effect on lag and runon is not obvious when the reagent is not heated.

[0078] 2. The new sequencing reaction reagent with only 1mM DMTU added can reduce both Lag and Runon by 0.01-0.02% when heated. The effect on lag and runon is not obvious when the reagent is not heated.

[0079] 3. When adding 1mM APDTC and 1mM DMTU to the new reaction reagent, Lag decreased by 0.03% and Runon decreased by about 0.04% under heating conditions, and the effect was better than adding APDTC or DMTU alone (adding them alone reduced lag and runon by 0.01%-0.02%). Adding APDTC and DMTU at the same time without heating the reagents can also reduce lag and runon by 0.01%.

[0080] Example 2 Test of the effect of new reaction sequencing reagents on the DIPSEQ sequencing platform

[0081] This example confirms the effect of APDTC / DMTU on the DIPSEQ platform. 1mM APDTC and 1mM DMTU reagents were added to the cPAS sequencing reagent (i.e., BGISEQ500-cPAS sequencing buffer) at the same time, and Hot and Cold were prepared, and the PE100 test was performed on the DIPSEQ T1 sequencing platform. During the test, the biochemical process was a total time of 4.5min / each cycle of the cPAS biochemical script. The sequencing Lag and Runon levels with or without APDTC / DMTU under the reaction conditions of the biochemical script were counted. It was found that APDTC / DMTU had significant improvements on lag1, lag2, Runon1 and Runon2. It is speculated that the reduction in Lag is due to the protective effect of DMTU on the enzyme, which improves the polymerization efficiency, and the reduction in Runon is due to the protective effect of APDTC and DMTU on the azide group at high temperatures.

[0082] Table 2 PE100 sequencing results of new sequencing reaction reagents on DIPSEQ sequencing platform

[0083]

[0084] Note: 1mM APDTC / DMTU means adding 1mM APDTC and 1mM DMTU to the sequencing reaction reagent. Slide ID refers to the serial number of the DIPSEQ T1 chip. Imager refers to the laser used to take pictures of the chip.

[0085] The above are only preferred embodiments 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of ammonium pyrrolidine dithiocarbamate and / or N,N'-dimethylthiourea in any of the following: (I), maintaining the high efficiency and / or specificity of the sequencing enzyme; and / or (II), opening the DNB special structure; and / or (III), increasing the specific binding of primers to templates; and / or (IV) facilitating the extension of the sequencing enzyme at the secondary structure; and / or (V), improving polymerization efficiency; and / or (VI), reducing the lag generated during the sequencing reaction; and / or (VII), improving sequencing quality; and / or (VIII), increasing sequencing read length and / or (IX) Expand the application scope of sequencing reagents.

2. A stabilizer composition, characterized in that These include ammonium pyrrolidinedithiocarbamate and / or N,N'-dimethylthiourea.

3. The stabilizer composition according to claim 2, characterized in that The stabilizer composition includes 1 mM ammonium pyrrolidinedithiocarbamate and 1 mM N,N'-dimethylthiourea.

4. Use of the stabilizer composition as claimed in claim 2 or 3 in preparing a sequencing reaction reagent or a sequencing kit.

5. A sequencing reaction reagent, characterized in that: The stabilizer composition comprises the stabilizer composition as claimed in claim 2 or 3, and acceptable auxiliary materials or additives.

6. A sequencing kit, characterized in that: It comprises the stabilizer composition as described in claim 2 or 3 and other reagents, wherein the other reagents include at least one of a sequencing buffer, dNTPs, DNA nanoballs, sequencing primers, probes and sequencing enzymes.

7. The sequencing kit according to claim 6, characterized in that The dNTPs are connected to fluorescent groups and / or blocking groups, and the sequencing enzyme includes DNB polymerase and / or DNA polymerase.

8. Use of the stabilizer composition according to claim 2 or 3, the sequencing reaction reagent according to claim 5, or the sequencing kit according to claim 6 or 7 in high-throughput sequencing.

9. A high-throughput sequencing method, characterized in that: Take the sample to be tested, mix it with any of the following items, and sequence it; (i), ammonium pyrrolidinedithiocarbamate and / or N,N'-dimethylthiourea; and / or (ii) a stabilizer composition as claimed in claim 2 or 3; and / or (iii) the sequencing reaction reagent according to claim 5; and / or (iv) The reagent in the sequencing kit as described in claim 6 or 7.

10. The high-throughput sequencing method according to claim 9, characterized in that: The high-throughput sequencing method comprises: taking a sample to be tested, (A), adding ammonium pyrrolidine dithiocarbamate and heating; and / or (B), adding N,N'-dimethylthiourea and heating; and / or (C) adding the stabilizer composition according to any one of claims 2 to 4, the sequencing reaction reagent according to claim 6, or the reagent in the sequencing kit according to any one of claims 7 to 9 without heating; and / or (D), adding the stabilizer composition according to any one of claims 2 to 4, the sequencing reaction reagent according to claim 6, or the reagent in the sequencing kit according to any one of claims 7 to 9, and heating; Preferably, the heating temperature is 60° C. and the heating time is 3 h.

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