Substrate additive for sequencing and application thereof

Abstract

The invention relates to the technical field of gene sequencing, in particular to a substrate additive for sequencing and application of the substrate additive. Wherein the stabilizer comprises potassium citrate, sodium salicylate, chitosan, sodium ascorbate, vitamin E, epigallocatechin gallate, glutathione, cysteine or a derivative thereof. The substrate additive provided by the invention can prevent a substrate from being oxidized to a certain extent and releasing free radicals, and can remove the free radicals generated in the reaction process, so that the substrate is more stable, and meanwhile, the generated free radicals are prevented from damaging to-be-detected DNA molecules, so that the sequencing quality is effectively improved.

Description

Technical Field

[0001] The present application relates to the field of gene sequencing technology, and particularly relates to a substrate additive for sequencing and its application. Background Art

[0002] With the rapid development of gene sequencing technology, in order to cooperate with fast, low-cost, and portable sequencers, self-luminescent sequencing methods have been widely used. In this method during the sequencing process, bases with special modifications at the ends are respectively labeled with different groups, and groups coupled with different target enzymes can specifically bind to the labeled group, and the enzyme can excite the corresponding substrate to emit signals to measure sequence information. In the Combinatorial Probe Anchor Synthesis (CPAS) sequencing technology based on DNA nanoballs (DNBs) using this method, DNA molecule anchors and modified probes are polymerized on the nanoballs, the enzyme binds to the modified probes, after introducing the substrate, light is emitted, and a CMOS imaging system collects the optical signals, and the gray values output by the CMOS system are analyzed to obtain the sequence to be measured.

[0003] However, in this technology, due to the specificity and complexity of the DNB structure, and at the same time the sequencing reaction process is complex, substrates such as coelenterazine substrates and fluorinated coelenterazine substrates will affect the quality of double-strand (Pair-end, PE) sequencing. These effects are mainly manifested as poor signal recovery of the two channels of the double strand and poor quality of double-strand sequencing.

[0004] Therefore, it is urgent to develop a substrate additive for sequencing to improve the sequencing quality. Summary of the Invention

[0005] The present application solves at least one of the problems of the related art from the following aspects.

[0006] To this end, an embodiment of the present application provides a substrate system for sequencing, the substrate system includes: a substrate and an additive, wherein the additive includes a stabilizer. Optionally, the substrate is coelenterazine or a derivative thereof. Optionally, the derivative is selected from the group consisting of fluorinated coelenterazine, bis-deoxycoelenterazine analog, coelenterazine cp, coelenterazine F, coelenterazine FCP, coelenterazine H, coelenterazine hcp, coelenterazine e, coelenterazine n, and dimethyl coelenterazine.

[0007] In some embodiments, the stabilizer is selected from at least one of the following: potassium citrate, sodium salicylate, chitosan, sodium ascorbate, vitamin E, epigallocatechin gallate, glutathione, cysteine or a derivative thereof. Optionally, the vitamin E is water-soluble vitamin E, and the cysteine is L-cysteine.

[0008] In some embodiments, the additive further comprises a chelating agent and / or a catalyst, wherein the chelating agent can be EDTA; the catalyst is one or more of the following: calcium ions, potassium ions, and sodium ions.

[0009] In some embodiments, the stabilizer is at least one of the following: sodium ascorbate, vitamin E, epigallocatechin gallate, glutathione, cysteine, or a derivative thereof. Preferably, the stabilizer is cysteine, and more preferably L-cysteine.

[0010] In some embodiments, the final concentration of sodium ascorbate in the substrate system is 100 - 500 mM, preferably 200 - 400 mM; the final concentration of vitamin E is 0.5 - 6 mM, preferably 1 - 6 mM; the final concentration of epigallocatechin gallate is 0.3 - 1 mM, preferably 0.5 - 0.8 mM; the final concentration of glutathione is 0.2 - 4 mM, preferably 1 - 2 mM, or the final concentration of cysteine is 0.2 - 4 mM and below, preferably 0.3 - 1 mM.

[0011] In some embodiments, the final concentration of sodium ascorbate in the substrate system is 400 mM; the final concentration of vitamin E is 4 mM; the final concentration of epigallocatechin gallate is 0.8 mM; the final concentration of glutathione is 1 mM, or the final concentration of cysteine is 0.5 mM.

[0012] In some embodiments, the final concentration of the substrate can be 300 μM.

[0013] The embodiments of the present application further provide a sequencing kit, which includes: a substrate system for sequencing as described in any of the above embodiments, and optionally a mediator sequence, a fluorescent enzyme, and a modified base. In some embodiments, the fluorescent enzyme includes luciferase. In some embodiments, the kit further includes a DNB preparation reagent.

[0014] The embodiments of the present application further provide a bioluminescent sequencing method, which includes: mixing a substrate with an additive to form a substrate system for sequencing, wherein the additive includes a stabilizer. In some embodiments, the substrate is coelenterazine or a derivative thereof. Optionally, the derivative is selected from the group consisting of fluorocoelenterazine, bis-deoxycoelenterazine analog, coelenterazine cp, coelenterazine F, coelenterazine FCP, coelenterazine H, coelenterazine hcp, coelenterazine e, coelenterazine n, and dimethylcoelenterazine.

[0015] In some embodiments, the stabilizer is selected from at least one of the following: potassium citrate, sodium salicylate, chitosan, sodium ascorbate, vitamin E, epigallocatechin gallate, glutathione, cysteine or its derivatives. Optionally, the vitamin E is water-soluble vitamin E, and the cysteine is L-cysteine.

[0016] In some embodiments, the additive further includes a chelating agent and / or a catalyst, wherein the chelating agent is EDTA; the catalyst is one or more of the following: calcium ions, potassium ions, and sodium ions.

[0017] In some embodiments, the method further includes: contacting the fluorescent enzyme with the substrate system so that the substrate binds to the fluorescent enzyme to obtain a sequencing signal. Specifically, photons are generated by the binding reaction of the substrate and the fluorescent enzyme, and the photons are then converted into electrons, which are received as a sequencing signal.

[0018] The embodiments of the present application also propose an application of the substrate system for sequencing as described in any of the above embodiments in sequencing, and the sequencing is single-end sequencing or paired-end sequencing, preferably paired-end sequencing.

[0019] The embodiments of the present application achieve the following beneficial effects:

[0020] The substrate additives for sequencing and the related sequencing methods proposed in the embodiments of the present application can produce high-quality sequencing data. The addition of the additives can avoid a certain degree of oxidation of the substrate and the release of free radicals, and can scavenge the free radicals generated during the reaction, thereby making the substrate more stable. At the same time, the generated free radicals are prevented from damaging the DNA molecule to be tested, thereby effectively improving the sequencing quality. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 Sequencing signal values using different concentrations of additive 1 according to Embodiment 1 of the present application;

[0023] Figure 2 Curve of the sequencing quality value varying with the number of cycles at each concentration of additive 1 according to Embodiment 1 of the present application;

[0024] Figure 3 Curve of the error rate varying with the number of cycles at each concentration of additive 1 according to Embodiment 1 of the present application;

[0025] Figure 4 is the sequencing signal value using different concentrations of additive 2 according to Embodiment 2 of the present application;

[0026] Figure 5 is the curve of the sequencing quality value varying with the number of cycles at each concentration of additive 2 according to Embodiment 2 of the present application;

[0027] Figure 6 is the curve of the error rate varying with the number of cycles at each concentration of additive 2 according to Embodiment 2 of the present application;

[0028] Figure 7 is the sequencing signal value using different concentrations of additive 3 according to Embodiment 3 of the present application;

[0029] Figure 8 is the curve of the sequencing quality value varying with the number of cycles at each concentration of additive 3 according to Embodiment 3 of the present application;

[0030] Figure 9 is the curve of the error rate varying with the number of cycles at each concentration of additive 3 according to Embodiment 3 of the present application;

[0031] Figure 10 is the sequencing signal value using different concentrations of additive 4 according to Embodiment 4 of the present application;

[0032] Figure 11 is the curve of the sequencing quality value varying with the number of cycles at each concentration of additive 4 according to Embodiment 4 of the present application;

[0033] Figure 12 is the curve of the error rate varying with the number of cycles at each concentration of additive 4 according to Embodiment 4 of the present application;

[0034] Figure 13 is the sequencing signal value based on additive 5 according to Embodiment 5 of the present application;

[0035] Figure 14 is the curve of the sequencing quality value based on additive 5 varying with the number of cycles according to Embodiment 5 of the present application;

[0036] Figure 15 is the curve of the error rate based on additive 5 varying with the number of cycles according to Embodiment 5 of the present application;

[0037] Figure 16 is the sequencing signal value based on additives 1 - 5 according to Embodiment 6 of the present application;

[0038] Figure 17 is the curve of the sequencing quality value based on additives 1 - 5 varying with the number of cycles according to Embodiment 6 of the present application;

[0039] Figure 18 It is a curve showing the change of error rate based on Additive 1-5 with the number of cycles according to Embodiment 6 of the present application. Detailed implementation manners

[0040] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention and do not limit the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0041] The present application is made by the inventors based on the following understanding:

[0042] In the related art, bases with special modifications at the ends are labeled with different groups, and these groups will specifically bind to the groups coupled with different target enzymes to connect the enzymes to the bases. Under the action of the substrate, they react with the enzymes to emit light signals (i.e., bioluminescence). However, during the sequencing process, especially during the double-stranded sequencing of paired-end sequencing, the sequencing quality using substrates such as coelenterazine substrates and fluorinated coelenterazine substrates is not good. Through a large number of experiments, the inventors surprisingly found that adding additives to the substrate can effectively stabilize the substrate and protect the DNA molecules to be tested, and produce high-quality sequencing data. The addition of additives can avoid a certain degree of oxidation of the substrate and the release of free radicals, and can scavenge the free radicals generated during the reaction process, thereby making the substrate more stable. At the same time, it avoids the damage of the generated free radicals to the DNA molecules to be tested, thereby effectively improving the sequencing quality.

[0043] The substrate additives proposed in the embodiments of the present application can improve the sequencing quality of the sequencing process, especially the reverse sequencing in paired-end sequencing. Specifically, it can improve the recovery multiple of the double-stranded signal (reverse sequencing signal) in paired-end sequencing (that is, the ratio of the signal value of the first cycle of the double strand to the signal value of the first cycle of the single strand. The higher the recovery multiple, the better the quality of the double strand, and the higher the data quality of the double strand will be), making the sequencing reaction more stable, thereby improving the sequencing quality of the reverse strand in paired-end sequencing.

[0044] In the embodiments of the present application, a "substrate" is a molecule or compound on which an enzyme acts, including naturally and non-naturally occurring proteins and chemical compounds that emit light when acted on by a luminescent enzyme. Substrate molecules include substrate molecules that undergo a catalytic reaction with an enzyme to emit light under suitable conditions. Examples of substrate molecules include luciferin, coelenterazine, luminol, isoluminol, or their derivatives or analogs.

[0045] In embodiments of the present application, a "derivative" or "analogue" refers to a compound or molecule having the same or very similar core structure to the parent compound but with chemical or physical modifications, such as having different or additional side groups, or 2' and / or 3' protecting groups. For example, the term includes derivatives or analogues of a substrate molecule (e.g., analogues and derivatives of coelenterazine), which produce a signal after being exposed to a catalyst (e.g., luciferase) under suitable conditions.

[0046] In some embodiments, the substrate molecule is coelenterazine or a derivative or analogue thereof. Coelenterazine (e.g., C 26 H 21 N3O3; CAS NO: 55779-48-1) is present in aquatic organisms, including squid, shrimp, ctenophores, and radiolarians. Coelenterazine is a substrate for luciferase.

[0047] Derivatives of coelenterazine are known in the art. For example, Vece and Vuocolo substituted the C-3 position to obtain three novel derivatives (Tetrahedron, 71(46): 8781-85 (2015)); while Yuan et al. replaced the methylene at C-8 with an electron conjugate, resulting in a 63 nm shift in the emission signal relative to native coelenterazine (Chinese Chemical Letters, 27(4): 550-554 (2016)).

[0048] Derivatives of coelenterazine include coelenterazine cp (luminescence intensity 15 times higher than native coelenterazine), coelenterazine F (luminescence intensity 20 times higher than native coelenterazine, emission spectrum about 8 nm longer than native coelenterazine), coelenterazine FCP (luminescence intensity 135 times higher than native coelenterazine), coelenterazine H (luminescence intensity 10 times higher than native coelenterazine), coelenterazine hcp (luminescence intensity 190 times higher than native coelenterazine), all of which can be obtained from Biotium, Fremont, CA. Other derivatives of coelenterazine may also include coelenterazine e, coelenterazine n, dimethyl coelenterazine, etc.

[0049] Other derivatives of coelenterazine include, but are not limited to, bis-deoxycoelenterazine analogues (see, e.g., Nakamura et al., Tetrahedron Letters, 38: 6405 (1997)) and coelenterazine analogues described in U.S. Patent No. 8,809,529, U.S. Patent Publication 2017 / 0217969, PCT Publication WO2018 / 022865, and Japanese Patent No. 6,160,716.

[0050] In some embodiments, the derivative of coelenterazine can be fluorocoelenterazine.

[0051] In the embodiments of the present application, "luminescence" refers to the light (detectable signal) emitted by a substance caused by a chemical reaction. Luminescence is generated by the action of an enzyme or a catalyst (such as luciferase or horseradish peroxidase) on a substrate molecule (such as coelenterazine or luminol). As used herein, luminescence includes chemiluminescence and bioluminescence, preferably bioluminescence. The reaction that generates luminescence can be referred to as a "luminescence reaction". The enzymes involved in these reactions can be referred to as "luminescent enzymes" or "enzymes that generate luminescence".

[0052] In the embodiments of the present application, a "luminescent enzyme", "fluorescent enzyme" or "enzyme that generates luminescence" refers to an enzyme that catalyzes a reaction in which a substrate molecule releases a fluorescent group to generate light. Examples of luminescent enzymes include firefly luciferase, Renilla luciferase, Cypridina luciferase, Aequorin photoprotein, Obelin luminescent protein, peroxidase, horseradish peroxidase, etc. In some embodiments, the fluorescent enzyme can be luciferase.

[0053] In the embodiments of the present application, the additive can include a stabilizer and an optional chelating agent and / or catalyst. In some embodiments, the stabilizer is selected from at least one of the following: potassium citrate, sodium salicylate, chitosan, sodium ascorbate (VCNa), vitamin E, epigallocatechin gallate (EGCG), glutathione (GSH), cysteine or its derivatives. In some embodiments, vitamin E can be water-soluble vitamin E (Trolox). In some embodiments, cysteine can be L-cysteine (L-Cysteine).

[0054] In some embodiments, the chelating agent can be ethylenediaminetetraacetic acid (EDTA), etc. In some embodiments, the catalyst can be one or more of the following: calcium ions, potassium ions, sodium ions, etc.

[0055] It can be understood that the addition of the additive avoids a certain degree of oxidation of the substrate and the release of free radicals, and scavenges the free radicals generated during the reaction through reduction or chelation, etc., thereby making the substrate more stable, and at the same time avoiding the damage of the generated free radicals to the DNA molecule to be detected, thus effectively improving the sequencing quality.

[0056] In the embodiments of the present application, the substrate may be present in an amount corresponding to the additive. In some embodiments, the final concentration of sodium ascorbate in the substrate system may be 100 - 500 mM, preferably 200 - 400 mM; the final concentration of vitamin E may be 0.5 - 6 mM, preferably 1 - 6 mM; the final concentration of epigallocatechin gallate may be 0.3 - 1 mM, preferably 0.5 - 0.8 mM; the final concentration of glutathione may be 0.2 - 4 mM, preferably 1 - 2 mM or the final concentration of cysteine may be 0.2 - 4 mM, preferably 0.3 - 1 mM. In some embodiments, the final concentration of sodium ascorbate in the substrate system may be 400 mM. In some embodiments, the final concentration of vitamin E may be 4 mM. In some embodiments, the final concentration of epigallocatechin gallate may be 0.8 mM. In some embodiments, the final concentration of glutathione may be 1 mM. In some embodiments, the final concentration of cysteine may be 0.5 mM.

[0057] In the embodiments of the present application, the DNA molecule to be tested may be a polymer of nucleotide monomers. As used herein, the term may refer to single-stranded or double-stranded forms. In some embodiments, the DNA molecule to be tested may be a library molecule, which contains the nucleotide sequence to be tested and the adapter sequence for sequencing. In some embodiments, the DNA molecule to be tested may be a DNB molecule obtained after rolling circle amplification. In some embodiments, the DNA molecule to be tested may also be a double-stranded DNA molecule (also referred to as an MDA strand) synthesized based on DNB in paired-end sequencing.

[0058] In the embodiments of the present application, "recovery" refers to the ratio of the signal value of the first cycle of the double-strand to the signal value of the first cycle of the single-strand. The higher the recovery multiple, the better the quality of the double-strand, and the higher the data quality of the double-strand will be accordingly.

[0059] In the embodiments of the present application, the "sequencing kit" may include reagents for sequencing. In some embodiments, the sequencing kit includes the substrate system for sequencing proposed in any of the above embodiments. In some embodiments, the kit may further include an enzyme that acts on the substrate system. In some embodiments, based on bioluminescence, the kit may contain a fluorescent enzyme, such as luciferase. It can be understood that the specific type of enzyme used can be determined according to actual needs and the type of substrate.

[0060] In some embodiments, the kit may further include DNB preparation reagents for obtaining DNBs based on rolling circle amplification of ordinary double-stranded and / or single-stranded libraries. In some embodiments, the kit may further include a mediation sequence (also referred to as a molecular anchor) and modified bases. The mediation sequence can bind to the DNA molecule to be detected to serve as a primer. Under its guidance, modified bases (dNTPs) with different labeling groups are loaded onto the DNA molecule to be detected. Subsequently, the labeling groups on the dNTPs specifically bind to the groups coupled to different target enzymes, thereby specifically linking different target enzymes to the corresponding bases. Then, based on the binding between the different target enzymes linked to the bases and the substrate and the luminescence situation, and based on the conversion of photoelectric signals, the capture of sequencing signals is completed. It can be understood that during this sequencing process, by adding additives to the substrate, a certain degree of oxidation of the substrate and the release of free radicals can be avoided, and the free radicals generated during the reaction can be scavenged. Thereby, the substrate is made more stable, and at the same time, the generated free radicals are prevented from damaging the DNA molecule to be detected, thereby achieving high-quality sequencing.

[0061] In the embodiments of the present application, the sequencing can be single-end sequencing or paired-end sequencing, preferably paired-end sequencing.

[0062] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0063] Unless otherwise specified, in the following embodiments, the quantitative analysis tests are all set with three repeated experiments, and the results are averaged.

[0064] Example 1

[0065] In this example, sodium ascorbate (VCNa) was used as an additive (hereinafter referred to as additive 1), and paired-end sequencing was performed on the standard library reagent (PCR product) based on the ordinary substrate in the prior art (i.e., signal reagent 1 and signal reagent 2 in the following DNBSEQ-E5RS sequencing kit). The read length of the first strand sequencing was 101 cycles (one cycle corresponds to the sequencing of one base, i.e., 101 bp), and the read length of the second strand sequencing was 24 cycles (i.e., 24 bp).

[0066] 1 Experimental materials

[0067] a. Standard library reagent (PCR product) V3.0 (purchased from MGI, product number: 1000005038)

[0068] b. DNBSEQ-E5RS sequencing kit (purchased from MGI, product number: 1000026249)

[0069] c. DNBSEQ One-step DNB Preparation Kit V2.0 (OS-SB) (purchased from MGI, product number: 940-000035-00)

[0070] d. Sodium ascorbate (VCNa) (sigma)

[0071] 2 Experimental equipment

[0072] a. DNBSEQ-E5RS Genetic Sequencer (MGI, product number 900-000388-00)

[0073] b. DNBSEQ-E5RS Integrated Sequencing Slide (MGI, product number 930-000040-00)

[0074] c. PCR Amplifier (Life)

[0075] 3 Experimental procedures

[0076] 3.1 Referring to the instructions of the DNBSEQ one-step DNB preparation kit, the double-stranded DNA of the standard library reagent (PCR product) was subjected to rolling circle amplification to prepare DNA nanoballs. The time for DNA rolling circle replication was 40 min.

[0077] 3.2 Chip loading: Referring to the usage instructions of the DNBSEQ-E5 sequencing slide, the prepared DNA nanoballs were loaded onto the chip.

[0078] 3.3 Sequencing reagent loading: Referring to the instructions of the DNBSEQ-E5RS sequencing kit, the DNBSEQ-E5RS sequencing reagent was placed in the DNBSEQ-E5RS sequencer and the sequencing reagent was loaded.

[0079] 3.4 Sequencing: Referring to the instructions of the DNBSEQ-E5RS sequencing kit, the corresponding built-in control program (paired-end sequencing) of the sequencer was started for sequencing.

[0080] 3.5 Off-machine data analysis: The ImageE module of the sequencer was used to perform base call on the off-machine data, and at the same time, data filtering was not performed, and the output data was subjected to quality assessment.

[0081] 4. Experimental results and analysis

[0082] In this example, the effect of additive 1 was measured. Based on the experimental method of this example, the concentration gradient of additive 1 was further set, that is, 100 mM, 150 mM, 200 mM, 300 mM, and 400 mM of additive 1 were added to the substrate respectively, and the sequencing data produced was analyzed. The results are shown in Table 1.

[0083] Table 1 Quality Report of Double - end Sequencing Data of Additive 1 (VCNa)

[0084]

[0085]

[0086] As can be seen from Table 1, with the addition of Additive 1, the total number of reads, Q30, alignment rate, etc. of the output data are stable, and the signal recovery multiples of the two channels show an upward trend, indicating that the addition of Additive 1 can improve the sequencing quality. At the same time, the Q30, alignment rate, and signal recovery multiples of the two channels of the data increase with the increase in the concentration of Additive 1, and the average error rate decreases with the increase in concentration. Therefore, compared with lower concentrations, 300 mM and 400 mM of Additive 1 show a better effect on stabilizing and improving the data quality.

[0087] Figure 1 This is the sequencing signal value of using different concentrations of Additive 1 in this embodiment, where the vertical axis represents the signal value intensity and the horizontal axis represents the sequencing cycle number. Among them, the first 124 are the signal values of Channel 1 with 100 cycles of the first strand plus 24 cycles of the second strand, and the last 124 are the signal values of Channel 2 with 100 cycles of the first strand plus 24 cycles of the second strand. As Figure 1 can be seen, in the second - strand sequencing (starting from the 102nd cycle), for different concentrations of Additive 1, the signal value changes with the increase in the concentration of Additive 1, and 200 mM, 300 mM, and 400 mM of Additive 1 show a higher signal recovery ratio.

[0088] Figure 2 This is the curve of the sequencing quality value of Additive 1 changing with the cycle number at each concentration, where the horizontal axis is the sequencing cycle number and the vertical axis is the Q30 value. As Figure 2 shown, each group with the addition of Additive 1 shows an extremely high Q30 quality in the first 101 cycles, indicating that the Additive 1 proposed in this embodiment can effectively guarantee the quality value of the data; at the same time, the groups with 200 mM, 300 mM, and 400 mM of Additive 1 show a higher Q30 quality value and can stably maintain this high quality value with the increase in the cycle number.

[0089] Figure 3 This is the curve of the error rate of Additive 1 changing with the cycle number at each concentration, where the horizontal axis is the sequencing cycle number and the vertical axis is the error rate value. As Figure 3 shown, each group with the addition of Additive 1 shows a lower error rate in the first 101 cycles, indicating that the Additive 1 proposed in this embodiment can effectively guarantee the accuracy of the sequencing data; at the same time, the groups with 200 mM, 300 mM, and 400 mM of Additive 1 show a lower error rate and can stably maintain this low error rate with the increase in the cycle number.

[0090] In summary, considering various sequencing quality index parameters comprehensively, generally speaking, adding Additive 1 to the substrate can stabilize the substrate and improve the sequencing quality. At the same time, adding 200 mM, 300 mM, and 400 mM of Additive 1 to the substrate can increase the double-strand signal recovery multiple and the sequencing quality of the double strand. With the increase in the concentration of Additive 1, the sequencing quality shows a gradually increasing trend. Additive 1 at 300 mM and 400 mM shows better effects, and 400 mM is better.

[0091] Example 2

[0092] In this example, water-soluble vitamin E (Trolox) was used as an additive (hereinafter referred to as Additive 2) for relevant experiments. The experimental materials, experimental equipment, operation steps, etc. were the same as those in Example 1. The experimental results and analysis are as follows.

[0093] Table 2 Quality Report of Paired-End Sequencing Data of Additive 2 (Trolox)

[0094]

[0095] As can be seen from Table 2, compared with the data produced by the ordinary substrate without adding any additives (i.e., the control group, whose experimental operations are exactly the same as those of each group with additives except for not adding additives), with the addition of Additive 2, the Q30, alignment rate, etc. of the produced data show obvious improvement, and the double-channel signal recovery multiples are equivalent to or higher than those of the control, and the average error rate is reduced, indicating that the addition of Additive 2 can improve the sequencing quality. At the same time, the Q30, alignment rate, and double-channel signal recovery multiples of the data are higher in the groups with 2 mM and 4 mM of Additive 2, and the average error rate is lower. Therefore, Additive 2 at 2 mM and 4 mM shows a more excellent effect on stabilizing and improving the data quality.

[0096] Figure 4 This is the sequencing signal value of using different concentrations of Additive 2 in this example, where the vertical axis represents the signal value intensity and the horizontal axis represents the sequencing cycle number. Among them, the first 124 are the signal values of Channel 1 for 100 cycles of the first strand plus 24 cycles of the second strand, and the last 124 are the signal values of Channel 2 for 100 cycles of the first strand plus 24 cycles of the second strand. Figure 4 It can be seen that in the second-strand sequencing (i.e., starting from the 102nd cycle), for different concentrations of Additive 2, the signal value changes with the increase in the concentration of Additive 2. Compared with the control group, Additive 2 at 2 mM and 4 mM shows a higher signal recovery ratio.

[0097] Figure 5 This is the curve of the sequencing quality value changing with the cycle number at each concentration of Additive 2, where the horizontal axis is the sequencing cycle number and the vertical axis is the Q30 value. AsFigure 5 As shown, each group added with additive 2 showed extremely high Q30 quality in the first 101 cycles, and still showed stable Q30 values after 101 cycles (i.e., the double-stranded sequencing process) compared with the control group, indicating that additive 2 proposed in this embodiment can effectively ensure the quality value of data, especially double-stranded data, throughout the sequencing process; at the same time, compared with the control group, the groups added with 2 mM and 4 mM additive 2 showed higher Q30 quality values and could stably maintain this high quality value as the number of cycles increased.

[0098] Figure 6 It is a curve of the error rate varying with the number of cycles at each concentration of additive 2, where the horizontal axis is the number of sequencing cycles and the vertical axis is the error rate value. As Figure 6 shown, except for the 6 mM group, each group added with additive 2 showed a lower error rate in the first 101 cycles and still maintained an error rate lower than that of the control after 101 cycles (i.e., the double-stranded sequencing process), indicating that additive 2 proposed in this embodiment can effectively ensure the accuracy of sequencing data, especially double-stranded data; at the same time, the groups added with 2 mM and 4 mM additive 2 showed even lower error rates and could stably maintain this low error rate as the number of cycles increased.

[0099] In summary, considering all sequencing quality index parameters comprehensively, generally speaking, adding additive 2 to the substrate can stabilize the substrate and improve the sequencing quality; at the same time, adding 2 mM, 4 mM, and 6 mM of additive 2 to the substrate can increase the double-stranded signal recovery multiple and the sequencing quality of double-stranded. Additive 2 at 2 mM and 4 mM shows better effects, and 4 mM is even better.

[0100] Example 3

[0101] In this example, epigallocatechin gallate (EGCG) was used as an additive (hereinafter referred to as additive 3) for relevant experiments. The experimental materials, experimental equipment, operation steps, etc. were the same as those in Example 1. The experimental results and analysis are as follows.

[0102] Table 3 Quality Report of Double-End Sequencing Data of Additive 3 (EGCG)

[0103]

[0104]

[0105] As can be seen from Table 3, compared with the data produced by the ordinary substrate without any additives (i.e., the control group, whose experimental operations are exactly the same as those of the groups with additives except for the absence of additives), with the addition of Additive 3, the total data volume (i.e., the total number of reads), Q30, ESR value, alignment rate, etc. of the output data show obvious improvement. Moreover, the signal recovery multiples of the two channels are higher than those of the control, and the average error rate is significantly reduced, indicating that the addition of Additive 3 can improve the sequencing quality. At the same time, the Q30, ESR value, alignment rate, and signal recovery multiples of the two channels of the data all show relatively high levels in the groups with Additive 2 concentrations of 0.8 mM and 0.5 mM, and the average error rate is relatively low. Therefore, Additive 2 at 0.8 mM and 0.5 mM demonstrates a strong effect on stabilizing and improving the data quality.

[0106] Figure 7 Sequencing signal values of different concentrations of Additive 3 in this embodiment, where the vertical axis represents the signal value intensity and the horizontal axis represents the sequencing cycle number. Among them, the first 124 are the signal values of Channel 1 with 100 cycles of the first strand plus 24 cycles of the second strand, and the last 124 are the signal values of Channel 2 with 100 cycles of the first strand plus 24 cycles of the second strand. As Figure 7 can be seen, among different concentrations of Additive 3, the signal value changes with the increase in the concentration of Additive 2. Compared with the control group, Additive 3 at 0.8 mM shows a higher signal recovery ratio.

[0107] Figure 8 Curves showing the change of sequencing quality values with the cycle number at different concentrations of Additive 3, where the horizontal axis is the sequencing cycle number and the vertical axis is the Q30 value. As Figure 8 shown, other groups with Additive 3 added show extremely high Q30 quality in the first 101 cycles, and compared with the control group, they still show stable Q30 values after 101 cycles (i.e., during the second-strand sequencing process), indicating that Additive 3 proposed in this embodiment can effectively guarantee the quality value of data throughout the sequencing process. At the same time, compared with the control group, the groups with 0.8 mM and 0.5 mM of Additive 3 added both show higher Q30 quality values and can stably maintain this high-quality value with the increase in the cycle number.

[0108] Figure 9 Curves showing the change of error rate with the cycle number at different concentrations of Additive 3, where the horizontal axis is the sequencing cycle number and the vertical axis is the error rate value. As Figure 9 shown, the groups with Additive 3 added, especially the group with 0.5 mM, show a lower error rate in the first 101 cycles, and after 101 cycles (i.e., during the second-strand sequencing process), the groups with Additive 3 added still maintain an error rate lower than that of the control, indicating that Additive 3 proposed in this embodiment can effectively guarantee the accuracy of sequencing data, especially the second-strand sequencing data (reverse sequencing data).

[0109] In summary, considering all sequencing quality index parameters, generally speaking, adding additive 3 to the substrate can stabilize the substrate and improve the sequencing quality; at the same time, adding 0.5 mM and 0.8 mM of additive 3 to the substrate can increase the double-strand signal recovery multiple and the sequencing quality of the double strand. Comparing the two, 0.8 mM of additive 3 shows better effects.

[0110] Example 4

[0111] In this example, glutathione (GSH) was used as an additive (hereinafter referred to as additive 4) for relevant experiments. Except that the double-strand sequencing read length became 23 cycles (i.e., 23 bp), other experimental materials, experimental equipment, operation steps, etc. were the same as in Example 1. The experimental results and analysis are as follows.

[0112] Table 4 Quality Report of Double-End Sequencing Data of Additive 4 Glutathione (GSH)

[0113]

[0114] As can be seen from Table 4, compared with the data produced by the ordinary substrate without any additive (i.e., the control group, whose experimental operations are exactly the same as those of each group with additive except that no additive is added), with the addition of additive 4, the Q30, alignment rate, etc. of the produced data show an increase, and the double-channel signal recovery multiple is higher than that of the control, and the average error rate is reduced, indicating that the addition of additive 4 can improve the sequencing quality. At the same time, the Q30, alignment rate, and double-channel signal recovery multiple of the data are relatively high in the groups with 1 mM and 2 mM of additive 4 and the average error rate is relatively low. Therefore, 1 mM and 2 mM of additive 4 show excellent effects on stabilizing and improving the data quality.

[0115] Figure 10 This is the sequencing signal value of using different concentrations of additive 4 in this example, where the vertical axis represents the signal value intensity and the horizontal axis represents the sequencing cycle number. Among them, the first 123 are the signal values of channel 1 of 100 cycles of the first strand plus 23 cycles of the second strand, and the last 123 are the signal values of channel 2 of 100 cycles of the first strand plus 23 cycles of the second strand. As Figure 10 can be seen, in the second-strand sequencing (i.e., starting from the 102nd cycle), for different concentrations of additive 4, the signal value changes with the increase of the concentration of additive 4. Compared with the control group, both 1 mM and 2 mM of additive 4 show higher signal recovery ratios.

[0116] Figure 11 This is the curve of the sequencing quality value changing with the cycle number at each concentration of additive 4, where the horizontal axis is the sequencing cycle number and the vertical axis is the Q30 value. As Figure 11As shown, each group added with additive 4 showed extremely high Q30 quality in the first 101 cycles, and still showed stable and higher Q30 values after 101 cycles (i.e., during the double-strand sequencing process) compared with the control group, indicating that the additive 4 proposed in this embodiment can effectively guarantee the quality value of data, especially double-strand data, throughout the sequencing process and can stably maintain this high-quality value as the number of cycles increases.

[0117] Figure 12 Figure 4 shows the curves of error rates at various concentrations of additive 4 changing with the number of cycles, where the horizontal axis is the number of sequencing cycles and the vertical axis is the error rate value. As Figure 12 shown, each group added with additive 4 showed lower error rates in the first 101 cycles and still maintained error rates lower than those of the control after 101 cycles (i.e., during the double-strand sequencing process), indicating that the additive 4 proposed in this embodiment can effectively guarantee the accuracy of sequencing data, especially double-strand data.

[0118] In summary, considering all sequencing quality index parameters comprehensively, generally speaking, adding additive 4 to the substrate can stabilize the substrate and improve the sequencing quality, especially can significantly improve the double-strand signal recovery multiple and the sequencing quality of double strands. Additive 4 at both 1 mM and 2 mM showed better effects, with 1 mM being better.

[0119] Example 5

[0120] In this example, L-Cysteine was used as an additive (hereinafter referred to as additive 5) for relevant experiments. Except that the double-strand sequencing read length became 28 cycles (i.e., 28 bp), other experimental materials, experimental equipment, operation steps, etc. were the same as those in Example 1. The experimental results and analysis are as follows.

[0121] Table 5 Quality Report of Double-End Sequencing Data of Additive 5 L-Cysteine

[0122]

[0123]

[0124] As can be seen from Table 5, compared with the data produced by the ordinary substrate without adding any additives (i.e., the control group, whose experimental operations are exactly the same as those of each group added with additives except for not adding additives), with the addition of additive 5, the total data volume (i.e., the total number of reads), Q30, alignment rate, etc. of the produced data showed obvious improvement, and the double-channel signal recovery multiple was higher than that of the control, indicating that the addition of additive 5 can improve the sequencing quality and demonstrated a strong role in stabilizing and improving the data quality.

[0125] Figure 13The sequencing signal values based on additive 5 in this embodiment, where the vertical axis represents the signal value intensity and the horizontal axis represents the sequencing cycle number. Among them, the first 128 are the signal values of channel 1 for 100 cycles of the first strand plus 28 cycles of the second strand, and the last 128 are the signal values of channel 2 for 100 cycles of the first strand plus 28 cycles of the second strand. From Figure 13 It can be seen that for the signal values produced by adding additive 5, the signal recovery multiple is significantly higher than that of the control group during the second-strand sequencing (i.e., starting from the 102nd cycle), indicating that the addition of additive 5 is beneficial to the growth of the second strand and the increase of signal values during the sequencing process.

[0126] Figure 14 This is the curve of the sequencing quality values based on additive 5 in this embodiment changing with the cycle number, where the horizontal axis is the sequencing cycle number and the vertical axis is the Q30 value. As Figure 14 shown, the group with additive 5 shows extremely high Q30 quality in the first 101 cycles, and compared with the control group, it still shows a stable and higher Q30 value after 101 cycles (i.e., during the second-strand sequencing process), indicating that the additive 5 proposed in this embodiment can effectively ensure the quality value of data throughout the sequencing process.

[0127] Figure 15 This is the curve of the error rate based on additive 5 in this embodiment changing with the cycle number, where the horizontal axis is the sequencing cycle number and the vertical axis is the error rate value. As Figure 15 shown, the group with additive 5 shows a lower error rate in the first 101 cycles, and the group still maintains an error rate comparable to that of the control after 101 cycles (i.e., during the second-strand sequencing process), indicating that the addition of additive 5 proposed in this embodiment does not affect the accuracy of the data and effectively ensures the accuracy of the sequencing data, especially the second-strand sequencing data (reverse sequencing data).

[0128] In summary, considering all the sequencing quality index parameters comprehensively, generally speaking, adding additive 5 to the substrate does not affect other sequencing indicators. At the same time, the addition of additive 5 can stabilize the substrate and can increase the signal recovery multiple of the second strand and the sequencing quality of the second strand. As an example, 0.5 mM of additive 5 shows excellent effects.

[0129] Verified by the above embodiments, the additives 1-5 proposed in the embodiments of this application can stabilize the sequencing process with the increase of the sequencing cycle number and produce high-quality sequencing data. Especially during the sequencing of the single-end 100 bp and the second strand of the paired-end, it shows extremely high stability compared with the control. Therefore, the additives proposed in the embodiments of this application can be effectively used in the substrate system for sequencing.

[0130] Example 6

[0131] Based on the optimal concentrations in Embodiments 1-5, further comparative experiments were conducted on Additives 1-5. The experimental materials, experimental equipment, operating procedures, etc. were the same as those in Embodiment 1. The concentrations of Additives 1-5 used and the data output are shown in Table 6.

[0132] Table 6 Quality Report of Double-End Sequencing Data of Additives 1-5

[0133]

[0134] As can be seen from Table 6, compared with the data produced by the ordinary substrate without any additives (i.e., the control group, whose experimental operations are exactly the same as those of the groups with additives except for no additives), with the addition of Additives 1-5, the total data volume (i.e., the total number of reads), Q30, ESR value, alignment rate, etc. of the output data showed significant improvement, and the signal recovery multiples of the two channels were higher than those of the control, indicating that the addition of Additives 1-5 can effectively improve the sequencing quality and demonstrate a strong role in stabilizing and improving the data quality.

[0135] Figure 16 This is the sequencing signal value based on Additives 1-5 in this embodiment, where the vertical axis represents the signal value intensity and the horizontal axis represents the sequencing cycle number.

[0136] Figure 17 This is the curve of the sequencing quality value based on Additives 1-5 changing with the cycle number in this embodiment, where the horizontal axis is the sequencing cycle number and the vertical axis is the Q30 value.

[0137] Figure 18 This is the curve of the error rate based on Additive 5 changing with the cycle number in this embodiment, where the horizontal axis is the sequencing cycle number and the vertical axis is the error rate value.

[0138] As Figures 16 - 18 shown, the signal recovery multiples of the double-stranded signals of Additives 1-5 are all higher than those of the control group, indicating that the addition of Additives 1-5 is beneficial to stabilizing the oxidation of the substrate itself and avoiding some substances generated during the reaction from affecting the growth of the double strand and further affecting the sequencing quality of the double strand; at the same time, the groups with the addition of Additives 1-5 showed extremely high Q30 quality in the first 100 cycles, and compared with the control group, they still showed stable and higher Q30 values after 100 cycles (i.e., during the double-stranded sequencing process), indicating that the Additives 1-5 proposed in this embodiment can effectively ensure the quality value of the data throughout the sequencing process; moreover, the groups with the addition of Additives 1-5 showed a lower error rate in the first 100 cycles, and the groups still maintained an error rate significantly lower than that of the control after 100 cycles (i.e., during the double-stranded sequencing process), indicating that the addition of the Additives 1-5 proposed in this embodiment did not affect the accuracy of the data and effectively ensured the accuracy of the sequencing data, especially the double-stranded sequencing data (reverse sequencing data).

[0139] In summary, considering various sequencing quality index parameters comprehensively, generally speaking, adding additives 1-5 to the substrate does not affect other sequencing indexes. At the same time, the addition of additives 1-5 can stabilize the substrate and improve the double-strand signal recovery multiple and the sequencing quality of double-strands. As an example, 0.5 mM of additive 5 shows more excellent effects compared with the corresponding concentrations of additives 1-4.

[0140] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0141] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A substrate system for sequencing, characterized in that, The substrate system includes: a substrate and an additive, wherein the additive includes a stabilizer. Optionally, the substrate is coelenterazine or a derivative thereof. Optionally, the derivative is selected from the group consisting of fluorocoelenterazine, bis-deoxycoelenterazine analog, coelenterazine cp, coelenterazine F, coelenterazine FCP, coelenterazine H, coelenterazine hcp, coelenterazine e, coelenterazine n, and dimethylcoelenterazine.

2. The substrate system according to claim 1, characterized in that, The stabilizer is selected from at least one of the following: potassium citrate, sodium salicylate, chitosan, sodium ascorbate, vitamin E, epigallocatechin gallate, glutathione, cysteine or a derivative thereof. Optionally, the vitamin E is water-soluble vitamin E, and the cysteine is L-cysteine. Optionally, the additive further includes a chelating agent and / or a catalyst. The chelating agent is EDTA; the catalyst is one or more of the following: calcium ion, potassium ion, and sodium ion.

3. The substrate system according to claim 1 or 2, characterized in that, The stabilizer is at least one of the following: sodium ascorbate, vitamin E, epigallocatechin gallate, glutathione, cysteine or a derivative thereof. Preferably, the stabilizer is cysteine, and more preferably L-cysteine.

4. The substrate system according to any one of claims 1 to 3, characterized in that, In the substrate system, the final concentration of sodium ascorbate is 100 - 500 mM, preferably 200 - 400 mM; the final concentration of vitamin E is 0.5 - 6 mM, preferably 1 - 6 mM; the final concentration of epigallocatechin gallate is 0.3 - 1 mM, preferably 0.5 - 0.8 mM; the final concentration of glutathione is 0.2 - 4 mM, preferably 1 - 2 mM; or the final concentration of cysteine is 0.2 - 4 mM or less, preferably 0.3 - 1 mM. More preferably, in the substrate system, the final concentration of sodium ascorbate is 400 mM, the final concentration of vitamin E is 4 mM, the final concentration of epigallocatechin gallate is 0.8 mM, the final concentration of glutathione is 1 mM, or the final concentration of cysteine is 0.5 mM.

5. The substrate system according to any one of claims 1 to 4, characterized in that, The final concentration of the substrate is 300 μM.

6. A sequencing kit, characterized in that, The kit includes: a substrate system for sequencing as described in any one of claims 1 to 5, and optionally a mediator sequence, a luciferase, and a modified base. Optionally, the luciferase includes luciferase. Optionally, the kit further includes a DNB preparation reagent.

7. A biological self-luminescent sequencing method, characterized in that, The method includes: mixing a substrate with an additive to form a substrate system for sequencing, wherein the additive includes a stabilizer. Optionally, the substrate is coelenterazine or a derivative thereof. Optionally, the derivative is selected from the group consisting of fluorocoelenterazine, bis-deoxycoelenterazine analog, coelenterazine cp, coelenterazine F, coelenterazine FCP, coelenterazine H, coelenterazine hcp, coelenterazine e, coelenterazine n, and dimethylcoelenterazine.

8. The method according to claim 7, characterized in that, The stabilizer is selected from at least one of the following: potassium citrate, sodium salicylate, chitosan, sodium ascorbate, vitamin E, epigallocatechin gallate, glutathione, cysteine or a derivative thereof. Optionally, the vitamin E is water-soluble vitamin E, and the cysteine is L-cysteine. Optionally, the additive further comprises a chelating agent and / or a catalyst, wherein the chelating agent is EDTA; the catalyst is one or more of the following: calcium ions, potassium ions, and sodium ions.

9. The method according to claim 7 or 8, characterized in that, The method further comprises: contacting the fluorescent enzyme with the substrate system to bind the substrate to the fluorescent enzyme to obtain a sequencing signal.

10. The application of the substrate system for sequencing according to any one of claims 1 - 5 in sequencing, characterized in that, The sequencing is single-end sequencing or paired-end sequencing, preferably paired-end sequencing.

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