WO2025007252A1 - Repeated sequencing method - Google Patents

Abstract

Provided is a repeated sequencing method, comprising: on the basis of a first sequencing primer, performing first sequencing on a sample to obtain a first sequencing sequence based on a first sequencing synthetic strand; bringing an elution reagent into contact with the first sequencing synthetic strand so as to remove the first sequencing synthetic strand; on the basis of the first sequencing primer, performing second sequencing on the sample to obtain a second sequencing sequence based on a second sequencing synthetic strand; and comparing the first sequencing sequence with the second sequencing sequence to determine sequence information of the sample.

Description

Repeat sequencing method Technical Field

The present application relates to the field of biological sequencing technology, and in particular to a method for repeated sequencing.

Background Art

In recent years, with the development of the gene sequencing industry, the technology of gene sequencing has advanced by leaps and bounds, from the first-generation Sanger sequencing method, the second-generation sequencing by synthesis (SBS) method to the third-generation single-molecule sequencing method. These sequencing technologies will inevitably have sequencing errors during the sequencing process due to various reasons such as sequencing reagents, sequencing samples, sequencing instruments and external environment. In particular, the second-generation sequencing technology is based on the PCR principle. During the process of sequencing by synthesis, sequencing errors are inevitable due to internal and external environments, resulting in reduced sequencing accuracy.

In order to improve sequencing accuracy, various sequencing platforms have proposed relevant solutions. For example, Illumina recently announced "Chemistry X", a new chemical method technology with high sequencing accuracy, including new dyes, linkers, blockers, and polymerases; Singular released the "Sequencing Engine" technology; Sena Bio combined Fluorogenic fluorescence sequencing chemistry technology and ECC (Error-Correction Code) error correction coding technology to improve sequencing accuracy. However, the optimization of these platforms focuses on biochemical reactions and signal acquisition, such as improving fluorescence intensity, improving the fidelity of polymerases, improving elution efficiency, or improving the accuracy of optical systems, which increases sequencing costs and is highly dependent on sequencing platforms.

Therefore, there is an urgent need to provide a simple, easy, low-cost, and high-accuracy sequencing method suitable for multiple platforms.

Summary of the invention

To this end, an embodiment of the present application provides a repeated sequencing method.

The first aspect of the present application proposes a repeated sequencing method, comprising: performing a first sequencing on a sample based on a first sequencing primer to obtain a first sequencing sequence based on a first sequencing synthetic chain; contacting an elution reagent with the first sequencing synthetic chain to remove the first sequencing synthetic chain; performing a second sequencing on the sample based on the first sequencing primer to obtain a second sequencing sequence based on a second sequencing synthetic chain; and comparing the first sequencing sequence and the second sequencing sequence to determine the sequence information of the sample.

In some embodiments, the first sequencing or the second sequencing of the sample based on the first sequencing primer includes: annealing the first sequencing primer to the sample and performing multiple base sequencing cycles, wherein each cycle completes signal detection of one base, thereby obtaining an optical signal of the first sequencing synthesis chain or the second sequencing synthesis chain, and obtaining the first sequencing sequence or the second sequencing sequence based on the optical signal of the first sequencing synthesis chain or the second sequencing synthesis chain.

In some embodiments, performing a first sequencing on the sample based on the first sequencing primer further comprises: fixing the sample on a solid phase carrier for sequencing. In some embodiments, the solid phase carrier is a bead or a chip.

In some embodiments, the elution reagent is one or more of the following: a DNA denaturant and/or an exonuclease, wherein the DNA denaturant includes methanol, ethanol, urea, formamide, and sodium hydroxide, and the exonuclease includes snake venom phosphodiesterase, Escherichia coli exonuclease I, Escherichia coli exonuclease II, Escherichia coli exonuclease III, spleen phosphodiesterase, and Lactobacillus acidophilus nuclease. In some embodiments, the DNA denaturant is formamide and/or sodium hydroxide, and the exonuclease is Escherichia coli exonuclease III.

In some embodiments, the concentration of formamide is 30%-100%. In some embodiments, the concentration of formamide is 40-100%. In some embodiments, the concentration of E. coli exonuclease III is 1-50 U/μL. In some embodiments, the concentration of E. coli exonuclease III is 1-50 U/μL. The concentration of exonuclease III is 1-10 U/μL.

In some embodiments, the elution time of the elution reagent is 0-20 min. In some embodiments, the elution time of the elution reagent is 5-15 min. In some embodiments, the elution time of the elution reagent is 5-10 min.

In some embodiments, the elution temperature of the elution reagent is 10-60° C. In some embodiments, the elution temperature of the elution reagent is 20-50° C. In some embodiments, the elution temperature of the elution reagent is 25-45° C.

In some embodiments, the first sequencing sequence and the second sequencing sequence are compared to determine the sequence information of the sample, including: comparing the bases at corresponding positions of the first sequencing sequence and the second sequencing sequence; in response to the bases at corresponding positions of the first sequencing sequence and the second sequencing sequence being the same, determining the same base as the base type at the position; in response to the bases at corresponding positions of the first sequencing sequence and the second sequencing sequence being different, determining the base type at the position by statistical data.

In some embodiments, the statistical data is a Q value.

In some embodiments, determining the base type at the position by using statistical data specifically includes: comparing the Q values of bases at corresponding positions of the first sequencing sequence and the second sequencing sequence, and determining the base x as the base type at the position based on the fact that the Q value of the base x at the position of the first sequencing sequence is greater than the Q value of the base y at the position of the second sequencing sequence.

In some embodiments, the repeated sequencing method further includes: repeating the sequencing process based on the first sequencing primer, including: sequencing the sample m times again to obtain m sequencing sequences based on m sequencing synthesis chains; allowing the elution reagent to contact the m or m-1 sequencing synthesis chains respectively to remove the m or m-1 sequencing synthesis chains; and comparing the first sequencing sequence, the second sequencing sequence and the m sequencing sequences to determine the sequence information of the sample.

In some embodiments, m is a positive integer greater than 0. In some embodiments, 1≤m≤10.

In some embodiments, the comparing the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences to determine the sequence information of the sample includes: comparing the bases at corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences; in response to the bases at corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences being the same, determining the same bases as the base type at the position; in response to the bases at corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences being different, determining the base with the highest occurrence frequency among m+2 bases as the base type at the position, or in response to the bases at corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences being different, determining the base type at the position by statistical data.

In some embodiments, the repeated sequencing method further comprises: in response to the first sequencing sequence, the second sequencing sequence and the m sequencing sequences having different bases at corresponding positions, and the m+2 bases having base types with the same frequency of occurrence, determining the base type at the position by using statistical data. In some embodiments, the statistical data is a Q value.

In some embodiments, the first sequencing primer is a forward sequencing primer or a reverse sequencing primer.

On the other hand, an embodiment of the present application also proposes a method for double-end repeated sequencing, comprising: based on a first sequencing primer, performing first-end repeated sequencing on a sample according to the repeated sequencing method as described in any of the above embodiments of the present application to obtain first-end sequence information of the sample; based on a second sequencing primer, performing second-end sequencing on the sample to obtain second-end sequence information of the sample; and analyzing the first-end sequence information and the second-end sequence information to determine the sequence information of the sample, wherein the first sequencing primer is one of a forward sequencing primer or a reverse sequencing primer, and the second sequencing primer is the other of the forward sequencing primer or the reverse sequencing primer.

On the other hand, an embodiment of the present application also proposes a method for double-end repeated sequencing, comprising: based on a first sequencing primer, performing first-end repeated sequencing on a sample according to a repeated sequencing method as described in any of the above embodiments of the present application to obtain first-end sequence information of the sample; based on a second sequencing primer, performing second-end repeated sequencing on the sample according to a repeated sequencing method as described in any of the above embodiments of the present application to obtain second-end sequence information of the sample; and analyzing the first-end sequence information and the second-end sequence information to determine the sequence information of the sample, wherein the first sequencing primer is a forward sequencing primer, and the first-end sequence information is forward-read sequence information, and the second sequencing primer is a reverse sequencing primer, and the second-end sequence information is reverse-read sequence information.

In some embodiments, performing second end repeat sequencing on the sample based on the second sequencing primer includes: generating a complementary strand of the sample, and performing the second end repeat sequencing based on the complementary strand.

In some embodiments, after generating the complementary chain of the sample, the method further comprises: removing the sample, and performing second-end sequencing or second-end repeated sequencing based on the complementary chain.

In some embodiments, the sample is a DNA nanoball, and the complementary strand is an MDA strand.

In some embodiments, the sample contains barcode 1 and optionally barcode 2, and the method further includes: sequencing the barcode 1 and the barcode 2 respectively to obtain sequence information of the barcode 1 and the barcode 2; and screening out the sequence information of the sample based on the sequence information of the barcode 1 and the barcode 2.

On the other hand, the present application also proposes a repeated sequencing system, including: a sequencing reagent and an elution reagent, wherein the elution reagent is one or more of the following: a DNA denaturant and/or a nuclease, wherein the DNA denaturant includes: methanol, ethanol, urea, formamide, sodium hydroxide, and the nuclease includes: snake venom phosphodiesterase, Escherichia coli nuclease I, Escherichia coli nuclease II, Escherichia coli nuclease III, spleen phosphodiesterase, and Lactobacillus acidophilus nuclease. In some embodiments, the DNA denaturant is formamide and/or sodium hydroxide, and the exonuclease is Escherichia coli exonuclease III.

In some embodiments, the sequencing reagent comprises a first sequencing primer, a reaction enzyme and a dNTP. In some embodiments, a fluorescent group is attached to the dNTP for base reporting. In some embodiments, the sequencing reagent further comprises a second sequencing primer. In some embodiments, the sequencing reagent further comprises a first barcode sequencing primer and optionally a second barcode sequencing primer.

In some embodiments, the reaction enzymes include a DNA polymerase and optionally a DNA ligase.

Another aspect of the present application also provides a sequencing kit, comprising a repeated sequencing system as described in any of the above embodiments of the present application.

The technical solution of this application achieves the following technical effects:

The repeated sequencing method and its application in the embodiment of the present application improve the sequencing accuracy from the sequencing logic, by sequencing the same nucleotide sequence template multiple times to obtain multiple sequencing sequences, and then using bioinformatics analysis to mutually correct the obtained multiple sequencing sequences, so as to achieve effective detection of sequence errors, effectively reduce the impact of sequencing errors caused by internal and external environments on sequencing results during sequencing, and significantly improve sequencing accuracy; at the same time, there is no need to perform multiple independent repeated sequencing processes, but only multiple sequencing operations for the same nucleotide sequence template in a single experiment, so it has the advantages of simple operation and low cost. In addition, this method is applicable to multiple sequencing platforms and can be widely used to improve the sequencing accuracy of multiple platforms.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a schematic diagram of a repeated sequencing method according to an embodiment of the present application;

FIG. 2 is a schematic diagram of another repeated sequencing method according to an embodiment of the present application.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and are not intended to limit the present invention in any way.

This application is made based on the following knowledge of the inventor:

In the related art, the second-generation sequencing platforms all use the SBS method, and their sequencing processes are similar, including injecting the reaction solution into the chip by pressure, generating a polymerization reaction, collecting light signals, and then eluting to sequence the next base. Such a process based on the PCR principle is affected by multiple factors such as the external environment, the instrument itself, sequencing reagents, and samples. For example, base mismatches are prone to occur, so sequencing errors cannot be avoided, and with the accumulation of sequencing read lengths, the probability of sequencing errors increases, resulting in a high error rate at the end of the sequencing fragment. Reading bases more accurately is an important development direction for sequencing development. At present, the optimization of related platforms focuses on biochemical reactions and signal acquisition, such as improving fluorescence intensity, improving the fidelity of polymerases, improving the efficiency of elution, or improving the accuracy of optical systems. These improvements increase sequencing costs and increase dependence on dedicated sequencing reagents and sequencing platforms. In addition, in the related art, multiple independent repeated sequencing is often performed for the same sample to increase the amount of data to increase the sequencing depth, thereby improving the accuracy of sample sequence reduction. However, such multiple independent repeated sequencing requires a large sample input, and the consumption of sequencing reagents is large, and the sequencing cost is high.

Based on this, the inventors have developed a method of repeated sequencing after many experiments and tests. This method optimizes the sequencing scheme in terms of sequencing logic. By repeatedly sequencing the same nucleotide sequence template in a single sequencing, multiple sequencing sequences are obtained, and then these multiple sequencing sequences are mutually corrected, thereby effectively improving the sequencing accuracy.

In the embodiments of the present application, by sequencing the same DNA template for the second or third time, the factors affecting the sequencing errors mentioned above can be effectively reduced. For example, based on multiple sequencing, the bases with errors in the first sequencing can be sequenced correctly in the second or third sequencing, or the bases with errors in the second sequencing can be sequenced correctly in the first or third sequencing, and the identification and correction of the wrong bases can be effectively completed, that is, based on sequencing the same DNA template twice or more, the base sequence with the same result twice or more at the same position is determined to be the true correct sequence, thereby effectively improving the sequencing accuracy. At the same time, compared with multiple independent sequencing processes, such a repeated detection method based on single sequencing is simple to operate and has low cost.

In the embodiments of the present application, "sample", "sequence to be tested", "sample to be tested", and "nucleotide sequence template to be tested" refer to nucleic acid samples to be tested. In some embodiments, the sample can be a library to be tested applied to various sequencing platforms, such as single-stranded DNBs, double-stranded DNA libraries, etc. In some embodiments, the sample contains a primer binding sequence that can bind to a sample sequencing primer. In some implementations, the sample also contains a barcode primer binding sequence that can bind to a barcode sequencing primer, so as to determine the forward/reverse sequence based on the sequencing results of the barcode (barcode or index), or to distinguish between different samples. In some embodiments, the sample also contains a sequence that can bind to a solid phase carrier to fix the sample on the solid phase carrier.

In the present application embodiment, "solid phase carrier" refers to a solid phase medium on which a nucleic acid sample to be tested can be fixed for subsequent sequencing. In certain embodiments, a sequence that can identify and bind to the nucleic acid sample to be tested is fixed on the solid phase carrier to fix the nucleic acid sample to be tested thereon. In other embodiments, a groove of a prefabricated size is provided on the solid phase carrier to match the size of the nucleic acid sample to be tested to fix it thereon. In other embodiments, a chemical group is attached to the solid phase carrier to be connected to the nucleic acid sample to be tested by chemical bonds, van der Waals forces, etc. to fix it thereon. In certain embodiments, the solid phase carrier can be a bead, a chip, etc.

In some embodiments, the solid phase carrier can be a chip. In some embodiments, the chip is used to fix a conventional double-stranded DNA library or a single-stranded DNA library. In some embodiments, the chip is also used for DNA nanoballs (DNBs) prepared based on the double-stranded DNA library or the single-stranded DNA library. In the case where the nucleic acid sample to be tested is DNB, the DNB can be fixed on the chip by means of, for example, probe technology, and the sequencing chip is sequenced based on cPAL (combined probe anchoring ligation method) or CPAS (combined probe anchoring polymerization technology), so as to obtain the nucleic acid information of the DNA nanoball. Specifically, for example, four different color-labeled probes can be used to read the base, and then the DNA ligase binds the four different color-labeled probes to the corresponding bases of the template, and the base type is judged by imaging the fluorescent group. In some embodiments, the DNA nanoball is fixed on the chip through a mesh hole or hexamethyldisilazane.

In the embodiments of the present application, "repeated sequencing" refers to a single independent sequencing process, for the same sample to be tested, by eluting the synthetic chain generated in the sequencing process and restarting the sequencing process while synthesizing, so as to perform multiple repeated sequencing processes, such as performing the first sequencing, the second sequencing or more repeated sequencing. In some embodiments, the same sample can also be sequenced m times on the basis of two sequencings to obtain m sequencing sequences based on m sequencing synthetic chains; using elution reagents to remove the m or m-1 sequencing synthetic chains respectively; and comparing the first sequencing sequence, the second sequencing sequence and the m sequencing sequences to determine the sequence information of the sample, wherein m can be any positive integer greater than 0, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times of repeated sequencing. It can be understood that two or three times of repeated sequencing effectively improves the accuracy of the results, and as the number of repeated sequencing increases, the accuracy of the sequencing results will also be improved to a certain extent, and the number of repeated sequencing can be determined according to the actual needs of the user. In addition, it is understood that when sequencing is repeated multiple times (twice or more than twice), the last generated sequencing synthesis chain can be selectively removed.

In the embodiments of the present application, "sequencing primers" may include sample sequencing primers, such as a forward sample sequencing primer for forward sequencing (Forward strand) and a reverse sample sequencing primer for reverse sequencing (Reverse strand), i.e., a first sequencing primer and/or a second sequencing primer. In some embodiments, "sequencing primers" may also include barcode sequencing primers, such as a forward barcode sequencing primer for forward sequencing (Forward strand) and a reverse barcode sequencing primer for reverse sequencing (Reverse strand), i.e., a barcode 1 sequencing primer and the barcode 2 sequencing primer.

In the embodiment of the present application, "obtaining a sequencing sequence based on a sequencing synthetic chain" refers to the principle of sequencing while synthesizing: by adding DNA polymerase, primers and four dNTPs with base-specific fluorescent labels to the reaction system at the same time, the 3'-OH of these dNTPs is protected by chemical methods, so that only one dNTP can be added at a time, which ensures that only one base will be added at a time during the sequencing process. At the same time, after the dNTP is added to the synthetic chain, all unused free dNTPs and DNA polymerase will be washed away. The recording of the fluorescence signal is completed by optical equipment, and finally the optical signal is converted into a sequencing base by computer analysis. After the fluorescence signal is recorded, a chemical reagent is added to quench the fluorescence signal and remove the dNTP 3'-OH protecting group so that the next round of sequencing reaction (that is, the reading of the next base) can be carried out. Therefore, it can be understood that "obtaining a sequencing sequence based on a sequencing synthetic chain" means that a sequencing synthetic chain is generated when the sample is sequenced, and the base reading can be completed by optical signal reading and conversion based on the specific optical signal (fluorescent signal) released when the synthetic chain is synthesized, thereby obtaining a sequencing sequence.

In the embodiments of the present application, "elution of synthetic chains" refers to the use of elution reagents to remove the synthetic chains generated during the sequencing process. In some embodiments, the elution reagent can be a reagent that breaks the hydrogen bonds between the base pairs of the DNA double strands, thereby converting the double strands into single strands. In some embodiments, the elution reagent can be a DNA denaturant and/or an exonuclease, wherein the DNA denaturant can be an organic or inorganic reagent that can destroy the double helix structure; the exonuclease hydrolyzes the DNA molecule chain into single nucleotides by sequentially hydrolyzing the phosphodiester bonds at the ends of the DNA molecule chain. In some embodiments, the DNA denaturant can be: methanol, ethanol, urea, formamide, sodium hydroxide, etc. In some embodiments, the exonuclease can be: snake venom phosphodiesterase, Escherichia coli exonuclease I, Escherichia coli exonuclease II, Escherichia coli exonuclease II, Bacillus exonuclease III, spleen phosphodiesterase and Lactobacillus acidophilus nuclease (Lac-tobacillus acidophilus), etc. In some embodiments, the exonuclease may include snake venom phosphodiesterase, Escherichia coli exonuclease II and/or Escherichia coli exonuclease III.

In the embodiments of the present application, the concentration of DNA denaturing agents such as formamide or sodium hydroxide can be 30%-100%, preferably 40-100%. In some embodiments, the concentration of DNA denaturing agents is 50% or 100%. In some embodiments, the concentration of exonuclease can be 1-50U/μL, preferably 1-10U/μL. In some embodiments, the concentration of exonuclease can be 4-8U/μL and any concentration value within the range thereof, such as 4U/μL, 5U/μL, 6U/μL, 7U/μL, 8U/μL, etc. In some embodiments, the elution time of the elution reagent can be 0-20min, preferably 5-15min, more preferably 5-10min, for example, 5min, 6min, 7min, 8min, 9min, 10min. In some embodiments, the elution temperature of the elution reagent can be 10-60°C, preferably 20-50°C, and more preferably any value between 25-45°C, for example, it can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 42, 43, 44, 45 or any value therebetween.

In the embodiment of the present application, the sequencing with a library amount of 10fmol per single channel (4 channels on a slide, and a corresponding sequencing system volume of 40μL for a single channel) is taken as an example, and the concentration of formamide used can be adjusted according to the temperature and time of the reaction (the concentration of formamide is 100%). In some embodiments, when the elution time is 2min-20min and the elution temperature is 25°C-40°C, the concentration of formamide can be any value between 25%-100%. In some embodiments, also taking the sequencing with a library amount of 10fmol per single channel (4 channels on a slide, and a corresponding sequencing system volume of 40μL for a single channel) as an example, based on the mother liquor concentration of Escherichia coli exonuclease III of 100U/μL, the reaction conditions can be a final concentration of Escherichia coli of 1U/μL-2U/μL, a reaction temperature of 25-37°C, and a reaction time of 2-10min.

In the embodiments of the present application, the comparison between sequences obtained by repeated sequencing (such as the comparison between the first sequencing sequence and the second sequencing sequence) refers to comparing multiple sequences measured by repeated sequencing for the same sample. The comparison can determine the position of each base of each sequence obtained by repeated sequencing based on the coordinate information of the same reference sequence, and perform statistics on each base at the same position to analyze the true type of the base at the position. In other embodiments, the comparison can be performed directly between sequences obtained by repeated sequencing without the need for the reference sequence to provide coordinate information.

In the embodiment of the present application, the sequence information of the sample is determined based on the comparison of the sequencing sequences obtained by repeated sequencing. Specifically, when the number of repeated sequencing is 2, the bases at the same position (also the corresponding position) of the two sequences obtained by repeated sequencing are counted, and in response to the same bases at the corresponding positions of the first sequencing sequence and the second sequencing sequence, the same base is determined as the base type at the position; in response to the different bases at the corresponding positions of the first sequencing sequence and the second sequencing sequence, the base type at the position is determined by statistical data. In some embodiments, the statistical data is a Q value (Q-score/Q phred, i.e., base quality value, which reflects the quality value score of the measured base. Generally, the larger the Q value, the smaller the possibility of identification error and the higher the credibility). In some embodiments, the Q values of the bases at the corresponding positions of the first sequencing sequence and the second sequencing sequence are compared. According to the Q value weighting method, based on the fact that the Q value of the base x at the position of the first sequencing sequence is greater than the Q value of the base y at the position of the second sequencing sequence, the base x is determined as the base type at the position.

In some embodiments, when the number of repeated sequencing is 2+m (where m is a positive integer greater than 0), the comparison between the repeated sequencing sequences includes: comparing the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences to determine the sequence information of the sample. In some embodiments, the process specifically includes: comparing the bases at the corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences; in response to the bases at the corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences being the same, determining the same base as the base type at the position; in response to the bases at the corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences Different, the frequencies of m+2 bases are counted, and the base with the highest frequency among the m+2 bases is determined as the base type at the position. Further, in response to the different bases at the corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences, and there are base types with the same frequency of occurrence among the m+2 bases, the base type at the position is determined by statistical data. In some embodiments, the statistical data is a Q value, that is, the base type with a larger Q value is determined as the base type at the position. It is understandable that by performing error detection and correction on multiple sequencing of the same position, sequencing errors caused by environmental factors inside and outside the sequencing can be effectively reduced.

In the embodiments of the present application, the above-mentioned repeated sequencing method can be applied to single-end sequencing (Single-end sequencing) and/or double-end sequencing (Pair-end sequencing). In some embodiments, based on the sequencing primer being a forward sequencing primer or a reverse sequencing primer, the above-mentioned repeated sequencing method can be applied to single-end sequencing, and this single-end sequencing can be forward sequencing or reverse sequencing. It can be understood that the repeated sequencing method proposed in the embodiments of the present application will not affect the sequencing of the other end when applied to single-end sequencing, that is, after the repeated sequencing of the single end is performed, the sequencing of the other end can be performed normally. In some embodiments, the sequencing of the other end can be a single sequencing, or the other end can be repeatedly sequenced based on the sequencing primer at the other end according to the repeated sequencing method described in any of the above embodiments of the present application.

Thus, the present application embodiment proposes a double-end sequencing method. In some embodiments, when the other end is normal sequencing (i.e., single sequencing), the double-end sequencing method includes: based on the second sequencing primer, the sample is sequenced at the second end to obtain the second end sequence information of the sample; and the first end sequence information and the second end sequence information obtained by single-end repeated sequencing are analyzed to determine the sequence information of the sample, wherein the first sequencing primer is one of the forward sequencing primer or the reverse sequencing primer, and the second sequencing primer is the other of the forward sequencing primer or the reverse sequencing primer. It is understandable that the accuracy of the sequencing data can be effectively improved by repeated sequencing of the single end and normal sequencing of the other end.

The embodiment of the present application also proposes another double-end sequencing method. In other embodiments, when the other end is repeated sequencing, the double-end sequencing method includes: based on the first sequencing primer, the sample is repeated sequencing at the first end according to the repeated sequencing method described in any of the above embodiments of the present application to obtain the first end sequence information of the sample; based on the second sequencing primer, the sample is repeated sequencing at the second end according to the repeated sequencing method described in any of the above embodiments of the present application to obtain the second end sequence information of the sample; and the first end sequence information and the second end sequence information are analyzed to determine the sequence information of the sample, wherein the first sequencing primer is a forward sequencing primer, the first end sequence information is the forward reading sequence information, and the second sequencing primer is a reverse sequencing primer, and the second end sequence information is the reverse reading sequence information. It can be understood that the double-end sequencing method proposed in the embodiment of the present application includes two single-end repeated sequencing, and the base sequence is determined based on multiple sequences of repeated sequencing, thereby effectively improving the accuracy of the sequencing results.

It is understood that in the embodiments of the present application, the other-end sequencing refers to the sequencing relative to the first single-end sequencing. In other words, when the first single-end sequencing is forward sequencing, the other-end sequencing refers to reverse sequencing; when the first single-end sequencing is reverse sequencing, the other-end sequencing refers to forward sequencing.

In some embodiments, reverse sequencing the sample may further include: generating a complementary strand (i.e., a second strand) of the sample, and performing reverse sequencing based on the complementary strand. In some embodiments, after generating the complementary strand of the sample, the sample may be removed to perform reverse sequencing based on the complementary strand.

It can be understood that when the sample is DNB, the generation of the complementary chain (second chain) is based on multiple displacement amplification (MDA) of DNA polymerase, so the complementary chain can also be called MDA chain.

In the embodiment of the present application, when performing single-end sequencing, the method further includes: sequencing the barcode sequence in the sample, wherein the barcode sequence has sample specificity to distinguish different samples. In some embodiments, the barcode sequence can also be repeatedly sequenced.

In the embodiment of the present application, when performing double-end sequencing, the method further includes: respectively sequencing barcode 1 and barcode 2 contained in the sample Sequencing is performed to obtain the sequence information of barcode 1 and the barcode 2; and the sequence information of the sample is screened out according to the sequence information of barcode 1 and barcode 2. In some embodiments, barcode 1 and barcode 2 are direction-specific. It is understandable that by combining the sequence information of barcode 1 and barcode 2, the direction of the sequence information of the sample can be distinguished, that is, a certain sequence is determined to be a forward read sequence or a reverse read sequence. Therefore, in some embodiments, the sequence information of the sample may include the first end sequence information (forward read sequence information) and the second end sequence information (reverse read sequence information).

In the embodiments of the present application, the later statistics and analysis of the sequencing data can be implemented by analysis software, scripts, command lines, etc., as long as the sequence alignment, Q value statistics, base determination and other steps in the embodiments of the present application can be completed.

It can be understood that the repeated sequencing method proposed in the embodiments of the present application is applicable to multiple sequencing platforms, and can be applied to the sequencing of DNB templates based on the principle of Rolling Circle Replication, and can also be applied to the sequencing of linear DNA templates based on the principle of Bridge PCR. It is based on the template and/or the reverse complementary chain of the template, and the sequencing synthetic chain generated during the sequencing process is eluted, which effectively improves the accuracy of the sequence output of multiple platforms.

It should be noted that for the double-end sequencing method proposed in the embodiments of the present application, when the sequencing at the other end is also repeated sequencing, the operation and data processing of the repeated sequencing at the other end are the same as those of the single-end repeated sequencing method proposed in any of the above embodiments of the present application, and will not be repeated here.

The repeated sequencing method in the embodiment of the present application improves the sequencing accuracy from the sequencing logic, by sequencing the same nucleotide sequence template multiple times to obtain multiple sequencing sequences, and then using bioinformatics analysis to mutually correct the obtained multiple sequencing sequences, so as to achieve effective detection of sequence errors, effectively reduce the impact of sequencing errors caused by internal and external environments on sequencing results during sequencing, and significantly improve sequencing accuracy; at the same time, there is no need to perform multiple independent repeated sequencing processes, but only multiple sequencing operations for the same nucleotide sequence template in a single experiment, so it has the advantages of simple operation and low cost. In addition, this method is applicable to multiple sequencing platforms and can be widely used to improve the sequencing accuracy of multiple platforms.

The present application embodiment also proposes a repeated sequencing system, including: sequencing reagents and elution reagents, wherein the elution reagent is one or more of the following: DNA denaturants and/or exonucleases, wherein the DNA denaturants include: methanol, ethanol, urea, formamide, sodium hydroxide; the exonucleases include: snake venom phosphodiesterase, Escherichia coli exonuclease I, Escherichia coli exonuclease II, Escherichia coli exonuclease III, spleen phosphodiesterase and Lactobacillus acidophilus nuclease. In some embodiments, the DNA denaturant is formamide and/or sodium hydroxide, and the exonuclease is Escherichia coli exonuclease III.

In some embodiments, the sequencing reagent includes a sequencing primer, such as a forward sequencing primer and/or a reverse sequencing primer (corresponding to the first sequencing primer and/or the second sequencing primer), a reaction enzyme, and a dNTP. In some embodiments, a fluorescent group is attached to the dNTP for base reporting to read the base type based on different fluorescent colors to obtain the sequencing sequence of the sample. In some embodiments, the sequencing reagent also includes a barcode sequencing primer for reading the barcode information in single-end or double-end sequencing.

In some embodiments, the reaction enzyme in the sequencing reagent is used for amplification of the synthetic chain, etc., and the reaction enzyme may include DNA polymerase and optionally DNA ligase.

The embodiments of the present application also provide a sequencing kit, comprising the repeated sequencing system provided in any of the above embodiments.

It should be noted that the above explanation of the embodiment of the repeated sequencing method is also applicable to the repeated sequencing system and sequencing kit in the above embodiment, and will not be repeated here.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, according to the techniques or conditions described in the literature in the field. Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

Example 1

Figure 1 is a schematic diagram of a repeated sequencing method according to Example 1 of the present application. As shown in Figure 1, the method may include i. a biochemical experiment scheme and ii. a bioinformatics analysis scheme, wherein the biochemical experiment scheme includes:

a. Fix the nucleotide sequence template to be tested on the chip, introduce sequencing primers for annealing, and then perform the first sequencing. Detect and record the fluorescence information of multiple rounds of SBS reactions in the first sequencing, and obtain the first base sequence information of the nucleotide template to be tested through the fluorescence information.

b. An organic solvent capable of denaturing the DNA double-strand or an enzyme with 3' end exo-cleavage function is introduced to remove the first sequencing chain described in (a) above, and sequencing primers are introduced again for annealing, and a second sequencing is performed. The fluorescence information of multiple rounds of SBS reactions in the second sequencing is detected and recorded, and the second base sequence information of the nucleotide template to be tested is obtained through the fluorescence information.

c. An organic solvent capable of denaturing the DNA double-strand or an enzyme with 3' end exo-cleavage function is introduced to remove the second sequencing chain described in (b) above, and sequencing primers are introduced again for annealing, and a third sequencing is performed, and the fluorescence information of multiple rounds of SBS reactions in the third sequencing is detected and recorded, and the third base sequence information of the nucleotide template to be tested is obtained through the fluorescence information.

d. Repeat more times: remove sequencing chain → annealing of sequencing primers → sequencing, so as to obtain multiple sequencing base sequence information of the same nucleotide sequence template.

The bioinformatics analysis scheme includes the following steps: bioinformatics analysis is performed on the sequences obtained by sequencing three times for each nucleotide sequence template: first, the same base is read in the three sequencings of the same alignment site, and the three identical bases are identified as the correct bases of the site; the bases of the same alignment site are the same twice and different once, and the two identical bases are identified as the correct bases of the site; if the bases of the same alignment site are different three times, further correction is performed, specifically: the Q value weight method is used to determine the base with the largest Q value as the correct base of the site. For example, in the three sequencings, if the measured bases are the same twice and different once, the minority obeys the majority method is adopted; if the measured bases are different in the three sequencings, the Q value weight method is adopted to take the base with the largest Q value. Finally, the correctly read bases and the corrected bases are spliced into a complete sequence to improve the accuracy of the sequencing sequence.

The repeated sequencing method in this embodiment effectively improves the sequence accuracy by sequencing the same template twice or more (sequencing-removing the sequencing chain-resequencing) and performing mutual comparison and correction analysis on the two or more sequencing results.

Example 2

Figure 2 is a schematic diagram of another repeated sequencing method according to Example 2 of the present application. As shown in Figure 2, the method can be applied to related sequencing technologies based on bridge amplification, including i. a biochemical experimental scheme and ii. a bioinformatics analysis scheme, wherein the biochemical experimental scheme includes:

1. Fix the DNA molecules of the sample to be tested on a flow cell covered with connectors.

2. Use the adapters on the flow cell as primers to perform multiple rounds of bridge amplification to form a Cluster, which is the nucleotide sequence template to be tested.

3. Introduce sequencing primers for annealing, and then perform multiple rounds of SBS reactions for the first sequencing (one base is measured in each round of SBS reactions), detect and record the fluorescence information of the multiple rounds of SBS reactions in the first sequencing, and obtain the first sequencing base sequence information through the fluorescence information.

4. Use NaOH alkaline solution to remove the sequencing chain generated by the first sequencing.

5. The sequencing primers are introduced again for annealing, followed by multiple rounds of SBS reactions for the second sequencing (one base is measured in each round of SBS reactions), and the fluorescence information of the multiple rounds of SBS reactions in the second sequencing is detected and recorded, and the base sequence of the second sequencing is obtained through the fluorescence information. information.

6. Repeat steps 4 and 5 to detect and record the fluorescence information of multiple rounds of SBS reactions in the third sequencing (one base is measured in each round of SBS reaction), and obtain the base sequence information of the third sequencing through the fluorescence information.

The bioinformatics analysis scheme includes the following steps: bioinformatics analysis is performed on the sequence obtained by three sequencing of each nucleotide sequence template: first, the same base is read in three sequencings of the same comparison site, and the three identical bases are identified as the correct bases of the site; the bases of the same comparison site are the same twice and different once, and the two identical bases are identified as the correct bases of the site; if the bases of the same comparison site are different three times, further correction is performed, specifically: the Q value weight method is used to determine the base with the largest Q value as the correct base of the site. For example, in the three sequencings, the measured bases are the same twice and different once, the minority obeys the majority method is adopted; if the measured bases are different in the three sequencings, the Q value weight method is adopted to take the base with the largest Q value. Finally, the correctly read bases and the corrected bases are spliced into a complete sequence to improve the accuracy of the sequencing sequence.

The repeated sequencing method based on bridge amplification in this embodiment may also optionally include reverse sequencing of the two strands to obtain double-end sequencing results, and the method may include: synthesizing a complementary strand (i.e., a second strand) of each nucleotide sequence template; removing each nucleotide sequence template; and reverse sequencing based on the complementary strand. Its bioinformatics analysis scheme is the same as the analysis scheme of the above-mentioned single-end sequencing.

The repeated sequencing method based on bridge amplification in this embodiment effectively improves the sequence accuracy by sequencing the same template twice or more (sequencing-removal of sequencing chain-resequencing) and performing mutual comparison and correction analysis on the two or more sequencing results.

Example 3

Using E. coli PCR free samples (MGI), using the MGISEQ-2000RS high-throughput sequencing kit (MGI, catalog number 1000012552) on the MGISEQ-2000 platform, according to the instructions for use of the kit and sequencing platform, three SE100 sequencings (i.e., single-end 100bp repeated sequencing) were performed using the nucleotide sequence of the sample as a template:

1) First, the nucleotide sequence template of the E. coli PCR free sample is fixed on the chip, and the CPAS AD153 sequencing primer working solution in the sequencing kit above is passed through for annealing, and then the first SE100 sequencing is performed, and the fluorescence information of multiple rounds of SBS reactions in the first sequencing is detected and recorded (one base is measured in each round of SBS reaction), and the first base sequence information of the nucleotide template is obtained through the fluorescence information.

2) Pass 40 μL of 100% formamide and react at 40°C for 8 minutes (single lane dosage) to remove the first sequencing chain (i.e., sequencing synthesis chain) described in 1) above, pass CPAS AD153 sequencing primer working solution again for annealing, perform a second SE100 sequencing, detect and record the fluorescence information of multiple rounds of SBS reactions in the second sequencing (one base is measured in each round of SBS reaction), and obtain the second base sequence information of the nucleotide template through the fluorescence information.

3) Pass 40 μL of 100% formamide again and react at 40°C for 8 minutes to remove the second sequencing chain described in 2) above, pass CPAS AD153 sequencing primer working solution again for annealing, perform SE100 sequencing for the third time, detect and record the fluorescence information of multiple rounds of SBS reactions in the third sequencing (one base is measured in each round of SBS reaction), and obtain the third base sequence information of the nucleotide template through the fluorescence information.

4) Analyze the base sequence information obtained three times to obtain a highly accurate base sequence.

Table 1 shows the results of the first SE100 sequencing (i.e., 1st) obtained in step 1); Table 2 shows the results of the second (2nd) and third SE100 sequencing (3rd) obtained in steps 2) and 3). As can be seen from Tables 1 and 2, the quality index Q30 of the first sequencing is 91.03%. After the first formamide reaction to remove the sequencing chain, the quality index Q30 of the second sequencing is 83.46%. After the second formamide reaction to remove the sequencing chain, the quality index Q30 of the third sequencing is 81.07%. It can be seen that the use of formamide to remove the sequencing chain will affect the production of DNB templates. Some damage occurred, resulting in a decrease in the second and third sequencing quality, but the data as a whole still maintained a high quality, that is, the three rounds of sequencing maintained a high data quality output.

Table 1: Results of the first SE100 sequencing

Table 2: Second and third SE100 sequencing results

The BWA MEM alignment algorithm (https://doi.org/10.48550/arXiv.1303.3997) was used to calibrate the sequences obtained from the three SE100 sequencing runs: first, the same base was read in three sequencing runs at the same alignment site, and the three identical bases were identified as the correct bases for the site; the same alignment site had the same base twice and a different base once, and the two identical bases were identified as the correct bases for the site; if the three sequencing bases at the same alignment site were different, further correction was performed: the Q value weighting method was used to determine the base with the largest Q value as the correct base for the site.

Table 3 shows the bioinformatics analysis results of the first SE100 sequencing of this embodiment; Table 4 shows the results of mutual calibration of the three SE100 sequencings of this embodiment.

Table 3: Bioinformatics analysis results of the first SE100 sequencing - single sequencing

Table 4: Bioinformatics analysis results of three SE100 sequencing runs - Mutual correction of three sequencing runs

From the comparison of the single sequencing analysis results of the first SE100 and the results after mutual calibration of the three SE100 sequencings shown in Tables 3 and 4, it can be seen that after three sequencings, the data volume indicator BaseNum shows a downward trend. The BaseNum in the first sequencing is about 47.8 Gb. After the sequencing chain is removed by formamide, the data volume of the second and third sequencing is reduced. Finally, the BaseNum used in the mutual calibration of the three sequencings is about 27.9 Gb. It can be seen that even after three repeated sequencing, the total data volume generated by the repeated sequencing method of the embodiment of the present application is still small in volume, which is easy to store and process later.

At the same time, after three sequencings, the error rate index Mismatch rate! N (%) showed a downward trend, and the Mismatch rate! N (%) during the first sequencing was 0.48%. After the three sequencings were mutually corrected, the Mismatch rate! N (%) was 0.12%, which was 75% lower than the error rate of the first sequencing; in addition, compared with the single sequencing, the data quality indexes Q20 and Q30 values after the integrated analysis of the three sequencings were significantly improved, and the mapping rate (%) also increased, indicating that the repeated sequencing method of the embodiment of the present application can ensure the quality of the sequencing results and effectively improve the accuracy of the sequencing results.

Example 4

Using E. coli PCR free samples (MGI), using the MGISEQ-2000RS high-throughput sequencing kit (MGI, catalog number 1000012551) on the MGISEQ-2000 platform, according to the instructions for use of the kit and sequencing platform, three SE50 sequencing (i.e., single-end 50bp repeated sequencing) was performed using the nucleotide sequence of the sample as a template:

1) First, the nucleotide sequence template of the E. coli PCR free sample is fixed on the chip, and the CPAS AD153 sequencing primer working solution is passed through for annealing. Then, the first SE50 sequencing is performed, and the fluorescence information of multiple rounds of SBS reactions in the first sequencing is detected and recorded (one base is measured in each round of SBS reaction), and the first base sequence information of the nucleotide template is obtained through the fluorescence information.

2) Pass 40 μL of 100% formamide and react at 40°C for 8 minutes to remove the first sequencing chain (i.e., the sequencing synthesis chain) described in 1) above, pass CPAS AD153 sequencing primer working solution again for annealing, perform a second SE50 sequencing, detect and record the fluorescence information of multiple rounds of SBS reactions in the second sequencing (one base is measured in each round of SBS reaction), and obtain the second base sequence information of the nucleotide template through the fluorescence information.

3) Pass 40 μL of 100% formamide again and react at 40°C for 8 minutes to remove the second sequencing chain described in 2) above, pass CPAS AD153 sequencing primer working solution again for annealing, perform SE50 sequencing for the third time, detect and record the fluorescence information of multiple rounds of SBS reactions in the third sequencing (one base is measured in each round of SBS reaction), and obtain the third base sequence information of the nucleotide template through the fluorescence information.

4) Analyze the base sequence information obtained three times to obtain a highly accurate base sequence.

Table 5 shows the results of the first SE50 sequencing (i.e., 1st) obtained in step 1); Table 6 shows the results of the second (2nd) and third SE50 sequencing (3rd) obtained in steps 2) and 3). As can be seen from Tables 5 and 6, the quality index Q30 of the first sequencing is 95.34%. After the first formamide reaction to remove the sequencing chain, the quality index Q30 of the second sequencing is 93.82%. After the second formamide reaction to remove the sequencing chain, the quality index Q30 of the third sequencing is 93.71%. It can be seen that the use of formamide to remove the sequencing chain will affect the DNB template production. The second and third rounds of sequencing were damaged to a certain extent, resulting in a slight decrease in the quality of the second and third rounds of sequencing, but the data as a whole still maintained a high quality. In addition, compared with the sequencing results of SE100 in Example 3, the sequencing read length of SE50 in this example is shorter, its overall Q30 is improved, and the decrease in the second and third rounds of sequencing quality is not obvious, that is, the three rounds of sequencing maintain extremely high data quality output.

Table 5: First SE50 sequencing results

Table 6: Second and third SE50 sequencing results

The BWA MEM alignment algorithm was used to calibrate the sequences obtained from the three SE50 sequencing runs: first, the same base was read in three sequencing runs at the same alignment site, and the three identical bases were identified as the correct bases for the site; the same alignment site had the same base twice and a different base once, and the two identical bases were identified as the correct bases for the site; if the three sequencing bases at the same alignment site were different, further correction was performed: the Q value weighting method was used to determine the base with the largest Q value as the correct base for the site.

Table 7 is the bioinformatics analysis result of the first SE50 sequencing of this embodiment; Table 8 is the result of mutual calibration of the three SE50 sequencings of this embodiment.

Table 7: Bioinformatics analysis results of the first SE50 sequencing - single sequencing

Table 8: Bioinformatics analysis results of three SE50 sequencings - three sequencings were mutually corrected

From the comparison of the single sequencing analysis results of the first SE50 and the results after the three SE50 sequencings are mutually corrected as shown in Tables 7 and 8, it can be seen that after the three sequencings, the data volume indicator BaseNum shows a downward trend. The BaseNum of the first sequencing is about 27.9Gb. After the sequencing chain is removed by formamide, the data volume of the second and third sequencings is slightly reduced. Finally, the BaseNum used in the mutual correction of the three sequencings is about 26.1Gb. It can be seen that even after three repeated sequencings, the total data volume generated by the repeated sequencing method of the embodiment of the present application is still small in volume, which is easy to store and process later.

At the same time, after three sequencings, the error rate index Mismatch rate! N (%) showed a downward trend, and the Mismatch rate! N (%) during the first sequencing was 0.2%. After the three sequencings were mutually corrected, the Mismatch rate! N (%) was 0.06%, which was 60% lower than the error rate of the first sequencing; in addition, compared with the single sequencing, the data quality indexes Q20 and Q30 values after the integrated analysis of the three sequencings were significantly improved, and the mapping rate (%) also increased, indicating that the repeated sequencing method of the embodiment of the present application can ensure the quality of the sequencing results and effectively improve the accuracy of the sequencing results.

Example 5

Using E. coli PCR free (MGI) and E. coli PCR samples (MGI, catalog number: 1000005033), using the BGISEQ-500RS high-throughput sequencing kit (MGI, catalog number 1000005485) on the BGISEQ-500 platform, according to the instructions for the kit and sequencing platform, the nucleotide sequences of the two samples were used as templates for three SE50 sequencing (i.e., single-end 50bp repeated sequencing):

1) First, the nucleotide sequence templates of the two samples, E. coli PCR free and E. coli PCR, were fixed on the chip, and the CPAS AD153 sequencing primer working solution was introduced for annealing. Then, the first SE50 sequencing was performed, and the fluorescence information of multiple rounds of SBS reactions in the first sequencing was detected and recorded (one base was measured in each round of SBS reaction), and the first base sequence information of the two nucleotide templates was obtained through the fluorescence information.

2) Pass 60 μL of Exonuclease III at a concentration of 4 U/μL and react at 37°C for 10 min to remove the first sequencing chain (i.e., the sequencing synthesis chain) described in 1) above, pass CPAS AD153 sequencing primer working solution again for annealing, perform a second SE50 sequencing, detect and record the fluorescence information of multiple rounds of SBS reactions in the second sequencing (one base is measured in each round of SBS reaction), and obtain the second base sequence information of the two-nucleotide template through the fluorescence information.

3) Pass 60 μL of 4 U/μL Exonuclease III again and react at 37°C for 10 min to remove the second sequencing chain described in 2) above, pass CPAS AD153 sequencing primer working solution again for annealing, perform the third SE50 sequencing, detect and record the fluorescence information of multiple rounds of SBS reactions in the third sequencing (one base is measured in each round of SBS reaction), and obtain the third base sequence information of the two-nucleotide template through the fluorescence information.

4) Analyze the base sequence information of the three measurements of the two samples to obtain a base sequence with high accuracy.

Table 9 shows the results of the first SE50 sequencing of the two samples obtained in step 1) (i.e., 1st); Table 10 shows the results of the second (2nd) and third SE50 sequencing (3rd) of the two samples obtained in steps 2) and 3). As can be seen from Table 9, for the quality index Q30 of the first sequencing, the PCR free sample is 92.75%; the PCR sample is 92.71%. As can be seen from Table 10, after the first Exonuclease III reaction to remove the sequencing chain, the quality index Q30 for the second sequencing is 86.33% for the PCR free sample; the PCR sample is 85.42%. After the second Exonuclease III reaction to remove the sequencing chain, the quality index Q30 for the third sequencing is 82.82% for the PCR free sample; the PCR sample is 82.35%. It can be seen that the use of Exonuclease III to remove the sequencing chain will cause certain damage to the DNB template, resulting in the second and third The sequencing quality of each sequencing was reduced, but the data as a whole still maintained a high quality, that is, the three rounds of sequencing maintained a high data quality output.

Table 9: First SE50 sequencing results

Table 10: Second and third SE50 sequencing results

The BWA MEM alignment algorithm was used to calibrate the sequences obtained from the three SE50 sequencing of the above two samples: first, the three sequencing reads of the same alignment site were the same base, and the three identical bases were identified as the correct bases for the site; the same alignment site had the same base twice and a different base once, and the two identical bases were identified as the correct bases for the site; if the three sequencing bases of the same alignment site were different, further correction was performed: the Q value weight method was used to determine the base with the largest Q value as the correct base for the site.

Table 11 shows the bioinformatics analysis results of the first SE50 sequencing of the two samples of this embodiment; Table 12 shows the results of mutual calibration of the three SE50 sequencing of the two samples of this embodiment.

Table 11: Bioinformatics analysis results of the first SE50 sequencing - single sequencing

Table 12: Bioinformatics analysis results of three SE50 sequencings - three sequencings were mutually corrected

From the comparison of the single sequencing analysis results of the first SE50 of the two samples and the results after mutual calibration of the three SE50 sequencings shown in Tables 11 and 12, it can be seen that after three sequencings, the data volume index BaseNum of the two samples showed a downward trend. The BaseNum of the first sequencing was about 10.9Gb for the PCR free library and about 11.1Gb for the PCR library. After Exonuclease III removed the sequencing chain, the amount of data for the second and third sequencing was reduced. Finally, the BaseNum used in the mutual calibration of the three sequencings was about 6.8Gb for the PCR free library and about 6.6Gb for the PCR library. It can be seen that even after three repeated sequencings, the total amount of data generated by the repeated sequencing method of the embodiment of the present application is still small in size and easy to store and process later.

At the same time, after three sequencings, the error rate indicator Mismatch rate! N (%) showed a downward trend, that is, for the Mismatch rate! N (%) during the first sequencing, the PCR free library was 0.04%; the PCR library was 0.06%. After the three sequencings were mutually corrected, the Mismatch rate! N (%) of the PCR free library was 0.03%, which was a 25% decrease compared with the error rate of the first sequencing; the PCR library was 0.05%, which was a 17% decrease compared with the error rate of the first sequencing. In addition, compared with a single sequencing, the data quality indicators Q20 and Q30 values of the two samples after the integrated analysis of the three sequencings were significantly improved, indicating that the repeated sequencing method of the embodiment of the present application can ensure the quality of the sequencing results and effectively improve the accuracy of the sequencing results.

Example 6

Using E. coli PCR sample (MGI), using MGISEQ-2000RS high-throughput sequencing kit (MGI, catalog number 1000012551) on the MGISEQ-2000 platform, according to the instructions for use of the kit and sequencing platform, use the nucleotide sequence of the sample as a template to perform SE50 sequencing (i.e., single-end 50bp sequencing), and after removing the sequencing synthesis chain, perform PE50 sequencing (i.e., double-end 50bp sequencing):

1) First, the nucleotide sequence template of the E. coli PCR sample was fixed on the chip, and the CPAS AD153 sequencing primer working solution was introduced for annealing. According to the operating instructions of MGISEQ-2000, SE50 sequencing was performed once, and the fluorescence information of multiple rounds of SBS reactions in this SE50 sequencing was detected and recorded (one base was measured in each round of SBS reaction). The base sequence information of the nucleotide template in this SE50 sequencing was obtained through the fluorescence information.

2) 40 μL of 50% formamide was introduced and reacted at 40° C. for 8 min to remove the sequencing chain (ie, the sequencing synthesis chain) of SE50 described in 1) above.

3) Perform PE50 sequencing: introduce the sequencing primer of the first chain Read1 in the sequencing kit for annealing, perform 50bp sequencing of the first chain Read1 according to the operating instructions of the sequencing platform, detect and record the fluorescence information of multiple rounds of SBS reactions in Read1 sequencing, and obtain the 50 base sequence information of the first chain nucleotide template through the fluorescence information; then introduce the high-fidelity polymerase and dNTP substrate with chain displacement function in the kit, synthesize the second chain sequencing template (i.e., the complementary chain of the first chain) under the action of this polymerase, then introduce the sequencing primer of the second chain Read2 in the sequencing kit for annealing, and then perform 50bp sequencing of the second chain Read2 according to the operating instructions of the sequencing platform. Sequencing, detect and record the fluorescence information of multiple rounds of SBS reactions in Read2 sequencing, and obtain the 50-base sequence information of the second-chain nucleotide template through the fluorescence information.

4) Analyze the measured base sequence information to obtain the base sequence.

Table 13 shows the results of a single SE50 sequencing (i.e., 1st) obtained in step 1); Table 14 shows the results of PE50 sequencing obtained in step 3).

Table 13: Single SE50 sequencing results

Table 14: PE50 sequencing results

It can be seen from Tables 13 and 14 that the quality index Q30 of the first single SE sequencing is 96.1%. After the first formamide reaction to remove the sequencing chain, the quality index Q30 of the subsequent PE50 sequencing is 96.37% for Read1, 96.0% for Read2, and 96.19% for Total. It can be seen that the use of formamide to remove the sequencing chain of SE50 and then perform PE50 sequencing did not show that formamide had any damage to the DNB template, and the data maintained a relatively high quality overall.

The BWA MEM alignment algorithm was used to perform bioinformatics analysis on the above SE50 and PE50 sequencing results. The data statistics are shown in Tables 15 and 16.

Table 15: Bioinformatics analysis results of SE50 sequencing

Table 16: Bioinformatics analysis results of PE50 sequencing

From the bioinformatics analysis results of SE50 sequencing and the subsequent PE50 sequencing shown in Tables 15 and 16, respectively, it can be seen that the data volume indicator BaseNum shows a downward trend. The BaseNum during the first SE50 sequencing is about 27.78 Gb. After formamide removal of the sequencing chain, the data volume of PE50-Read1 during the second PE50 sequencing is equivalent to that of SE50, which is 26.46 Gb.

At the same time, by comparing the results of SE sequencing and PE sequencing, it was found that the error rate indicator Mismatch rate! N (%), data quality indicators Q20 and Q30 values, and mapping rate (%) indicators were comparable, all indicating high data quality, indicating that after SE50 sequencing and removal of the sequencing chain according to the repeated sequencing method proposed in the embodiment of the present application, PE sequencing can be performed normally afterwards, and the single or multiple single-end sequencing performed previously will not affect the subsequent double-end sequencing. Therefore, the repeated sequencing method proposed in the embodiment of the present application can be flexibly combined with normal single-end/double-end sequencing, and can obtain high-quality and high-accuracy sequencing results.

Example 7

Using E. coli PCR sample (MGI, catalog number: 1000005033), using MGISEQ-2000RS high-throughput sequencing kit (MGI, catalog number 1000012551) on the MGISEQ-2000 platform, according to the instructions for use of the kit and sequencing platform, the second strand (Reverse Strand) of the sample was used as a template to perform four single-end 50bp sequencing (i.e., single-end 50bp repeated sequencing):

1) First, the nucleotide sequence template of the E. coli PCR sample is fixed on the chip, and the high-fidelity polymerase and dNTP substrate with chain displacement function in the kit are introduced. Under the action of this polymerase, the second chain sequencing template (i.e., the complementary chain of the first chain) is synthesized.

2) Introduce the second-chain sequencing primer (CPAS AD153 sequencing primer 2 working solution) in the sequencing kit for annealing, and then perform the first 50bp sequencing of the second chain according to the operating instructions of the sequencing platform. Detect and record the fluorescence information of multiple rounds of SBS reactions during sequencing, and obtain the first 50 base sequence information of the second-chain nucleotide template through the fluorescence information.

3) 40 μL of 50% formamide was introduced and reacted at 40° C. for 8 min to remove the first 50-base sequencing chain (ie, the sequencing synthesis chain) described in 2) above.

4) The CPAS AD153 sequencing primer 2 working solution was introduced again for annealing, and a second 50-base sequencing was performed. The fluorescence information of the multiple rounds of SBS reactions in the second sequencing was detected and recorded, and the second 50-base sequence information based on the second chain nucleotide template was obtained through the fluorescence information.

5) Repeat steps 3) and 4), detect and record the fluorescence information of multiple rounds of SBS reactions in the third 50 bp sequencing, and obtain the third 50 base sequence information of the second chain nucleotide template through the fluorescence information.

6) Repeat steps 3) and 4), detect and record the fluorescence information of multiple rounds of SBS reactions in the fourth 50 bp sequencing, and obtain the fourth 50 base sequence information of the second chain nucleotide template through the fluorescence information.

7) Analyze the base sequence information obtained four times to obtain a base sequence with high accuracy.

Table 17 shows the results of the first 50bp sequencing obtained in step 2) (i.e., 1st); Table 18 shows the results of the second (2nd), third (3rd), and fourth (4th) 50bp sequencing obtained in steps 4) and 5).

Table 17: First 50bp sequencing results

Table 18: Second, third and fourth 50bp sequencing results

As can be seen from Tables 17 and 18, the quality index Q30 of the first 50bp sequencing is 94.7%, after the first formamide reaction to remove the sequencing chain, the quality index Q30 of the second 50bp sequencing is 94.7%, after the second formamide reaction to remove the sequencing chain, the quality index Q30 of the third 50bp sequencing is 94.79%, and after the third formamide reaction to remove the sequencing chain, the quality index Q30 of the fourth 50bp sequencing is 93.18%. It can be seen that when formamide is used to remove the sequencing chain for repeated sequencing, the second and third sequencing quality do not show a significant decline, indicating that the method of this embodiment can be effectively used for elution of the sequencing chain and can effectively ensure the sequencing quality; in addition, formamide removal of the sequencing chain will cause certain damage to the DNB template, resulting in a slight decline in the fourth sequencing quality, but the data as a whole still maintains a high quality, that is, the four rounds of sequencing maintain extremely high data quality output.

The BWA MEM alignment algorithm was used to calibrate the sequences obtained from the above four 50 bp double-strand sequencing, including the mutual calibration between the three sequencing results (Triple) and the mutual calibration between the four sequencing results (Quadruple), as follows:

The mutual correction process of three sequencing runs (the first, third, and fourth sequencing data are analyzed): First, the same base is read in three sequencing runs for the same alignment site, and the three identical bases are identified as the correct bases for the site; the bases read in two sequencing runs for the same alignment site are the same and one is different, and the identical bases read in the two reads are identified as the correct bases for the site; if the bases read in three sequencing runs for the same alignment site are different, the Q value weighting method is used to select the base with the largest Q value as the correct base for the site.

The mutual correction process of four sequencings: First, the same base is read in four sequencings of the same alignment site, and the four identical bases are identified as the correct bases for the site; the bases read in three sequencings of the same alignment site are the same and one is different, and the identical bases read twice are identified as the correct bases for the site; if the bases read in two sequencings of the same alignment site are the same and two are different, or the bases read in four sequencings are different, further correction is performed: the Q value weight method is used to take the base with the largest Q value as the correct base for the site.

Table 19: Bioinformatics analysis results of the first 50bp sequencing - single sequencing

Table 20: Bioinformatics analysis results of four 50 bp sequencing runs - mutual calibration of three runs and mutual calibration of four runs

Table 19 is the bioinformatics analysis result of the first 50bp sequencing of this embodiment; Table 20 is the result of mutual correction of three 50bp sequencing and four 50bp sequencing of this embodiment. It can be seen from the results shown in Tables 19 and 20 that after four times of two-chain sequencing, the data volume indicator BaseNum shows a downward trend. The BaseNum of the first 50bp sequencing of the second chain is about 26.87Gb. After the sequencing chain is removed by formamide, the second, third and fourth 50bp sequencing data volume is slightly reduced. Finally, the BaseNum used in the mutual correction of three 50bp sequencing is about 25.48Gb, and the BaseNum used in the mutual correction of four 50bp sequencing is about 25.36Gb. It can be seen that even after three repeated sequencing, the total data volume generated by the repeated sequencing method of the embodiment of the present application is still small in volume, which is easy to store and process later.

At the same time, after four times of two-strand sequencing, the error rate indicator Mismatch rate! N (%) showed a downward trend, and the Mismatch rate! N (%) of the first 50bp sequencing of the second strand was 0.18%. The Mismatch rate! N (%) after three 50bp sequencings were mutually corrected was 0.08%, which was 55.6% lower than the error rate of the first 50bp sequencing; the Mismatch rate! N (%) after four 50bp sequencings were mutually corrected was 0.07%, which was 61.1% lower than the error rate of the first 50bp sequencing, indicating that the repeated sequencing method of the embodiment of the present application can ensure the quality of the sequencing results and effectively improve the accuracy of the sequencing results.

The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (21)

A repeated sequencing method comprising: Based on the first sequencing primer, performing a first sequencing on the sample to obtain a first sequencing sequence based on the first sequencing synthesis chain; contacting the first sequencing synthesized strand with an elution reagent to remove the first sequencing synthesized strand; Based on the first sequencing primer, performing a second sequencing on the sample to obtain a second sequencing sequence based on a second sequencing synthesis chain; and The first sequencing sequence and the second sequencing sequence are compared to determine the sequence information of the sample. The method according to claim 1, wherein the first sequencing or the second sequencing of the sample based on the first sequencing primer comprises: The first sequencing primer is annealed to the sample and multiple base sequencing cycles are performed, wherein each cycle completes signal detection of one base, thereby obtaining an optical signal of the first sequencing synthesis chain or the second sequencing synthesis chain, and obtaining the first sequencing sequence or the second sequencing sequence based on the optical signal of the first sequencing synthesis chain or the second sequencing synthesis chain. The method according to claim 1 or 2, wherein performing a first sequencing on the sample based on the first sequencing primer further comprises: The sample is fixed on a solid phase carrier for sequencing. Optionally, the solid phase carrier is a bead or a chip. The method according to claim 1, wherein the elution reagent is one or more of the following: DNA denaturants and/or exonucleases, wherein the DNA denaturants include: methanol, ethanol, urea, formamide and sodium hydroxide, and the exonucleases include: snake venom phosphodiesterase, Escherichia coli exonuclease I, Escherichia coli exonuclease II, Escherichia coli exonuclease III, spleen phosphodiesterase and Lactobacillus acidophilus nuclease, Preferably, the DNA denaturant is formamide and/or sodium hydroxide, and the exonuclease is Escherichia coli exonuclease III. The method according to claim 3, wherein the concentration of formamide is 30%-100%, preferably 40-100%; the concentration of Escherichia coli exonuclease III is 1-50U/μL, preferably 1-10U/μL, Optionally, the elution time of the elution reagent is 0-20 min, preferably 5-15 min, more preferably 5-10 min. Optionally, the elution temperature of the elution reagent is 10-60°C, preferably 20-50°C, and more preferably 25-45°C. The method according to claim 1, wherein comparing the first sequencing sequence and the second sequencing sequence to determine the sequence information of the sample comprises: Comparing the bases at corresponding positions of the first sequencing sequence and the second sequencing sequence; In response to the bases at corresponding positions of the first sequencing sequence and the second sequencing sequence being identical, determining the identical base as the base type at the position; In response to the difference in bases at corresponding positions of the first sequencing sequence and the second sequencing sequence, determining the type of the base at the position by using statistical data, Optionally, the statistical data is a Q value. The method according to claim 6, wherein determining the base type at the position by statistical data specifically comprises: The Q values of bases at corresponding positions of the first sequencing sequence and the second sequencing sequence are compared, and based on the fact that the Q value of base x at the position of the first sequencing sequence is greater than the Q value of base y at the position of the second sequencing sequence, the base x is determined as the base type at the position. The method according to any one of claims 1 to 7, further comprising: repeating the sequencing process based on the first sequencing primer, comprising: The sample is sequenced again m times to obtain m sequencing sequences based on the m sequencing synthesis chains; contacting the elution reagent with the m or m-1 sequencing synthesis chains respectively to remove the m or m-1 sequencing synthesis chains; and The first sequencing sequence, the second sequencing sequence and the m sequencing sequences are compared to determine sequence information of the sample. The method according to claim 8, wherein m is a positive integer greater than 0, preferably, 1≤m≤10. The method according to claim 8 or 9, wherein comparing the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences to determine the sequence information of the sample comprises: Comparing the bases at corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences; In response to the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences having the same base at the corresponding position, determining the same base as the base type at the position; In response to the fact that the bases at corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences are different, the base with the highest occurrence frequency among the m+2 bases is determined as the base type at the position; or in response to the fact that the bases at corresponding positions of the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences are different, the base type at the position is determined by statistical data. The method according to claim 10, further comprising: In response to the first sequencing sequence, the second sequencing sequence, and the m sequencing sequences having different bases at corresponding positions, and the m+2 bases having base types with the same occurrence frequency, determining the base type at the position by using statistical data, Optionally, the statistical data is a Q value. The method according to any one of claims 1 to 11, wherein the first sequencing primer is a forward sequencing primer or a reverse sequencing primer. A method for double-end repeat sequencing, comprising: Based on the first sequencing primer, performing first-end repetitive sequencing on the sample according to the repetitive sequencing method according to any one of claims 1 to 12 to obtain first-end sequence information of the sample; Based on the second sequencing primer, performing second-end sequencing on the sample to obtain second-end sequence information of the sample; and analyzing the first end sequence information and the second end sequence information to determine the sequence information of the sample, The first sequencing primer is one of a forward sequencing primer and a reverse sequencing primer, and the second sequencing primer is the other of the forward sequencing primer and the reverse sequencing primer. A method for double-end repeat sequencing, comprising: Based on the first sequencing primer, performing first-end repetitive sequencing on the sample according to the repetitive sequencing method according to any one of claims 1 to 12 to obtain first-end sequence information of the sample; Based on the second sequencing primer, performing second-end repetitive sequencing on the sample according to the repetitive sequencing method according to any one of claims 1 to 12 to obtain second-end sequence information of the sample; and analyzing the first end sequence information and the second end sequence information to determine the sequence information of the sample, wherein the first sequencing primer is a forward sequencing primer, the first end sequence information is a forward read sequence information, and The second sequencing primer is a reverse sequencing primer, and the second end sequence information is reverse reading sequence information. The method according to claim 14, wherein performing second end repeat sequencing on the sample based on the second sequencing primer comprises: generating a complementary strand of the sample, and performing second end repeat sequencing based on the complementary strand, Optionally, the sample is a DNA nanoball, and the complementary chain is an MDA chain. The method according to claim 15, wherein after generating the complementary strand of the sample, the method further comprises: The sample is removed, and the second end repeat sequencing is performed based on the complementary strand. The method according to claim 16, wherein the sample contains barcode 1 and optionally barcode 2, the method further comprising: Sequencing the barcode 1 and the barcode 2 respectively to obtain sequence information of the barcode 1 and the barcode 2; and The sequence information of the sample is screened out according to the sequence information of the barcode 1 and the barcode 2. A repeated sequencing system comprises: a sequencing reagent and an elution reagent, wherein the elution reagent is one or more of the following: DNA denaturants and/or exonucleases, wherein the DNA denaturants include: methanol, ethanol, urea, formamide, sodium hydroxide, and the exonucleases include: snake venom phosphodiesterase, Escherichia coli exonuclease I, Escherichia coli exonuclease II, Escherichia coli exonuclease III, spleen phosphodiesterase, and Lactobacillus acidophilus nuclease, Preferably, the DNA denaturant is formamide and/or sodium hydroxide, and the exonuclease is Escherichia coli exonuclease III. The system according to claim 18, wherein the sequencing reagent comprises a first sequencing primer, a reaction enzyme and dNTPs, Preferably, a fluorescent group is attached to the dNTP for base reporting. Optionally, the sequencing reagent further comprises a second sequencing primer, Optionally, the sequencing reagents further include a first barcode sequencing primer and optionally a second barcode sequencing primer. The system according to claim 19, wherein the reaction enzyme comprises DNA polymerase and optionally DNA ligase. A sequencing kit comprising the repeated sequencing system according to any one of claims 18 to 20.