Multi-system coding and reading method for composite base DNA (Deoxyribose Nucleic Acid) storage

Abstract

The invention discloses a multi-system coding and reading method for compound base DNA (deoxyribonucleic acid) storage, which combines multi-system error correction coding and symbol mapping methods to code digital information and map the digital information into a DNA chain containing a compound base and a standard base, and adopts a high-reliability short grouping error correction code to code an address sequence and add the coded address sequence to two ends of a data DNA sequence; when the data are read out, a short grouping error correction code is utilized, a unique address of a sequencing read section is achieved, rapid clustering is achieved, entropy-driven set division and maximum likelihood-based letter detection are combined, original characters of each synthesis site are accurately detected, and original data are recovered through error correction decoding. According to the method, the separable characteristic of the information entropy is utilized, the composite bases are subjected to diversity, the distinction degree between the concentrated characters is increased, the detection capacity is enhanced, the base frequency information in the sequencing data can be effectively utilized, and the coverage degree needed by composite base detection is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA data storage, and in particular to a multi-base encoding and reading method for composite base DNA storage. Background Art

[0002] With the continuous growth of global data volume, traditional storage media based on magnetism, light, and electricity are difficult to meet the needs of long-term data storage. Therefore, exploring new storage media has become a hot research field. In 2012, researchers proposed to store digital information in synthetic DNA with the help of high-throughput DNA synthesis technology and high-throughput sequencing technology, which is called DNA data storage. The main advantages of DNA storage are high storage density and long media availability. It is considered to be a data storage technology with great potential. Using a large number of shorter oligonucleotides to form an oligonucleotide pool for data storage has become the main mode of DNA data storage. DNA data storage includes two basic steps. The first is to write data into synthetic DNA, and the second is to read data from synthetic oligonucleotides. The writing step mainly includes the addition of coding redundancy for error correction, the conversion of information sequence to 4-base sequence, and the synthesis of specific DNA chains; the reading step is to use sequencing technology to obtain sequencing reads from DNA samples, and reconstruct the original base sequence from multiple sequencing copies through a merging algorithm, and then decode and recover the information.

[0003] The core of DNA data storage technology is to use DNA synthesizer to synthesize artificially designed DNA sequences to store data. Its cost is limited by the cost of DNA synthesis. However, there are only four natural bases. In the process of converting computer binary data to DNA, the theoretical information capacity of each base is 2 bits (for example, A=00, T=01, G=10, C=11). The information capacity here refers to the amount of digital information that can be stored in a single synthesis cycle. The type of base and the encoding method are key factors affecting the storage density of DNA. A study by Erlich et al. on DNA storage fountain coding based on the quaternary coding system has achieved the highest information capacity of 1.57 bits based on standard bases and confirmed that the storage density of DNA can reach 215PB / g.

[0004] DNA synthesis technology based on phosphoramidite chemistry synthesizes a large number of identical molecules simultaneously during the synthesis process, and synthesizes multiple copies for each designed DNA sequence. These copies represent the same information, which will lead to information redundancy. The method of mixing nucleotides in a specific ratio during the synthesis process to generate "composite DNA letters" can effectively store more information in each synthesis cycle and reduce the number of synthesis cycles. Writing composite bases at fixed positions in the DNA sequence is equivalent to multiple oligonucleotide copies of the synthetic sequence. The generation of composite bases has unique advantages in the high-throughput synthesis process. First, it combines four standard DNA nucleotides (adenine A, thymine T, guanine G, cytosine C) and generates mixed sites with different base ratios in the synthesized DNA chain by precisely controlling their ratios. This means that at each base position, a certain ratio of mixed bases can replace the original single base. This method allows multiple bases to participate simultaneously, thereby bringing higher information density. The randomness and diversity of these composite bases make them very suitable for application in high-throughput sequencing and complex information encoding, representing multiple possibilities at the same time at fixed positions, greatly increasing the redundancy of information representation and the diversity of sequences.

[0005] In 2019, researchers proposed to use composite bases generated by mixing in the same proportion to increase information capacity. They introduced 11 composite bases mixed in equal proportions into DNA coding, effectively increasing the information capacity to 3.37 bits / symbol (Scientific Reports, 2019). After synthesis, composite bases present base diversity at the same position in different sequences. When reading data, if an appropriate composite base reconstruction algorithm is not adopted, the composite bases and standard bases are distinguished only based on the ratio of the four bases observed at the same position, which requires a higher sequencing coverage. In their composite base recovery experiment, they did not use a special algorithm, but only judged the original base letters based on the most frequently observed base type, which resulted in the need for at least 250× sequencing depth for error-free recovery, which increased the sequencing cost. A recent study suggested using composite DNA letters mixed in arbitrary proportions to increase logical density. The core of the alphabet is composed of a set of four standard DNA nucleotides combined in a predetermined free proportion (Nature Biotechnology, 2019). In theory, the composite alphabet can be infinitely enlarged. However, due to the limitations of synthesis technology and precision, it is not practical to synthesize in exact proportion. In their work, 6 composite letters were used to achieve a logical density of 1.96 bits / nt, and 20 composite letters were used to achieve a logical density of 4.29 bits / nt. However, the scale of the experiment was small, and only a composite letter sequence of 42 nt was encoded and designed. In their work, a relative entropy-based method was designed to distinguish different composite letters, which reduced the sequencing coverage to a certain extent and achieved 100% error-free recovery of data at 48× coverage. Summary of the invention

[0006] The present invention provides a multi-base encoding and readout method for composite base DNA storage, which realizes the matching of the multi-base encoding model and the composite base, breaks through the existing storage limit, and increases the theoretical limit of the information capacity of each synthesis cycle from 2 bits to 3 bits, as described below:

[0007] A multi-base encoding and reading method for composite base DNA storage, the method comprising the following steps:

[0008] (1) At the composite base data writing end, a group error correction code is used to encode the user data as a whole to obtain a codeword, and the codeword is converted into a multi-base symbol sequence according to a mapping method in which each m bits corresponds to a multi-base symbol d, and then divided into short segments. The standard base letters and composite base letters corresponding to the multi-base symbols are selected according to a user-defined method to obtain a DNA sequence containing standard bases and composite bases, and a highly reliable address sequence and primer sequence are added at both ends to obtain a composite base oligonucleotide pool for storing data;

[0009] (2) At the composite base data reading end, with the help of second-generation high-throughput sequencing, the composite bases of each synthesis site are converted into sequencing reads composed of standard bases. According to the primer sequence and address sequence, the unique position of the sequencing read is determined and clustered. Then, the base observation frequency in each address cluster is counted and converted into information entropy. Based on the separable property of entropy, the character set of each synthesis site is divided into groups. Furthermore, within each character set, the maximum likelihood is calculated for the observation frequency and the candidate character set, and the standard bases and composite bases are detected. Finally, the inferred characters are sent to the error correction decoder to correct the remaining substitution errors and erasure errors and restore the original data.

[0010] The step (1) is specifically:

[0011] (1.1) Using a binary or multi-bit block error correction code long code C1(n1,k1), the digital file of length k1 bits is encoded as a whole into a codeword of length n1 bits. The encoding process of the binary code and the multi-bit code is calculated in terms of bits, where k1 and n1 are both positive integers, representing the length of the information bit and the length of the coding bit, respectively;

[0012] (1.2) Divide each m bits of the codeword into a group and map them into a multi-ary symbol d according to a predetermined mapping rule, where the multi-ary symbols have a total of 2 m , a multi-ary symbol sequence of length n1 / m is obtained, which is then divided into S symbol sequences of length L, corresponding to S payload DNA sequences, where m is a positive integer not less than 3, d∈[0,2 m -1], S*L=n1 / m;

[0013] (1.3) According to the mapping relationship between the predetermined symbols and DNA bases, the standard base set {A, T, G, C}, the composite base {Y, M, K, R, S, W, R, B, V, H, D, N} and the composite bases {Y i ,M i ,K i ,R i ,S i ,W i ,R i ,B i ,V i ,H i ,D i ,N i}, i represents a variety of composite bases mixed with standard bases in different proportions, and 2 m The letters are used to represent the encoded polynomial symbol sequence, and the payload DNA sequence is obtained, and the payload DNA sequence contains standard bases and composite bases. The vector representing the mixing ratio of all four standard DNA nucleotides in the composite base during the synthesis process can be expressed as:

[0014] σ=(σ A ,σ T ,σ G ,σ C ),

[0015] Among them, the vector σ satisfies σ A +σ T +σ G +σ C =1;

[0016] (1.4) adding address sequences and primer sequences to both ends of the S data-bearing sequences of length L to generate an oligonucleotide pool containing standard bases and composite bases, wherein the address sequences and primer sequences are generated by four standard bases {A, T, G, C};

[0017] (1.5) According to the ratio of the standard bases corresponding to the composite bases or a single standard base, each site of each information sequence is synthesized in sequence by DNA synthesis, and all the synthesized sequences are mixed into a pool to obtain a composite oligonucleotide pool.

[0018] Wherein, the step (1.5) is specifically as follows:

[0019] According to the vector σ=(σ A ,σ T ,σ G ,σ C ), synthesize each site in turn, for the standard base, A = (1,0,0,0), T = (0,1,0,0), G = (0,0,1,0), C = (0,0,0,1), composite base It means that the composite base Y is synthesized by mixing the standard base T and the standard base C in the same proportion. It is synthesized by mixing the standard base T and the standard base C in a ratio of 2:1, and the same applies to other cases;

[0020] The step (2) is specifically:

[0021] (2.1) amplifying the synthesized composite oligonucleotide pool, preparing the library and performing second-generation high-throughput sequencing to obtain sequencing reads composed of standard bases;

[0022] (2.2) Identify the primer sequence and address sequence of the sequencing read, determine the unique address number of the sequencing read, obtain all DNA sequences corresponding to the same primer and address sequence, and aggregate them into clusters;

[0023] (2.3) Align the r data-bearing region sequences in each cluster, and count the frequency of occurrence of the four standard bases {A, T, G, C} at each different synthesis site i corresponding to the r sequences, denoted as: r i,A ,r i,T ,r i,G ,r i,C , where the observation frequency satisfies

[0024] (2.4) Based on the base frequencies observed at each synthesis site, calculate the frequency probability distribution p of the four standard bases i =(p i,A ,p i,T ,p i,G ,p i,C ), and calculate the information entropy, and then divide different types of characters into different sets based on the separable characteristics of entropy;

[0025] (2.5) Within each set, the normalized maximum log-likelihood method is used to compare the observed frequencies with the candidate character set to accurately detect standard bases and composite bases. Each inferred character is then demapped into m bits in turn and connected into a block code of length n1 bits. A highly reliable error correction decoding method is then used to correct the remaining substitution errors and erasure errors and restore the original digital file.

[0026] The step (1.4) is specifically:

[0027] (3.1) Select address information of length k2 in the range [1, S] to represent the order information of S multi-ary sequences, where Then, the short block error correction code C2(n2,k2) is used to encode the address bits of length k2 to generate a short block error correction code codeword of length n2 bits, and a check bit that can represent the parity check is added to form a coded address bit sequence of length L1;

[0028] (3.2) Group every two bits and map the encoded address bit sequence of length L1 into an address DNA sequence of length L2 according to the mapping rules: 00→A, 01→T, 10→G, 11→C, and add it to the 5' end of the data-bearing sequence;

[0029] (3.3) Generate a check bit that can represent the parity check based on the address bits of length k2, cyclically shift the original address bits of length k2 to the left by one bit to obtain the address bits of length k2 after the left shift, and then obtain the coded address bit sequence of length L1 according to the concatenation rule of "check bit - original address bit - check bit - left-shifted address bit";

[0030] (3.4) According to the same mapping rules as step (3.2), the encoded address bit sequence obtained in step (3.3) is converted into an address DNA sequence of length L2, which is added to the 3' end of the data-bearing sequence, and primer sequences for amplification are added at both ends. The address sequence and primer sequence only use the standard four bases (A, T, G, C) to generate an oligonucleotide pool containing standard bases and composite bases.

[0031] The step (2.4) is specifically:

[0032] (4.1) According to the observed base frequency distribution r of the r data-bearing sequences in each cluster i , calculate the frequency probability distribution of the four standard bases, expressed as p i =(p i,A ,p i,T ,p i,G ,p i,C ), and then calculate the information entropy at each fixed point based on the observed frequency distribution The frequency probability distribution satisfies p i,α represents the probability of the character α appearing at the synthesis position i in the r data-bearing sequence;

[0033] (4.2) Based on the separable property of information entropy, the set segmentation method is adopted. According to the information entropy results at each site, the three character types of standard bases, composite bases, and composite bases in different proportions are classified. And when m=3, all characters are divided into three character sets according to the information entropy results, namely, the standard base character set S1={A,T,G,C}, the two-base hybrid character set S2={Y,M,K,R,S,W} and the three-base hybrid character set S3={H,B,V,D}.

[0034] The step (2.5) is specifically:

[0035] (5.1) In each character set, according to the observed frequency distribution r i , and then the standard vectors σ=(σ A ,σ T ,σ G ,σ C ), calculate the log-likelihood function l(σ,r i ), which is used to infer the character in the set that is closest to the encoded character, and can be expressed as:

[0036]

[0037] (5.2) The log-likelihood function l(σ,r i) is normalized, the character with the smallest result is regarded as the detected character, and then each detected character is mapped to m bits, and a high-reliability error correction decoding algorithm is executed to correct the remaining replacement errors and erasure errors and restore the original digital file.

[0038] The technical solution provided by the present invention has the following beneficial effects: the present invention proposes a polybinary encoding method and an information representation model that match composite bases, which can realize highly reliable data recovery; proposes a highly reliable address sequence design method based on short block error correction code, which can resist synthesis and sequencing errors and realize more accurate multi-copy read segment clustering; when reading data, it is proposed to use the observed frequency distribution to calculate information entropy for composite base character set segmentation to realize reliable classification of different types of characters, and in each character set, based on the log-likelihood method, infer the original polybinary character, and combine linear block code decoding to perform residual error correction to realize error-free reading of user data. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A block diagram of a multi-base encoding and readout method for composite base DNA storage;

[0040] Figure 2 A flow chart of the multi-bit group error correction coding method designed for the present invention;

[0041] Figure 3 A schematic diagram of selecting standard bases and composite bases as storage media for the present invention;

[0042] Figure 4 Schematic diagram of the address DNA sequence based on the short packet error correction code designed for the present invention;

[0043] Figure 5 A schematic diagram of the structure of an oligonucleotide molecule comprising a standard base and a composite base designed for the present invention;

[0044] Figure 6 It is a schematic diagram of the DNA synthesis process based on the introduction of bases of the present invention;

[0045] Figure 7 A flowchart for performing double-ended primer identification and high-reliability label identification in the present invention;

[0046] Figure 8 A flow chart of the composite base detection method based on set segmentation designed for the present invention;

[0047] Fig. 9 A result diagram of the effective read ratio of the sequencing read primer identification and address identification provided by the present invention;

[0048] Fig.10 A length distribution diagram of the sequencing reads provided by the present invention after primer truncation;

[0049] Fig.11 A distribution diagram of the number of copies within a cluster after the read segment is clustered after label identification provided by the present invention;

[0050] Fig.12 A distribution diagram of information entropy provided by the present invention;

[0051] Fig.13 A normalized log-likelihood distribution graph provided by the present invention;

[0052] Fig.14 This is a bit error rate distribution diagram after reconstruction of the composite base character provided by the present invention. DETAILED DESCRIPTION

[0053] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention are described in further detail below.

[0054] The present invention combines the characteristics of DNA high-throughput synthesis technology with a large number of synthetic molecules at each synthesis site to propose a poly-binary encoding and reading method for composite base DNA storage, breaking through the existing storage limit and increasing the theoretical information capacity limit of 2 bits per synthesis cycle to 3 bits. The present invention uses poly-binary encoding to map user data into poly-binary symbol sequences, and then converts them into oligonucleotide pools containing standard bases and composite bases as storage media. When the data is read out, multiple sequencing copies are clustered based on the designed highly reliable address sequence, combined with the multi-copy characteristics of the encoded poly-binary character sequence, based on the frequency distribution of the four standard bases observed at each fixed site, a method for calculating information entropy using frequency probability distribution is proposed, which is used to realize the set distinction of different types of composite bases in a set segmentation manner, and in each letter set, based on the logarithmic likelihood method, the original poly-binary letters are inferred from the candidate alphabet table. Compared with directly judging the original base letter based on the most frequently observed base type, it has the advantage of lower inference error rate, combined with the designed highly reliable error correction decoding, to achieve data recovery under low coverage.

[0055] The present invention proposes a multi-base encoding and reading method for composite base DNA storage, designs a multi-base encoding method and an information representation model, and can achieve matching with composite bases; proposes an address sequence design method with strong error correction capability based on short block error correction code, which can resist synthesis and sequencing errors and achieve more accurate multi-copy read segment clustering; when reading data, it is proposed to use the observed frequency distribution to calculate information entropy for composite base character set segmentation to achieve reliable character classification of different types, and in each character set, based on the log-likelihood method, infer the original multi-base character, combine linear block code decoding to perform residual error correction, and achieve error-free reading of user data.

[0056] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0057] This embodiment describes in detail a multi-base encoding and reading method for composite base DNA storage proposed by the present invention, see Figure 1 , specifically including the following steps:

[0058] (1) At the composite base data writing end, a group error correction code is used to encode the user data as a whole to obtain a codeword, and the codeword is converted into a multi-base symbol sequence according to a mapping method in which each m bits corresponds to a multi-base symbol d, and then divided into short segments. The standard base letters and composite base letters corresponding to the multi-base symbols are selected according to a user-defined method to obtain a DNA sequence containing standard bases and composite bases, and a highly reliable address sequence and primer sequence are added at both ends to obtain a composite base oligonucleotide pool for storing data;

[0059] (2) At the composite base data reading end, with the help of second-generation high-throughput sequencing, the composite bases of each synthesis site are converted into sequencing reads composed of standard bases. According to the primer sequence and address sequence, the unique position of the sequencing read is determined and clustered. Then, the base observation frequency in each address cluster is counted and converted into information entropy. Based on the separable property of entropy, the character set of each synthesis site is divided into groups. Furthermore, within each character set, the maximum likelihood is calculated for the observation frequency and the candidate character set, and the standard bases and composite bases are detected. Finally, the inferred characters are sent to the error correction decoder to correct the remaining substitution errors and erasure errors and restore the original data.

[0060] The step (1) is specifically:

[0061] (1.1) Figure 2 As shown, a binary or multi-bit block error correction code long code C1(n1,k1) is used to encode a digital file with a length of k1 bits as a whole into a codeword with a length of n1 bits. The binary code and multi-bit code encoding process are calculated according to bits, wherein k1 and n1 are both positive integers, representing the length of the information bit and the length of the coding bit, respectively;

[0062] (1.2) Divide each m bits of the codeword into a group and map them into a multi-ary symbol d according to a predetermined mapping rule, where the multi-ary symbols have a total of 2 m , a multi-ary symbol sequence of length n1 / m is obtained, which is then divided into S symbol sequences of length L, corresponding to S payload DNA sequences, where m is a positive integer not less than 3, d∈[0,2 m -1], S*L=n1 / m;

[0063] (1.3) Figure 3As shown, according to the mapping relationship between the predetermined symbols and DNA bases, the standard base set {A, T, G, C}, the composite base {Y, M, K, R, S, W, R, B, V, H, D, N} and the composite bases {Y i ,M i ,K i ,R i ,S i ,W i ,R i ,B i ,V i ,H i ,D i ,N i}, i represents a variety of composite bases mixed with standard bases in different proportions, select 2 m The letters are used to represent the encoded polynomial symbol sequence, and the payload DNA sequence is obtained, and the payload DNA sequence contains standard bases and composite bases. The vector representing the mixing ratio of all four standard DNA nucleotides in the composite base during the synthesis process can be expressed as:

[0064] σ=(σ A ,σ T ,σ G ,σ C ),

[0065] The vector σ satisfies σ A +σ T +σ G +σ C =1;

[0066] (1.4) Figure 4 As shown, address sequences and primer sequences are added to both ends of S data-bearing sequences of length L to generate an oligonucleotide pool containing standard bases and composite bases, wherein the address sequence and primer sequence are generated by four standard bases {A, T, G, C}, and the structure of the oligonucleotide molecule is as follows Figure 5 As shown;

[0067] (1.5) Figure 6 As shown, according to the ratio of the standard bases corresponding to the composite bases or a single standard base, each site of each information sequence is synthesized in sequence by DNA synthesis, and all the synthesized sequences are mixed into a pool to obtain a composite oligonucleotide pool.

[0068] Wherein, the step (1.5) is specifically as follows:

[0069] According to the vector σ=(σ A ,σ T ,σ G ,σ C), synthesize each site in turn, for the standard base, A = (1,0,0,0), T = (0,1,0,0), G = (0,0,1,0), C = (0,0,0,1), composite base It means that the composite base Y is synthesized by mixing the standard base T and the standard base C in the same proportion. It is synthesized by mixing the standard base T and the standard base C in a ratio of 2:1, and the same applies to other cases;

[0070] The step (2) is specifically:

[0071] (2.1) amplifying the synthesized composite oligonucleotide pool, preparing the library and performing second-generation high-throughput sequencing to obtain sequencing reads composed of standard bases;

[0072] (2.2) Figure 7 As shown, the primer sequence and address sequence of the sequencing read are identified, the unique address number of the sequencing read is determined, and all DNA sequences corresponding to the same primer and address sequence are obtained and aggregated into clusters;

[0073] (2.3) Align the r data-bearing region sequences in each cluster, and count the frequency of occurrence of the four standard bases {A, T, G, C} at each different synthesis site i corresponding to the r sequences, denoted as: r i,A ,r i,T ,r i,G ,r i,C , where the observation frequency satisfies

[0074] (2.4) Figure 8 As shown, based on the base frequencies observed at each synthetic site, the frequency probability distribution p of the four standard bases is calculated. i =(p i,A ,p i,T ,p i,G ,p i,C ), and calculate the information entropy, and then divide different types of characters into different sets based on the separable characteristics of entropy;

[0075] (2.5) Within each set, the normalized maximum log-likelihood method is used to compare the observed frequencies with the candidate character set to accurately detect standard bases and composite bases. Each inferred character is then demapped into m bits in turn and connected into a block code of length n1 bits. A highly reliable error correction decoding method is then used to correct the remaining substitution errors and erasure errors and restore the original digital file.

[0076] The step (1.4) is specifically:

[0077] (3.1) Select address information of length k2 in the range [1, S] to represent the order information of S multi-ary sequences, where Then, the short block error correction code C2(n2,k2) is used to encode the address bits of length k2 to generate a short block error correction code codeword of length n2 bits, and a check bit that can represent the parity check is added to form a coded address bit sequence of length L1;

[0078] (3.2) Group every two bits and map the encoded address bit sequence of length L1 into an address DNA sequence of length L2 according to the mapping rules: 00→A, 01→T, 10→G, 11→C, and add it to the 5' end of the data-bearing sequence;

[0079] (3.3) Generate a check bit that can represent the parity check based on the address bits of length k2, cyclically shift the original address bits of length k2 to the left by one bit to obtain the address bits of length k2 after the left shift, and then obtain the coded address bit sequence of length L1 according to the concatenation rule of "check bit - original address bit - check bit - left-shifted address bit";

[0080] (3.4) According to the same mapping rules as step (3.2), the encoded address bit sequence obtained in step (3.3) is converted into an address DNA sequence of length L2, which is added to the 3' end of the data-bearing sequence, and primer sequences for amplification are added at both ends. The address sequence and primer sequence only use the standard four bases (A, T, G, C) to generate an oligonucleotide pool containing standard bases and composite bases.

[0081] The step (2.4) is specifically:

[0082] (4.1) According to the observed base frequency distribution r of the r data-bearing sequences in each cluster i , calculate the frequency probability distribution of the four standard bases, expressed as p i =(p i,A ,p i,T ,p i,G ,p i,C ), and then based on the observed frequency distribution, the information entropy at each fixed point is calculated, which can be expressed as:

[0083]

[0084] Among them, the frequency probability distribution satisfies p i,α represents the probability of the character α appearing at the synthesis position i in the r data-bearing sequence;

[0085] (4.2) Based on the separable property of information entropy, the set segmentation method is adopted. According to the information entropy results at each site, the three character types of standard bases, composite bases, and composite bases in different proportions are classified. And when m=3, all characters are divided into three character sets according to the information entropy results, namely, the standard base character set S1={A,T,G,C}, the two-base hybrid character set S2={Y,M,K,R,S,W} and the three-base hybrid character set S3={H,B,V,D}.

[0086] The step (2.5) is specifically:

[0087] (5.1) In each character set, according to the observed frequency distribution r i , and then the standard vectors σ=(σ A ,σ T ,σ G ,σ C ), calculate the log-likelihood function l(σ,r i ), which is used to infer the character in the set that is closest to the encoded character, and can be expressed as:

[0088]

[0089] (5.2) The log-likelihood function obtained in step (5.1) is normalized, and the character with the smallest result is regarded as the detected character. Each detected character is then mapped to m bits, and a highly reliable error correction decoding algorithm is executed to correct the remaining substitution errors and erasure errors and restore the original digital file. Specific embodiments

[0091] Specific examples are given below to illustrate the feasibility of the multi-base encoding and reading method for composite base DNA storage provided by the present invention.

[0092] This embodiment uses a multi-base LDPC code defined in the Galois field GF (64) with strong error correction capability, the number of symbols is 3780, the degree of the variable node is 2, the error correction performance is excellent, and an octal symbol mapping is designed. Every 3 bits are mapped to an octal symbol and then mapped to a standard base or a composite base. In the design of the present invention, the conversion from quaternary encoding to octal encoding does not bring about the loss of bit and symbol conversion, the coding efficiency is 1, and the theoretical information capacity of each synthesis cycle is increased from 2 bits per base to 3 bits per base. Specifically, the present invention designs a NB-LDPC (22680, 7560) codeword defined in the Galois field GF (64) with a code rate of 1 / 3, and a digital information of size 7560 bits is added with 15120 bits of redundant bits to generate a long codeword. Then, according to the rule that every 3 bits are mapped to 1 character, 7560 coded characters containing standard bases and composite bases are obtained.

[0093] Furthermore, the present invention divides the designed 7560 coded characters into 126 sequence fragments with a length of 60 characters, uses 8 kinds of nucleotides including standard bases and compound bases to represent the polynomial sequence, adds address sequences and primer sequences at both ends of the 126 polynomial sequences, and generates an oligonucleotide pool including standard bases and compound bases.

[0094] The specific steps of the address sequence designed in this embodiment are as follows:

[0095] (1.1) Select address information ranging from 1 to 126 with a length of 7 bits to represent the sequence information of the designed 126 multi-ary sequences, where 2 7 >126, then use the short block error correction code convolution code BCH (15, 7) to encode the address information with a length of 7 bits to generate a short block error correction code codeword with a length of 15 bits, and further add a check bit that can represent a parity check to the codeword to form a coded address bit sequence with a length of 16 bits;

[0096] (1.2) Group every two bits and map the coded address bit sequence of 16 bits into an address DNA sequence of 8 bases in length according to the mapping rules: 00→A, 01→T, 10→G, 11→C, and add it to the 5' end of the data-bearing sequence;

[0097] (1.3) Generate a check bit that can represent the parity check based on the original address information with a length of 7 bits, then cyclically shift the original address bits with a length of 7 bits to the left by one bit, and obtain the address information with a length of 7 bits after the left shift, and further obtain the coded address bit sequence with a length of 15 bits according to the splicing rule of "check bit-original bit original address bit-check bit-left shifted address bit";

[0098] (1.4) According to the same mapping rules as step (1.2), the encoded address bit sequence obtained in step (1.3) is converted into an address DNA sequence with a length of 8 bases, which is added to the 3' end of the data-bearing sequence, and a primer sequence for amplification with a length of 20 bases is added at both ends to generate an oligonucleotide pool containing standard bases and composite bases.

[0099] The present invention synthesizes an oligonucleotide pool containing standard bases and composite bases by a high-throughput parallel synthesis method, and realizes the synthesis of composite bases by adjusting the reagent ratio of standard DNA and adopting fuzzy synthesis. In the specific scheme, the present invention designs two design schemes of composite bases according to different standard DNA nucleotide ratios, as shown in Table 1. Scheme 1 is a composite base constructed by two standard bases in a ratio of 1:1, and scheme 2 is a composite base constructed by three standard bases in a ratio of 1:1:1. Six composite bases (R / Y / M / K / S / W) are designed in scheme 1, and an octal model is formed with the standard base (A / T); in scheme 2, the composition mode of the composite base is adjusted, and a composite base (H / B / V / D) is constructed, which forms an octal model with the standard base (A / T / G / C).

[0100] The present invention uses a synthetic oligonucleotide pool as a storage medium, and generates a sequencing read segment with a length of 150 nucleotides through multiple rounds of amplification reaction, library construction and second-generation double-end sequencing. The method of recovering data from the double-end sequencing read segment of the present invention is specifically as follows:

[0101] (2.1) Splicing the double-ended sequencing reads and aligning them with the designed 20-nucleotide double-ended primers, selecting the sequencing reads with the correct primers, and intercepting the address sequence part and the data-bearing sequence part according to the boundary position of the double-ended primers;

[0102] (2.2) Compare the double-ended address sequence with the designed address DNA sequence of 16 nucleotides after the two-end 8-nucleotide address sequences are combined, select the sequencing reads with the correct address sequence, and intercept the data-bearing sequence according to the boundary position of the address sequence, cluster the reads, and obtain multiple copies of sequencing reads for each specific sequence. Fig. 9 The ratio of primer identification to address identification is shown;

[0103] (2.3) Align the r data-carrying sequences in the cluster corresponding to each address sequence, and count the frequency distribution of the four standard bases at each fixed site r i (r i,A ,r i,T ,r i,G ,r i,C ),in, Fig.10The length distribution of sequencing reads after primer truncation is shown. Fig.11 The distribution results of the number of copies within a cluster after read segment clustering after label identification are shown.

[0104] Table 1 Types of designed composite bases and their corresponding relationships with standard bases

[0105]

[0106] Furthermore, based on the observed base frequency distribution, the set segmentation method is used to classify different types of characters, and then the normalized log-likelihood method is used to distinguish different characters in each type of character set, reconstruct standard bases and composite bases, and further, each reconstructed character is mapped to 3 bits, and high-reliability error correction decoding is performed to correct the remaining substitution errors and erasure errors, and restore the original digital file. The specific steps are:

[0107] (3.1) According to the observed base frequency distribution r of the r data-bearing sequences in each cluster i , calculate the frequency probability distribution of the four standard bases, expressed as p i =(p i,A ,p i,T ,p i,G ,p i,C ),in p i,α Indicates the probability of character α appearing at synthetic position i in the r data-carrying sequence; then calculates the information entropy at each fixed position The information entropy result is as follows Fig.12 As shown;

[0108] (3.2) Using the set segmentation method, according to the information entropy results at each site, the three character types of standard bases, composite bases, and composite bases with different proportions are classified. When m = 3, all characters are segmented into three character sets according to the information entropy results, namely, the standard base character set S1 = {A, T, G, C}, the two-base hybrid character set S2 = {Y, M, K, R, S, W}, and the three-base hybrid character set S3 = {H, B, V, D};

[0109] (3.3) In each character set, according to the observed frequency distribution r i , and then the standard vectors σ=(σ A ,σ T ,σ G ,σ C ) Calculate the log-likelihood function Used to infer the character in the set that is closest to the encoded character;

[0110] (3.4) Normalize the log-likelihood function obtained in step (3.3) and regard the character with the smallest result as the reconstructed character. Fig.13 The normalized log-likelihood results of the characters "K" and "H" are shown. Each reconstructed character is then mapped to 3 bits, and a highly reliable error correction decoding algorithm is performed to correct the remaining substitution errors and erasure errors and restore the original digital file.

[0111] Fig.14 The number of missing bits and the number of erroneous bits of two example storage schemes at different sequencing coverages are shown respectively. When the coverage is 10x, the missing error is as high as 10%, which is not within the erasure correction capability of the LDPC code designed by the present invention, and the character derivation error rate of the coding scheme of the three base mixtures at this coverage is significantly higher than that of the coding scheme of the composite bases mixed with two bases. With the increase of sequencing coverage, erasure errors and substitution errors gradually decrease, and at 50x sequencing coverage depth, erasure errors and substitution errors are already as low as 1% or even lower.

[0112] Those skilled in the art will appreciate that the accompanying drawing is only a schematic diagram of a preferred embodiment, and the serial numbers of the embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A multi-base encoding and reading method for composite base DNA storage, characterized in that: The method comprises the following steps: (1) At the composite base data writing end, a group error correction code is used to encode the user data as a whole to obtain a codeword, and the codeword is converted into a multi-base symbol sequence according to a mapping method in which each m bits corresponds to a multi-base symbol d, and then divided into short segments, and standard base letters and composite base letters corresponding to the multi-base symbols are selected to obtain a DNA sequence containing standard bases and composite bases, and a highly reliable address sequence and primer sequence are added at both ends to obtain a composite base oligonucleotide pool for storing data; (2) At the composite base data reading end, the second-generation high-throughput sequencing converts the composite bases of each synthesis site into sequencing reads composed of standard bases. According to the primer sequence and address sequence, the unique position of the sequencing read is determined and clustered. Then, the base observation frequency in each address cluster is counted and converted into information entropy. Based on entropy separability, the characters of each synthesis site are divided into subsets. In each character set, the maximum likelihood is calculated for the observation frequency and the candidate character set, and the standard bases and composite bases are detected. Finally, the inferred characters are sent to the error correction decoder to correct the remaining substitution errors and erasure errors and restore the original data.

2. The multi-base encoding and reading method for composite base DNA storage according to claim 1, characterized in that: The step (1) is specifically: (1.1) Using a binary or multi-bit block error correction code long code C1(n1,k1), the digital file of length k1 bits is encoded as a whole into a codeword of length n1 bits. The encoding process of the binary code and the multi-bit code is calculated in terms of bits, where k1 and n1 are both positive integers, representing the length of the information bit and the length of the coding bit, respectively; (1.2) Divide each m bits of the codeword into a group and map them into a multi-ary symbol d according to a predetermined mapping rule, where the multi-ary symbols have a total of 2 m , a multi-ary symbol sequence of length n1 / m is obtained, which is then divided into S symbol sequences of length L, corresponding to S payload DNA sequences, where m is a positive integer not less than 3, d∈[0,2 m -1], S*L=n1 / m; (1.3) According to the mapping relationship between the predetermined symbols and DNA bases, the standard base set {A, T, G, C}, the composite base {Y, M, K, R, S, W, R, B, V, H, D, N} and the composite bases {Y i ,M i ,K i ,R i ,S i ,W i ,R i ,B i ,V i ,H i ,D i ,N i }, i represents a variety of composite bases mixed with standard bases in different proportions, and 2 m The letters are used to represent the encoded polynomial symbol sequence to obtain the payload DNA sequence, and the payload DNA sequence includes standard bases and composite bases. The vector representing the mixing ratio of all four standard DNA nucleotides in the composite base during the synthesis process can be expressed as σ = (σ A ,σ T ,σ G ,σ C ), the vector σ satisfies σ A +σ T +σ G +σ C =1; (1.4) adding address sequences and primer sequences to both ends of the S data-bearing sequences of length L to generate an oligonucleotide pool containing standard bases and composite bases, wherein the address sequences and primer sequences are generated by four standard bases {A, T, G, C}; (1.5) synthesizing each site of each information sequence in sequence by DNA synthesis according to the ratio of the standard bases corresponding to the composite bases or a single standard base, and mixing all the synthesized sequences into a pool to obtain a composite oligonucleotide pool; Wherein, the step (1.5) is specifically as follows: According to the vector σ=(σ A ,σ T ,σ G ,σ C ), synthesize each site in turn, for the standard base, A = (1,0,0,0), T = (0,1,0,0), G = (0,0,1,0), C = (0,0,0,1), composite base It means that the composite base Y is synthesized by mixing the standard base T and the standard base C in the same proportion. It is synthesized by mixing the standard base T and the standard base C in a ratio of 2:

1.

3. The multi-base encoding and reading method for composite base DNA storage according to claim 1, characterized in that: The step (2) is specifically: (2.1) amplifying the synthesized composite oligonucleotide pool, preparing the library and performing second-generation high-throughput sequencing to obtain sequencing reads composed of standard bases; (2.2) Identify the primer sequence and address sequence of the sequencing read, determine the unique address number of the sequencing read, obtain all DNA sequences corresponding to the same primer and address sequence, and aggregate them into a cluster; (2.3) Align the r data-bearing region sequences in each cluster, and count the frequency of occurrence of the four standard bases {A, T, G, C} at each different synthesis site i corresponding to the r sequences, denoted as: r i,A ,r i,T ,r i,G ,r i,C , where the observation frequency satisfies (2.4) Based on the base frequencies observed at each synthesis site, calculate the frequency probability distribution p of the four standard bases i =(p i,A ,p i,T ,p i,G ,p i,C ), and calculate the information entropy, and then divide different types of characters into different sets based on the separable characteristics of the information entropy; (2.5) Within each set, the normalized maximum log-likelihood method is used to compare the observed frequencies with the candidate character set to accurately detect standard bases and composite bases. Each inferred character is then demapped into m bits in turn and connected into a block code of length n1 bits. A highly reliable error correction decoding method is then used to correct the remaining substitution errors and erasure errors and restore the original digital file.

4. The multi-base encoding and reading method for composite base DNA storage according to claim 2, characterized in that: The step (1.4) is specifically: (3.1) Select address information of length k2 in the range [1, S] to represent the order information of S multi-ary sequences, where Then, the short block error correction code C2(n2,k2) is used to encode the address bits of length k2 to generate a short block error correction code codeword of length n2 bits, and a check bit representing a parity check is added to form a coded address bit sequence of length L1; (3.2) Grouping every two bits, according to the mapping rule MR = {00 → A, 01 → T, 10 → G, 11 → C}, the encoded address bit sequence of length L1 is mapped into an address DNA sequence of length L2, and added to the 5' end of the data bearing sequence; (3.3) Generate a check bit that can represent parity check based on the address bits of length k2, cyclically shift the original address bits of length k2 left by one bit, obtain the address bits of length k2 after left shift, and then obtain the coded address bit sequence of length L1 according to the concatenation rule of "check bit - original address bit - check bit - left shifted address bit"; (3.4) According to the mapping rule MR, the encoded address bit sequence obtained in step (3.3) is converted into an address DNA sequence of length L2, which is added to the 3' end of the data-bearing sequence, and primer sequences for amplification are added at both ends. The address sequence and primer sequence use the standard four bases (A, T, G, C) to generate an oligonucleotide sequence containing standard bases and composite bases.

5. The multi-base encoding and reading method for composite base DNA storage according to claim 3, characterized in that: The step (2.4) is specifically: (4.1) Based on the observed base frequency distribution r of the r data-bearing sequences in each cluster i , calculate the frequency distribution of the four standard bases, expressed as p i =(p i,A ,p i,T ,p i,G ,p i,C ), then based on the observed frequency distribution, the information entropy at each fixed point is calculated, expressed as Frequency distribution satisfies p i,α represents the probability of the character α appearing at the synthesis position i in the r data-bearing sequence; (4.2) Based on the information entropy, the set segmentation method is separable and the three character types of standard bases, composite bases, and composite bases in different proportions are classified according to the information entropy results at each site; when m=3, all characters are divided into three character sets according to the information entropy results, namely, the standard base character set S1={A, T, G, C}, the two-base hybrid character set S2={Y, M, K, R, S, W} and the three-base hybrid character set S3={H, B, V, D}.

6. The multi-base encoding and reading method for composite base DNA storage according to claim 3, characterized in that: The step (2.5) is specifically: (5.1) In each character set, according to the observed frequency distribution r i , and then the standard vectors σ=(σ A ,σ T ,σ G ,σ C ) calculates the log-likelihood function l(σ,r i ) Estimate the character in the set that is closest to the encoded character. The log-likelihood ratio function is expressed as (5.2) The log-likelihood function l(σ,r i ) is normalized, the character with the smallest result is regarded as the detected character, and then each detected character is mapped to m bits, and a high-reliability error correction decoding algorithm is executed to correct the remaining replacement errors and erasure errors and restore the original digital file.

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