Primer design method, device, electronic device and storage medium

Abstract

The primer design method, device, electronic device and storage medium provided by the embodiments of the present disclosure include: obtaining a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing, generating an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing, wherein the difference fragment pair includes a pre-editing fragment and a post-editing fragment in which the reference sequence after gene editing undergoes gene editing relative to the reference sequence before gene editing, and the difference fragment pair sequence is composed of at least one difference fragment pair arranged in order from front to back according to the starting base sites of the pre-editing fragment in the difference fragment pair, and generating a target difference fragment pair sequence according to the initial difference fragment pair sequence and the preset difference fragment pair merging condition. In this way, the post-editing fragment in which gene editing occurs in the post-editing reference sequence can be automatically found through the reference sequence before gene editing and the reference sequence after gene editing.

Description

Technical Field

[0001] Embodiments of the present disclosure relate to the technical field of bioinformatics analysis, and in particular to a primer design method, device, electronic device, and storage medium. Background Art

[0002] After gene editing of a DNA sequence (for example, knocking out a segment of bases at a specific position in a DNA sequence, or replacing a segment of bases at a specific position in a DNA sequence with bases of the same length or a different length), the edited DNA sequence needs to be verified to determine whether the gene editing operation of the DNA sequence has been successful and whether the original DNA sequence has been successfully modified. At present, the methods for verifying the edited DNA sequence mainly include whole genome sequencing of the DNA sequence and PCR (Polymerase Chain Reaction) experiments on the edited region of the DNA sequence.

[0003] However, the above two methods cannot directly find the difference between the original DNA sequence and the edited DNA sequence, and the above methods require the experimenter to manually design the PCR primers. When conducting large-scale gene editing projects or involving multiple editing types (such as knockout, knock-in, promoter replacement, etc.), there are high requirements for the experimenter's professional knowledge and experience. In addition, the manual design of PCR primers has low design efficiency and may also lead to inaccurate primer design, affecting the reliability of the verification results.

[0004] Therefore, it is necessary to propose a primer design method to solve at least one of the above technical problems. Summary of the invention

[0005] The embodiments of the present disclosure provide a primer design method, device, electronic device and storage medium.

[0006] In a first aspect, the present disclosure provides a primer design method, comprising:

[0007] Obtaining a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing;

[0008] Generate an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing, wherein the difference fragment pair includes a pre-edited fragment and a post-edited fragment of the reference sequence after gene editing relative to the reference sequence before gene editing, and the difference fragment pair sequence is composed of at least one difference fragment pair arranged in order from front to back according to the starting base sites of the pre-edited fragment in the difference fragment pair;

[0009] A target difference segment pair sequence is generated according to the initial difference segment pair sequence and a preset difference segment pair merging condition.

[0010] In some optional embodiments, generating an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing comprises:

[0011] Comparing the reference sequence before gene editing and the reference sequence after gene editing to obtain matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing;

[0012] Obtaining non-matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing according to the matching base pairs;

[0013] Obtaining, according to the non-matching base pairs, a post-edited segment of the reference sequence after gene editing and a pre-edited segment corresponding to the post-edited segment in the reference sequence before gene editing;

[0014] The initial difference fragment pair sequence is generated according to the edited fragment of the reference sequence after gene editing and the pre-edited fragment corresponding to the edited fragment in the reference sequence before gene editing.

[0015] In some optional implementations, generating a target difference segment pair sequence according to the initial difference segment pair sequence and a preset difference segment pair merging condition includes:

[0016] For any two adjacent difference segment pairs in the initialized difference segment pair sequence, in response to determining that the two adjacent difference segment pairs satisfy a preset difference segment pair merging condition, merge the two adjacent difference segment pairs to generate a merged difference segment pair, and replace the two adjacent difference segment pairs in the initial difference segment pair sequence with the merged difference segment pair, until no two adjacent difference segment pairs satisfying the preset difference segment pair merging condition exist in the initial difference segment pair sequence;

[0017] A target difference segment pair sequence is generated based on the difference segment pairs and / or the merged difference segment pairs in the initialization difference segment pair sequence.

[0018] In some optional implementations, in response to determining that the two adjacent difference segment pairs satisfy a pre-difference segment pair merging condition, the method includes:

[0019] In response to the number of bases between the edited segment in the first difference segment pair that is ranked higher and the edited segment in the second difference segment pair that is ranked lower in the two adjacent difference segment pairs being within a preset matching base number range, it is determined that the two adjacent difference segment pairs meet the pre-difference segment pair merging condition.

[0020] In some optional implementations, merging the two adjacent difference segment pairs to generate a merged difference segment pair includes:

[0021] The starting base of the edited fragment in the merged difference fragment pair is the starting base of the edited fragment in the first difference fragment pair ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the edited fragment in the second difference fragment pair ranked later among the two adjacent difference fragment pairs;

[0022] The starting base of the pre-editing fragment in the merged difference fragment pair is the starting base of the pre-editing fragment in the first difference fragment pair ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the pre-editing fragment in the second difference fragment pair ranked later among the two adjacent difference fragment pairs.

[0023] In some optional embodiments, the method further comprises:

[0024] For each target difference fragment pair in the target difference fragment pair sequence, a primer pair set corresponding to the target difference fragment pair is generated according to the target difference fragment pair.

[0025] In some optional embodiments, generating a primer pair set corresponding to the target difference fragment pair according to the target difference fragment pair comprises:

[0026] generating at least one first primer pair based on the target edited fragment in the target differential fragment pair;

[0027] generating at least one second primer pair based on the target pre-editing fragment in the target differential fragment pair;

[0028] A primer pair set corresponding to the target differential fragment pair is generated based on the first primer pair and the second primer pair.

[0029] In a second aspect, the present disclosure provides a primer design device, comprising:

[0030] A sequence acquisition unit, used to acquire a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing;

[0031] an initial difference fragment pair generating unit, configured to generate an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing, wherein the difference fragment pair comprises a pre-editing fragment and a post-editing fragment of the reference sequence after gene editing relative to the reference sequence before gene editing, and the difference fragment pair sequence is formed by arranging at least one difference fragment pair in the order of the starting base sites of the pre-editing fragment in the difference fragment pair from front to back;

[0032] The target difference segment pair generating unit is used to generate a target difference segment pair sequence according to the initial difference segment pair sequence and a preset difference segment pair merging condition.

[0033] In a third aspect, the present disclosure provides an electronic device, including:

[0034] one or more processors;

[0035] a storage device having one or more programs stored thereon,

[0036] When the one or more programs are executed by the one or more processors, the one or more processors implement the method described in any embodiment of the first aspect and / or the second aspect of the present disclosure.

[0037] In a fourth aspect, the present disclosure provides a computer-readable storage medium having a computer program stored thereon, wherein the computer program, when executed by one or more processors, implements the method described in any embodiment of the first aspect and / or second aspect of the present disclosure.

[0038] In a fifth aspect, the present disclosure provides a computer program product, comprising a computer program / instruction, which, when executed by a processor, implements the method described in any embodiment of the first aspect and / or second aspect of the present disclosure.

[0039] The primer design method, device, electronic device and storage medium provided by the embodiments of the present disclosure first obtain a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing, then generate an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing, wherein the difference fragment pair includes the pre-editing fragment and the post-editing fragment in which the reference sequence after gene editing undergoes gene editing relative to the reference sequence before gene editing, and the difference fragment pair sequence is composed of at least one difference fragment pair arranged in order from front to back according to the starting base site of the pre-editing fragment in the difference fragment pair, and finally, generate a target difference fragment pair sequence according to the initial difference fragment pair sequence and the preset difference fragment pair merging condition. In this way, the post-editing fragment in which gene editing occurs in the post-editing reference sequence can be automatically found through the reference sequence before gene editing and the reference sequence after gene editing to obtain the difference between the reference sequence before gene editing and the reference sequence before gene editing, and PCR primers can be automatically designed through the target difference fragment pair, without the need to manually design PCR primers through experiments, thereby improving the efficiency of PCR primer design and the accuracy of PCR primer design, and improving the reliability of experimental verification results. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Other features, objects and advantages of the present disclosure will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings. The drawings are only for the purpose of illustrating specific embodiments and are not to be considered as limiting the present invention. In the drawings:

[0041] Figure 1 is a system architecture diagram of one embodiment to which the primer design method disclosed herein can be applied;

[0042] Figure 2 is a flow chart of one embodiment of a primer design method according to the present disclosure;

[0043] Figure 3 is a decomposed flow chart of one embodiment of step 202 according to the present disclosure;

[0044] Figure 4 is a decomposed flow chart of one embodiment of step 203 according to the present disclosure;

[0045] Figure 5 is a schematic structural diagram of an embodiment of a primer design device according to the present disclosure;

[0046] Figure 6 It is a structural diagram of a computer system of an electronic device suitable for implementing an embodiment of the present disclosure. DETAILED DESCRIPTION

[0047] The present disclosure is further described in detail below in conjunction with the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the relevant invention are shown in the accompanying drawings.

[0048] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present disclosure may be combined with each other. The present disclosure will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0049] Figure 1 An exemplary system architecture 100 is shown to which embodiments of the primer design method, apparatus, electronic device, and storage medium of the present disclosure can be applied.

[0050] like Figure 1 As shown, the system architecture 100 may include terminal devices 101, 102, 103, a network 104 and a server 105. The network 104 is used to provide a medium for communication links between the terminal devices 101, 102, 103 and the server 105. The network 104 may include various connection types, such as wired, wireless communication links or optical fiber cables, etc.

[0051] The user can use the terminal devices 101, 102, 103 to interact with the server 105 through the network 104 to receive or send messages, etc. Various communication client applications can be installed on the terminal devices 101, 102, 103, such as primer design applications, voice interaction applications, video conferencing applications, short video social applications, web browser applications, shopping applications, search applications, instant messaging tools, email clients, social platform software, etc.

[0052] Terminal devices 101, 102, 103 can be hardware or software. When terminal devices 101, 102, 103 are hardware, they can be various electronic devices with microphones and speakers, including but not limited to smart phones, tablet computers, e-book readers, MP3 players (Moving Picture Experts Group Audio Layer III, Moving Picture Experts Compression Standard Audio Layer 3), MP4 players (Moving Picture Experts Group Audio Layer IV, Moving Picture Experts Compression Standard Audio Layer 4), portable computers and desktop computers, etc. When terminal devices 101, 102, 103 are software, they can be installed in the electronic devices listed above. It can be implemented as multiple software or software modules, or as a single software or software module. No specific limitation is made here.

[0053] The server 105 may be a server that provides various services, such as a background server that processes the reference sequence before gene editing and the reference sequence after gene editing corresponding to the reference sequence before gene editing obtained on the terminal devices 101, 102, and 103. The background server may perform corresponding processing based on the reference sequence before gene editing obtained by the terminal device and the reference sequence after gene editing corresponding to the reference sequence before gene editing.

[0054] In some cases, the primer design method provided in the present disclosure can be jointly performed by the terminal devices 101, 102, 103 and the server 105. For example, the step of "obtaining a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing" can be performed by the terminal devices 101, 102, 103, and the step of "generating an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing" can be performed by the server 105. The present disclosure does not limit this. Accordingly, the primer design device can also be respectively set in the terminal devices 101, 102, 103 and the server 105.

[0055] In some cases, the primer design method provided in the present disclosure may be executed by the server 105 , and accordingly, the primer design device may also be set in the server 105 . In this case, the system architecture 100 may not include the terminal devices 101 , 102 , and 103 .

[0056] In some cases, the primer design method provided by the present disclosure may be executed by the terminal devices 101 , 102 , 103 , and accordingly, the primer design apparatus may also be disposed in the terminal devices 101 , 102 , 103 . In this case, the system architecture 100 may not include the server 105 .

[0057] It should be noted that the server 105 can be hardware or software. When the server 105 is hardware, it can be implemented as a distributed server cluster consisting of multiple servers, or it can be implemented as a single server. When the server 105 is software, it can be implemented as multiple software or software modules (for example, for providing distributed services), or it can be implemented as a single software or software module. No specific limitation is made here.

[0058] It should be understood that Figure 1 The number of terminal devices, networks and servers in the embodiment is only for illustration. Any number of terminal devices, networks and servers may be provided according to implementation requirements.

[0059] Continue to refer Figure 2 , Figure 2 200 shows a flow chart of one embodiment of a primer design method according to the present disclosure. Figure 2 The primer design method shown can be applied to Figure 1 The process 200 includes the following steps:

[0060] Step 201, obtain a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing.

[0061] In this embodiment, the reference sequence before gene editing may refer to a DNA sequence that has not undergone gene editing, and the reference sequence after gene editing corresponding to the reference sequence before gene editing may refer to a DNA sequence after gene editing of the reference sequence before gene editing. Specifically, the gene editing operation of the reference sequence before gene editing may include at least one of knock-out, knock-in and promoter exchange.

[0062] Among them, knock-out may refer to removing part of the DNA sequence from a specific position in the reference sequence before gene editing, knock-in may refer to replacing part of the DNA sequence at a specific position in the reference sequence before gene editing with a new DNA sequence of the same or different length, and promoter exchange may refer to replacing the promoter DNA sequence in the reference sequence before gene editing with a different promoter DNA sequence.

[0063] In this embodiment, the reference sequence after gene editing may include multiple edited fragments, that is, the reference sequence after gene editing may include multiple DNA fragments that have been modified relative to the reference sequence before gene editing.

[0064] Here, the user can perform at least one gene editing operation such as knock-out, knock-in and promoter exchange on the pre-gene editing reference sequence to obtain a post-gene editing reference sequence corresponding to the pre-gene editing reference sequence, and then input the pre-gene editing reference sequence and the post-gene editing reference sequence corresponding to the pre-gene editing reference sequence into the terminal device, so that the terminal device obtains the pre-gene editing reference sequence and the post-gene editing reference sequence corresponding to the pre-gene editing reference sequence. Alternatively, the user inputs the pre-gene editing reference sequence and the post-gene editing reference sequence corresponding to the pre-gene editing reference sequence into the terminal device, and then the server obtains the pre-gene editing reference sequence and the post-gene editing reference sequence corresponding to the pre-gene editing reference sequence through the terminal device.

[0065] Step 202, generating an initial difference fragment pair sequence based on the reference sequence before gene editing and the reference sequence after gene editing.

[0066] In this embodiment, the initial difference fragment pair sequence includes a difference fragment pair, wherein the difference fragment pair includes a pre-edited fragment and an edited fragment in which gene editing has occurred in the reference sequence after gene editing relative to the reference sequence before gene editing, that is, the difference fragment pair includes a post-edited fragment in which gene editing has occurred in the reference sequence after gene editing relative to the reference sequence before gene editing and a pre-edited fragment corresponding to the edited fragment in the reference sequence before gene editing.

[0067] Here, the edited fragment may refer to a fragment that has been gene-edited in a reference sequence after gene editing relative to the reference sequence before gene editing, and the pre-edited fragment may refer to the fragment corresponding to the edited fragment in the reference sequence before gene editing.

[0068] The differential fragment pair sequence is formed by arranging at least one differential fragment pair in order from front to back according to the starting base sites of the pre-editing fragments in the differential fragment pair.

[0069] In some optional embodiments, obtaining a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing also includes obtaining the site of each base in the reference sequence before gene editing and the site of each base in the reference sequence after gene editing.

[0070] The difference fragment pair sequences can be arranged in order from front to back according to the start base sites of the pre-editing fragments in the difference fragment pair.

[0071] For example, if the starting base site of the pre-editing fragment in the difference fragment pair 1 corresponds to the base site in the reference sequence before gene editing and is located before the starting base site of the pre-editing fragment in the difference fragment pair 2 corresponds to the base site in the reference sequence before gene editing, then the order of the difference fragment pair 1 in the difference fragment pair sequence is located before the difference fragment 2.

[0072] Since the reference sequence before gene editing and the reference sequence after gene editing are in a corresponding relationship, in some optional embodiments, the difference fragment pair sequence can also be arranged in order from front to back according to the starting base sites of the edited fragments in the difference fragment pair.

[0073] Since the reference sequence after gene editing may include multiple gene editing fragments, that is, multiple post-editing fragments, correspondingly, the initial difference fragment pair sequence may include multiple difference fragment pairs consisting of post-editing fragments and pre-editing fragments.

[0074] Specifically, step 202, generating an initial difference fragment pair sequence according to a reference sequence before gene editing and a reference sequence after gene editing, may include steps 2021 to 2024.

[0075] refer to Figure 3 , Figure 3 is a decomposed flow chart of an embodiment of step 202 according to the present disclosure. Figure 3 As shown,

[0076] Step 2021, align the reference sequence before gene editing and the reference sequence after gene editing to obtain matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing.

[0077] Here, various existing alignment algorithms can be used to align the reference sequence before gene editing and the reference sequence after gene editing to obtain the matching part between the reference sequence before gene editing and the reference sequence after gene editing.

[0078] For example, a Sequence Alignment algorithm can be used to align the reference sequence before gene editing and the reference sequence after gene editing to obtain matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing.

[0079] For example, by aligning the reference sequence before gene editing and the reference sequence after gene editing through the global alignment method of the Sequence Alignment algorithm, each base of the reference sequence before gene editing and the reference sequence after gene editing can be aligned, and by comparing the entire reference sequence before gene editing and the reference sequence after gene editing, the longest continuous matching part between the reference sequence before gene editing and the reference sequence after gene editing can be found, and further, the matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing can be obtained.

[0080] It should be understood that the matching base pairs here are calculated by the algorithm, and whether the status matches is determined based on the local maximum likelihood of the reference sequence before gene editing and the reference sequence after gene editing, rather than the bases corresponding to the reference sequence after gene editing and the reference sequence before gene editing are exactly the same.

[0081] Step 2022, obtaining non-matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing based on the matching base pairs.

[0082] In this embodiment, since the matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing are obtained, the non-matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing can be obtained by reverse calculation through the matching base pairs, that is, the remaining base pairs after removing the matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing are the non-matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing.

[0083] Step 2023, obtaining the edited fragment of the reference sequence after gene editing and the corresponding pre-edited fragment of the edited fragment in the reference sequence before gene editing according to the non-matching base pairs.

[0084] In this embodiment, multiple edited fragments present in the reference sequence after gene editing and the pre-edited fragments of the reference sequence before gene editing corresponding to each edited fragment can be obtained through the non-matching base pairs in the reference sequence before gene editing and the reference sequence after gene editing.

[0085] In some optional embodiments, various existing algorithms can be used to merge the non-matching base pairs to generate edited fragments, as well as the corresponding pre-edited fragments of each edited fragment in the reference sequence before gene editing.

[0086] Step 2024, generating an initial difference fragment pair sequence according to the edited fragment of the reference sequence after gene editing and the corresponding pre-edited fragment of the edited fragment in the reference sequence before gene editing.

[0087] Here, each difference fragment pair can be composed of each edited fragment and the corresponding pre-edited fragment of the edited fragment in the reference sequence before gene editing, and the initial difference fragment pair sequence can be generated by arranging the difference fragment pairs from front to back according to the starting base sites of the pre-edited fragment in the difference fragment pairs by at least one difference fragment pair.

[0088] Step 203: Generate a target difference segment pair sequence according to the initial difference segment pair sequence and a preset difference segment pair merging condition.

[0089] Here, since the reference sequence before gene editing is subjected to gene editing, some DNA fragments in an edited fragment may match or be identical to the corresponding DNA fragments in the reference sequence before gene editing.

[0090] However, among the edited segments obtained in step 202, one edited segment may be mistakenly identified as two different edited segments. Therefore, through step 203, different edited segments that actually belong to one edited segment can be merged according to the preset difference segment pair merging condition to obtain the target difference segment pair sequence.

[0091] Specifically, step 203 , generating a target difference segment pair sequence according to the initial difference segment pair sequence and a preset difference segment pair merging condition, may include steps 2031 to 2032 .

[0092] refer to Figure 4 , Figure 4 is a decomposition flow chart of an embodiment of step 203 of the present disclosure. Figure 4 As shown,

[0093] Step 2031, for any two adjacent difference segment pairs in the initialized difference segment pair sequence, in response to determining that the two adjacent difference segment pairs satisfy a preset difference segment pair merging condition, the two adjacent difference segment pairs are merged to generate a merged difference segment pair, and the merged difference segment pair is used to replace the two adjacent difference segment pairs in the initial difference segment pair sequence until there are no two adjacent difference segment pairs satisfying the preset difference segment pair merging condition in the initial difference segment pair sequence.

[0094] In some optional embodiments, in response to determining that the two adjacent difference fragment pairs satisfy the pre-difference fragment pair merging condition, it can be that in response to the number of bases between the edited fragment in the first difference fragment pair that is ranked higher among the two adjacent difference fragment pairs and the edited fragment in the second difference fragment pair that is ranked lower among the two adjacent difference fragment pairs is within a preset matching base number range, determining that the two adjacent difference fragment pairs satisfy the pre-difference fragment pair merging condition.

[0095] Here, the initialized difference segment pair sequence may have multiple difference segment pairs arranged in sequence, the first difference segment pair may be a difference segment pair ranked first between any two adjacent difference segment pairs, the second difference segment pair may be a difference segment pair ranked last between any two adjacent difference segment pairs, and determining that two adjacent difference segment pairs meet the pre-difference segment pair merging condition may be that the number of bases between the edited segment in the first difference segment pair and the edited segment in the second difference segment pair ranked last is within a preset matching base number range, that is, if the number of bases between two adjacent edited segments is within a certain range, then the two adjacent edited segments may be the same edited segment, and the two edited segments are merged, and the pre-edited segments corresponding to the two edited segments are merged, that is, the two adjacent difference segment pairs are merged to generate a merged difference segment pair, and the merged difference segment pair is used to replace the two adjacent difference segment pairs in the initial difference segment pair sequence, and then determine whether the number of bases between the difference segment pairs adjacent to the merged difference segment pair is within the preset matching base number range, so as to merge other difference segment pairs, until there are no two adjacent difference segment pairs meeting the preset difference segment pair merging condition in the initial difference segment pair sequence.

[0096] In some optional embodiments, the number of bases between the edited fragment in the first difference fragment pair that is ranked first and the edited fragment in the second difference fragment pair that is ranked later in two adjacent difference fragment pairs may refer to the number of bases between the end base of the edited fragment in the first difference fragment pair that is ranked first and the start base of the edited fragment in the second difference fragment pair that is ranked later.

[0097] Here, the number of bases between the edited fragment in the first difference fragment pair that is ranked higher in two adjacent difference fragment pairs and the edited fragment in the second difference fragment pair that is ranked lower in two adjacent difference fragment pairs is within a preset matching base number range, which may mean that the number of bases between the edited fragment in the first difference fragment pair that is ranked higher in two adjacent difference fragment pairs and the edited fragment in the second difference fragment pair that is ranked lower in two adjacent difference fragment pairs is less than or equal to the preset matching base number.

[0098] In some optional implementations, for any two adjacent difference segment pairs in the initialized difference segment pair sequence, in response to determining that the two adjacent difference segment pairs do not satisfy a preset difference segment pair merging condition, the two adjacent difference segment pairs are not merged.

[0099] For example, the initialized difference segment pair sequence includes the pre-editing segment 1, segment 2 and segment 3 and the post-editing segment a, segment b and segment c arranged in the following order, wherein segment 1 and segment a, segment 2 and segment b, and segment 3 and segment c correspond to each other one by one. That is, segment 1 and segment a, segment 2 and segment b, and segment 3 and segment c are three pairs of difference segment pairs, respectively.

[0100] When the number of bases between fragment a and fragment b is less than or equal to the preset number of matching bases, fragment a and fragment b are merged, and fragment 1 and fragment 2 are merged to generate a merged difference fragment pair.

[0101] Furthermore, when the number of bases between the edited fragment and fragment c in the merged difference fragment pair is greater than the preset number of matching bases, the edited fragment and fragment c in the merged difference fragment pair are not merged, indicating that the two fragments belong to different edited fragments.

[0102] In some optional embodiments, the starting base of the edited fragment in the merged difference fragment pair is the starting base of the edited fragment in the first difference fragment pair that is ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the edited fragment in the second difference fragment pair that is ranked later among the two adjacent difference fragment pairs.

[0103] Here, merging the edited segments in the difference segment pairs includes merging the bases between two adjacent edited segments in the two adjacent difference segment pairs, that is, merging the bases between two adjacent edited segments in the two adjacent difference segment pairs as partial segments in the edited segments in the merged difference segment pairs.

[0104] For example, if there are three bases between fragment a and fragment b, these three bases are merged together with fragment a and fragment b.

[0105] In this way, the starting base of the edited fragment in the entire merged difference fragment pair is the starting base of the edited fragment in the first difference fragment pair that is ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the edited fragment in the second difference fragment pair that is ranked later among the two adjacent difference fragment pairs.

[0106] In some optional embodiments, the starting base of the pre-editing fragment in the merged difference fragment pair is the starting base of the pre-editing fragment in the first difference fragment pair that is ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the pre-editing fragment in the second difference fragment pair that is ranked later among the two adjacent difference fragment pairs.

[0107] Similarly, if there are three bases between fragment 1 and fragment 2, these three bases are merged together with fragment 1 and fragment 2.

[0108] In this way, the starting base of the pre-editing fragment in the entire merged difference fragment pair is the starting base of the pre-editing fragment in the first difference fragment pair that is ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the pre-editing fragment in the second difference fragment pair that is ranked later among the two adjacent difference fragment pairs.

[0109] Step 2032: Generate a target difference segment pair sequence based on the difference segment pairs in the initialization difference segment pair sequence and / or the merged difference segment pairs.

[0110] Here, after the difference segment pairs in the initialization difference segment pair sequence are merged, there may be unmerged difference segment pairs and / or merged difference segment pairs in the initialization difference segment pair sequence. In step 2032, a target difference segment pair sequence is generated according to the difference segment pairs and / or merged difference segment pairs in the initialization difference segment pair sequence.

[0111] The target difference fragment pair sequence may include the current pre-editing fragment and the target post-editing fragment in which the post-gene editing reference sequence undergoes gene editing relative to the pre-gene editing reference sequence.

[0112] In some optional implementations, step 204 may be further included after step 203 .

[0113] Step 204: for each target difference fragment pair in the target difference fragment pair sequence, generate a primer pair set corresponding to the target difference fragment pair according to the target difference fragment pair.

[0114] Here, primer pairs of target differential fragment pairs can be generated based on various existing primer design algorithms. For example, primer 3 algorithm can be used to generate primer pairs of target differential fragment pairs.

[0115] Here, the primer is a short DNA fragment, which is mainly used to guide the replication or amplification of the target pre-editing fragment or the target post-editing fragment. It can bind to the complementary region on the target pre-editing fragment or the target post-editing fragment and serve as the starting point of DNA replication.

[0116] Generally, primers are present in the form of a primer pair, which includes an upstream primer and a downstream primer.

[0117] The upstream primer usually binds complementary to the 5' end of the target pre-editing fragment or the target post-editing fragment, and the downstream primer usually binds complementary to the 3' end of the target pre-editing fragment or the target post-editing fragment.

[0118] In some optional embodiments, first, at least one first primer pair can be generated based on the target post-editing fragment in the target difference fragment pair, and at least one second primer pair can be generated based on the target pre-editing fragment in the target difference fragment pair, and then, a primer pair set corresponding to the target difference fragment pair can be generated based on the first primer pair and the second primer pair.

[0119] In some optional embodiments, two first primer pairs can be generated based on the target fragment after editing, and two second primer pairs can be generated based on the target fragment before editing.

[0120] Here, the first primer pair is mainly used to guide the replication or amplification of the target fragment after editing, but cannot be used to guide the replication or amplification of the target fragment before editing. The second primer pair is mainly used to guide the replication or amplification of the target fragment before editing, but cannot be used to guide the replication or amplification of the target fragment after editing.

[0121] In this way, when a subsequent PCR experiment is performed, replication or amplification can be achieved based on whether the first primer pair or the second primer pair can bind to the target post-editing fragment or the target pre-editing fragment to verify whether the reference sequence before gene editing has been successfully modified.

[0122] In some optional embodiments, the standard reference primer length of each first primer pair and second primer pair may also be obtained, wherein the standard reference length is the length of each first primer pair or second primer pair actually output by the algorithm.

[0123] Here, at least one first primer pair can be generated according to a preset primer length and a target edited fragment, and a standard reference primer length of each first primer pair can be obtained.

[0124] Here, the preset primer length may be the desired primer length input by the user. Generally, there is a certain length difference between the preset primer length input by the user and the standard reference primer length.

[0125] For example, the preset primer length may be 500, and the standard reference primer length may be 550, which means that the length of the primer expected by the user may be 500 bases, while the length of the primer actually output by the algorithm may be 550 bases.

[0126] In some optional embodiments, each target post-editing fragment, each target pre-editing fragment, each first primer pair, each second primer pair, and the standard reference length of each first primer and the standard reference length of each second primer pair can also be sent to the user's terminal device, so that the user can directly select the desired target post-editing fragment, target pre-editing fragment, first primer pair and second primer pair through the user terminal for PCR experiment.

[0127] In some optional implementations, the user may also set the length of the DNA fragment to be replicated or amplified through the terminal device.

[0128] The primer design method provided by the embodiment of the present disclosure first obtains a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing, and then generates an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing, wherein the difference fragment pair includes the pre-editing fragment and the post-editing fragment in which the reference sequence after gene editing undergoes gene editing relative to the reference sequence before gene editing, and the difference fragment pair sequence is composed of at least one difference fragment pair arranged in order from front to back according to the starting base site of the pre-editing fragment in the difference fragment pair, and finally, generates a target difference fragment pair sequence according to the initial difference fragment pair sequence and the preset difference fragment pair merging condition. In this way, the post-editing fragment in which gene editing occurs in the post-editing reference sequence can be automatically found through the reference sequence before gene editing and the reference sequence after gene editing to obtain the difference between the reference sequence before gene editing and the reference sequence before gene editing, and PCR primers can be automatically designed through the target difference fragment pair, without the need to manually design PCR primers through experiments, thereby improving the efficiency of PCR primer design and the accuracy of PCR primer design, and improving the reliability of experimental verification results.

[0129] Further references Figure 5 As an implementation of the methods shown in the above figures, the present disclosure provides an embodiment of a primer design device, which is similar to Figure 2 Corresponding to the method embodiment shown, the apparatus can be specifically applied to various terminal devices.

[0130] like Figure 5 As shown, the primer design device of this embodiment, the device 500 includes: a sequence acquisition unit 501, an initial difference fragment pair generation unit 502 and a target difference fragment pair generation unit 503. Among them, the sequence acquisition unit 501 is used to obtain a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing; the initial difference fragment pair generation unit 502 is used to generate an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing, wherein the difference fragment pair includes a pre-editing fragment and a post-editing fragment of the reference sequence after gene editing relative to the reference sequence before gene editing, and the difference fragment pair sequence is composed of at least one difference fragment pair arranged in order from front to back according to the starting base sites of the pre-editing fragment in the difference fragment pair; the target difference fragment pair generation unit 503 is used to generate a target difference fragment pair sequence according to the initial difference fragment pair sequence and a preset difference fragment pair merging condition.

[0131] In this embodiment, the specific processing of the sequence acquisition unit 501, the initial difference segment pair generation unit 502 and the target difference segment pair generation unit 503 and the technical effects thereof can be referred to in Figure 2The relevant descriptions of step 201 to step 204 in the corresponding embodiment are not repeated here.

[0132] In some optional implementations, the initial difference segment pair generating unit 502 may be further configured as follows:

[0133] Comparing the reference sequence before gene editing and the reference sequence after gene editing to obtain matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing;

[0134] Obtaining non-matching base pairs of a reference sequence before gene editing and a reference sequence after gene editing according to the matching base pairs;

[0135] Obtaining a post-edited segment of the reference sequence after gene editing and a pre-edited segment corresponding to the post-edited segment in the reference sequence before gene editing according to the non-matching base pairs;

[0136] An initial differential fragment pair sequence is generated according to the edited fragment of the reference sequence after gene editing and the pre-edited fragment corresponding to the edited fragment in the reference sequence before gene editing.

[0137] In some optional implementations, the target difference segment pair generation unit 503 may be further configured as follows:

[0138] For any two adjacent difference segment pairs in the initialized difference segment pair sequence, in response to determining that the two adjacent difference segment pairs satisfy a preset difference segment pair merging condition, merge the two adjacent difference segment pairs to generate a merged difference segment pair, and replace the two adjacent difference segment pairs in the initial difference segment pair sequence with the merged difference segment pair, until no two adjacent difference segment pairs satisfying the preset difference segment pair merging condition exist in the initial difference segment pair sequence;

[0139] A target difference segment pair sequence is generated based on the difference segment pairs in the initialization difference segment pair sequence and / or the merged difference segment pairs.

[0140] In some optional implementations, the target difference segment pair generation unit 503 may be further configured as follows:

[0141] In response to the number of bases between the edited segment in the first difference segment pair ranked higher and the edited segment in the second difference segment pair ranked lower in the two adjacent difference segment pairs being within a preset matching base number range, it is determined that the two adjacent difference segment pairs meet the pre-difference segment pair merging condition.

[0142] In some optional implementations, the target difference segment pair generation unit 503 may be further configured as follows:

[0143] The starting base of the edited fragment in the merged difference fragment pair is the starting base of the edited fragment in the first difference fragment pair ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the edited fragment in the second difference fragment pair ranked later among the two adjacent difference fragment pairs;

[0144] The starting base of the pre-editing fragment in the merged difference fragment pair is the starting base of the pre-editing fragment in the first difference fragment pair ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the pre-editing fragment in the second difference fragment pair ranked later among the two adjacent difference fragment pairs.

[0145] In some optional embodiments, the method further comprises:

[0146] The primer generating unit 504 is used to generate a primer pair set corresponding to each target difference fragment pair in the target difference fragment pair sequence according to the target difference fragment pair.

[0147] In some optional implementations, the primer generation unit 504 may be further configured as follows:

[0148] generating at least one first primer pair based on the target edited fragment in the target differential fragment pair;

[0149] generating at least one second primer pair based on the target pre-editing fragment in the target differential fragment pair;

[0150] A primer pair set corresponding to the target differential fragment pair is generated based on the first primer pair and the second primer pair.

[0151] Reference below Figure 6 , which shows a schematic diagram of the structure of a computer system 600 of a terminal device suitable for implementing the present disclosure. Figure 6 The computer system 600 shown is only an example and should not bring any limitation to the functions and scope of use of the embodiments of the present disclosure.

[0152] like Figure 6 As shown, the computer system 600 may include a processing device (e.g., a central processing unit, a graphics processing unit, etc.) 601, which can perform various appropriate actions and processes according to a program stored in a read-only memory (ROM) 602 or a program loaded from a storage device 608 to a random access memory (RAM) 603. Various programs and data required for the operation of the computer system 600 are also stored in the RAM 603. The processing device 601, the ROM 602, and the RAM 603 are connected to each other via a bus 604. An input / output (I / O) interface 605 is also connected to the bus 604.

[0153] Typically, the following devices may be connected to the I / O interface 605: input devices 606 including, for example, a touch screen, a touch pad, a keyboard, a mouse, a camera, a microphone, etc.; output devices 607 including, for example, a liquid crystal display (LCD), a speaker, a vibrator, etc.; storage devices 608 including, for example, a magnetic tape, a hard disk, etc.; and communication devices 609. The communication devices 609 may allow the computer system 600 to communicate with other devices wirelessly or by wire to exchange data. Although Figure 6 The computer system 600 of an electronic device having various devices is shown, but it should be understood that it is not required to implement or possess all the devices shown. More or fewer devices may be implemented or possessed instead.

[0154] In particular, according to an embodiment of the present disclosure, the process described above with reference to the flowchart can be implemented as a computer software program. For example, an embodiment of the present disclosure includes a computer program product, which includes a computer program carried on a computer-readable medium, and the computer program contains program code for executing the method shown in the flowchart. In such an embodiment, the computer program can be downloaded and installed from a network through a communication device 609, or installed from a storage device 608, or installed from a ROM 602. When the computer program is executed by the processing device 601, the above-mentioned functions defined in the method of the embodiment of the present disclosure are executed.

[0155] It should be noted that the computer-readable medium disclosed above may be a computer-readable signal medium or a computer-readable storage medium or any combination of the above two. The computer-readable storage medium may be, for example, but not limited to, an electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, device or device, or any combination of the above. More specific examples of computer-readable storage media may include, but are not limited to: an electrical connection with one or more wires, a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disk read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the above. In the present disclosure, a computer-readable storage medium may be any tangible medium containing or storing a program that may be used by or in combination with an instruction execution system, device or device. In the present disclosure, a computer-readable signal medium may include a data signal propagated in a baseband or as part of a carrier wave, in which a computer-readable program code is carried. This propagated data signal may take a variety of forms, including but not limited to electromagnetic signals, optical signals, or any suitable combination of the above. The computer readable signal medium may also be any computer readable medium other than a computer readable storage medium, which may send, propagate or transmit a program for use by or in conjunction with an instruction execution system, apparatus or device. The program code contained on the computer readable medium may be transmitted using any suitable medium, including but not limited to: wires, optical cables, RF (radio frequency), etc., or any suitable combination of the above.

[0156] The computer-readable medium may be included in the electronic device, or may exist independently without being incorporated into the electronic device.

[0157] The computer readable medium carries one or more programs. When the one or more programs are executed by the electronic device, the electronic device implements the following Figure 2 The illustrated examples and their alternative embodiments illustrate methods for designing primers.

[0158] Computer program code for performing the operations of the present disclosure may be written in one or more programming languages, or a combination thereof, including object-oriented programming languages, such as Java, Smalltalk, C++, and conventional procedural programming languages, such as "C" or similar programming languages. The program code may be executed entirely on the user's computer, partially on the user's computer, as a separate software package, partially on the user's computer and partially on a remote computer, or entirely on a remote computer or server. In cases involving a remote computer, the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or may be connected to an external computer (e.g., through the Internet using an Internet service provider).

[0159] The flow chart and block diagram in the accompanying drawings illustrate the possible architecture, function and operation of the system, method and computer program product according to various embodiments of the present disclosure. In this regard, each square box in the flow chart or block diagram can represent a module, a program segment or a part of a code, and the module, the program segment or a part of the code contains one or more executable instructions for realizing the specified logical function. It should also be noted that in some implementations as replacements, the functions marked in the square box can also occur in a sequence different from that marked in the accompanying drawings. For example, two square boxes represented in succession can actually be executed substantially in parallel, and they can sometimes be executed in the opposite order, depending on the functions involved. It should also be noted that each square box in the block diagram and / or flow chart, and the combination of the square boxes in the block diagram and / or flow chart can be implemented with a dedicated hardware-based system that performs a specified function or operation, or can be implemented with a combination of dedicated hardware and computer instructions.

[0160] The units involved in the embodiments described in the present disclosure may be implemented by software or hardware. The name of the unit does not limit the unit itself in some cases. For example, the sequence acquisition unit may also be described as a "unit for a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing".

[0161] The above description is only a preferred embodiment of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of disclosure involved in the present disclosure is not limited to the technical solutions formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above disclosed concept. For example, the above features are replaced with the technical features with similar functions disclosed in the present disclosure (but not limited to) by each other to form a technical solution.

Claims

1. A primer design method, characterized in that: The method comprises: Obtaining a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing; Generate an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing, wherein the difference fragment pair includes a pre-edited fragment and a post-edited fragment of the reference sequence after gene editing relative to the reference sequence before gene editing, and the difference fragment pair sequence is composed of at least one difference fragment pair arranged in order from front to back according to the starting base sites of the pre-edited fragment in the difference fragment pair; generating a target difference segment pair sequence according to the initial difference segment pair sequence and the preset difference segment pair merging condition, wherein, for any two adjacent difference segment pairs in the initial difference segment pair sequence, in response to the number of bases between the edited segment in a first difference segment pair ranked higher and the edited segment in a second difference segment pair ranked lower among the two adjacent difference segment pairs being within a preset matching base number range, determining that the two adjacent difference segment pairs meet the preset difference segment pair merging condition; The method further comprises: For each target difference fragment pair in the target difference fragment pair sequence, a primer pair set corresponding to the target difference fragment pair is generated according to the target difference fragment pair.

2. The method according to claim 1, characterized in that The step of generating an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing comprises: Comparing the reference sequence before gene editing and the reference sequence after gene editing to obtain matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing; Obtaining non-matching base pairs of the reference sequence before gene editing and the reference sequence after gene editing according to the matching base pairs; Obtaining, according to the non-matching base pairs, a post-edited segment of the reference sequence after gene editing and a pre-edited segment corresponding to the post-edited segment in the reference sequence before gene editing; The initial difference fragment pair sequence is generated according to the edited fragment of the reference sequence after gene editing and the pre-edited fragment corresponding to the edited fragment in the reference sequence before gene editing.

3. The method according to claim 1, characterized in that The step of generating a target difference segment pair sequence according to the initial difference segment pair sequence and a preset difference segment pair merging condition includes: For any two adjacent difference segment pairs in the initialized difference segment pair sequence, in response to determining that the two adjacent difference segment pairs satisfy a preset difference segment pair merging condition, merge the two adjacent difference segment pairs to generate a merged difference segment pair, and replace the two adjacent difference segment pairs in the initial difference segment pair sequence with the merged difference segment pair, until no two adjacent difference segment pairs satisfying the preset difference segment pair merging condition exist in the initial difference segment pair sequence; A target difference segment pair sequence is generated based on the difference segment pairs and / or the merged difference segment pairs in the initialization difference segment pair sequence.

4. The method according to claim 3, characterized in that The two adjacent difference segment pairs are merged to generate a merged difference segment pair, including: The starting base of the edited fragment in the merged difference fragment pair is the starting base of the edited fragment in the first difference fragment pair ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the edited fragment in the second difference fragment pair ranked later among the two adjacent difference fragment pairs; The starting base of the pre-editing fragment in the merged difference fragment pair is the starting base of the pre-editing fragment in the first difference fragment pair ranked earlier among the two adjacent difference fragment pairs, and the ending base is the ending base of the pre-editing fragment in the second difference fragment pair ranked later among the two adjacent difference fragment pairs.

5. The method according to claim 1, characterized in that The step of generating a primer pair set corresponding to the target difference fragment pair according to the target difference fragment pair comprises: generating at least one first primer pair based on the target edited fragment in the target differential fragment pair; generating at least one second primer pair based on the target pre-editing fragment in the target differential fragment pair; A primer pair set corresponding to the target differential fragment pair is generated based on the first primer pair and the second primer pair.

6. A primer design device comprising: A sequence acquisition unit, used to acquire a reference sequence before gene editing and a reference sequence after gene editing corresponding to the reference sequence before gene editing; an initial difference fragment pair generating unit, configured to generate an initial difference fragment pair sequence according to the reference sequence before gene editing and the reference sequence after gene editing, wherein the difference fragment pair comprises a pre-editing fragment and a post-editing fragment of the reference sequence after gene editing relative to the reference sequence before gene editing, and the difference fragment pair sequence is formed by arranging at least one difference fragment pair in the order of the starting base sites of the pre-editing fragment in the difference fragment pair from front to back; a target difference segment pair generation unit, configured to generate a target difference segment pair sequence according to the initial difference segment pair sequence and a preset difference segment pair merging condition, wherein, for any two adjacent difference segment pairs in the initial difference segment pair sequence, in response to the number of bases between the edited segment in a first difference segment pair ranked higher and the edited segment in a second difference segment pair ranked lower among the two adjacent difference segment pairs being within a preset matching base number range, it is determined that the two adjacent difference segment pairs meet the preset difference segment pair merging condition; The primer generating unit is used for generating a primer pair set corresponding to each target difference fragment pair in the target difference fragment pair sequence according to the target difference fragment pair.

7. An electronic device comprising: one or more processors; A storage device for storing one or more programs; When the one or more programs are executed by the one or more processors, the one or more processors Implement the method as claimed in any one of claims 1 to 5.

8. A computer readable medium having a computer program stored thereon, wherein: When the program is executed by a processor, the method according to any one of claims 1 to 5 is implemented.

9. A computer program product, comprising a computer program / instruction, wherein when the computer program / instruction is executed by a processor, the method according to any one of claims 1 to 5 is implemented.

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