Amplicon sequence demultiplexing method, device, electronic device and storage medium

Abstract

The embodiments of the present disclosure provide an amplicon sequence demultiplexing method, device, electronic device, and storage medium, the method comprising: obtaining a sample identifier of each sample included in a target mixed sample and a corresponding first forward identification label and a first reverse identification label, obtaining a set of sequencing sequences to be resolved, wherein the set of sequencing sequences to be resolved is generated based on sequencing results obtained by multiplexing the mixed sample to be sequenced, for each sequencing sequence to be resolved, respectively resolving the sequencing sequence to be resolved according to preset forward identification label position information and preset reverse identification label position information, obtaining a corresponding second forward identification label and a second reverse identification label, and determining the sample identifier corresponding to the sequencing sequence to be resolved according to the second forward identification label and the second reverse identification label. The sequencing of amplicon sequences in multiple experimental plates is achieved simultaneously, while reducing the waste of sequencing data.

Description

Technical Field

[0001] Embodiments of the present disclosure relate to the technical field of bioinformatics analysis, and in particular to an amplicon sequence demultiplexing method, device, electronic device, and storage medium. Background Art

[0002] High-throughput sequencing technology has become an important tool for biological research because it can generate a large amount of genetic information in a short period of time. In high-throughput sequencing technology, multiplexing technology allows multiple sequencing samples to be processed simultaneously in the same sequencing. This technology is achieved by assigning a unique DNA barcode to each sequencing sample, so that different samples can be distinguished after mixing. After sequencing is completed, the sequencing data of the mixed sequencing samples are usually separated according to their respective barcodes through demultiplexing technology.

[0003] However, existing sequencing technologies usually support no more than 96 mixed sequencing samples (a single 96-well experimental plate) in the same sequencing run, which to a certain extent limits the further improvement of sequencing throughput. In addition, since the base length of sequencing samples is relatively short, the sequencing data required to analyze each sequencing sample is also relatively small. When the total amount of sequencing data produced by the sequencer each time is fixed, a large amount of sequencing data may be wasted in the sequencing experiment.

[0004] Therefore, it is necessary to propose an amplicon sequence demultiplexing method to solve at least one of the above technical problems. Summary of the invention

[0005] The embodiments of the present disclosure provide an amplicon sequence demultiplexing method, device, electronic device and storage medium.

[0006] In a first aspect, the present disclosure provides a method for demultiplexing an amplicon sequence, comprising:

[0007] Obtaining a sample identifier and a corresponding first forward identification label and a first reverse identification label of each sample included in a target mixed sample, wherein the target mixed sample is a mixture of at least two samples;

[0008] A set of sequencing sequences to be analyzed is obtained, wherein the set of sequencing sequences to be analyzed is generated based on sequencing results obtained by performing multiplex sequencing on the mixed samples to be sequenced, and before the multiplex sequencing, a first forward identification label and a first reverse identification label corresponding to a sample identifier of the sample are respectively added to the front end and the rear end of the amplicon sequence of each of the samples; for each sequencing sequence to be analyzed, the sequencing sequence to be analyzed is analyzed according to preset forward identification label position information and preset reverse identification label position information to obtain corresponding second forward identification label and second reverse identification label, and the sample identifier corresponding to the sequencing sequence to be analyzed is determined according to the second forward identification label and the second reverse identification label.

[0009] In some optional embodiments, determining the sample identifier corresponding to the sequencing sequence to be parsed according to the second forward identification tag and the second reverse identification tag includes:

[0010] The first forward identification tag and the first reverse identification tag in the mixed sample to be sequenced are respectively the sample identifiers of the second forward identification tag and the second reverse identification tag, and are determined as the sample identifier corresponding to the sequencing sequence to be analyzed.

[0011] In some optional embodiments, the first positive identification label and the first reverse identification label corresponding to the sample identification of each sample are used to characterize the experimental plate where the sample is located and the well position in the experimental plate, respectively.

[0012] In some optional embodiments, each sample in the mixed sample to be sequenced is set in a different well position in at least one experimental plate, and the front end and the back end of the amplicon sequence of each sample are respectively bound to the first forward identification label and the first reverse identification label corresponding to the sample through a PCR experiment.

[0013] In some optional embodiments, the step of parsing the sequencing sequence to be parsed according to the preset forward identification tag position information and the preset reverse identification tag position information to obtain the corresponding second forward identification tag and the second reverse identification tag comprises:

[0014] Parsing a first preset forward length from the front end of the sequencing sequence to be parsed to obtain a corresponding second forward identification tag;

[0015] A second preset forward length is parsed from the rear end of the sequencing sequence to be parsed to obtain a corresponding second reverse identification tag.

[0016] In a second aspect, the present disclosure provides an amplicon sequence demultiplexing device, comprising:

[0017] A target mixed sample acquisition unit, used to acquire a sample identifier of each sample included in the target mixed sample and a corresponding first positive identification label and a first reverse identification label, wherein the target mixed sample is a mixture of at least two samples;

[0018] The unit for acquiring sequencing sequences to be analyzed is used to acquire a set of sequencing sequences to be analyzed, wherein the set of sequencing sequences to be analyzed is generated based on sequencing results obtained by performing multiplex sequencing on the mixed samples to be sequenced, and before the multiplex sequencing, a first forward identification label and a first reverse identification label corresponding to the sample identifier of the sample are respectively added to the front end and the rear end of the amplicon sequence of each of the samples; for each sequencing sequence to be analyzed, the sequencing sequence to be analyzed is analyzed according to preset forward identification label position information and preset reverse identification label position information to obtain corresponding second forward identification label and second reverse identification label, and the sample identifier corresponding to the sequencing sequence to be analyzed is determined according to the second forward identification label and the second reverse identification label.

[0019] In a third aspect, the present disclosure provides an electronic device, including:

[0020] one or more processors;

[0021] a storage device having one or more programs stored thereon,

[0022] 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.

[0023] 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.

[0024] 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.

[0025] The embodiment of the present disclosure provides an amplicon sequence demultiplexing method, first, obtaining a sample identifier and a corresponding first forward identification label and a first reverse identification label of each sample included in a target mixed sample, wherein the target mixed sample is mixed with at least two samples, and then obtaining a set of sequencing sequences to be analyzed, wherein the set of sequencing sequences to be analyzed is generated based on sequencing results obtained by performing multiplex sequencing on the mixed sample to be sequenced, and before the multiplex sequencing, the first forward identification label and the first reverse identification label corresponding to the sample identifier of the sample are added to the front end and the back end of the amplicon sequence of each sample, respectively; for each sequencing sequence to be analyzed, the sequencing sequence to be analyzed is analyzed according to preset forward identification label position information and preset reverse identification label position information, respectively, to obtain corresponding second forward identification labels and second reverse identification labels, and the sample identifier corresponding to the sequencing sequence to be analyzed is determined according to the second forward identification label and the second reverse identification label. In this way, since the sample identifier added to each sample corresponds to the first forward identification label and the first reverse identification label, after each sequencing sequence to be analyzed is analyzed, the sample identifier corresponding to the sequencing sequence to be analyzed can be determined according to the second forward identification label and the second reverse identification label of the analyzed sequencing sequence to be analyzed, thereby realizing the simultaneous sequencing of amplicon sequences in multiple experimental plates, improving the sequencing throughput, and reducing the waste of sequencing data when the total amount of sequencing data is constant. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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:

[0027] Figure 1 is a system architecture diagram of an embodiment to which the amplicon sequence demultiplexing method disclosed herein can be applied;

[0028] Figure 2 is a flow chart of an embodiment of the amplicon sequence demultiplexing method according to the present disclosure;

[0029] Figure 3 It is a structural schematic diagram of an embodiment of an amplicon sequence demultiplexing method and device according to the present disclosure;

[0030] Figure 4 It is a structural diagram of a computer system of an electronic device suitable for implementing an embodiment of the present disclosure. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] Figure 1 An exemplary system architecture 100 is shown to which embodiments of the amplicon sequence demultiplexing method, apparatus, electronic device, and storage medium of the present disclosure may be applied.

[0034] 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.

[0035] 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 amplicon sequence demultiplexing 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.

[0036] 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.

[0037] The server 105 may be a server that provides various services, such as a background server that processes the sample identification of each sample included in the target mixed sample obtained on the terminal devices 101, 102, and 103 and the corresponding first forward identification tag and first reverse identification tag. The background server may perform corresponding processing based on the sample identification of each sample included in the target mixed sample of the terminal device and the corresponding first forward identification tag and first reverse identification tag.

[0038] In some cases, the amplicon sequence demultiplexing 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 the sample identification of each sample included in the target mixed sample and the corresponding first forward identification label and first reverse identification label" can be performed by the terminal devices 101, 102, 103, and the step of "obtaining the set of sequencing sequences to be parsed" can be performed by the server 105. The present disclosure does not limit this. Accordingly, the amplicon sequence demultiplexing method device can also be respectively set in the terminal devices 101, 102, 103 and the server 105.

[0039] In some cases, the amplicon sequence demultiplexing method provided in the present disclosure may be executed by the server 105 , and accordingly, the amplicon sequence demultiplexing method 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 .

[0040] In some cases, the amplicon sequence demultiplexing method provided in the present disclosure may be executed by the terminal devices 101 , 102 , 103 , and accordingly, the amplicon sequence demultiplexing 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 .

[0041] 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.

[0042] 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.

[0043] Continue to refer Figure 2 , Figure 2 A flowchart 200 of an embodiment of an amplicon sequence demultiplexing method according to the present disclosure is shown. Figure 2 The amplicon sequence demultiplexing method shown can be applied to Figure 1 The process 200 includes the following steps:

[0044] Step 201: Obtain a sample identifier of each sample included in a target mixed sample and a corresponding first forward identification label and first reverse identification label.

[0045] In this embodiment, the target mixed sample is a mixture of at least two samples.

[0046] The target mixed sample may refer to an amplicon sequence that needs to be sequenced, wherein each sample needs to be placed in a sequencing chip for sequencing before conducting a sequencing experiment. In order to confirm the identity of each sample, that is, to determine the amplicon sequence of which well of which experimental plate the sample is located in before sequencing, a first forward identification label and a first reverse identification label may be added to each sample.

[0047] Here, the sample identifier is used to determine the identity of the sample, that is, which amplicon sequence the sample belongs to. Each sample identifier corresponds to a first forward identification label and a first reverse identification label.

[0048] In some optional embodiments, the first positive identification label and the first reverse identification label corresponding to the sample identification of each sample are used to characterize the experimental plate where the sample is located and the well position in the experimental plate, respectively.

[0049] It should be noted that before conducting the sequencing experiment on the machine, each sample exists in each well position in at least one experimental plate, and each sample is arranged in the order of the well positions in each experimental plate. Here, the sample identifier can be used to identify the experimental plate where the sample is located and the corresponding well position. In this way, the first forward identification label and the first reverse identification label can be used to determine the experimental plate and the corresponding well position where the corresponding sample is located before the sequencing experiment to determine the identity of the sample.

[0050] In some optional embodiments, the first forward identification tag may refer to a forward barcode sequence, and the first reverse identification tag may refer to a reverse barcode sequence.

[0051] In high-throughput sequencing, barcode generally refers to a short, unique DNA sequence used to distinguish and identify different samples or molecules. In this embodiment, barcode can be used to distinguish and identify amplicon sequences.

[0052] Among them, the forward barcode is usually attached to the 5' end or upstream end of the amplicon sequence, and the reverse barcode is usually attached to the 3' end or downstream end of the amplicon sequence.

[0053] In some optional embodiments, each sample in the mixed sample to be sequenced is set in a different well position in at least one experimental plate, and the front end and the back end of the amplicon sequence of each sample are respectively bound to the first forward identification label and the first reverse identification label corresponding to the sample through a PCR (Polymerase Chain Reaction Experiment).

[0054] Here, the front end of the amplicon sequence may refer to the 5' end of the amplicon sequence, and the back end of the amplicon sequence may refer to the 3' end of the amplicon sequence.

[0055] Specifically, the first forward identification tag of each sample can be bound to the 5' end of the amplicon sequence through a PCR experiment, and the first reverse identification tag of each sample can be bound to the 3' end of the amplicon sequence through a PCR experiment.

[0056] In some optional embodiments, the 5' end and 3' end of each amplicon sequence to be tested have the same DNA fragment, that is, the 5' end of each amplicon sequence to be tested has an identical DNA fragment, and the 3' end of each amplicon sequence to be tested has an identical DNA fragment. Therefore, a pair of universal binding sites can be designed based on the two identical DNA fragments, wherein the forward binding site is bound to the 5' end of the amplicon sequence, and the reverse binding site is bound to the 3' end of the amplicon sequence.

[0057] Here, the forward and reverse binding sites can be used to connect the first forward recognition tag, the first reverse recognition tag and the specific DNA sequence region of the amplicon sequence to be tested, ensuring that the first forward recognition tag, the first reverse recognition tag and the amplicon sequence to be tested can be efficiently and specifically connected together by DNA ligase to form a new fusion sequence (i.e., the amplicon sequence to be tested bound to the first forward recognition tag and the first reverse recognition tag).

[0058] Here, the forward and reverse binding sites and the first forward recognition tag and the first reverse recognition tag can be uniformly bound to each amplicon sequence through a PCR experiment.

[0059] The front end of the amplicon sequence is a forward binding site and a first forward recognition tag in sequence, and the rear end of the amplicon sequence is a reverse binding site and a first reverse recognition tag in sequence.

[0060] In order to ensure that the first forward identification tag and the first reverse identification tag can be accurately identified and to avoid incorrect identification of the first forward identification tag and the first reverse identification tag due to other sequences being similar to the first forward identification tag and the first reverse identification tag, in some optional embodiments, the first forward identification tag also includes a first forward filling sequence, and the first reverse identification tag also includes a first reverse filling sequence.

[0061] Among them, the first forward filling sequence is an extended sequence of the first forward identification tag, and the first reverse filling sequence is an extended sequence of the first reverse identification tag. By extending the first forward identification tag and the first reverse identification tag, the recognition error rate of the first forward identification tag and the first reverse identification tag can be reduced, and the real first forward identification tag and the first reverse identification tag can be found.

[0062] Step 202: Obtain a set of sequencing sequences to be parsed.

[0063] Among them, the set of sequencing sequences to be analyzed is generated based on the sequencing results obtained by multiplexing sequencing the mixed samples to be sequenced. Before the multiplexing sequencing, the first forward identification label and the first reverse identification label corresponding to the sample identifier of the sample are added to the front and back ends of the amplicon sequence of each sample respectively; for each sequencing sequence to be analyzed, the sequencing sequence to be analyzed is analyzed according to the preset forward identification label position information and the preset reverse identification label position information to obtain the corresponding second forward identification label and the second reverse identification label, and the sample identifier corresponding to the sequencing sequence to be analyzed is determined according to the second forward identification label and the second reverse identification label.

[0064] Here, the mixed sample to be sequenced may refer to part or all of the samples in the target mixed sample, and the set of sequencing sequences to be resolved generated by the sequencing results obtained by performing multiple sequencing on the mixed sample to be sequenced includes multiple sequencing sequences to be resolved, wherein each sequencing sequence to be resolved may be derived from each mixed sample to be sequenced, and each mixed sample to be sequenced may generate at least one sequencing sequence to be resolved after multiple sequencing, and each sequencing sequence to be resolved may cover the same or similar sequence as the mixed sample to be sequenced.

[0065] For each sequencing sequence to be analyzed, the sequencing sequence to be analyzed is analyzed according to the preset forward identification label position information and the preset reverse identification label position information, respectively, to obtain a second forward identification label and a second reverse identification label corresponding to the sequencing sequence to be analyzed, wherein the second forward identification label can be used to indicate the experimental plate where the sequencing sequence to be analyzed is located, and the second reverse identification label can be used to indicate the well position in the experimental plate where the sequencing sequence to be analyzed is located.

[0066] After the second forward identification tag and the second reverse identification tag of the sequencing sequence to be analyzed are obtained, the sample identifier corresponding to the sequencing sequence to be analyzed can be determined according to the second forward identification tag and the second reverse identification tag.

[0067] In some optional embodiments, the sample identifier of the sample to be sequenced in which the first forward identification label and the first reverse identification label in the mixed sample to be sequenced are respectively the second forward identification label and the second reverse identification label can be determined as the sample identifier corresponding to the sequencing sequence to be analyzed.

[0068] Specifically, the second forward identification label of the sequencing sequence to be analyzed can be matched with the first forward identification label in the mixed sample to be sequenced, and the second reverse identification label can be matched with the first reverse identification label in the mixed sample to be sequenced. If the second forward identification label and the second reverse identification label of the sequencing sequence to be analyzed and the first forward identification label and the first reverse identification label in one of the mixed samples to be sequenced are the same, it means that the sequencing sequence to be analyzed is obtained by sequencing the mixed sample to be sequenced, that is, the sample identifier of the mixed sample to be sequenced is determined as the sample identifier corresponding to the sequencing sequence to be analyzed.

[0069] In some optional embodiments, the sequencing sequence to be analyzed is analyzed according to the preset forward identification tag position information and the preset reverse identification tag position information, respectively, to obtain a second forward identification tag and a second reverse identification tag corresponding to the sequencing sequence to be analyzed. Specifically, the first preset forward length can be analyzed from the front end of the sequencing sequence to be analyzed to obtain the corresponding second forward identification tag, and the second preset forward length can be analyzed from the back end of the sequencing sequence to be analyzed to obtain the corresponding second reverse identification tag.

[0070] Here, the first preset length may be greater than or equal to the total length of the first forward filling sequence, the first forward recognition tag and the forward binding site. The second preset length may be greater than or equal to the total length of the first reverse filling sequence, the first reverse recognition tag and the reverse binding site.

[0071] For example, the sequencing sequence to be analyzed can be analyzed from the front end until the base corresponding to the sequencing sequence to be analyzed with a first preset length is located, and the second positive identification tag corresponding to the sequencing sequence to be analyzed can be obtained. The sequencing sequence to be analyzed can be analyzed from the back end until the base corresponding to the sequencing sequence to be analyzed with a second preset length is located, and the second positive identification tag corresponding to the sequencing sequence to be analyzed can be obtained.

[0072] In some optional implementations, the first positive identification label and the first reverse identification label may be generated in advance by various existing barcode design algorithms.

[0073] For example, if the experimenter needs to sequence 96 (96 experimental plates)*96 (number of experimental plate wells), a total of 9216 amplicon sequences, then 96 first forward identification tags and 96 reverse identification tags can be generated by the barcode design algorithm, wherein each first forward identification tag can be used to identify one experimental plate, and each first reverse identification tag can be used to identify one well position. In this way, 9261 subsequences to be amplified can be identified by 96 first forward identification tags and 96 first reverse identification tags.

[0074] Among them, the base length of each first forward identification label can be the same, the base length of each first reverse identification label can be the same, the base length of the first forward identification label and the length of the first reverse identification label can be different, generally, the base length of the first forward identification label and the first reverse identification label can be between 20 and 30 bases, wherein each first forward identification label and each first reverse identification label needs to ensure a low bit error rate, so that the amplicon sequence can be accurately identified in the subsequent demultiplexing process.

[0075] The embodiment of the present disclosure provides an amplicon sequence demultiplexing method, first, obtaining a sample identifier and a corresponding first forward identification label and a first reverse identification label of each sample included in a target mixed sample, wherein the target mixed sample is mixed with at least two samples, and then obtaining a set of sequencing sequences to be analyzed, wherein the set of sequencing sequences to be analyzed is generated based on sequencing results obtained by performing multiplex sequencing on the mixed sample to be sequenced, and before the multiplex sequencing, the first forward identification label and the first reverse identification label corresponding to the sample identifier of the sample are added to the front end and the back end of the amplicon sequence of each sample, respectively; for each sequencing sequence to be analyzed, the sequencing sequence to be analyzed is analyzed according to preset forward identification label position information and preset reverse identification label position information, respectively, to obtain corresponding second forward identification labels and second reverse identification labels, and the sample identifier corresponding to the sequencing sequence to be analyzed is determined according to the second forward identification label and the second reverse identification label. In this way, since the sample identifier added to each sample corresponds to the first forward identification label and the first reverse identification label, after each sequencing sequence to be analyzed is analyzed, the sample identifier corresponding to the sequencing sequence to be analyzed can be determined according to the second forward identification label and the second reverse identification label of the analyzed sequencing sequence to be analyzed. In this way, in a sequencing experiment, the experimental plate where the amplicon sequence is located and the corresponding well position in the experimental plate can be simultaneously identified through the first forward identification label and the first reverse identification label, and the amplicon sequences in multiple experimental plates can be identified, thereby improving the sequencing throughput. When the total amount of sequencing data is constant, the waste of sequencing data can also be reduced.

[0076] Further references Figure 3 As an implementation of the methods shown in the above figures, the present disclosure provides an embodiment of an amplicon sequencing multiplexing and demultiplexing device, and the device embodiment is similar to Figure 2 Corresponding to the method embodiment shown, the apparatus can be specifically applied to various terminal devices.

[0077] like Figure 3 As shown, an amplicon sequence demultiplexing device of the present embodiment, the device 300 includes: a target mixed sample acquisition unit 301 and a sequencing sequence acquisition unit 302 to be analyzed, wherein the target mixed sample acquisition unit 301 is used to obtain the sample identification of each sample included in the target mixed sample and the corresponding first forward identification label and first reverse identification label, wherein the target mixed sample is mixed with at least two samples; the sequencing sequence acquisition unit 302 to be analyzed is used to obtain a set of sequencing sequences to be analyzed, wherein the set of sequencing sequences to be analyzed is generated based on the sequencing results obtained by multiplexing sequencing the mixed samples to be sequenced, and before the multiplexing sequencing, the first forward identification label and the first reverse identification label corresponding to the sample identification of the sample are added to the front end and the back end of the amplicon sequence of each sample respectively; for each sequencing sequence to be analyzed, the sequencing sequence to be analyzed is analyzed according to the preset forward identification label position information and the preset reverse identification label position information, respectively, to obtain the corresponding second forward identification label and the second reverse identification label, and the sample identification corresponding to the sequencing sequence to be analyzed is determined according to the second forward identification label and the second reverse identification label.

[0078] In this embodiment, the specific processing of the target mixed sample acquisition unit 301 and the to-be-analyzed sequencing sequence acquisition unit 302 and the technical effects thereof can be referred to in Figure 2 The relevant descriptions of step 201 to step 202 in the corresponding embodiment are not repeated here.

[0079] In some optional implementations, the to-be-parsed sequencing sequence obtaining unit 302 may be further configured as follows:

[0080] The first forward identification tag and the first reverse identification tag in the mixed sample to be sequenced are respectively the sample identifiers of the second forward identification tag and the second reverse identification tag, and are determined as the sample identifier corresponding to the sequencing sequence to be analyzed.

[0081] In some optional embodiments, the first positive identification label and the first reverse identification label corresponding to the sample identification of each sample are used to characterize the experimental plate where the sample is located and the well position in the experimental plate, respectively.

[0082] In some optional embodiments, each sample in the mixed sample to be sequenced is set in a different well position in at least one experimental plate, and the front end and the back end of the amplicon sequence of each sample are respectively bound to the first forward identification label and the first reverse identification label corresponding to the sample through a PCR experiment.

[0083] In some optional implementations, the to-be-parsed sequencing sequence obtaining unit 302 may be further configured as follows:

[0084] Parsing a first preset forward length from the front end of the sequencing sequence to be parsed to obtain a corresponding second forward identification tag;

[0085] A second preset forward length is parsed from the rear end of the sequencing sequence to be parsed to obtain a corresponding second reverse identification tag.

[0086] Reference below Figure 4 , which shows a schematic diagram of the structure of a computer system 400 of a terminal device suitable for implementing the present disclosure. Figure 4 The computer system 400 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.

[0087] like Figure 4 As shown, the computer system 400 may include a processing device (e.g., a central processing unit, a graphics processing unit, etc.) 401, which can perform various appropriate actions and processes according to a program stored in a read-only memory (ROM) 402 or a program loaded from a storage device 408 to a random access memory (RAM) 403. Various programs and data required for the operation of the computer system 400 are also stored in the RAM 403. The processing device 401, the ROM 402, and the RAM 403 are connected to each other via a bus 404. An input / output (I / O) interface 405 is also connected to the bus 404.

[0088] Typically, the following devices may be connected to the I / O interface 404: input devices 406 including, for example, a touch screen, a touch pad, a keyboard, a mouse, a camera, a microphone, etc.; output devices 407 including, for example, a liquid crystal display (LCD), a speaker, a vibrator, etc.; storage devices 408 including, for example, a magnetic tape, a hard disk, etc.; and communication devices 409. The communication devices 409 may allow the computer system 400 to communicate with other devices wirelessly or by wire to exchange data. Although Figure 4 The computer system 400 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.

[0089] 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 includes a 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 409, or installed from a storage device 408, or installed from a ROM 402. When the computer program is executed by the processing device 401, the above-mentioned functions defined in the method of the embodiment of the present disclosure are executed.

[0090] 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.

[0091] The computer-readable medium may be included in the electronic device, or may exist independently without being installed in the electronic device.

[0092] 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 2The illustrated embodiment and its alternative implementations illustrate a method for demultiplexing amplicon sequences.

[0093] 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).

[0094] 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.

[0095] The units involved in the embodiments described in the present disclosure may be implemented by software or hardware. The name of a unit does not limit the unit itself in some cases. For example, a target mixed sample acquisition unit may also be described as a "unit for acquiring a target mixed sample".

[0096] 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 method for demultiplexing an amplicon sequence, characterized in that: The method comprises: Obtaining a sample identifier and a corresponding first forward identification label and a first reverse identification label of each sample included in a target mixed sample, wherein the target mixed sample is a mixture of at least two samples; A set of sequencing sequences to be analyzed is obtained, wherein the set of sequencing sequences to be analyzed is generated based on sequencing results obtained by performing multiplex sequencing on a mixed sample to be sequenced, and before the multiplex sequencing, a first forward identification label and a first reverse identification label corresponding to a sample identifier of the sample are respectively added to the front end and the rear end of the amplicon sequence of each of the samples; for each sequencing sequence to be analyzed, the sequencing sequence to be analyzed is analyzed according to preset forward identification label position information and preset reverse identification label position information to obtain corresponding second forward identification label and second reverse identification label, and the sample identifier corresponding to the sequencing sequence to be analyzed is determined according to the second forward identification label and the second reverse identification label.

2. The method according to claim 1, characterized in that The step of determining the sample identifier corresponding to the sequencing sequence to be analyzed according to the second forward identification tag and the second reverse identification tag includes: The second forward identification tag of the sequencing sequence to be analyzed is matched with the first forward identification tag in the mixed sample to be sequenced, and the second reverse identification tag of the sequencing sequence to be analyzed is matched with the first reverse identification tag in the mixed sample to be sequenced. If the second forward identification tag and the second reverse identification tag of the sequencing sequence to be analyzed are the same as the first forward identification tag and the first reverse identification tag of one of the mixed samples to be sequenced, the sample identifier of the mixed sample to be sequenced is determined as the sample identifier corresponding to the sequencing sequence to be analyzed.

3. The method according to claim 1, characterized in that The first positive identification label and the first reverse identification label corresponding to the sample identification of each sample are used to respectively characterize the experimental plate where the sample is located and the well position in the experimental plate.

4. The method according to claim 1, characterized in that Each sample in the mixed sample to be sequenced is set in a different well position in at least one experimental plate, and the front end and the back end of the amplicon sequence of each sample are respectively bound to the first forward identification tag and the first reverse identification tag corresponding to the sample through a PCR experiment.

5. The method according to claim 1, characterized in that The method of parsing the sequencing sequence to be parsed according to the preset forward identification tag position information and the preset reverse identification tag position information to obtain the corresponding second forward identification tag and the second reverse identification tag comprises: Parsing a first preset forward length from the front end of the sequencing sequence to be parsed to obtain a corresponding second forward identification tag; A second preset forward length is parsed from the rear end of the sequencing sequence to be parsed to obtain a corresponding second reverse identification tag.

6. An amplicon sequence demultiplexing device, comprising: A target mixed sample acquisition unit, used to acquire a sample identifier of each sample included in the target mixed sample and a corresponding first positive identification label and a first reverse identification label, wherein the target mixed sample is a mixture of at least two samples; The unit for acquiring sequencing sequences to be analyzed is used to acquire a set of sequencing sequences to be analyzed, wherein the set of sequencing sequences to be analyzed is generated based on sequencing results obtained by performing multiplex sequencing on a mixed sample to be sequenced, and before the multiplex sequencing, a first forward identification label and a first reverse identification label corresponding to a sample identifier of the sample are respectively added to the front end and the rear end of the amplicon sequence of each sample; for each sequencing sequence to be analyzed, the sequencing sequence to be analyzed is analyzed according to preset forward identification label position information and preset reverse identification label position information to obtain corresponding second forward identification label and second reverse identification label, and the sample identifier corresponding to the sequencing sequence to be analyzed is determined according to the second forward identification label and the second reverse identification label.

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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