A method for analyzing gene expression of plant-pathogen interaction based on umi tag

Abstract

The present application relates to the field of biotechnology and molecular detection, in particular to a plant and pathogenic bacteria interaction gene expression analysis method based on UMI tag, comprising: obtaining interaction sample total nucleic acid extract; and constructing a molecular hybridization system with host-specific chimeric blocking probes containing locked nucleic acid modification and 3' end dideoxy modification, and reverse transcription primers; through temperature rising and denaturation and temperature reduction and annealing, the blocking probes are combined with host high abundance nucleic acid to form a hybridization blocking complex, and the primers are combined with pathogenic bacteria nucleic acid; template switching oligonucleotides with unique molecular identifier sequences and reverse transcriptase are added, the host extension is blocked, and UMI is introduced into the pathogenic bacteria complementary chain, and an interaction expression library is generated; after sequencing, the pathogenic bacteria effective reads and the host invalid reads are counted, and the effective interaction signal signal-to-noise ratio is output; the present application can preferentially capture pathogenic bacteria transcripts in the high background of host RNA.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and molecular detection, specifically to a method for analyzing the expression of plant-pathogen interaction genes based on UMI tags. Background Technology

[0002] In the analysis of gene expression in plant-pathogen interactions, how to accurately capture pathogen-derived expression signals under high host background conditions has always been a key issue in molecular detection of interactions and early disease monitoring. Currently, when performing transcriptional expression analysis on plant-pathogen interaction samples, the common approach is to directly construct libraries and sequence total nucleic acid or total RNA. If the abundance of pathogen nucleic acid in the sample is low, it is often necessary to increase the sequencing depth or perform subsequent bioinformatics screening to obtain pathogen-related reads. However, when analyzing interacting samples using the above methods, the proportion of ribosomal nucleic acids and other high-abundance transcripts in the plant host is higher than the preset proportion. This causes them to preferentially consume reaction resources during reverse transcription, library construction, and sequencing, resulting in insufficient capture of low-abundance transcripts from pathogens. This leads to problems such as the proportion of effective reads from pathogens not reaching the detection limit and signals being masked by the host background. At the same time, the lack of single-molecule identifiers during conventional library construction makes it difficult to eliminate the influence of amplification bias on quantitative results, and it is difficult to stably count the true number of pathogen molecules, resulting in low quantitative accuracy. Furthermore, existing methods typically lack a unified process judgment mechanism in terms of host background suppression effectiveness, library construction integrity, and subsequent signal separation quality. This makes it difficult to identify abnormal sources in a timely manner in cases of sample degradation, insufficient background suppression, or label collisions, resulting in insufficient interpretability of library quality and poor consistency of results. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a method for analyzing the expression of plant-pathogen interaction genes based on UMI tags. Specifically, the technical solution of this invention includes: Obtain total nucleic acid extracts from plant-pathogen interaction samples; The extract, host-specific chimeric blocking probe, and reverse transcription primers were mixed to construct a molecular hybridization reaction system. The probe contained locked nucleic acid modified structure and 3' dideoxynucleotide modified structure. The system was subjected to heating to break down the molecules and cooling to anneal them, so that the probe could bind to the host's abundant nucleic acid molecules to form a hybridization blocking complex, and the reverse transcription primers could bind to the pathogen's nucleic acid molecules. The template-switching oligonucleotide with a unique molecular identifier (UMI) sequence and reverse transcriptase were added to the system; the reverse transcriptase had terminal transferase activity and template switching activity; the 3' end of the template-switching oligonucleotide contained a nucleotide sequence segment that paired with a non-template polycytosine tail. The reverse transcriptase is used to initiate an enzymatic polymerization and extension reaction, which causes the reverse transcriptase to extend along the pathogen nucleic acid molecule and add a non-template polycytosine tail to the end of its complementary strand. Based on the steric hindrance of the complex, the polymerization and extension of the host high-abundance nucleic acid molecule is blocked. Based on template-converting oligonucleotides, a unique molecular identifier sequence is introduced into the complementary strand of the pathogen nucleic acid molecule to generate an interaction expression library. High-throughput sequencing was performed on the library to obtain a set of sequencing reads; Extract the number of valid reads that carry unique molecular identifier sequences and belong to pathogenic nucleic acid molecules from this set, and the number of invalid reads that belong to high-abundance host nucleic acid molecules; When the number of valid reads is greater than zero and the number of invalid reads is greater than zero, the effective interaction signal-to-noise ratio is calculated by dividing the number of valid reads by the number of invalid reads. When the number of valid reads is greater than zero and the number of invalid reads is equal to zero, the maximum value of the preset signal-to-noise ratio representing the background below the sequencing detection limit is output as the effective interaction signal-to-noise ratio. When the number of valid reads is equal to zero, the effective interaction signal-to-noise ratio is zero.

[0004] Optionally, before the step of mixing the extract, the host-specific chimeric blocking probe, and the reverse transcription primer to construct the molecular hybridization reaction system, the method further includes: Obtain high-abundance nucleic acid sequences from the host; Design complementary nucleic acid sequences for the host with high abundance of nucleic acid sequences; The ribosomal loop of the complementary nucleic acid sequence is subjected to a methylene bridging locking operation to generate the locked nucleic acid modified structure; The 3' end of the complementary nucleic acid sequence is dehydroxylated to generate the 3' dideoxynucleotide modified structure, thus obtaining the host-specific chimeric blocking probe.

[0005] Optionally, the steps of heating to dechain and cooling to anneal the system specifically include: Based on the base sequence, nucleotide length, and locked nucleic acid modification structure of the host-specific chimeric blocking probe, the probe annealing temperature is calculated. The primer annealing temperature is calculated based on the nucleotide composition of the reverse transcription primer, wherein the probe annealing temperature is greater than the primer annealing temperature; The system is controlled to cool down to the probe annealing temperature after the heating and de-milking process, thereby promoting the binding of the probe to the host high-abundance nucleic acid molecules; The system is further cooled to the primer annealing temperature to induce the reverse transcription primer to bind to the pathogen nucleic acid molecule.

[0006] Optionally, the step of using reverse transcriptase to initiate the enzymatic polymerization and elongation reaction, and blocking the polymerization and elongation of high-abundance host nucleic acid molecules based on the steric hindrance of the complex, specifically includes: In the enzyme-catalyzed polymerization and extension reaction, the reverse transcriptase polymerizes and extends along the pathogenic nucleic acid molecule to generate a complementary strand of the pathogenic nucleic acid molecule; When the reverse transcriptase polymerizes and extends along the host's abundant nucleic acid molecule and encounters the hybridization blocking complex, the polymerization and extension of the reverse transcriptase is terminated and it detaches from the template due to the high affinity steric hindrance generated by the locked nucleic acid modification structure and the dehydroxylation chain-breaking effect caused by the 3' dideoxynucleotide modification structure, thereby blocking the reverse transcription process of the host's abundant nucleic acid molecule.

[0007] Optionally, the introduction of a unique molecular identifier sequence into the complementary strand of the pathogenic nucleic acid molecule based on template-conversion oligonucleotides specifically includes: In the enzymatic polymerization extension reaction, the reverse transcriptase extends along the pathogen nucleic acid molecule to the 5' end of the pathogen nucleic acid molecule and adds a non-template polycytosine tail to the end of the complementary strand of the pathogen nucleic acid molecule; The template-converting oligonucleotide is used to pair and bind with the non-template polycytosine tail; The reverse transcriptase is guided to perform a template switching operation, transcribing the unique molecular identifier sequence into the complementary strand of the pathogen's nucleic acid molecule.

[0008] Optionally, after the step of generating the interaction expression library, the method further includes: The fragment distribution map of the total nucleic acid extract was obtained by a nucleic acid electrophoresis analysis platform, and the nucleic acid integrity index was used as a unified evaluation criterion; the first nucleic acid integrity value of the total nucleic acid extract before constructing the molecular hybridization reaction system was extracted. Extract the second nucleic acid integrity value of the library nucleic acid molecules after generating the interaction expression library; Compare the integrity values ​​of the first nucleic acid and the second nucleic acid; When the difference between the first nucleic acid integrity value and the second nucleic acid integrity value is less than or equal to a preset degradation threshold, it is determined that the pathogenic nucleic acid molecule has not undergone non-specific physical degradation in the enzymatic polymerization extension reaction and a normal state marker is generated; when the difference between the first nucleic acid integrity value and the second nucleic acid integrity value is greater than the preset degradation threshold, it is determined that the pathogenic nucleic acid molecule has undergone non-specific physical degradation and a reaction system optimization prompt data package is generated.

[0009] Optionally, after determining that the pathogenic nucleic acid molecule has not undergone non-specific physical degradation in the enzymatic polymerization extension reaction, the method further includes: The interaction expression library was subjected to real-time quantitative polymerase chain reaction (qPCR) detection to obtain the first cycle threshold of the host high-abundance nucleic acid molecules and the second cycle threshold of the pathogen nucleic acid molecules; The fluorescence quantitative polymerase chain reaction detection data of the control library were obtained. The control library was constructed using homologous samples without the addition of the host-specific chimeric blocking probe. The third cycle threshold corresponding to the host high-abundance nucleic acid molecules and the fourth cycle threshold corresponding to the pathogen nucleic acid molecules were extracted from the control library. When the first cyclic threshold is greater than the third cyclic threshold, and the absolute value of the difference between the second cyclic threshold and the fourth cyclic threshold is less than or equal to a preset error threshold, the background suppression effectiveness of the interaction expression library is confirmed and a suppression success label is generated; otherwise, the background suppression of the interaction expression library is confirmed to be invalid and a suppression failure label is generated.

[0010] Optionally, after the step of calculating the signal-to-noise ratio of the output effective interaction signal, the method further includes: Extract the set of unique molecular identifier sequences from sequencing reads that carry the unique molecular identifier sequence and belong to the nucleic acid molecule of the pathogen; The molecular collision rate is calculated by counting the number of repeating sequences in the set of unique molecular identifier sequences and dividing the number of repeating sequences by the total number of sequences in the set of unique molecular identifier sequences. When the molecular collision rate is lower than the preset collision threshold, the pathogen signal separation of the interaction expression library is deemed qualified and a qualified data tag is output; when the molecular collision rate is greater than or equal to the preset collision threshold, the pathogen signal separation of the interaction expression library is deemed unqualified and a calibration operation command for the probe annealing temperature is triggered.

[0011] Optionally, the plant host in the plant-pathogen interaction sample includes potato or rapeseed, and the host's high-abundance nucleic acid molecules include ribosomal nucleic acid, starch synthesis-related transcripts, or lipid synthesis-related transcripts.

[0012] Compared with the prior art, the present invention has the following beneficial effects: 1. This method employs a host-specific chimeric blocking probe containing locked nucleic acid modification and a 3' dideoxynucleotide modification, which can bind to high-abundance host nucleic acid molecules to generate a highly stable hybridization blocking complex. When reverse transcriptase initiates the enzymatic polymerization extension reaction, the high affinity, steric hindrance, and dehydroxylation chain-breaking effect generated by this complex directly terminate the polymerization extension of reverse transcriptase on the host template. This mechanism preferentially allocates reaction resources to pathogen nucleic acid molecules, solving the problem that the pathogen signal is easily submerged due to excessive host background in the interaction sample, and significantly improving the signal-to-noise ratio of the effective interaction signal. 2. This method comprehensively calculates the probe annealing temperature and the primer annealing temperature, ensuring that the probe annealing temperature is higher than the primer annealing temperature. After heating and denaturation, a stepwise cooling annealing strategy is implemented. This mechanism promotes the blocking probe to preferentially bind to the host's high-abundance nucleic acid molecules, and then the reverse transcription primer binds to the pathogen's nucleic acid molecules. This effectively avoids disordered competition between the probe and primer in the reaction system, achieving pre-suppression of the host's high-abundance background before reverse transcription initiation, and further improving the capture rate of low-abundance nucleic acid molecules from pathogens. 3. This method introduces template-converting oligonucleotides with unique molecular identifier sequences into the reaction system, and uses reverse transcriptase with terminal transferase and template-switching activities to add non-template polycytosine tails to the complementary strands of pathogen nucleic acid molecules. Through pairing and template-switching operations, the unique molecular identifier sequence is successfully transcribed into the complementary strands of pathogen nucleic acid molecules. This embeds a non-replicable molecular identity tag in the interaction expression library construction stage, effectively overcoming the influence of traditional amplification bias on quantitative results and achieving accurate counting of pathogen nucleic acid molecules. 4. After generating the interaction expression library, this method extracts and compares the integrity values ​​of the first and second nucleic acids to determine whether non-specific physical degradation has occurred and generates a reaction system optimization prompt data package as needed. Subsequently, real-time polymerase chain reaction (PCR) detection is performed, and the effectiveness of background suppression is confirmed by comparing the cycle thresholds in the interaction expression library and the control library. This closed-loop verification mechanism solves the problem of insufficient interpretability of library quality in existing technologies, can promptly identify process anomalies, and ensure the consistency of detection results. 5. This method calculates the molecular collision rate by extracting a set of unique molecular identifier sequences from the sequencing reads and counting the number of repetitive sequences. This index is used to determine whether the pathogen signal separation of the interaction expression library is qualified. When the molecular collision rate is greater than or equal to the preset collision threshold, the system can determine that the separation is unqualified and trigger a calibration operation command for the probe annealing temperature. This mechanism realizes the automatic correction of parameters from the back end of the sequencing results to the front end of the reaction system, avoids data distortion caused by insufficient tag discrimination ability, and ensures the stability of the detection system. Attached Figure Description

[0013] The present invention will be further explained below with reference to the accompanying drawings and embodiments: Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0015] like Figure 1 As shown, a method for analyzing the expression of plant-pathogen interaction genes based on UMI tags includes: Obtain total nucleic acid extracts from plant-pathogen interaction samples; The extract, host-specific chimeric blocking probe, and reverse transcription primers were mixed to construct a molecular hybridization reaction system. The probe contained locked nucleic acid modified structure and 3' dideoxynucleotide modified structure. The system was subjected to heating to break down the molecules and cooling to anneal them, so that the probe could bind to the host's abundant nucleic acid molecules to form a hybridization blocking complex, and the reverse transcription primers could bind to the pathogen's nucleic acid molecules. The template-switching oligonucleotide with a unique molecular identifier (UMI) sequence and reverse transcriptase were added to the system; the reverse transcriptase had terminal transferase activity and template switching activity; the 3' end of the template-switching oligonucleotide contained a nucleotide sequence segment that paired with a non-template polycytosine tail. The reverse transcriptase is used to initiate an enzymatic polymerization and extension reaction, which allows the reverse transcriptase to extend along the pathogen nucleic acid molecule and add a non-template polycytosine tail to the end of its complementary strand. Based on the steric hindrance of the complex, the polymerization and extension of the host high-abundance nucleic acid molecule is blocked. Based on template-converting oligonucleotides, a unique molecular identifier sequence is introduced into the complementary strand of the pathogen nucleic acid molecule to generate an interaction expression library. High-throughput sequencing was performed on the library to obtain a set of sequencing reads; Extract the number of valid reads that carry unique molecular identifier sequences and belong to pathogenic nucleic acid molecules from this set, and the number of invalid reads that belong to high-abundance host nucleic acid molecules; When both the number of valid reads and the number of invalid reads are greater than zero, the effective interaction signal-to-noise ratio (SNR) is calculated by dividing the number of valid reads by the number of invalid reads. When both the number of valid reads and the number of invalid reads are equal to zero, the maximum SNR value representing a background below the sequencing detection limit is output as the effective interaction signal SNR. When both the number of valid reads and the number of invalid reads are equal to zero, the effective interaction signal SNR is zero. The specific formula for calculating the effective interaction signal SNR is as follows: ,in To the number of valid segments read, Invalid segment reads The expected value of background free nucleic acid noise. and This is a non-negative correction coefficient set based on sequencing depth.

[0016] This embodiment provides a mechanism for analyzing the gene expression of plant-pathogen interaction based on UMI tags. Specifically, the application scenarios of the method include molecular monitoring of plant tissues in the early stage of late blight infection. The goal is to preferentially capture pathogenic Phytophthora transcripts and establish a quantitatively comparable interaction library in the context of high abundance of host ribonucleic acid (RNA). Specifically, total nucleic acid was extracted from infected potato tissue. The extract contained both host-derived and pathogen-derived nucleic acids. The total nucleic acid extract, host-specific chimeric blocking probes, and reverse transcription primers were then placed in the same reaction tube to form a single-tube molecular hybridization reaction system. The host-specific chimeric blocking probes were used to target high-abundance nucleic acids in potatoes, such as 18S rRNA, 28S rRNA, or starch synthesis-related high-expression transcripts. The reverse transcription primers preferentially targeted the pathogen nucleic acid template to initiate reverse transcription. The reaction system was first heated to denature and dissolve the original secondary structures and local double-stranded regions in the sample. Then, it was cooled according to a preset program so that the blocking probes first occupied the host high-abundance template, while the reverse transcription primers subsequently bound to the pathogen template. After annealing, template-converting oligonucleotides with unique molecular identifier sequences and reverse transcriptase are added to the same reaction system. The reverse transcriptase extends along the pathogenic nucleic acid molecule that has already bound the primer, forming a complementary strand. When it advances to the vicinity of the 5' end of the pathogenic template, it forms a structure at the end of the nascent strand that can be paired with template-converting oligonucleotides, thereby introducing the UMI sequence into the pathogenic complementary strand. In contrast, the host's abundant nucleic acid has formed a hybridization blocking complex with the chimeric blocking probe. Once the reverse transcriptase advances to this region, it is blocked by the combined steric hindrance of the highly stable hybridization structure and the non-extending end structure, thus terminating its extension. Therefore, the enzymatic reaction capacity and subsequent sequencing read capacity in the system are preferentially allocated to the pathogenic source molecule. For clarity, a specific quantitative model is used to illustrate this. Assume the reaction system of the same batch of total nucleic acid extract contains only 6 template molecules, where H1, H2, H3, and H4 represent 4 host high-abundance templates, and P1 and P2 represent 2 pathogen templates. Without blocking, reverse transcription may yield 6 complementary deoxyribonucleic acid (cDNA) molecules, of which only P1 and P2 are relevant to the target analysis. With the addition of blocking probes, 3 of H1-H4 are completely blocked, and only H4 produces 1 host background cDNA due to insufficient opening of local secondary structures, while P1 and P2 successfully complete UMI introduction. At this point, the effective reads can be abstractly represented as 200, and the ineffective host high-abundance reads as 4, resulting in an effective interaction signal-to-noise ratio of 200 / 4 = 50. If no host high-abundance ineffective reads are detected in subsequent sequencing, a preset maximum value, such as 1000, is output to indicate that the background at this sequencing depth is below the detection limit. Furthermore, if the sample degradation exceeds the preset degradation threshold, causing both the reverse transcription primers and blocking probes to fail to bind stably, the batch of samples can be directly marked as low-confidence samples and will not be included in subsequent quantitative alignment; if the sequencing read contains both pathogen sequences and host sequences but neither carries a UMI, it can be identified as a library construction failure byproduct and removed from the read set; if the number of invalid reads is zero, no division by zero is performed, and the preset maximum signal-to-noise ratio is directly output to avoid calculation anomalies. In the actual monitoring process, three leaf perforation samples were collected from the edge of the same lesion. The sequencing results of the first sample showed that there were 1280 effective reads carrying UMI and belonging to the pathogenic Phytophthora effector protein gene, and 16 invalid reads belonging to potato 18S rRNA, so the effective interaction signal-to-noise ratio was 80. The second sample had 910 effective reads and 0 invalid reads, so the maximum output value was 1000. The third sample had 120 effective reads and 60 invalid reads, so the signal-to-noise ratio was only 2, indicating that the background suppression of this sample was insufficient and the reaction conditions needed to be verified. The purpose of this step is to first physically suppress the host high-abundance template and then preferentially introduce the pathogen UMI response resource redistribution at the key biochemical node of reverse transcription, so as to make the expression signals of low-abundance pathogens in the interacting samples measurable, countable and comparable. The effective identification structure standard for the unique molecular identifier sequence in this embodiment is: it meets the preset nucleotide sequence length and completely contains the sequence structure that can be used for template conversion of oligonucleotide pairing; It should be further noted that, in order to maintain consistency in terminology, the unique molecular identifier sequence, UMI sequence, UMI tag, and effective UMI in this embodiment all refer to the same type of molecular identification sequence; the effective interaction signal-to-noise ratio and the signal-to-noise ratio referred to below are also the same indicator, and will not be distinguished unless otherwise specified. Furthermore, the total nucleic acid extract referred to in this embodiment refers to the total nucleic acid obtained from the interacting sample, which may include RNA, deoxyribonucleic acid (DNA), and their complex forms; however, the RNA template or its equivalent transcription template that can be recognized by reverse transcription primers and extended by reverse transcriptase, especially pathogen transcripts, is preferred to enter the reverse transcription, template switching, and UMI introduction processes and be counted as effective reads of pathogens. Similarly, the host high-abundance nucleic acid molecules are preferably high-abundance RNA templates that actually form background competition in the reverse transcription system, such as rRNA or highly expressed transcripts. With this definition, the concept of total nucleic acid extract as a sample source is consistent with the concept of reverse transcription template as a reaction object, and non-target nucleic acids that are difficult to enter the reverse transcription process are not directly mixed into the subsequent counting caliber. It is also necessary to specify that the statistical definitions of the number of valid and invalid reads remain fixed throughout the text: the number of valid reads refers to the number of reads that simultaneously meet the two conditions of carrying a valid UMI structure and being classified as a pathogenic nucleic acid molecule after alignment; the number of invalid reads refers to the number of background reads that are classified as high-abundance host nucleic acid molecules after alignment. If such reads lack a complete UMI but still form a recognizable host background signal, they are also counted as invalid reads in the background burden. Correspondingly, reads that cannot confirm pathogen attribution or high-abundance host attribution, or those with incomplete library structures, are uniformly removed as failure byproducts and are not included in the above two counts. Thus, the numerator and denominator of the signal-to-noise ratio have stable and verifiable statistical boundaries. In this embodiment, before the step of mixing the extract, the host-specific chimeric blocking probe, and the reverse transcription primer to construct the molecular hybridization reaction system, the following steps are also included: Obtain high-abundance nucleic acid sequences from the host; Design complementary nucleic acid sequences targeting high-abundance nucleic acid sequences in the host; Methylene bridging was performed on the ribose loops of complementary nucleic acid sequences to generate locked nucleic acid modified structures; the substitution rate of the locked nucleic acid modified structures in host-specific chimeric blocking probes was measured. Satisfying the formula: ,in To lock the number of nucleotides modified by nucleic acid, The total nucleotide length of the probe is defined as follows: ; The 3' end of the complementary nucleic acid sequence is dehydroxylated to generate a 3' dideoxynucleotide modified structure, thus obtaining a host-specific chimeric blocking probe.

[0017] This embodiment provides a mechanism for constructing a host-specific chimeric blocking probe. Specifically, in the aforementioned potato late blight monitoring scenario, relying solely on ordinary DNA probes is prone to two defects: first, the binding strength to the host's high-abundance RNA is insufficient; second, it may be replaced by enzymes or misextended in the reverse transcription environment, causing the host background to re-enter the library. Therefore, the blocking probe itself is specially designed before entering the library construction. Specifically, host high-abundance nucleic acid sequences are first screened from the potato reference transcriptome or previously measured high-expression panels; 18S rRNA fragments, 28S rRNA fragments, and high-expression regions of starch synthase-related transcripts can be selected; for each target sequence, a complementary sequence of 15 to 25 bases in length is designed; subsequently, locking nucleic acid modifications are introduced at certain sites of the complementary sequence to bridge and lock the corresponding ribose loop, thereby enhancing hybridization affinity with the RNA template; furthermore, dideoxynucleotides are introduced at the 3' end of the probe to remove the 3' hydroxyl group (3'-OH), thus preventing it from serving as the starting point for polymerization extension; For ease of explanation, assuming the high-abundance fragment in the target host is 5-AUGCUAACCGGAUUA-3, a complementary probe of the corresponding form 3-TACGATTGGCCTAAT-5 can be designed, with locked nucleic acid modifications at positions 3, 7, and 11, and a dideoxy modification at the terminal base. This treatment not only provides higher hybridization affinity for stable binding to host RNA, but also prevents enzymes from initiating polymerization and extension reactions using this probe as a primer, even if the enzyme is near the region. Furthermore, using two probes targeting different regions of the same highly expressed transcript can create a dual-site synergistic blockade, enhancing the completeness of the blockade. Furthermore, if multiple closely related homologous regions exist in the high-abundance sequence of the target host, regions without continuous high homology in the pathogen transcriptome should be prioritized to avoid off-target effects. If a candidate probe is predicted to have more than 80% continuous complementarity with important pathogen transcripts, the probe should be discarded and the region selection design should be redesigned. If there are obvious secondary structures near the target site in the same host, a second adjacent probe can be configured as a supplement to avoid the single probe reducing its effectiveness due to local inaccessibility. During the process development phase, three candidate probes, B1, B2, and B3, were designed for potato 18S rRNA. B1 is a standard DNA probe, B2 is a probe with three locked nucleic acid sites but without dehydroxylation at the ends, and B3 is a chimeric probe with three locked nucleic acid sites and dideoxy modification at the 3' end. After testing on homologous samples, the host background of the B1 group decreased by about 4-fold, the B2 group by about 15-fold, but a small amount of abnormal extension products were still observed. The host background of the B3 group decreased by about 300-fold and no detectable extension byproducts were observed. Therefore, B3 was selected for the formal monitoring process. The purpose of this step is to first endow the blocking probe with two properties at the molecular structure level: high affinity binding and non-extensionability, so as to achieve a stable blocking basis in the subsequent single-tube reverse transcription reaction. In this embodiment, the system undergoes heating to dissociate and cooling to anneal, specifically including: Based on the host-specific chimeric blocking probe's base sequence, nucleotide length, and locked nucleic acid modification structure, the probe annealing temperature was calculated. The calculation model is as follows: ,in For hybridization enthalpy change, For hybridization entropy change, Let be the ideal gas constant. The probe concentration, The temperature compensation constant resulting from single-base locked nucleic acid modification; The primer annealing temperature is calculated based on the nucleotide composition of the reverse transcription primers, wherein the probe annealing temperature is greater than the primer annealing temperature; The system is controlled to cool down to the probe annealing temperature after heating and de-milking, thereby promoting the binding of the probe to the host's high-abundance nucleic acid molecules; The system was kept cooled to the primer annealing temperature to induce the reverse transcription primers to bind to the pathogen nucleic acid molecules.

[0018] This embodiment provides a thermodynamic timing control mechanism. Specifically, in the aforementioned process, even if the blocking probe structure is optimized, if it and the reverse transcription primer simultaneously compete disorderly for the template, the host template may be preferentially bound by the reverse transcriptase and cause polymerization extension before the pathogen primer is bound. Therefore, this embodiment sets the annealing temperature in stages to allow the blocking action to occur first, followed by the binding of the pathogen template primer. Specifically, the annealing temperature of the blocking probe is first determined based on its base composition, length, and number of locked nucleic acid sites. Then, the annealing temperature of the reverse transcription primer is determined based on its GC content and length. Since the melting temperature of the probe is usually higher than that of ordinary primers after the introduction of locked nucleic acids, a clear temperature difference can be formed between the two. For example, the annealing temperature of a host blocking probe can be set to 68℃, and the annealing temperature of the reverse transcription primer can be set to 55℃. The reaction system is first heated to 95℃ for melting for 2 minutes, then lowered to 68℃ and held for 3 minutes to allow the blocking probe to preferentially occupy the high-abundance template of the host. Then, it is lowered to 55℃ and held for 5 minutes to allow the reverse transcription primer to bind to the unblocked pathogen template. By simplifying the model, the reaction template can be abstracted into two categories: the host template region (H region) and the pathogen template region (P region). If the temperature is directly reduced from 95℃ to 55℃ in one step, both the H and P regions will expose binding sites, and the blocking probe and reverse transcription primer will compete simultaneously. It is possible that 20% of the sites in the H region will be occupied by the primer first. If a two-stage cooling process of 68℃ and 55℃ is used, at 68℃, only the high-Tm blocking probe can stably occupy the sites, and ordinary primers cannot bind efficiently. When the temperature is reduced to 55℃, most sites in the H region have been occupied, and the remaining free template is mainly the P region. Therefore, the reverse transcription primer is more biased towards the pathogen template. Thus, it can be seen that the above-mentioned time-sequence control mechanism achieves physical isolation of the binding steps of different target molecules through the difference in thermodynamic annealing temperature. Under special operating conditions, if the probe annealing temperature is too close to the primer annealing temperature, for example, the difference is less than 3°C, the priority of occupancy may be lost. In this case, the probe length should be adjusted or a locking nucleic acid modification site should be added to increase the probe Tm. If the probe annealing temperature is too high, causing some RNA template secondary structures to reform, it can also be corrected by extending the high-temperature buffer time after melting or adding a compatible structural relaxation component. If the sample is a low-concentration pathogen sample, the primer annealing time can be appropriately extended to increase the binding probability of low-abundance templates. In the same potato lesion sample, researchers compared single-stage and two-stage cooling programs. In the single-stage program, direct annealing at 55℃ resulted in 240 background reads of host 18S rRNA and 720 effective reads of pathogens, with a signal-to-noise ratio of 3. In the two-stage program, the blocking probe was annealed first at 68℃, followed by annealing of the reverse transcription primers at 55℃. This resulted in a reduction of the host background reads to 18, an increase in the effective pathogen reads to 900, and an improvement in the signal-to-noise ratio to 50. The purpose of this step is to utilize the annealing temperature difference between the blocking probe and the reverse transcription primer to construct a reaction sequence of blocking first and then initiating, thereby achieving pre-suppression of the high-abundance background in the host. In this embodiment, reverse transcriptase is used to initiate the enzymatic polymerization and elongation reaction, and the polymerization and elongation steps of high-abundance host nucleic acid molecules are blocked based on the steric hindrance of the complex. Specifically, it includes: In the enzymatic polymerization and extension reaction, reverse transcriptase polymerizes and extends along the pathogenic nucleic acid molecules to generate complementary strands of the pathogenic nucleic acid molecules; When reverse transcriptase polymerizes and elongates along a host high-abundance nucleic acid molecule and encounters a hybridization blocking complex, the polymerization and elongation of the reverse transcriptase is terminated and it detaches from the template due to the high affinity steric hindrance caused by the locked nucleic acid modification structure and the dehydroxylation chain-breaking effect caused by the 3' dideoxynucleotide modification structure, thereby blocking the reverse transcription process of the host high-abundance nucleic acid molecule; the blocking efficiency of polymerization and elongation termination. Binding energy subject to steric hindrance of high affinity Regulation, when combined with energy At that time, it was determined that the dehydroxylation chain scission effect and steric hindrance worked together to achieve absolute blocking.

[0019] This embodiment provides a polymerization extension blocking mechanism; specifically, the aforementioned annealing control only solves the problem of which occupies the site first, but under extreme conditions, reverse transcriptase may still undergo non-specific strand substitution and bypass the already bound probe by relying on its own strand substitution ability; therefore, this embodiment further relies on the physical spatial steric hindrance and non-extending ends formed by the chimeric probe to achieve in-situ termination in the enzyme propagation process. Specifically, when reverse transcriptase extends on a pathogenic template, since the template is not blocked, the enzyme can continuously read the template and synthesize complementary strands. However, on a host template with high abundance, if the enzyme advances along the template to the blocking probe binding region, it will encounter a highly stable hybridization blocking complex. Locked nucleic acid modification causes the probe and host RNA to form a double-stranded structure with a higher melting temperature and greater steric hindrance, increasing the dissociation barrier when the enzyme advances. Dideoxy modification at the 3' end of the probe further prevents it from being used as an extendable primer. After the two are combined, reverse transcriptase usually terminates and detaches at the blocking site, thus not producing complete host cDNA. This can be elucidated using a microscopic reaction model. It is assumed that the reverse transcriptase extends from position 1 to position 50 on the host template, blocking the probe binding at positions 51 to 68. With a regular DNA probe, the enzyme may continue to advance to position 60 or even completely bypass the probe, eventually forming a long host cDNA. However, when using a chimeric probe with locked nucleic acid and dideoxy ends, the enzyme stops near position 51, ultimately producing only a short fragment of less than 50 nucleotides. This short fragment will be rejected in subsequent library amplification and effective read screening due to insufficient length or lack of complete template transition structure, thus not forming usable host background data. It should be noted that the actual mechanism of the dehydroxylation chain breakage effect caused by the 3' dideoxynucleotide modification structure mentioned above in this embodiment is mainly to prevent the blocking probe itself from providing a 3-hydroxyl initiation site that can be utilized by polymerase, thereby avoiding the probe being mistakenly used as a primer for extension; the termination of reverse transcriptase on the host template is still mainly due to the propulsion barrier formed by the highly stable probe-template hybridization structure after the locked nucleic acid enhancement, and the in-situ blocking caused by the probe's non-extension property, rather than meaning that the host template itself undergoes chemical breakage; therefore, host background inhibition can be understood as the synergistic result of highly stable occupancy barrier + probe non-extension. Furthermore, when determining whether a penetration event has occurred, the frequency of host-derived long-fragment cDNA can be used as an auxiliary observation indicator: if the host background reads are present, but their length is mainly concentrated in the short fragment region and lacks a complete template transition structure, it can be attributed to residual truncated products after blocking; only when the host-derived reads simultaneously exhibit amplifiable length and carry a complete library structure can they be preferentially judged as partially penetrated by the blocking site; this treatment not only maintains consistency with the aforementioned blocking mechanism, but also facilitates the differentiation between true penetration and invalid fragments that have been truncated during process optimization. Under special operating conditions, if the chain substitution activity of a certain type of reverse transcriptase is greater than the preset activity threshold, causing a small number of penetration events, the free region in which the enzyme may continuously advance can be shortened by increasing the coverage density of the blocking probe. If the probe binding site on the host template is located downstream of the reverse transcription initiation site and the distance is greater than the preset length threshold, the enzyme may still synthesize a longer byproduct in front of the probe. In this case, the probe can be moved forward to a position downstream of the primer binding site and less than the preset length threshold to terminate the enzyme as early as possible. If there is a local conserved region between the pathogen target template and the host template, the blocking probe should be designed to be placed in the conserved region to avoid blocking the pathogen signal at the same time. In the actual detection of late blight samples, ordinary DNA blocking probes and chimeric blocking probes were used in the same reaction system. The former yielded 320 background reads of the host 18S, with a large number of them concentrated in the 250 to 400 bp range. The latter yielded only 12 background reads, and the length of the residual fragments was mainly less than 80 bp, which could not form an effective library. At the same time, the effective reads of the pathogen effector protein transcripts remained at a similar level, indicating that the inhibitory effect mainly occurred on the host high-abundance template. The purpose of this step is to further advance host background inhibition from binding competition to enzyme-driven termination, thereby achieving physical-level truncation of the reverse transcription stage; It should also be noted that, in order to maintain consistency in the terminology and mechanism descriptions throughout the text, the physical spatial steric hindrance, propulsion barrier, in situ blockade, and enzyme propulsion termination in this embodiment all refer to the same type of blocking phenomenon, namely, the difficulty of reverse transcriptase to continue extending after encountering a stable probe-template hybridization region, and do not represent different independent biochemical mechanisms. Furthermore, the aforementioned dehydroxylation chain scission effect is used in this specification only as a general description of the result state in which the probe loses its extendable hydroxyl group at the 3' end. Its technical focus is always on blocking the probe from being extended due to dideoxy modification at the 3' end, rather than referring to the cleavage of the host template, the cleavage of the pathogen template, or the occurrence of an independent chemical cleavage reaction in the system. This avoids the misunderstanding of this term as a chemical degradation mechanism and distinguishes it from the quality judgment concept of non-specific physical degradation in the examples: the former is the non-extension property of the probe end, while the latter is the phenomenon of decreased sample or library integrity. The two have different meanings and different criteria. Furthermore, in actual read discrimination, if the host-derived sequence only appears as a short truncated fragment after being blocked, and does not possess a complete template switching structure, amplified adapter structure, or effective sequencing length, it is considered a residual byproduct after successful blocking and is not regarded as evidence of successful penetration. Only when the host-derived sequence crosses the blocking site, forms continuous amplifiable cDNA, and enters the effective sequencing structure is it regarded as a background signal of insufficient blocking. Through this discrimination boundary, the blocking mechanism, library interpretation, and subsequent optimization actions can be kept consistent. In this embodiment, the step of introducing a unique molecular identifier sequence into the complementary strand of a pathogenic nucleic acid molecule based on template-conversion oligonucleotides specifically includes: In the enzymatic polymerization extension reaction, reverse transcriptase extends along the pathogen nucleic acid molecule to the 5' end of the pathogen nucleic acid molecule and adds a non-template polycytosine tail to the end of the complementary strand of the pathogen nucleic acid molecule. Template-converted oligonucleotides are used to pair and bind with non-template polycytosine tails; The reverse transcriptase is guided to perform a template switching operation, transcribing a unique molecular identifier sequence into the complementary strand of the pathogen's nucleic acid molecule; the effective switching probability of template-converting oligonucleotides. Length of non-template polycytosine tail Establish a connection, and when This is the process by which transcription is activated and transcribed into the complementary strand of the pathogen's nucleic acid molecule.

[0020] This embodiment provides a UMI introduction mechanism based on template switching. Specifically, in the aforementioned scheme, if only the reverse transcription of the pathogen template is completed without the unique molecular identifier being stably introduced to the single molecule level, the subsequent PCR amplification bias will still amplify the quantitative error. Therefore, in this embodiment, after the pathogen template has been extended, the UMI sequence is written into the nascent complementary strand through template switching oligonucleotides. Specifically, when reverse transcriptase extends along the pathogen RNA template to near its 5' end, it adds several non-template cytosine residues to the end of the newly formed cDNA, forming a non-template polycytosine tail. One end of the template-converting oligonucleotide contains a guanine segment that pairs with this tail, and the other end contains the UMI sequence and the adapter sequence for subsequent amplification. Through this pairing, the reverse transcriptase performs template switching, transcribing the UMI sequence on the template-converting oligonucleotide into the head end of the pathogen cDNA. Thus, in principle, no matter how many times the same original pathogen RNA molecule is amplified subsequently, it can be considered to belong to the same UMI family, and thus be used for actual molecule counting. This is explained through a specific quantitative model. Assuming that after reverse transcription of pathogen template P1, CCC forms at the end of the nascent strand; the template conversion oligonucleotide has a GGG front end, an 8-position UMI (e.g., ATCGTGCA) in the middle, and a universal adapter sequence at the rear end; after pairing, reverse transcriptase continues synthesis, causing the cDNA corresponding to P1 to carry ATCGTGCA at its head end; if P1 subsequently amplifies 100 reads by PCR, and all of their UMIs are ATCGTGCA, they are still counted as one original molecule during quantification; assuming that the UMI of another template P2 is ATCGTGCT, then whether P2 amplifies 50 or 500 reads, it is counted as another independent molecule; this reduces the quantitative analysis error caused by PCR amplification bias. Furthermore, if the template switching efficiency is low, resulting in some pathogen cDNAs not carrying UMIs, these can be classified as unqualified reads during effective read screening and excluded from quantification. If the UMI length is less than the preset sequence length threshold, different molecules may share the same tag, which can be mitigated by increasing the number of UMI bits or reducing the amount of library loaded per read. If the pathogen template itself is severely degraded, and the reverse transcriptase cannot advance to near the 5' end, template switching events may decrease. In this case, the results should be combined with the sample integrity test results for a comprehensive judgment, rather than simply interpreting it as a failure of the blocking system. In this potato late blight sample, the transcript of a certain effector protein of the pathogenic Phytophthora blight was sequenced, yielding 1200 reads. Among them, 1000 reads carried valid UMIs and clustered into 25 UMI families, while 200 reads with missing UMIs or incomplete structures were removed. After deduplication, the original molecular count of this transcript in the sample was 25, not 1200. Compared with a regular library that did not use the UMI system, its molecular abundance is closer to the actual input level. The purpose of this step is to embed the single-molecule identity information of the pathogen's nucleic acid molecules into the library structure, thereby achieving subsequent quantitative de-amplification bias. In this embodiment, after the step of generating the interaction expression library, the method further includes: The fragment distribution map of the total nucleic acid extract was obtained by using a nucleic acid electrophoresis analysis platform, and the nucleic acid integrity index was used as a unified evaluation benchmark; the first nucleic acid integrity value of the total nucleic acid extract before constructing the molecular hybridization reaction system was extracted. The numerical value of the second nucleic acid integrity of the nucleic acid molecules in the library after generating the interaction expression library; Compare the integrity values ​​of the first and second nucleic acids; When the difference between the first nucleic acid integrity value and the second nucleic acid integrity value is less than or equal to the preset degradation threshold, it is determined that the pathogenic nucleic acid molecules have not undergone non-specific physical degradation in the enzymatic polymerization extension reaction and a normal state marker is generated; when the difference between the first nucleic acid integrity value and the second nucleic acid integrity value is greater than the preset degradation threshold, it is determined that the pathogenic nucleic acid molecules have undergone non-specific physical degradation and a reaction system optimization prompt data package is generated.

[0021] In this embodiment, after determining that the pathogenic nucleic acid molecules have not undergone non-specific physical degradation during the enzymatic polymerization extension reaction, the method further includes: The interaction expression library was subjected to real-time quantitative polymerase chain reaction (qPCR) detection to obtain the first cycle threshold of high-abundance host nucleic acid molecules and the second cycle threshold of pathogen nucleic acid molecules; The fluorescence quantitative polymerase chain reaction (qPCR) detection data of the control library were obtained. The control library was constructed using homologous samples without host-specific chimeric blocking probes. The third cycle threshold of the corresponding host high-abundance nucleic acid molecules and the fourth cycle threshold of the corresponding pathogen nucleic acid molecules were extracted from the control library. When the first loop threshold is greater than the third loop threshold, and the absolute value of the difference between the second loop threshold and the fourth loop threshold is less than or equal to the preset error threshold, the background suppression effectiveness of the interaction expression library is confirmed and a suppression success label is generated; otherwise, the background suppression of the interaction expression library is confirmed to be invalid and a suppression failure label is generated.

[0022] This embodiment provides a closed-loop verification mechanism for library quality. Specifically, although the aforementioned blocking and UMI introduction scheme can theoretically enhance pathogen signals, in real field samples, new problems may still arise due to sample background degradation, temperature control fluctuations, or excessively high probe concentrations: one type is the non-specific destruction of pathogen nucleic acids, and the other is that the host background is not truly suppressed. To avoid misjudging reaction errors as biological differences, this embodiment sets up two levels of quality assessment after library construction. Specifically, the first stage is integrity determination; firstly, the integrity value of the first nucleic acid before the total nucleic acid enters the reaction is extracted, and then the integrity value of the second nucleic acid of the library product is extracted; the integrity value here can be derived from nucleic acid electrophoresis peak shape, integrity score or similar instrument output indicators; the difference between the two is calculated, and if the difference is not greater than the preset degradation threshold, such as 0.8, it is determined that no significant non-specific physical degradation has occurred in the reaction stage, and a normal state label is generated; if the difference is greater than the threshold, a reaction system optimization prompt data package is generated, such as prompting to reduce the mixing intensity, shorten the high temperature exposure time or reduce the amount of probe added; The second stage is quantitative real-time polymerase chain reaction (qPCR) control verification. After passing the integrity test, quantitative real-time detection is performed on the interacting expression library, and a control library without blocking probes is constructed simultaneously. The detection targets must include at least one type of host high-abundance nucleic acid and one type of pathogen nucleic acid. If the cycle threshold of the host high-abundance nucleic acid in the current library is greater than the corresponding value in the control library, it indicates that the host background is suppressed. At the same time, if the absolute value of the difference between the cycle threshold of the pathogen nucleic acid and the corresponding value in the control library does not exceed the preset error threshold, such as one cycle, it indicates that the pathogen signal has not been significantly damaged. When both conditions are met, a successful inhibition label is generated; otherwise, a failed inhibition label is generated. It should be further explained that, to ensure the comparability of the two integrity values, the second nucleic acid integrity value is preferably obtained from the library nucleic acid molecules in the interacting expression library that can reflect the continuity of the inserted fragment, or from the main peak region of the library after removing free adapters, primer dimers, and byproducts with a length less than the preset fragment threshold. In other words, the second nucleic acid integrity value focuses on the integrity of the measurable inserted fragment in the library, rather than including all adapters, small oligonucleotides, and byproducts. After this treatment, although the first integrity value and the second integrity value belong to the total nucleic acid before the reaction and the library nucleic acid after the reaction, respectively, the basis for comparison lies in the continuity of the measurable nucleic acid, so it can be used as a process criterion for whether additional non-specific degradation has occurred. Furthermore, if instrumental scoring is used, the same detection platform, the same analysis algorithm, and the same fragment recognition window can be used to obtain two integrity values. If peak shape or fragment distribution indicators are used, it is preferable to compare similar parameters such as the position of the main peak, the width of the main peak, and the proportion of measurable fragments, rather than considering the fixed length difference of the linker and tail sequences themselves as degradation. This can avoid the situation where the library structure itself has a small inherent sequence length and is misjudged as degradation, making this level of judgment closer to the actual template damage in the reaction process. This is illustrated using a simplified model. Assuming a sample has a first integrity value of 8.6 and a second integrity value of 8.1, the difference is 0.5, which is less than the threshold of 0.8, thus proceeding to the second-level validation. Subsequent qPCR testing revealed: the first cycle threshold for the host 18S was 27, and the third cycle threshold for the control library was 19, a difference of 8, indicating a significant decrease in host background. The second cycle threshold for the pathogen effector protein gene was 24, and the fourth cycle threshold for the control library was 24.5, with an absolute difference of 0.5, not exceeding the threshold of 1, indicating that the pathogen signal was essentially preserved. Therefore, this batch of libraries was marked as successfully inhibited. Conversely, if the host cycle threshold (Ct) only increases from 19 to 20, or the pathogen Ct changes from 24 to 28, it indicates either insufficient blocking or damage to the pathogen signal, both of which should be considered failures. Under special conditions, if the difference in integrity before and after is greater than the preset degradation threshold, qPCR validation is unnecessary, and subsequent sequencing should be directly blocked to terminate the sequencing process of abnormal samples. If the host background decreases significantly in qPCR but the pathogen Ct also shifts significantly, it should be judged as an overly strong inhibition of the reaction system or template damage, rather than simply a successful conclusion. If both the host Ct and the pathogen Ct are close to the control, it indicates that the blocking probe has not worked, and the probe annealing step or the quality of the probe itself needs to be investigated. If the qPCR amplification curve is abnormal, there is no exponential phase, or the melting curve shows a non-specific peak, the validation is invalid, and the detection reaction should be prepared again. During continuous monitoring, among five samples from the same lesion edge in a batch, four samples had an integrity difference between 0.3 and 0.6, and the host 18S Ct was delayed by 7 to 9 cycles compared to the control, while the pathogen effector protein Ct changed by no more than 0.7 cycles. Therefore, these samples were uniformly tagged with "successful inhibition" and entered sequencing. Another sample, due to repeated freeze-thaw cycles during operation, had its integrity drop from 8.4 to 6.9, a difference of 1.5. The system directly generated an optimization prompt data package, indicating that this batch needed to be re-extracted for nucleic acid and was not included in subsequent analysis. The purpose of this mechanism is to verify whether the background stratification is degraded and whether it is truly suppressed, thereby achieving process interpretability of library quality and pre-blocking of abnormal batches. In this embodiment, after calculating the signal-to-noise ratio of the output effective interaction signal, the method further includes: Extract the set of unique molecular identifier sequences from sequencing reads that carry unique molecular identifier sequences and belong to the nucleic acid molecules of pathogens; The molecular collision rate is calculated by counting the number of repeating sequences in the set of unique molecular identifier sequences and dividing that number by the total number of sequences in the set. The theoretical baseline upper limit model is as follows: ,in The effective base length of the sequence that uniquely identifies a molecular identifier. This is an estimate of the true original total number of nucleic acid molecules belonging to this pathogen; When the molecular collision rate is lower than the preset collision threshold, the pathogen signal separation of the interaction expression library is deemed qualified and a qualified data label is output; when the molecular collision rate is greater than or equal to the preset collision threshold, the pathogen signal separation of the interaction expression library is deemed unqualified and a calibration operation command for the probe annealing temperature is triggered.

[0023] This embodiment provides a mechanism for determining the qualification of pathogen signal separation. Specifically, the aforementioned process can obtain pathogen reads with UMIs, but when the loading is high or the reaction window is offset, collisions may still occur where different original molecules share the same UMI, or the separation degree of pathogen reads may decrease because the host background is not suppressed below the target threshold. Therefore, this embodiment uses the UMI repetition as a posterior quality benchmark and performs reverse linkage annealing parameter calibration. Specifically, the process begins by extracting a set of UMI sequences from all sequencing reads belonging to pathogen nucleic acids and carrying valid UMIs. Then, the number of recurring UMIs is counted, and the molecular collision rate is obtained by dividing the number of recurring sequences by the total number of UMI sequences. The number of recurring sequences can be calculated based on identical UMI tag entries within the same batch of pathogen effective reads. If the collision rate is lower than a preset collision threshold, such as 0.1%, it indicates that most of the original pathogen molecules have acquired distinguishable identities, and the pathogen signal separation is satisfactory. If the threshold is reached or exceeded, it indicates insufficient UMI discrimination capability in the current library or that the effective binding window of the pathogen is affected, thus triggering a calibration command for the probe annealing temperature. It is necessary to further specify that, in this embodiment, the number of repetitive sequences is preferably not a direct count of identical UMIs appearing at the original read level. Instead, pathogenic reads are first clustered into families based on the same reference transcript affiliation, the same alignment start and end positions, or the same insertion fragment boundary. Identical UMIs appearing within the same cluster are generally considered as PCR repeats or sequencing repeats of the same original molecule and are not counted as collisions. Only when identical UMIs appear in different pathogenic molecule families, different insertion boundaries, or distinguishable independent molecular units are they counted as repetitive sequences. After this processing, the molecular collision rate reflects the probability that different original molecules share the same UMI, rather than misjudging normal amplified copies as UMI collisions. Furthermore, in actual statistical analysis, the PCR duplication preprocessing of effective reads from pathogens can be completed first, and then the UMI set can be extracted from the remaining molecular families. If a certain UMI appears multiple times, but the start position, end position, and library structure of these reads are consistent on the reference transcript, it is included in the same UMI family without adding extra collision counts. Only when the same UMI corresponds to two or more distinguishable pathogen molecular families is it identified as a repetitive sequence. Through this limitation, the collision rate can more accurately reflect whether the UMI space is sufficient, whether the template loading is too dense, and whether the front-end reaction leads to the abnormal enrichment of a few molecules. This can be illustrated using a specific data model. Suppose a sample yields 1000 effective reads of pathogens. After family clustering, 980 molecular families are formed. 975 families correspond to distinct UMIs, while 5 families have UMI sequence duplications with other independent families. The collision rate can be calculated as 5 / 980, approximately 0.51%. If the threshold is set to 0.1%, this batch of samples is unqualified and requires calibration. Further observation reveals that the host background of this batch is not high, but the effective reads of pathogens are concentrated in a few UMI families. This indicates that the problem may not be insufficient sequencing volume, but rather that the annealing window prevents some pathogen templates from being fully initiated, leading to over-amplification of a few templates. In this case, the probe annealing temperature can be lowered by 1°C or the probe priority occupancy time can be shortened to avoid competitive inhibition of low-abundance pathogen templates. Under special operating conditions, if the total number of valid reads from pathogens does not reach the statistically valid lower limit, such as less than 100, the collision rate is statistically unstable. In this case, it should be marked as insufficient sequencing depth rather than directly triggering calibration. If the collision rate increases and is accompanied by a significant increase in invalid host reads, it is more likely that the background blocking has failed. In this case, the calibration direction should prioritize increasing the probe annealing temperature or increasing probe coverage. If the collision rate increases but the host background remains extremely low, it is more likely that there is a problem with the UMI length or library loading. In this case, in addition to annealing calibration, the input volume can also be reduced or the UMI bit depth can be increased simultaneously. In a batch monitoring, sample A had 5000 effective reads of pathogens, which, after deduplication, resulted in 4900 pathogen molecular families. Only two families had cross-family UMI duplicates, with a collision rate of approximately 0.04%, thus the data was labeled as qualified. Sample B had 1800 effective reads of pathogens, which, after deduplication, resulted in 1760 pathogen molecular families. Nine families had cross-family UMI duplicates, with a collision rate of approximately 0.51%, and the host background reads were simultaneously increasing. The system triggered a calibration operation to increase the probe annealing temperature by 1°C and repeat the annealing test. Sample C only obtained 60 effective reads of pathogens. Even if one suspected cross-family duplicate UMI appeared, it was not directly judged as unqualified, but rather marked as insufficient sequencing depth. The purpose of this mechanism is to assess whether pathogen signals are truly effectively separated through the molecular-level posterior index of UMI collision, and thereby achieve closed-loop correction of the front-end hybridization parameters. It should also be noted that, to maintain the uniqueness of the statistical caliber, the total number of unique molecular identifier sequence sets in this paper preferably refers to the total number of UMI families to be identified after pathogen attribution determination, library structure screening, and merging with the same molecular family, rather than the total number of original sequencing reads; correspondingly, the number of repetitive sequences preferably refers to the number of UMI entries with duplicate numbers across independent molecular families at the UMI family level; thus, the molecular collision rate is always calculated at the family-level molecular unit and is not mixed with PCR repeats at the original read level. Furthermore, to avoid the misuse of terminology, in this embodiment, "pathogen signal separation qualified," "UMI discrimination ability sufficient," and "molecular collision rate below the threshold" are different expressions of the same result on the judgment link; "pathogen signal separation unqualified," "UMI discrimination ability insufficient," and "molecular collision rate reaching or exceeding the threshold" correspond to the same type of abnormal result; these expressions are only used to explain the same judgment conclusion from different observation perspectives and do not introduce additional evaluation indicators. Furthermore, the calibration operation command triggered for the probe annealing temperature is preferably understood as follows: the system selects the calibration direction based on other quality signals accompanying the increase in collision rate, rather than simply making a one-way temperature change; if the collision rate increases and is accompanied by an increase in host background, the probe annealing temperature is increased or the probe priority occupation time is extended; if the collision rate increases but the host background is still low and the effective initiation of pathogens is insufficient, the probe annealing temperature is decreased, the priority occupation time is shortened, or the pathogen primer binding window is expanded; after this processing, the calibration operation command in the embodiment and the two types of specific actions of adjustment and deregulation in the embodiment can be uniformly classified into the same calibration concept, avoiding inconsistencies in semantics; In this embodiment, the plant host in the plant-pathogen interaction sample includes potato or rapeseed, and the host's high-abundance nucleic acid molecules include ribosomal nucleic acid, starch synthesis-related transcripts or lipid synthesis-related transcripts.

[0024] This embodiment provides a host type adaptation mechanism. Specifically, the aforementioned monitoring main line takes the potato-pathogenic Phytophthora as an example. However, in actual agricultural disease detection platforms, the high abundance background composition of different crops varies. If the same set of host blocking targets is used, it may lead to insufficient background suppression for another crop. Therefore, this embodiment explicitly limits the applicable host to potato or rapeseed and constructs different blocking panels for their respective high abundance nucleic acids. Specifically, in the potato scenario, in addition to ribosomal RNA, starch synthesis-related transcripts often maintain a high background in leaf, stem, or tuber samples. Therefore, the blocking panel preferentially covers 18S rRNA, 28S rRNA, and starch synthase-related high-expression regions. For rapeseed samples, since lipid formation and storage-related transcripts often show high abundance in specific tissues or disease stages, the blocking panel, in addition to ribosomal RNA, can increase the target sites of lipid synthesis-related or oil body protein-related high-expression transcripts. This maintains the same biochemical principle while matching the blocked targets with the crop's background expression profile. To clarify this through a simplified model, if the platform processes two batches of samples: the first batch of potato leaf late blight samples and the second batch of rapeseed stem lesion samples; if the potato blocking panel is mistakenly used directly for rapeseed samples, even if ribosomal RNA decreases, rapeseed-specific high-abundance lipid synthesis-related transcripts will still occupy reverse transcription resources, resulting in a low proportion of effective reads from pathogens; conversely, by using potato panels and rapeseed panels respectively, the former can significantly suppress starch synthesis-related background, and the latter can significantly suppress lipid synthesis-related background, restoring the effective introduction window of pathogens to a similar level; In special circumstances, if the sample origin is unknown and it is impossible to determine in advance whether the background is mainly related to starch synthesis or lipid synthesis, a small-scale pre-scan can be performed first, and the appropriate panel can be selected based on the composition of the highly expressed host transcripts. If the same sample contains mixed sources such as roots, leaves and reproductive tissues, multiple host high-abundance target probes can be loaded in combination, but the total probe concentration must be controlled to avoid unexpected competition for the pathogen template. If the target is not a potato or rapeseed host, this panel should not be applied directly, but a chimeric blocking probe corresponding to the host's high-abundance nucleic acid should be redesigned according to the same idea. In the same agricultural pathogen monitoring platform, the spring batch mainly processed potato late blight leaf samples, and the blocking targets were mainly ribosomal RNA and starch synthesis-related transcripts; the autumn batch added rapeseed sclerotinia stem samples, and the platform switched the blocking targets to ribosomal RNA and lipid synthesis-related transcripts; both batches of samples used the same single-tube reverse transcription and UMI introduction process, and only the host blocking panel was adjusted to maintain a high effective interaction signal-to-noise ratio in different host backgrounds; The purpose of this mechanism is to extend the same blocking-reverse transcription-UMI mounting principle to high-abundance background conditions in different plant hosts, thereby achieving stable implementation of the solution in potato and rapeseed scenarios.

[0025] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for analyzing the expression of plant-pathogen interaction genes based on UMI tags, characterized in that, include: Obtain total nucleic acid extracts from plant-pathogen interaction samples; The extract, host-specific chimeric blocking probe, and reverse transcription primers were mixed to construct a molecular hybridization reaction system. The probe contained locked nucleic acid modified structure and 3' dideoxynucleotide modified structure. The system was subjected to heating to break down the molecules and cooling to anneal them, so that the probe could bind to the host's abundant nucleic acid molecules to form a hybridization blocking complex, and the reverse transcription primers could bind to the pathogen's nucleic acid molecules. The template-switching oligonucleotide with a unique molecular identifier (UMI) sequence and reverse transcriptase were added to the system; the reverse transcriptase had terminal transferase activity and template switching activity; the 3' end of the template-switching oligonucleotide contained a nucleotide sequence segment that paired with a non-template polycytosine tail. The reverse transcriptase is used to initiate an enzymatic polymerization and extension reaction, which causes the reverse transcriptase to extend along the pathogen nucleic acid molecule and add a non-template polycytosine tail to the end of its complementary strand. Based on the steric hindrance of the complex, the polymerization and extension of the host high-abundance nucleic acid molecule is blocked. Based on template-converting oligonucleotides, a unique molecular identifier sequence is introduced into the complementary strand of the pathogen nucleic acid molecule to generate an interaction expression library. High-throughput sequencing was performed on the library to obtain a set of sequencing reads; Extract the number of valid reads that carry unique molecular identifier sequences and belong to pathogenic nucleic acid molecules from this set, and the number of invalid reads that belong to high-abundance host nucleic acid molecules; When both the number of valid reads and the number of invalid reads are greater than zero, the effective interaction signal-to-noise ratio (SNR) is calculated by dividing the number of valid reads by the number of invalid reads. When both the number of valid reads and the number of invalid reads are equal to zero, the maximum preset SNR value representing a background below the sequencing detection limit is output as the effective interaction signal SNR. When the number of valid reads is equal to zero, the effective interaction signal SNR is zero. The specific formula for calculating the effective interaction signal SNR is as follows: ,in To the number of valid segments read, Invalid segment reads The expected value of background free nucleic acid noise. and This is a non-negative correction coefficient set based on sequencing depth.

2. The method for analyzing plant-pathogen interaction gene expression based on UMI tags according to claim 1, characterized in that, Before the step of constructing the molecular hybridization reaction system by mixing the extract, the host-specific chimeric blocking probe, and the reverse transcription primer, the following steps are also included: Obtain high-abundance nucleic acid sequences from the host; Design complementary nucleic acid sequences for the host with high abundance of nucleic acid sequences; The ribosomal loop of the complementary nucleic acid sequence is methylene-bridged and locked to generate the locked nucleic acid modified structure; the substitution rate of the locked nucleic acid modified structure in the host-specific chimeric blocking probe is... Satisfying the formula: ,in To lock the number of nucleotides modified by nucleic acid, The total nucleotide length of the probe is defined as follows: ; The 3' end of the complementary nucleic acid sequence is dehydroxylated to generate the 3' dideoxynucleotide modified structure, thus obtaining the host-specific chimeric blocking probe.

3. The method for analyzing plant-pathogen interaction gene expression based on UMI tags according to claim 2, characterized in that, The steps of heating to dechain and cooling to anneal the system specifically include: Based on the base sequence, nucleotide length, and locked nucleic acid modification structure of the host-specific chimeric blocking probe, the probe annealing temperature was calculated; the probe annealing temperature The calculation model is as follows: ,in For hybridization enthalpy change, For hybridization entropy change, Let be the ideal gas constant. The probe concentration, The temperature compensation constant resulting from single-base locked nucleic acid modification; The primer annealing temperature is calculated based on the nucleotide composition of the reverse transcription primer, wherein the probe annealing temperature is greater than the primer annealing temperature; The system is controlled to cool down to the probe annealing temperature after the heating and de-milking process, thereby promoting the binding of the probe to the host high-abundance nucleic acid molecules; The system is further cooled to the primer annealing temperature to induce the reverse transcription primer to bind to the pathogen nucleic acid molecule.

4. The method for analyzing plant-pathogen interaction gene expression based on UMI tags according to claim 1, characterized in that, The step of using reverse transcriptase to initiate an enzymatic polymerization and elongation reaction, and blocking the polymerization and elongation of high-abundance host nucleic acid molecules based on the steric hindrance of the complex, specifically includes: In the enzyme-catalyzed polymerization and extension reaction, the reverse transcriptase polymerizes and extends along the pathogenic nucleic acid molecule to generate a complementary strand of the pathogenic nucleic acid molecule; When the reverse transcriptase polymerizes and elongates along the host's abundant nucleic acid molecule and encounters the hybridization blocking complex, the polymerization and elongation of the reverse transcriptase is terminated and it detaches from the template due to the high affinity steric hindrance generated by the locked nucleic acid modification structure and the dehydroxylation chain-breaking effect caused by the 3' dideoxynucleotide modification structure, thereby blocking the reverse transcription process of the host's abundant nucleic acid molecule; the blocking efficiency of the termination of polymerization and elongation is... Binding energy subject to high affinity steric hindrance Regulation, when the binding energy When the dehydroxylation chain scission effect and steric hindrance work together to achieve absolute blocking, it is determined that the dehydroxylation chain scission effect and steric hindrance work together to achieve absolute blocking.

5. The method for analyzing plant-pathogen interaction gene expression based on UMI tags according to claim 1, characterized in that, The introduction of a unique molecular identifier sequence into the complementary strand of a pathogenic nucleic acid molecule based on template-conversion oligonucleotides specifically includes: In the enzymatic polymerization extension reaction, the reverse transcriptase extends along the pathogen nucleic acid molecule to the 5' end of the pathogen nucleic acid molecule and adds a non-template polycytosine tail to the end of the complementary strand of the pathogen nucleic acid molecule; The template-converting oligonucleotide is used to pair and bind with the non-template polycytosine tail; The reverse transcriptase is guided to perform a template switching operation, transcribing the unique molecular identifier sequence into the complementary strand of the pathogen's nucleic acid molecule; in this template switching operation, the effective switching probability of the template-converting oligonucleotide is... The length of the non-template polycytosine tail Establish a connection, and when The process of activating the transcription into the complementary strand of the pathogen's nucleic acid molecule.

6. The method for analyzing plant-pathogen interaction gene expression based on UMI tags according to claim 1, characterized in that, Following the step of generating the interaction expression library, the method further includes: The fragment distribution map of the total nucleic acid extract was obtained by a nucleic acid electrophoresis analysis platform, and the nucleic acid integrity index was used as a unified evaluation criterion; the first nucleic acid integrity value of the total nucleic acid extract before constructing the molecular hybridization reaction system was extracted. Extract the second nucleic acid integrity value of the library nucleic acid molecules after generating the interaction expression library; Compare the integrity values ​​of the first nucleic acid and the second nucleic acid; When the difference between the first nucleic acid integrity value and the second nucleic acid integrity value is less than or equal to a preset degradation threshold, it is determined that the pathogenic nucleic acid molecule has not undergone non-specific physical degradation in the enzymatic polymerization extension reaction and a normal state marker is generated; when the difference between the first nucleic acid integrity value and the second nucleic acid integrity value is greater than the preset degradation threshold, it is determined that the pathogenic nucleic acid molecule has undergone non-specific physical degradation and a reaction system optimization prompt data package is generated.

7. The method for analyzing plant-pathogen interaction gene expression based on UMI tags according to claim 6, characterized in that, After determining that the pathogenic bacterial nucleic acid molecule has not undergone non-specific physical degradation in the enzymatic polymerization extension reaction, the method further includes: The interaction expression library was subjected to real-time quantitative polymerase chain reaction (qPCR) detection to obtain the first cycle threshold of the host high-abundance nucleic acid molecules and the second cycle threshold of the pathogen nucleic acid molecules; The fluorescence quantitative polymerase chain reaction detection data of the control library were obtained. The control library was constructed using homologous samples without the addition of the host-specific chimeric blocking probe. The third cycle threshold corresponding to the host high-abundance nucleic acid molecules and the fourth cycle threshold corresponding to the pathogen nucleic acid molecules were extracted from the control library. When the first cyclic threshold is greater than the third cyclic threshold, and the absolute value of the difference between the second cyclic threshold and the fourth cyclic threshold is less than or equal to a preset error threshold, the background suppression effectiveness of the interaction expression library is confirmed and a suppression success label is generated; otherwise, the background suppression of the interaction expression library is confirmed to be invalid and a suppression failure label is generated.

8. The method for analyzing plant-pathogen interaction gene expression based on UMI tags according to claim 3, characterized in that, After the step of calculating the signal-to-noise ratio of the effective interactive signal output, the method further includes: Extract the set of unique molecular identifier sequences from sequencing reads that carry the unique molecular identifier sequence and belong to the nucleic acid molecule of the pathogen; The molecular collision rate is calculated by counting the number of repeating sequences in the unique molecular identifier sequence set and dividing that number by the total number of sequences in the unique molecular identifier sequence set. The theoretical baseline upper limit model is as follows: ,in The effective base length of the unique molecular identifier sequence. This is an estimate of the true original total number of nucleic acid molecules belonging to this pathogen; When the molecular collision rate is lower than the preset collision threshold, the pathogen signal separation of the interaction expression library is deemed qualified and a qualified data tag is output; when the molecular collision rate is greater than or equal to the preset collision threshold, the pathogen signal separation of the interaction expression library is deemed unqualified and a calibration operation command for the probe annealing temperature is triggered.

9. The method for analyzing plant-pathogen interaction gene expression based on UMI tags according to claim 1, characterized in that, The plant hosts in the plant-pathogen interaction samples include potatoes or rapeseed, and the host's high-abundance nucleic acid molecules include ribosomal nucleic acids, starch synthesis-related transcripts, or lipid synthesis-related transcripts.

Images