Design of multiplex amplicon primers and its application in detection of metastasis-related gene expression profile in nasopharyngeal carcinoma

Abstract

The present application relates to the technical field of molecular diagnosis, in particular to a multiplex amplicon primer design method in multiplex amplicon targeted sequencing, a primer combination and application thereof in detection of nasopharyngeal carcinoma metastasis related gene expression profile. The present application realizes reliable design of multiplex amplicon primer and obtains a primer system through a three-level progressive strategy of "calculation design-conflict screening-experiment verification", thereby realizing effective prediction of nasopharyngeal carcinoma metastasis risk.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics technology, specifically providing a method for designing multiplex amplicon primers for multiplex amplicon targeted sequencing, corresponding primer compositions, and their application in detecting gene expression profiles related to nasopharyngeal carcinoma metastasis. Background Technology

[0002] Currently, quantitative gene expression detection mainly relies on the following technology platforms: Real-time quantitative PCR (qRT-PCR) is the gold standard, with high sensitivity and wide dynamic range, but its throughput is limited, and the number of genes detected in a single run is usually no more than a few; Microarrays can detect tens of thousands of genes at once, but they rely on pre-designed probes, have blind spots in the detection of new transcripts, and are difficult to standardize between different batches; Whole transcriptome sequencing (RNA-seq) can detect all transcripts without bias and has become the benchmark technology for transcriptome research, but it requires a high starting amount for library construction (usually 1-4 μg of total RNA), the experimental procedure is complex (wet experiments take 1-2 days), the data analysis computation is large, and the cost per sample is high (about 3000-6000 yuan / sample), making it unsuitable for large-scale clinical testing scenarios; NanoString nCounter technology is based on direct hybridization detection using fluorescent barcode-labeled probes, which eliminates the need for reverse transcription and amplification steps, thus eliminating PCR amplification bias. It has the advantages of simple operation, low starting amount, and good reproducibility, and is widely used in gene expression panel detection. However, its reagents are expensive (more than 4,000 yuan per sample) and rely on specialized detection equipment, limiting its large-scale application in primary healthcare settings. The correlation between NanoString and RNA-seq expression quantification has been reported in the literature, with an R² of approximately 0.796.

[0003] Multiplex amplicon targeted sequencing (such as Thermo Fisher AmpliSeq and Illumina AmpliSeq compatible protocols) uses multiplex PCR to target and enrich genomic regions before sequencing. Since its introduction, this technology has primarily been applied to DNA-level detection scenarios such as somatic mutation detection (e.g., tumor hotspot mutation panels), pathogen identification, and genetic disease gene panel screening. In these applications, the detection target is a qualitative or semi-quantitative judgment such as "presence / absence" or "mutant / wildtype," and the requirements for amplification linearity are relatively relaxed. In recent years, this technology has begun to be explored for quantitative detection of gene expression at the RNA level. Compared with the aforementioned traditional methods, multiplex amplicon targeted sequencing for expression quantification has the following theoretical advantages: extremely low starting amount (1-10 ng RNA is sufficient for library construction, significantly lower than the 1-4 μg required for RNA-seq), simple experimental procedure (wet experiment 4-7 h), rapid data analysis (<30 min), and controllable cost per sample (approximately 800-1000 RMB / sample). These characteristics make it highly promising for large-sample clinical gene expression panel detection.

[0004] However, multiplex amplicon targeted sequencing was not originally designed for RNA expression quantification, and directly transplanting it to the expression quantification scenario presents the following fundamental problems.

[0005] 1) Lack of a systematic multiplex primer design and screening system. Existing primer design is mainly aimed at DNA mutation detection, using "whether the target region is amplified" as the criterion. However, expression quantification requires primers to meet the following more stringent conditions: a) High trial-and-error costs due to cross-interference: In multiplex PCR, the number of primer pairs increases, and cross-interference increases quadratically; b) Lack of selection rules in design strategies: RNA templates involve three strategies: cross-exon, junction, and unrestricted freedom. No priority rules have been reported in the literature for multiplex systems.

[0006] 2) Lack of a multi-dimensional comprehensive evaluation system. Existing evaluation systems mainly rely on single indicators (such as whether the target region was successfully amplified or the sequencing coverage), which cannot comprehensively reflect the performance of the expression quantification system. To meet the needs of RNA expression detection, there is an urgent need to establish evaluation indicators. Summary of the Invention

[0007] To address the aforementioned technical challenges, this invention employs a three-tiered progressive strategy of "computational design—conflict screening—experimental verification" to achieve the systematic design of multiplex amplicon primers. The nasopharyngeal carcinoma metastasis risk prediction panel primer combination designed based on this strategy showed high consistency with the expression profiles constructed by next-generation sequencing, RNA-seq (R2=0.92), and NanoString platform (R2=0.89), providing a reliable detection tool for assessing the metastasis risk of nasopharyngeal carcinoma patients.

[0008] Specifically, the detailed technical solution of this invention is as follows.

[0009] This invention first provides a method for designing multiplex amplicon primer compositions, the method comprising the following steps: Step 1) Candidate primer pair design: For N target genes, design multiple sets of candidate primer pairs independently, and mix the multiple sets of candidate primer pairs for all target genes to form a complete set of candidate primer pairs M; Step 2) Primer screening based on conflict matrix: Set M contains candidate primer pairs for all target genes, totaling m sets of candidate primer pairs; construct primer dimer conflict matrix, amplification region competition matrix and comprehensive conflict matrix, and obtain a set of multiplex PCR compatible primers through conflict elimination strategy; Step 3) Primer performance verification and evaluation: Integrate the selected primer sequences into sequencing adapters, construct a multiplex PCR amplification system for target region amplification, and perform high-throughput sequencing on the amplification products after library construction; perform quality control and primer source identification on sequencing data, and evaluate and screen candidate primers based on target amplification rate and primer combination crosstalk rate; preferably, for the same target gene containing multiple candidate primer pairs, after removing abnormal primers, sort and select the remaining candidate primers according to the principle of priority for target amplification rate and lowest primer crosstalk rate.

[0010] In some respects, in step 1), the candidate primer pair design rules satisfy the following conditions: The primer annealing temperature is 60±2℃; The length of the amplified product is 150±5bp; The primer pairs do not form dimers or hairpin structures; The primer sequence contains ≤1 SNP site; There were no multiple alignments when the primers were aligned back to the reference genome. Primer pairs designed across exons are given priority. If there are fewer than 3 primer pairs that meet the above conditions for a target gene, primer pairs that do not cross exons are included.

[0011] In some aspects, step 2) specifically involves: primer screening based on the conflict matrix: set M contains candidate primer pairs for all target genes, totaling m candidate primer pairs; matrix modeling is performed to obtain primer dimer conflict matrix, amplification region competition matrix and comprehensive conflict matrix for possible amplification conflicts, dimer conflicts and spatial competition relationships between candidate primer pairs, and the final set of multiplex PCR compatible primers is obtained through conflict elimination strategy.

[0012] Furthermore, the construction of the primer dimer conflict matrix includes: calculating the forward and reverse dimer free energies (ΔG) of any two sets of candidate primer sequences in turn, and taking the lowest value as the risk score; if the lowest ΔG ≤ -4.5 kcal / mol, a conflict is determined and recorded as 1, otherwise it is recorded as 0; accordingly, after evaluating each pair of m sets of primers, an m×m dimer conflict matrix is ​​generated.

[0013] Furthermore, the construction of the amplification region competition matrix includes: sequentially aligning each candidate primer of the same target gene to the target transcript reference sequence, obtaining the transcript position of each amplicon, and calculating the amplification region interval between adjacent primer pairs; if the interval is < 1000 bp, it is determined that there is a risk of regions being too close, and is recorded as 1; otherwise, it is recorded as 0; for any primer pair between different target genes, it is directly recorded as 0; accordingly, an m×m amplification region competition matrix is ​​generated.

[0014] Furthermore, the comprehensive conflict matrix is ​​constructed by summing the dimer conflict matrix and the amplified region competition matrix to generate the comprehensive conflict matrix.

[0015] Furthermore, the conflict logic elimination strategy includes: setting a minimum number of candidate primers to be retained for each target gene as one, ensuring that each target gene has usable detection primers; calculating the conflict score of each candidate primer based on the comprehensive conflict matrix, with higher scores indicating lower priority; and performing iterations in the following loop: each time, the candidate primer with the highest current conflict score is eliminated first. If, after elimination, the target gene containing that primer still retains at least one primer, then elimination is performed and the next round begins; otherwise, the primer is retained, and the next highest conflict primer is evaluated; the above process is repeated until the conflict scores of all primers in the set are zero, thus obtaining the optimal primer combination without conflict under the premise that at least one primer is retained for each target gene.

[0016] In some aspects, step 3) specifically includes: primer performance verification and evaluation: integrating the selected primer sequences into sequencing adapters, constructing a multiplex PCR amplification system for target region amplification, and performing high-throughput sequencing on the amplification products after library construction; quality control and primer source identification of sequencing data, and evaluating and screening candidate primers based on target amplification rate and primer combination crosstalk rate; preferably, for cases where the same target gene contains multiple candidate primer pairs, after removing abnormal primers, the remaining candidate primers are sorted and selected according to the principle of prioritizing target amplification rate and minimizing primer crosstalk rate.

[0017] Furthermore, the quality control and primer source identification include: performing quality control on the raw sequencing data; aligning valid sequencing reads to the target region and whole-genome reference sequence; extracting primer sequences based on the sequences at both ends of the reads; matching them with the designed primer set; and identifying forward primers, reverse primers, and reverse complementary sequences; and determining the amplification source corresponding to each sequencing read based on the primer combinations matched at both ends of the reads.

[0018] Furthermore, the target amplification rate is calculated as follows: , Where Ntarget,i is the number of reads correctly amplified by candidate primer pair i and located in the target region; Nprimer,i is the sum of all reads containing upstream or downstream primers in primer pair i.

[0019] Furthermore, the primer crosstalk rate is calculated as follows: , Where Ncross,i is the number of amplified reads generated by different primer pair combinations; Ncorrect,i is the number of reads generated by the correct primer pair combination.

[0020] Furthermore, the primers target the following genes: ANXA1, CBR3, CLASP1, CXCL10, DCTN1, FNDC3B, GRM4, HDLBP, LRIG1, POLR2M, RPL19, WNK1, WSB2, YBX3, ACTB, B2M, RPLP0, and PGK1. Furthermore, the specific sequences of the primers are shown in SEQ ID NO.1-36.

[0021] The present invention also provides a primer composition for multiplex amplicon, wherein the primer composition is obtained by any of the design methods described above; Furthermore, the primers target the following genes: ANXA1, CBR3, CLASP1, CXCL10, DCTN1, FNDC3B, GRM4, HDLBP, LRIG1, POLR2M, RPL19, WNK1, WSB2, YBX3, ACTB, B2M, RPLP0, and PGK1. Furthermore, the specific sequences of the primers are shown in SEQ ID NO.1-36.

[0022] The present invention also provides a detection product comprising any of the primer compositions described above.

[0023] The present invention also provides the use of any of the primer compositions described above in multiplex amplicon targeted sequencing; or in the preparation of multiplex amplicon targeted sequencing reagents.

[0024] The present invention also provides the application of any of the primer compositions described above in the detection of nasopharyngeal carcinoma metastasis-related gene expression profiles; or in the preparation of reagents for the detection of nasopharyngeal carcinoma metastasis-related gene expression profiles.

[0025] The present invention also provides a multiplex amplicon targeted sequencing method, comprising performing multiplex targeted amplification on a sample using any of the aforementioned primer compositions, and sequencing the amplified products after library construction.

[0026] Beneficial technical effects of the present invention: 1) This invention improves primer compatibility and stability in multiplex amplification systems. This invention designs multiple sets of candidate primers for multiple target genes and, by constructing a cross-dimer conflict matrix between candidate primers and an amplification region competition analysis model, screens and eliminates primer combinations with potential mutual interference, reducing non-specific binding, competitive amplification, and amplification bias among primers, thereby improving the stability of the multiplex PCR system.

[0027] 2) This invention establishes an experimental feedback optimization method based on primer source identification for sequencing reads, improving detection specificity and reliability. By identifying and tracing the primer sequences at both ends of sequencing reads, this invention can accurately determine the source of primer combinations for each type of amplification product. Based on this, evaluation indicators such as TargetRate and CrossRatio are established to quantitatively evaluate the actual amplification performance of candidate primers. This allows for the identification and elimination of abnormal primers with significant crosstalk or non-specific amplification, realizing the transformation from theoretical design to experimental data feedback optimization. This effectively increases the proportion of target amplification products, reduces detection background, and improves the accuracy and reliability of primer screening for RNA expression detection.

[0028] 3) This invention is applicable to the construction of panels for multi-gene RNA expression detection, improving detection flexibility and application scope. By setting multiple candidate primers for the same target gene and ranking and optimizing them based on actual amplification performance, the optimal detection primer can be effectively selected. This method is applicable to the construction of multiplex amplicon targeted sequencing panels for different numbers of target genes, improving the accuracy, stability, and reproducibility of RNA expression detection results.

[0029] 4) This invention provides a set of primer compositions specifically for nasopharyngeal carcinoma metastasis risk assessment, enabling high-performance detection based on next-generation sequencing platforms. Based on primer design methods, this invention develops primer combinations adapted to multiplex amplicon next-generation sequencing platforms targeting a set of characteristic genes associated with nasopharyngeal carcinoma metastasis. Compared to existing detection products based on the Nanostring platform, this invention significantly reduces the amount of starting RNA template used while maintaining detection accuracy, simplifies the operation process, and achieves parallel detection of more samples in a single run through the higher data throughput of the next-generation sequencing platform. This effectively reduces the cost per sample, providing a more accessible and scalable molecular detection solution for nasopharyngeal carcinoma metastasis risk assessment. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 Example 2: Primer dimer conflict matrix constructed.

[0032] Figure 2 Example 2: The amplified region competition conflict matrix constructed.

[0033] Figure 3 Example 2: The comprehensive conflict matrix constructed.

[0034] Figure 4 Scatter plot results of the 18-primer multiplex amplicon expression method of this invention vs. the transcriptome RNAseq method.

[0035] Figure 5 Scatter plot results of the 18-primer multiplex amplicon expression method of this invention versus the Nanostring method.

[0036] Figure 6 The triple-replication scatter plot and CV distribution of the standard UHRR.

[0037] Figure 7 Triple-replica scatter plot and CV distribution of clinical sample MixS1.

[0038] Figure 8 Triple-replica scatter plot and CV distribution of clinical sample MixS2. Detailed Implementation

[0039] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. Furthermore, the examples described are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] Some terminology is defined as follows: Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0041] The term "approximately" in this invention refers to an accuracy range that, as would be understood by those skilled in the art, still guarantees the technical effects of the features in question. This term typically indicates a deviation from the indicated value of ±10%, preferably ±5%.

[0042] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0043] Furthermore, the terms first, second, third, (a), (b), (c), and similar terms used in the specification and claims are for distinguishing similar elements and are not necessary for the order of description or chronological sequence. It should be understood that such terms are interchangeable in appropriate contexts, and the embodiments described in this invention can be implemented in a different order than that described or illustrated in this invention.

[0044] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0045] Example 1: Establishment of a Multiple Primer Design System Based on previous experimental experience, this invention has designed a three-level progressive strategy of "computational design - conflict screening - experimental verification" to systematically complete the design and screening of multiplex PCR primer pairs for RNA expression quantification.

[0046] 1. Candidate primer pair design Candidate primer pair design: For N target genes, multiple sets of candidate primer pairs are designed independently for each gene. These sets are then combined to form a complete candidate primer pair set M. Primer design for each target gene follows these constraints: Primer annealing temperature: 60±2℃; The length of the amplification product was controlled at 150±5bp. The primer pairs do not form dimers or hairpin structures; The primer sequence contains ≤1 SNP site; There were no multiple alignments when the primers were aligned back to the reference genome. Primer pairs designed across exons are given priority. If a gene has fewer than three primer pairs that meet the above criteria, primer pairs that do not cross exons may be included.

[0047] 2. Primer screening based on conflict matrix Set M contains candidate primer pairs for all target genes, totaling m candidate primer pairs. Matrix modeling is used to address potential amplification conflicts, dimer conflicts, and spatial competition among candidate primer pairs, and a conflict elimination strategy is employed to obtain the final set of multiplex PCR compatible primers.

[0048] 1) Construction of primer dimer conflict matrix To assess the risk of cross-dimerization between primers, the forward and reverse (original and reverse complementary) dimer free energies (ΔG) of any two candidate primer sequences are calculated sequentially, and the lowest value is taken as the risk score. If the lowest ΔG ≤ -4.5 kcal / mol, a conflict is identified and recorded as 1; otherwise, it is recorded as 0. Based on this, after pairwise evaluation of m primer groups, an m×m dimer conflict matrix is ​​generated.

[0049] 2) Construction of expanded regional competition matrix To assess the risk of amplification region competition among candidate primers, each candidate primer for the same target gene was sequentially aligned to the target transcript reference sequence to obtain the transcript position of each amplicon, and the amplification region interval between adjacent primer pairs was calculated. If the interval was < 1000 bp, a risk of regions being too close together (which may lead to competition in multiplex PCR amplification, template consumption, and decreased efficiency) was identified and recorded as 1; otherwise, it was recorded as 0. For any primer pair between different target genes, it was directly recorded as 0. Based on this, an m×m amplification region competition matrix was generated.

[0050] Summing the dimer conflict matrix and the amplified region competition matrix above generates a comprehensive conflict matrix. 3) Conflicting primer logical elimination strategy An iterative conflict elimination strategy was employed to optimize the candidate primer set. First, a minimum of one candidate primer was set for each target gene to ensure that each target gene had a usable detection primer. Then, a conflict score was calculated for each candidate primer based on a comprehensive conflict matrix. This score reflects the overall degree of conflict with other primers in the set; a higher score indicates a lower priority. The iterative process was executed cyclically as follows: The candidate primer with the highest current conflict score was eliminated first. If, after elimination, the target gene containing that primer still retained at least one primer, the elimination was performed and the process moved to the next round; otherwise, the primer was retained, and the next primer with the second highest conflict score was evaluated. This process was repeated until the conflict scores of all primers in the set were zero, thus obtaining the optimal conflict-free primer combination while ensuring that at least one primer was retained for each target gene.

[0051] 3. Primer performance verification and evaluation The final primer sequences obtained after screening were integrated with sequencing adapters to construct a multiplex PCR amplification system. The target region was amplified using the final primer pool, and the amplified products were then sequenced in high-throughput libraries. Quality control and alignment analysis were performed on the sequencing data. The origin of the amplified products was traced based on the primer sequence information at the ends of the sequencing reads. Candidate primers were analyzed based on the target amplification rate and primer combination crosstalk rate to evaluate the specificity, compatibility, and amplification uniformity of different primer pairs in the multiplex amplification system.

[0052] The specific evaluation process is as follows: 1) Sequencing data quality control and primer source identification Quality control was performed on the raw sequencing data, filtering out low-quality reads, reads with poor alignment quality, and abnormal alignment results. Valid sequencing reads were aligned to the target region and whole-genome reference sequence. Primer sequences were extracted from the sequences at both ends of the reads and matched with the designed primer set. To improve primer identification accuracy, a single base mismatch was allowed in the primer sequence, and the following were simultaneously identified: forward primer, reverse primer, and reverse complementary sequence. Based on the primer combinations matched at both ends of the reads, the amplification source corresponding to each sequencing read was determined.

[0053] 2) Target amplification rate The target amplification rate is used to evaluate whether the primer pair can correctly amplify the designed target region, reflecting the primer's target amplification ability; primer design specificity; and the proportion of the target product in the multiplex PCR system.

[0054] , Where: Ntarget,i is the number of reads correctly amplified by candidate primer pair i and located in the target region; Nprimer,i is the sum of all reads containing upstream or downstream primers in primer pair i.

[0055] 3) Primer crosstalk rate This indicator measures the occurrence of incorrect primer pair combinations in a multiplex PCR system. It is used to evaluate whether there is competition between primers, whether primers are incorrectly paired, and the compatibility of the multiplex PCR system.

[0056] , Where: Ncross,i is the number of amplified reads generated by different primer pair combinations; Ncorrect,i is the number of reads generated by the correct primer pair combination.

[0057] 4) Selection and elimination of abnormal primers The experimental performance of candidate primers was evaluated based on two metrics: target amplification rate and primer combination crosstalk rate. Abnormal primers with insufficient target amplification capacity or significant primer combination crosstalk were eliminated according to preset evaluation thresholds. For cases where multiple candidate primer pairs exist for the same target gene, after eliminating abnormal primers, the remaining candidate primers were ranked according to the principle of prioritizing target amplification rate and then selecting the primer pair with the best overall performance for final detection.

[0058] Example 2: Design of multiplex amplicon primer sequences This embodiment describes primer design and performance verification for a laboratory-developed panel for predicting the recurrence risk of nasopharyngeal carcinoma.

[0059] 1. Candidate primer design Our laboratory developed its own panel for predicting the recurrence and metastasis risk of nasopharyngeal carcinoma, which includes 18 target genes (see Table 1 for details). This embodiment strictly followed the protocol established in Example 1 for amplicon primer design. In the initial design phase, only one primer pair was designed for genes CXCL10 and RPL19; for each of the other genes, 2-4 candidate primer pairs were designed, resulting in a total of 50 primer pairs. Subsequently, the primer dimer conflict matrix and the amplification region competition conflict matrix were constructed and summed (see Table 1). Figure 1-3 Using a conflict primer logic elimination strategy, the 25 candidate primer pairs in Table 2 were ultimately filtered and retained for subsequent experimental evaluation.

[0060] 2. Primer screening and optimization Twenty-five primer pairs, selected through calculation and screening, were integrated with sequencing adapters at both ends and synthesized to construct a multiplex PCR amplification system. The final primer pool was used for target region amplification, and the amplified products were then used for library construction and high-throughput sequencing. Quality control and alignment analysis were performed on the sequencing data, and the origin of the amplified products was traced based on the primer sequence information at the ends of the sequencing reads to evaluate the specificity, compatibility, and amplification uniformity of different primer pairs in the multiplex amplification system. Evaluation indicators included: target amplification ratio (≥90%) and primer crosstalk ratio (<5%). Table 1 shows the measured results of the 25 candidate primer pairs validated by NGS sequencing.

[0061] Table 1 shows the target amplification rate and primer crosstalk rate results for 25 pairs of candidate primers.

[0062] Primer UP_06_FNDC3B was eliminated based on quality control standards; for the remaining target genes, when multiple candidate primer pairs were available, the pair with the best overall performance was selected: the pair with the highest priority targeting rate and the lowest secondary priority crosstalk rate. Ultimately, this invention retained one pair of preferred primers for each target gene, identifying a total of 18 primer pairs for subsequent expression assays. The specific primer sequences are shown in Table 2.

[0063] Table 2. The 18 identified primer pairs and their sequences

[0064] Example 3: Performance and repeatability verification of multiplex amplicon primers 1. Primer performance verification To verify the quantitative accuracy of the primer system established by the method of this invention, this embodiment used the RNA standard UHRR as the test sample. The sample was amplified using 18 pairs of primer sequences obtained through screening, and the entire multiplex amplicon sequencing and analysis were completed. Finally, the results were compared with those of RNAseq whole transcriptome sequencing and NanoString nCounter detection.

[0065] The specific sequencing results are shown in Table 3. It can be seen that the method of this invention obtained stable amplification signals for each target region, with an average targeting rate of 99.78% and an average crosstalk rate of only 0.22%. Among them, 17 genes achieved targeting rates exceeding 99%, with the highest reaching 99.98%, indicating that this method has excellent amplification specificity and multiplexing capability. Further comparison with RNA-seq and NanoString detection results revealed that the detection trends for high, medium, and low expression genes remained consistent. Effective sequencing signals could still be obtained for low-abundance transcripts (such as CXCL10, FPKM=1.03), showing that this method has higher target enrichment capability and lower low-abundance detection sensitivity compared to traditional RNA expression detection.

[0066] Table 3. Results of multiplex amplicon detection of 18 target genes and results of comparison methods.

[0067] According to statistics, a comparison of the results of this invention with RNAseq sequencing and NanoString nCounter can be found in [link to documentation]. Figure 4-5 It can be seen that the correlation coefficient R between the multiplex amplicon sequencing quantitative results based on the 18 pairs of primers of this invention and the expression levels of traditional RNAseq is... 2 =0.92, the correlation coefficient R with the detection results of the NanoString platform is 0.92. 2 =0.89. This result demonstrates that the 18 primer pairs of this invention maintain excellent quantitative performance in multiplex amplification systems, and also verifies the effectiveness and reliability of the computational compatibility screening strategy and NGS verification process established in this invention.

[0068] 2. Repeatability verification To evaluate the reproducibility of this method, 18 pairs of preferred primers were used. In this embodiment, three independent reproducible libraries were constructed and sequenced for each of the three samples: RNA standard UHRR and clinical samples (MixS1 and MixS2) (a total of 9 experiments). The quality control of the sequencing data was normal, as shown in Table 4. The targeting rate was 95-97%, the crosstalk rate was <0.3%, the alignment rate was >99%, and the Q30 was >95.7%. All indicators were highly stable among the three reproducibles within the samples, confirming the reliability and comparability of the sequencing data.

[0069] Table 4. Targeting rate, crosstalk rate, alignment rate, and Q30 data from three independent replicates of three samples.

[0070] Further analysis of log2 expression levels in the three replicate experiments was performed, and the mean and coefficient of variation (CV) for each sample were calculated. For details on the correlation and CV results of the three replicate experiments, please refer to [link to relevant documentation]. Figure 6-8And Table 5. It can be seen that the average R² among the three replicates of UHRR is 0.9990, the average R² of MixS1 is 0.9996, and the average R² of MixS2 is 0.9932. The median CV of UHRR is 0.8%, the median CV of MixS1 is 0.4%, and the median CV of MixS2 is 1.4%. These results demonstrate that this method has excellent reproducibility across all expression levels.

[0071] Table 5. Mean and coefficient of variation for each sample

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for designing multiplex amplicon primer compositions, characterized in that, The design method includes: Step 1) Candidate primer pair design: For N target genes, design multiple sets of candidate primer pairs independently, and mix the multiple sets of candidate primer pairs for all target genes to form a complete set of candidate primer pairs M; Step 2) Primer screening based on conflict matrix: Set M contains candidate primer pairs for all target genes, totaling m sets of candidate primer pairs; construct primer dimer conflict matrix, amplification region competition matrix and comprehensive conflict matrix, and obtain a set of multiplex PCR compatible primers through conflict elimination strategy; The construction of the primer dimer conflict matrix includes: calculating the forward and reverse dimer free energies ΔG of any two sets of candidate primer sequences in turn, and taking the lowest value as the risk score; if the lowest ΔG ≤ -4.5 kcal / mol, a conflict is determined and recorded as 1, otherwise it is recorded as 0; accordingly, after evaluating each pair of m sets of primers, an m×m dimer conflict matrix is ​​generated. The construction of the amplification region competition matrix includes: sequentially aligning each candidate primer of the same target gene to the target transcript reference sequence, obtaining the transcript position of each amplicon, and calculating the amplification region interval between adjacent primer pairs; if the interval is <1000 bp, it is determined that there is a risk of regions being too close, and is recorded as 1; otherwise, it is recorded as 0; for any primer pair between different target genes, it is directly recorded as 0; based on this, an m×m amplification region competition matrix is ​​generated. The construction of the comprehensive conflict matrix includes: summing the primer dimer conflict matrix and the amplification region competition matrix to generate the comprehensive conflict matrix; The conflict elimination strategy includes: setting a minimum number of candidate primers to be retained for each target gene as one; calculating the conflict score of each candidate primer based on the comprehensive conflict matrix, with higher scores indicating lower priority; and performing iterations in the following loop: each time, the candidate primer with the highest current conflict score is eliminated first. If, after elimination, the target gene containing that primer still retains at least one primer, then elimination is performed and the next round begins; otherwise, the primer is retained, and the next primer with the second highest conflict score is evaluated; the above loop is repeated until the conflict scores of all primers in the set are zero, thus obtaining the optimal primer combination without conflict under the premise that at least one primer is retained for each target gene. Step 3) Primer performance verification and evaluation: Integrate the selected primer sequences into sequencing adapters, construct a multiplex PCR amplification system for target region amplification, and perform high-throughput sequencing on the amplification products after library construction; perform quality control and primer source identification on sequencing data, and evaluate and screen candidate primers based on target amplification rate and primer combination crosstalk rate; for cases where the same target gene contains multiple candidate primer pairs, after removing abnormal primers, sort and select the remaining candidate primers according to the principle of prioritizing target amplification rate and lowest primer crosstalk rate.

2. The design method according to claim 1, characterized in that, In step 1), the candidate primer pair design rules meet the following conditions: primer annealing temperature is 60±2℃; amplification product length is 150±5bp; primer pairs do not form dimers or hairpin structures; the number of SNP sites in the primer sequence is ≤1; there are no multiple alignments when primers are aligned back to the reference genome; primer pairs designed across exons are preferred. If there are fewer than 3 primer pairs that meet the above conditions for a target gene, non-exon primer pairs are included.

3. The design method according to claim 1, characterized in that, In step 3), The quality control and primer source identification include: performing quality control on the raw sequencing data, aligning valid sequencing reads to the target region and whole genome reference sequence, extracting primer sequences based on the sequences at both ends of the reads, matching them with the designed primer set, and identifying forward primers, reverse primers, and reverse complementary sequences; and determining the amplification source corresponding to each sequencing read based on the primer combinations matched at both ends of the reads. The target amplification rate is calculated as follows: ， Where, N target,i N represents the number of reads correctly amplified by candidate primer pair i and located in the target region; primer,i This is the sum of all reads containing either an upstream or downstream primer in primer pair i; The primer crosstalk rate is calculated as follows: ， Where, N cross,i N represents the number of amplified reads generated by different primer pair combinations. correct,i The number of reads generated for the correct primer combination.

4. A primer composition for multiplex amplicon synthesis, characterized in that, The primer composition is obtained by the design method described in any one of claims 1-3, and the primers target the following genes: ANXA1, CBR3, CLASP1, CXCL10, DCTN1, FNDC3B, GRM4, HDLBP, LRIG1, POLR2M, RPL19, WNK1, WSB2, YBX3, ACTB, B2M, RPLP0, and PGK1.

5. The primer composition according to claim 4, characterized in that, The sequences of the primers are shown in SEQ ID NO.1-36.

6. A testing product, characterized in that, The detection product includes the primer composition according to any one of claims 4-5.

7. The use of the primer composition according to any one of claims 4-5 in the preparation of multiplex amplicon targeted sequencing reagents, or in the preparation of reagents for detecting gene expression profiles related to nasopharyngeal carcinoma metastasis.

8. A multiplex amplicon targeted sequencing method, characterized in that, The method includes using the primer composition described in any one of claims 4-5 to perform multiplex targeted amplification of a sample, followed by sequencing after library construction of the amplified products.

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