Joint primer group compatible with multiple PCR library establishment and application of joint primer group
By optimizing PCR primer design, the problem of sequencing channel inactivation caused by primer multimers was solved, achieving high-quality sequencing data output, which is suitable for multiplex PCR library preparation workflows.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WEST CHINA HOSPITAL SICHUAN UNIV
- Filing Date
- 2026-02-12
- Publication Date
- 2026-05-12
AI Technical Summary
Current PCR primer designs cannot effectively suppress the formation of primer multimers during multiple amplification processes, leading to sequencing channel inactivation and data quality degradation, especially in low-quality cDNA and complex clinical samples.
Design adapter primer sets compatible with multiplex PCR library construction. Optimize primer sets to block the generation of unexpected amplification products by removing complementary bases at the 3' end, extending primer length across complementary regions, and terminating at non-complementary sequences.
It effectively inhibits primer multimer formation, maintains the stability of sequencing wells, and improves sequencing data quality and yield, especially showing stronger applicability and stability in low-quality cDNA and complex clinical samples.
Smart Images
Figure CN122012681A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of biotechnology and molecular biology, and relates to a primer design optimization and verification technique for polymerase chain reaction (PCR), specifically to an adapter primer set compatible with multiplex PCR library construction and its application. Background Technology
[0002] PCR technology holds an irreplaceable core position in the wet sequencing process, its importance primarily manifested in two key applications: first, the efficient and specific addition of sequencing adapters to both ends of DNA fragments during library construction; and second, the effective enrichment of target regions (especially long fragments) to meet the high input volume and concentration requirements of sequencers. Whether it's short-read platforms like Illumina and MGI, or long-read technologies like PacBio and Nanopore, all rely on PCR amplification to amplify weak signals and improve detection sensitivity, becoming a decisive step in scenarios such as single-cell sequencing and low-frequency variant detection. Although amplification may introduce bias or even errors, currently no technology can completely replace PCR in achieving these functions while maintaining high efficiency and low cost. Therefore, PCR remains the most crucial wet experimental bridge connecting raw nucleic acids in samples with high-quality sequencing data.
[0003] To delve into the complexity of the human tumor microenvironment, this study integrated the advantages of single-cell RNA sequencing and Nanopore long-read sequencing technologies, introducing a long-fragment enrichment strategy into the experimental workflow. This process involved multiple rounds of PCR amplification, and to maximize amplification fidelity, the number of cycles in each PCR round was strictly limited. This design aimed to achieve balanced expression levels of all transcripts by precisely enriching transcript fragments of different lengths, thereby providing a high-quality data foundation for comprehensively analyzing cellular heterogeneity and gene expression dynamics in the tumor microenvironment. We found that during Nanopore sequencing, most clinical samples exhibited rapid well inactivation. After excluding conventional factors such as loading volume and voltage, the problem likely stemmed from the sample itself. Especially in experimental workflows requiring multiple PCR amplifications (such as long-fragment screening experiments), even with strict limitation of the number of cycles per round, the probability of primer multimer formation significantly increased with the number of PCR cycles. These multimers are relatively long and difficult to distinguish from the target product during fragment quality control (such as Q-sep), leading to their inclusion in the final sequencing library.
[0004] These multimer structures often contain nucleic acid strand breakpoints (Nick structures), which can cause serious problems during sequencing. On the one hand, Nick structures reduce the stability of DNA strands in nanopores, making them prone to dissociation and significantly reducing the number of effective sequencing wells. On the other hand, the short fragments generated by these breakpoints preferentially enter the well channels and are rapidly sequenced, not only consuming a large amount of sequencing resources but also causing premature inactivation of the sequencing wells. This ultimately manifests as a significant decrease in data yield and a sharp increase in the proportion of invalid reads. Therefore, fundamentally optimizing primer design and completely suppressing the formation of primer dimers and multimers are crucial prerequisites for preventing these problems and improving sequencing success rates and data quality.
[0005] Current conventional primer design methods for sequencing library construction largely rely on general principles to avoid primer dimer formation, such as controlling basic parameters like primer length, GC content, and Tm value, and using software to predict and evaluate primer complementary pairing. These methods typically assume good cDNA quality and do not adequately consider the severe multimer formation and sequencing performance degradation caused by primer structural interactions in low-quality cDNA or complex clinical samples. Existing technologies generally lack systematic analysis and targeted optimization of primer 3' end complementary structures, especially during multiple PCR amplification processes, and cannot effectively suppress rapid well inactivation and data quality degradation caused by primer multimers. Summary of the Invention
[0006] To address the shortcomings of existing technologies, the present invention aims to provide a primer design optimization method for Nanopore sequencing with severe pore inactivation that is compatible with multiple PCR library preparation processes.
[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a set of adapter primers compatible with multiplex PCR library construction, the adapter primer set comprising adapter primer P7-F, adapter primer P5-R, adapter primer P7-R1 and adapter primer P5-TSO.
[0008] In this invention, the sequence lengths of the adapter primers P7-F and P5-R are 30-35 bp, and the sequence lengths of the adapter primers P7-R1 and P5-TSO are 40-48 bp.
[0009] The adapter primers P7-F and P7-R1 contain nucleotide sequences as shown in SEQ ID NO.1.
[0010] The adapter primers P5-R and P5-TSO contain nucleotide sequences as shown in SEQ ID NO.2.
[0011] This invention, through systematic analysis of primers themselves or upstream and downstream primers, discovered the presence of complementary 3' ends. In order to prevent the formation of primer dimers and multimers from the source, the methods adopted in this invention include: removing complementary 3' ends, extending primer length to cross complementary regions, terminating at non-complementary sequences, etc., and designing a new adapter primer set compatible with multiplex PCR library construction, thereby blocking the generation of unexpected amplification products.
[0012] As a preferred embodiment of the present invention, the adapter primer P7-F contains the nucleotide sequence shown in SEQ ID NO.3.
[0013] Preferably, the adapter primer P5-R contains the nucleotide sequence shown in SEQ ID NO.4.
[0014] Preferably, the adapter primer P7-R1 comprises the nucleotide sequence shown in SEQ ID NO.5.
[0015] Preferably, the adapter primer P5-TSO comprises the nucleotide sequence shown in SEQ ID NO.6.
[0016] In traditional sequencing processes, the original adapter primers (as shown in SEQ ID NO.7 and 8) are only compatible with high-quality cDNA, resulting in relatively ideal sequencing data output. However, for low-quality cDNA, they are prone to forming a large number of primer polymers, leading to rapid inactivation of the sequencing wells and a significant decrease in data quality.
[0017] The sequences are summarized in Table 1 below: Table 1
[0018] In this invention, the adapter primer set obtained through primer optimization strategy effectively suppressed the formation of multimers, and even for low-quality cDNA, its sequencing quality was not affected by multimer contamination. The results show that the optimized primers exhibit stronger applicability and stability for clinical samples (which typically exhibit quality fluctuations).
[0019] Secondly, the present invention provides the application of adapter primer sets compatible with multiplex PCR library construction as described in the first aspect in the construction of sequencing libraries.
[0020] Thirdly, the present invention also provides a method for multiplex PCR library construction using the adapter primer set as described in the first aspect, characterized in that the method comprises the following steps: (1) Full-length cDNA was amplified and purified using the P7-R1 and P5-TSO primers from the adapter primer set; (2) The amplification product obtained in step (1) was amplified and purified using P7-F and P5-R from the adapter primer set; (3) Then use P7-F and P5-R in the adapter primer set to sort or enrich the amplification products obtained in step (2) to complete the multiplex PCR library construction.
[0021] As a preferred technical solution of the present invention, the concentration of each primer in the adapter primer set described in steps (1) and (2) is 0.4 µM to 1.6 µM, for example, it can be 0.4 µM, 0.5 µM, 0.6 µM, 0.7 µM, 0.8 µM, 0.9 µM, 1.0 µM, 1.1 µM, 1.2 µM, 1.3 µM, 1.4 µM, 1.5 µM or 1.6 µM, etc., preferably 0.8 µM.
[0022] As a preferred technical solution of the present invention, the annealing temperature for amplification in step (1) is 68°C.
[0023] Preferably, the number of amplification cycles in step (1) is 2 to 5 cycles, for example, 2, 3, 4 or 5 cycles.
[0024] As a preferred technical solution of the present invention, the annealing temperature for amplification in step (2) is 70°C.
[0025] Preferably, the number of amplification cycles in step (2) is 2 to 5 cycles, for example, 2, 3, 4 or 5 cycles.
[0026] Fourthly, the present invention also includes a sequencing method, the sequencing method comprising the following steps: constructing a library using the method described in the third aspect, and then sequencing the sequencing library.
[0027] Fifthly, the present invention also includes a kit for constructing sequencing libraries, the kit comprising adapter primers compatible with multiplex PCR library construction as described in the first aspect.
[0028] The numerical range described in this invention includes not only the point values listed above, but also any point values within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values included in the range.
[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes a novel adapter primer set for library construction and sequencing. This adapter primer set does not rely on global parameter adjustments or post-processing software filtering; instead, it is an effective method that proactively and structurally avoids primer dimer formation. Furthermore, this adapter primer set overcomes the limitations of traditional primers, which are sensitive to cDNA quality and perform poorly in low-quality samples, significantly enhancing compatibility with low-quality cDNA. Simultaneously, experiments show that even with poor-quality cDNA, the optimized primers effectively inhibit multimer formation and prevent rapid inactivation of sequencing wells, thereby ensuring the output of high-quality sequencing data, making it particularly suitable for clinical samples with varying quality. Attached Figure Description
[0030] Figure 1 Figure 1 shows the mass control results of the product before primer optimization in Example 1. Figure 1 is the distribution of sequencing data length, and Figure 2 is the real-time monitoring of effective wells during the sequencing process.
[0031] Figure 2 This is a diagram showing the complementary pairing results between the P7 and P5 extension regions before primer optimization in Example 1.
[0032] Figure 3 Figure 1 shows the quality control results after primer optimization in Example 1. Figure 1 is the sequencing data length distribution diagram, and Figure 2 is the real-time monitoring diagram of effective wells during the sequencing process. Detailed Implementation
[0033] The technical solution of the present invention will be further illustrated below through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be determined by the claims.
[0034] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.
[0035] Example 1: Comparison Experiment Before and After Optimization of Connector Primers To fundamentally eliminate primer-mediated dimer and multimer formation, this embodiment systematically optimized the P5, P7, and P7-R1 primers.
[0036] The optimization strategy mainly includes two key improvements: First, by truncating the two nucleotides at the end of the P7-R1 primer, the complementary structure that is easily formed between it and the P5-TSO primer was eliminated; Secondly, by extending the 3' ends of the P5 and P7 primers by 10-12 nucleotides respectively, the possibility of four bases pairing together in the extended region of the primers was effectively suppressed, thereby significantly reducing the generation of non-specific amplification products.
[0037] In this invention, before and after optimizing the P5 and P7 adapter primers, a quality control test was performed on the same sample. The multimer products present in the original protocol significantly interfered with the sequencing results, such as... Figure 1 As shown in Figure I, which is the sequencing data length distribution (N50 is 1.33 kb), the results indicate that a large number of reads below 600 bp appeared. Meanwhile, the real-time monitoring of effective wells during sequencing, as shown in Figure II, shows a rapid decrease in effective wells. Compositional analysis of the top 50 reads with the highest throughput in the sequencing data revealed that they were mainly composed of primer polymers. The presence of primer polymers caused a rapid decrease in effective wells, resulting in low sequencing quality and insufficient number of reads.
[0038] Based on the experimental procedure, it can be inferred that during multiple PCR cycles, primer dimers can further polymerize into higher-order primer multimers through 3' end extension and interstrand complementarity. These polymerized products are prone to forming multiple nucleic acid strand breaks (nick structures) in their structure. During nanopore sequencing, these nick structures prevent DNA strands from effectively occupying and stably passing through the nanopore, resulting in a sharp decrease in the number of usable channels in the sequencing chip, ultimately leading to a significant reduction in both sequencing data yield and quality.
[0039] (2) The primers were analyzed in this embodiment, such as Figure 2 As shown, a 4-base complementary pairing was found between the P7 and P5 extension regions. Simultaneously, the P5 primer itself also possesses a complementary region, with a complementary length of 6 bases, and the complementary types include not only AT pairing but also the more stable CG pairing. Subsequent sequencing results also clearly detected the corresponding fragment generated by the complementary extension of the P5 primer itself.
[0040] The sequences before and after optimization are shown in Table 2: Table 2
[0041] After primer optimization, such as Figure 3 As shown in Figure I, which is the distribution of sequencing data length (N50 is 1.92 kb), it can be seen that the generation of non-specific amplification fragments of about 600 bp has been significantly reduced. Combined with the real-time monitoring results of the effective wells in Figure II, it can be seen that the number of effective wells after optimization remains stable, indicating that primer multimer removal was successful, the number of effective wells remained stable, and the sequencing quality was high and the quantity was sufficient.
[0042] Example 2: Extraction and purification of cDNA This experiment involves a long-fragment enrichment method for single-cell RNA sequencing samples. This method regulates transcripts with uneven expression levels in the sample, specifically enriching low-expression transcripts (i.e., increasing their total nucleic acid content through screening and increasing their proportion in the final library preparation sample), thus achieving a relatively balanced expression level and improving the accuracy and reliability of subsequent transcript analysis. Because this experiment involves a large number of PCR iterations, the requirements for primers are relatively high.
[0043] 1. After recovering cDNA from 10X single cells, the cDNA is purified.
[0044] PCR was performed in 100 µl volume using 2X PCR Master Mix and P7-R1 / P5-TSO primers to amplify full-length cDNA. The reaction system (Table 3) and amplification program (Table 4) are shown below: Table 3
[0045] Table 4
[0046] 2. Perform cDNA purification (0.6X). Subsequently, perform PCR in 100 µl volume using 2X PCR Master Mix and P7-F / P5-R primers to amplify full-length cDNA. The amplification program (Table 5) is shown below.
[0047] Table 5
[0048] 3. Perform cDNA purification (0.6X volume, depending on the application) and analyze it using a Qubit or bioanalyzer to check the quantity and quality of DNA in the sample.
[0049] Example 3: Sorting and amplification of long cDNA fragments In this embodiment, the adapter primer pair provided in Example 1 is used to achieve the sorting and amplification of long fragments. The specific steps are as follows: First, dilute the CRStock Solution (stock reagent). The amount of NFW (nuclease-free water) can be set to 500 µl, with the volume of the reagent proportional to the volume of the NFW.
[0050] The components of the stocked reagents are shown in Table 6 below: Table 6
[0051] For example: 0.9X screening solution = 500 µl NFW + 450 µl stock reagent 0.8X screening solution = 500 µl NFW + 400 µl stock reagent Screening solutions of 0.9X, 0.8X, 0.75X, 0.725X, 0.7X, 0.69X and 0.68X were prepared according to this method.
[0052] Next, the long cDNA fragments are sorted and amplified to achieve long fragment enrichment. The specific steps are as follows: (1) Add 10-30 ng DNA to 50 µl of nuclease-free water (NFW), then add 50 µl of Clean-up magnetic beads, incubate at room temperature for 5 min, and remove the supernatant.
[0053] (2) Add 50 µl of diluted 0.9X stock reagent to the magnetic beads and incubate at room temperature for 5 min. Vortex for 10 s every 2 min during the process. Repeat the process three times and discard the supernatant. (3) Clean the magnetic beads twice with 80% ethanol, 30 seconds each time, to remove the ethanol; (4) Dry for 1 min, then elute with 42 µl NFW; (5) Long fragment enrichment was performed using P7-F / P5-R. The reaction volume (Table 7) and reaction procedure (Table 8) are shown in the following tables: Table 7
[0054] Table 8
[0055] Recommended PCR reaction cycles are shown in Table 9: Table 9
[0056] (6) Add 50 µl of Clean-up magnetic beads (0.5X) to the sample after PCR and incubate at room temperature for 5 min; (7) Wash the magnetic beads twice with 80% ethanol for 30 s each time, remove the ethanol and dry for 1 min, then elute with 25 µl NFW. (8) Use Qubit to determine the cDNA concentration. If the product concentration is too low, the PCR system of step 5 can be reconfigured as needed to perform an additional 3-4 cycles of PCR, and steps (6)-(7) can be repeated.
[0057] (9) Next, repeat the above experimental procedure using 0.8X stock reagent, and complete the experiment using 0.75X, 0.725X, 0.7X, 0.69X and 0.68X stock reagent in sequence.
[0058] It is important to note that long fragment enrichment must be performed sequentially, starting with 0.9X stock reagent.
[0059] (10) Take a certain amount of cDNA from the PCR products screened by the stock reagents of 0.9X, 0.8X, 0.75X, 0.725X, 0.7X, 0.69X and 0.68X respectively and mix them into a sample.
[0060] Recommended mixing ratios are shown in Table 10 (can be adjusted according to specific needs): Table 10
[0061] (11) Nanopore sequencing quality control (checking fragment size distribution).
[0062] Through this primer optimization, this invention successfully solved the following three key problems: primer multimer formation caused by over-amplification of low-quality cDNA, multimer-mediated nucleic acid strand breaks (Nick structures), and premature inactivation of sequencing channels as a result, thereby effectively improving the quality and output efficiency of sequencing data. For cDNA of relatively good quality, primer optimization has little impact on the results; however, the optimization effect is particularly significant for cDNA of poor quality. Given the considerable uncertainty in the quality of clinical samples, the optimized primers exhibit broader compatibility and reliability, making them more suitable for practical clinical testing applications.
[0063] 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; and these 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 set of adapter primers compatible with multiplex PCR library construction, characterized in that, The adapter primer set includes adapter primer P7-F, adapter primer P5-R, adapter primer P7-R1 and adapter primer P5-TSO. The sequence lengths of the adapter primers P7-F and P5-R are 30-35 bp, and the sequence lengths of the adapter primers P7-R1 and P5-TSO are 40-48 bp. The adapter primers P7-F and P7-R1 contain the nucleotide sequences shown in SEQ ID NO.1; The adapter primers P5-R and P5-TSO contain nucleotide sequences as shown in SEQ ID NO.
2.
2. The adapter primer set according to claim 1, characterized in that, The adapter primer P7-F contains the nucleotide sequence shown in SEQ ID NO. 3; The adapter primer P5-R contains the nucleotide sequence shown in SEQ ID NO.4; The adapter primer P7-R1 contains the nucleotide sequence shown in SEQ ID NO.5; The adapter primer P5-TSO contains the nucleotide sequence shown in SEQ ID NO.
6.
3. The application of the adapter primer set compatible with multiplex PCR library construction as described in claim 1 or 2 in the construction of sequencing libraries.
4. A method for multiplex PCR library construction using the adapter primer set as described in claim 1 or 2, characterized in that, The method includes the following steps: (1) Full-length cDNA was amplified and purified using the P7-R1 and P5-TSO primers from the adapter primer set; (2) The amplification product obtained in step (1) was amplified and purified using P7-F and P5-R from the adapter primer set; (3) Then use P7-F and P5-R in the adapter primer set to sort or enrich the amplification products obtained in step (2) to complete the multiplex PCR library construction.
5. The method according to claim 4, characterized in that, The concentration of each primer in the adapter primer set described in steps (1) and (2) is 0.4 µM to 1.6 µM, preferably 0.8 µM.
6. The method according to claim 4 or 5, characterized in that, The annealing temperature for amplification in step (1) is 68°C; Preferably, the number of amplification cycles in step (1) is 2 to 5 cycles.
7. The method according to any one of claims 4 to 6, characterized in that, The annealing temperature for amplification in step (2) is 70°C; Preferably, the number of amplification cycles in step (2) is 2 to 5 cycles.
8. A sequencing method, characterized in that, The sequencing method includes the following steps: constructing a library using the method described in any one of claims 4 to 7, and then sequencing the sequencing library.
9. A kit for constructing sequencing libraries, characterized in that, The kit includes the adapter primer set for multiplex PCR library construction as described in claim 1 or 2.