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CN-122012681-A - Joint primer group compatible with multiplex PCR library construction and application thereof

CN122012681ACN 122012681 ACN122012681 ACN 122012681ACN-122012681-A

Abstract

The invention provides a joint primer group compatible with multiplex PCR library construction and application thereof, wherein the joint primer group comprises joint primers P7-F, joint primers P5-R, joint primers P7-R1 and joint primers P5-TSO, and the sequences of the joint primers are shown as SEQ ID NO. 3-6. The adaptor primer set removes the 3' end complementary structure that may exist between itself or upstream and downstream primers and avoids primer dimer and multimer formation from the source by extending the primer length across the complementary region, terminating in a non-complementary sequence. Even for low-quality cDNA or library construction or sequencing steps with a large number of PCRs, the sequencing quality of the resulting product is not affected by the contamination of the multimers by the adapter primer set provided by the invention.

Inventors

  • LUO HAN

Assignees

  • 四川大学华西医院
  • 上海宁基之达生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260212

Claims (9)

  1. 1. The joint primer group compatible with multiplex PCR library establishment is characterized by comprising joint primers P7-F, joint primers P5-R, joint primers P7-R1 and joint primers P5-TSO; the sequence length of the joint primers P7-F and P5-R is 30-35 bp, and the sequence length of the joint primers P7-R1 and P5-TSO is 40-48 bp; The adaptor primer P7-F and P7-R1 comprise nucleotide sequences shown in SEQ ID NO. 1; The linker primers P5-R and P5-TSO comprise the nucleotide sequence shown as SEQ ID NO. 2.
  2. 2. The set of adaptor primers according to claim 1, wherein the adaptor primer P7-F comprises a nucleotide sequence as shown in SEQ ID No. 3; The adaptor primer P5-R comprises a nucleotide sequence shown as SEQ ID NO. 4; the adaptor primer P7-R1 comprises a nucleotide sequence shown as SEQ ID NO. 5; the linker primer P5-TSO comprises a nucleotide sequence as shown in SEQ ID NO. 6.
  3. 3. The use of a multiplex PCR-compatible pool of adaptor primer sets according to claim 1 or 2 in sequencing library construction.
  4. 4. A method for multiplex PCR pooling using the adapter primer set of claim 1 or 2, comprising the steps of: (1) Amplifying and purifying full-length cDNA by using P7-R1 and P5-TSO primers in the joint primer group; (2) Amplifying and purifying the amplified product obtained in the step (1) by using P7-F and P5-R in the adaptor primer set; (3) And (3) sorting or enriching the amplification product obtained in the step (2) by using P7-F and P5-R in the joint primer group to complete multiplex PCR library establishment.
  5. 5. The method according to claim 4, wherein the concentration of each primer in the adaptor primer set in step (1) and step (2) is 0.4. Mu.M to 1.6. Mu.M, preferably 0.8. Mu.M.
  6. 6. The method of claim 4 or 5, wherein the annealing temperature of the amplification in step (1) is 68 ℃; preferably, the number of cycles of amplification in the step (1) is 2 to 5 cycles.
  7. 7. The method of any one of claims 4-6, wherein the annealing temperature of the amplification in step (2) is 70 ℃; Preferably, the number of cycles of amplification in the step (2) is 2 to 5 cycles.
  8. 8. A sequencing method, which comprises the steps of constructing a library according to any one of claims 4 to 7 and sequencing the sequencing library.
  9. 9. A kit for sequencing library construction, comprising the adaptor primer set of claim 1 or 2 compatible with multiplex PCR library construction.

Description

Joint primer group compatible with multiplex PCR library construction and application thereof Technical Field The invention belongs to the fields of biotechnology and molecular biology, relates to a primer design optimization and verification technology for Polymerase Chain Reaction (PCR), and in particular relates to a joint primer group compatible with multiplex PCR library construction and application thereof. Background The PCR technology has irreplaceable core status in the wet experimental link of the sequencing technology, and the importance of the PCR technology is mainly reflected in two key applications, namely, the PCR technology adds sequencing joints to the two ends of a DNA fragment efficiently and specifically in the library construction stage, and the PCR technology effectively enriches a target area (especially a long fragment) so as to meet the high requirement of a sequencer on input quantity and concentration. Whether the technology is a short-reading long platform such as Illumina, MGI and the like or a PacBio, nanopore long-reading long technology, the technology depends on PCR amplification to amplify weak signals and improve detection sensitivity, and becomes a decisive step in scenes such as single-cell sequencing, low-frequency mutation detection and the like. Although amplification may introduce preference or even error, no technology is currently available that can fully replace PCR to achieve the above-described functions while maintaining high efficiency and low cost. Thus, PCR is still the most central wet experimental bridge between the original nucleic acid of the ligation sample and the high quality sequencing data. In order to deeply study the complexity of human tumor microenvironment, the study integrates the advantages of single-cell RNA sequencing technology and Nanopore long-reading long-sequencing technology, and introduces a long fragment enrichment strategy in an experimental flow. The process involves multiple rounds of PCR amplification, and we have severely limited the number of cycles per round of PCR in order to maximize amplification fidelity. The design aims to realize the equalization of the expression quantity of all transcripts finally by precisely enriching transcript fragments with different lengths, thereby providing a high-quality data base for comprehensively analyzing the cell heterogeneity and gene expression dynamics in the tumor microenvironment. We found that during the Nanopore sequencing process, most clinical samples show rapid inactivation of the available wells, and that after conventional factors such as loading, voltage, etc. have been eliminated, the problem is likely to originate from the sample itself. Particularly in the experimental procedure requiring multiple PCR amplifications (e.g., long fragment screening experiments), even if the number of single cycles is severely limited, the probability of primer multimer formation increases significantly with increasing PCR numbers. Such multimers are long in structure and difficult to distinguish efficiently from the target product during fragment quality control (e.g., Q-sep), resulting in their incorporation into the final sequencing library. The polymer structure often has a nucleic acid chain breaking point (a Nick structure) which causes serious problems in the sequencing process, namely, on one hand, the Nick structure causes the stability of a DNA chain in a nanopore to be reduced, the DNA chain is extremely easy to dissociate, the number of effective sequencing holes is greatly reduced, and on the other hand, short fragments generated by breaking preferentially enter a pore canal and are rapidly sequenced, so that a large amount of sequencing resources are occupied, and the sequencing holes are inactivated in advance. Which ultimately manifests itself as a significant drop in data throughput and a sharp rise in the proportion of invalid read lengths. Therefore, the method is a key premise for fundamentally optimizing primer design and thoroughly inhibiting the generation of primer dimers and multimers, blocking such problems and improving sequencing success rate and data quality. The conventional primer design method for sequencing library construction at present depends on general principles for avoiding primer dimer formation, such as control of primer length, GC content, tm value and other basic parameters, and prediction and evaluation of complementary pairing among primers by software. Such methods generally assume that the quality of the cDNA is good, and do not adequately address the problems of severe multimeric formation and sequencing performance degradation due to primer structure interactions in low quality cDNA or complex clinical samples. The prior art generally lacks systematic analysis and targeted optimization of the 3' -end complementary structure of the primer, and particularly cannot effectively inhibit rapid inactivation of a sequencing well and data quality de