阅读说明：本技术 一种提高多重pcr扩增文库质量的方法 (Method for improving quality of multiple PCR amplification library ) 是由 黄文潘 浦浩 张亚楠 于 2020-04-29 设计创作，主要内容包括：本发明提供了一种提高多重PCR扩增文库质量的方法,属于PCR扩增技术领域。该方法是根据多重PCR中每个扩增子的扩增靶标序列来设计、合成两端含有与扩增子对应引物结合位点的多重PCR扩增差异靶标,该差异靶标和理论的扩增靶标序列具有编辑距离大于5bp的差异,构建人工合成差异核苷酸序列集合IAC。将IAC加入PCR反应体系中进行多重PCR扩增反应,引物可以结合到IAC集合上,大大减少多重PCR扩增中非靶标引物形成引物二聚体和产生非特异性扩增的可能,从而提高多重PCR扩增文库的质量,为后续检测出待检靶标提供较好的基础。(The invention provides a method for improving the quality of a multiple PCR amplification library, belonging to the technical field of PCR amplification. The method comprises the steps of designing and synthesizing a multiple PCR amplification difference target with two ends containing primer binding sites corresponding to amplicons according to an amplification target sequence of each amplicon in multiple PCR, wherein the difference target and a theoretical amplification target sequence have a difference of an editing distance larger than 5bp, and constructing an artificially synthesized difference nucleotide sequence set IAC. The IAC is added into a PCR reaction system to carry out multiple PCR amplification reaction, and the primers can be combined to an IAC set, so that the possibility of primer dimer formation and non-specific amplification generation of non-target primers in multiple PCR amplification is greatly reduced, the quality of a multiple PCR amplification library is improved, and a better basis is provided for subsequent detection of a target to be detected.)

1. A method for improving the quality of a multiplex PCR amplification library, which is characterized by comprising the following steps:

1) artificially synthesizing a plurality of differential target sequences of which the two ends comprise primer complementary pairing fragments of the amplicons according to the amplification target sequence of each amplicon in the multiplex PCR, wherein the differential target sequences and the amplification target sequences have a difference sequence with an editing distance of more than 5 bp; the artificially synthesized differential target sequence is detected to be at least a differential sequence with more than 5bp in a sequencing read length range;

2) mixing not less than 10% of the total number of the artificially synthesized differential target sequences in the step 1) to construct a multiple PCR artificially synthesized sequence set with differences;

3) and taking a sample to be detected as a reaction template, and adding the multiple PCR artificially synthesized sequence sets with differences into a reaction system to perform multiple PCR amplification reaction to obtain a high-quality multiple PCR amplification library.

2. The method of claim 1, wherein the amplicons of step 1) are the amplification products of the candidate test organisms amplified by the respective primers of the multiplex PCR.

3. The method of claim 1, wherein the number of amplicons in the multiplex PCR of step 1) is determined based on the number of candidate test organisms.

4. The method according to claim 1, wherein the difference sequence in step 1) has a ratio of 4 bases that is not greater than 70% based on 100% of the sum of the ratios of 4 bases.

5. The method according to claim 1, wherein the form of the difference sequence in step 1) comprises one or more of insertion, deletion and mismatch.

6. The method according to claim 1 or 5, wherein the differential sequence in step 1) comprises inserting a 20bp nucleotide sequence shown as SEQ ID No.1 in the sequence Listing.

7. The method of claim 1 or 5, wherein the sequencing read in step 1) is 35-600 bp in length.

8. The method of claim 1, wherein in the step 2) the copy number of the target sequence of each of the sequences with difference in the multiple PCR synthetic pools of sequences with difference is not more than 10⁸copy/mL.

Technical Field

The invention belongs to the technical field of PCR amplification, and particularly relates to a method for improving the quality of a multiple PCR amplification library.

Background

The multiplex PCR is to add a plurality of pairs of primers into one reaction tube and carry out PCR reaction simultaneously; the number of primers added is small, and dozens of pairs, and many pairs are hundreds of pairs. Therefore, how to avoid the formation of dimers or non-specific amplification among a plurality of pairs of primers becomes a key technical point for improving the quality of an amplified library.

In addition to reducing the complementary pairing of the bases at the 3' ends of the primers in primer design and evaluation, the positions of the primers aligned pairwise are kept from alignment to multiple genomic positions, and other methods are still needed to avoid dimer formation or non-specific amplification among the multiple PCR primers and improve library quality.

In certain circumstances, such as where only a portion of the targets in the multiplex PCR primers are present in the environment to be detected. For example, a multiplex PCR primer mix contains primers for specific amplification of hundreds of microorganisms, but a test sample actually contains only one or two of the hundreds of microorganisms. In such an environment, only a certain pair of primers actually amplify in the hundreds of primers, and a large amount of other primers form a large amount of dimers or non-specific amplification because no amplification target is consumed, resulting in poor quality of an amplification library. Even subsequent purification by conventional magnetic beads does not achieve good results.

Disclosure of Invention

Accordingly, the present invention provides a method for improving the quality of a multiplex PCR amplification library, which can reduce the dimer generation and non-specific amplification of the multiplex PCR amplification product, thereby improving the quality of the multiplex PCR amplification library.

The invention provides a method for improving the quality of a multiplex PCR amplification library, which comprises the following steps:

1) artificially synthesizing a plurality of differential target sequences of which the two ends comprise primer complementary pairing fragments of the amplicons according to the amplification target sequence of each amplicon in the multiplex PCR, wherein the differential target sequences and the amplification target sequences have a difference sequence with an editing distance of more than 5 bp; the artificially synthesized differential target sequence is detected to be at least a differential sequence with more than 5bp in a sequencing read length range;

2) mixing not less than 10% of the total number of the artificially synthesized differential target sequences in the step 1) to construct a multiple PCR artificially synthesized sequence set with differences;

3) and taking a sample to be detected as a reaction template, and adding the multiple PCR artificially synthesized sequence sets with differences into a reaction system to perform multiple PCR amplification reaction to obtain a high-quality multiple PCR amplification library.

Preferably, the amplicons in the multiplex PCR in step 1) are amplification products obtained by amplifying candidate detection organisms respectively by the multiplex PCR primers.

Preferably, the number of amplicons in the multiplex PCR of step 1) is determined according to the number of candidate test organisms.

Preferably, in the differential sequence in step 1), the number of 4 bases is not more than 70% based on 100% of the sum of the number of 4 bases.

Preferably, the difference sequence in step 1) consists of insertions, deletions or mismatches.

Preferably, the differential sequence in step 1) comprises a 20bp nucleotide sequence shown in SEQ ID No.1 of the sequence table.

Preferably, the sequencing read length in the step 1) is 35-600 bp.

Preferably, in the step 2) of synthesizing the sequence sets with difference by multiplex PCR, the copy number of the target sequence of each sequence with difference is not more than 10⁸copy/mL.

Preferably, the sequence set of the multiple PCR artificial synthesis differences in the step 2) comprises 15 sequences shown in SEQ ID No. 2-SEQ ID No.16 and/or 16 sequences shown in SEQ ID No. 111-SEQ ID No.126 in the sequence table.

Preferably, the procedure of the multiplex PCR amplification reaction in the step 3) is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 20 s; annealing at 63 deg.C for 2 min; extending for 72 ℃ for 2 min; 45 cycles; final extension at 72 deg.C for 5 min;

step (ii) of3) The system of the multiplex PCR amplification reaction in (1) is a primer set of 4 mu l, ddH of the multiplex PCR₂O6. mu.l, 3 × TENzyme mix 10. mu.l, artificially synthesized differential sequence set 2. mu.l, sample template 8. mu.l.

The method for improving the quality of the multiple PCR amplification library provided by the invention designs and synthesizes multiple PCR amplification difference target sequences with primer binding sites corresponding to the amplicons at two ends according to the amplification target sequence of each amplicon in the multiple PCR, the difference target sequences and the theoretical amplification target sequences have the difference of editing distance larger than 5bp, and not less than 10% of the total number of artificially synthesized difference target sequences are constructed into an artificially synthesized sequence set (IAC set) with difference. The IAC is added into a PCR reaction system to carry out multiple PCR amplification reaction, and the primers can be combined to an IAC set, so that the possibility of primer dimer formation and non-specific amplification generation of non-target primers in multiple PCR amplification is greatly reduced, the quality of a multiple PCR amplification library is improved, and a better basis is provided for subsequent detection of a target to be detected. Experiments show that the multiplex PCR amplification library prepared without adding the IAC set is taken as a control (the proportion of the Q30 mass value is only 3.73 percent, the proportion of the dimer is as high as 95.31 percent, the final effective reads of 71316 reads are only 290 and only 0.41 percent), the Q30 mass value of the multiplex PCR amplification library prepared with adding the IAC set is more than 95 percent, the proportion of the dimer is less than 0.5 percent, the number of the effective reads is as high as 89 percent, and the quality of the library is far higher than that of the control. Therefore, under the condition that only part or a small part of amplification targets exist in a sample, after the artificially synthesized IAC set is added, the dimer and the non-specific amplification of the multiplex PCR amplification library are obviously reduced, the library quality of the multiplex PCR amplification library is obviously improved, and meanwhile, the multiplex PCR primers can be enriched and amplified to increase the library concentration.

Drawings

FIG. 1 is a schematic diagram of the construction of synthetic differenced sequence sets (IAC sets);

FIG. 2 is a schematic diagram of an artificially synthesized set of sequences with differences (IAC set) participating in a multiplex PCR reaction;

FIG. 3 is a schematic diagram of the on-machine sequencing of nucleotide sequences of artificially synthesized differential target sequences;

FIG. 4 is an illustration of the alignment of the amplification target sequence to the sequences in the IAC pool.

Detailed Description

The invention provides a method for improving the quality of a multiplex PCR amplification library, which comprises the following steps:

1) artificially synthesizing a plurality of differential target sequences of which the two ends comprise primer complementary pairing fragments of the amplicons according to the amplification target sequence of each amplicon in the multiplex PCR, wherein the differential target sequences and the amplification target sequences have a difference sequence with an editing distance of more than 5 bp; the artificially synthesized differential target sequence is detected to be at least a differential sequence with more than 5bp in a sequencing read length range;

2) mixing not less than 10% of the total number of the artificially synthesized differential target sequences in the step 1) to construct a multiple PCR artificially synthesized sequence set with differences;

3) and taking a sample to be detected as a reaction template, and adding the multiple PCR artificially synthesized sequence sets with differences into a reaction system to perform multiple PCR amplification reaction to obtain a high-quality multiple PCR amplification library.

According to the amplification target sequence of each amplicon in the multiplex PCR, artificially synthesizing a plurality of differential target sequences of which two ends comprise complementary pairing fragments with primers of the amplicons, wherein the differential target sequences and the amplification target sequences have a differential sequence with an editing distance of more than 5 bp; the artificially synthesized differential target sequence is detected to be at least a differential sequence of more than 5bp in the sequencing read length range.

A schematic diagram of the construction of artificially synthesized differenced sequence sets (IA sets) is shown in FIG. 1. According to the target region of the amplicon corresponding to the primer _ cluster, P1 and P2 … Pi … Pn (if a target virus is designed, the region is the sequence of the virus region which can be amplified by the pair of primers) T1, T2, Ti.. Tn, each target synthesizes a nucleotide sequence (several targets can be combined to synthesize An artificial sequence) A1, A2, … Ai and … An which are similar to the target (only the primer binding region is needed). Artificially synthesized differential target sequences A1, A2, … Ai and … An are enriched, quantified and diluted and then mixed together according to a certain proportion to construct An IAC set. The IAC pool can be amplified by the multiplex primer pool P.

In the present invention, the amplicons in the multiplex PCR are amplification products obtained by amplifying candidate detection organisms respectively by the multiplex PCR primers. The candidate test sample includes various types of pathogens, such as viruses, bacteria, fungi, or the like. The number of amplicons in the multiplex PCR is preferably determined based on the number of candidate test organisms, each of which can be extended using one or more primer pairs, preferably one organism using one primer extension. The method is not limited by the number of amplicons in the multiplex PCR, namely the method provided by the invention is not limited by the number of primer types and different target sequences in the artificially synthesized sequence sets with differences in the multiplex PCR, but the quality of the multiplex PCR amplification library obtained by the increase of the number of the amplicons is better. In order to illustrate the amplification target sequences of the multiple PCR amplicons, the amplification target sequences of the amplicons obtained by amplification of 15 multiple PCR primer sets (the nucleotide sequences are SEQ ID No.17 to SEQ ID No.51) are SEQ ID No.47 to SEQ ID No. 61.

In the present invention, in the differential sequence, the ratio of the number of 4 bases is not more than 70%, more preferably 20% to 40%, and most preferably 25%, based on 100% of the sum of the number of 4 bases. The difference sequence is preferably obtained by combining one or more of insertion, deletion or mismatch. And when the length of the artificially synthesized target sequence is shorter than that of the amplified target sequence, deleting partial internal bases of the nucleotide sequence of the amplified target sequence, and reserving primer complementary pairing fragments at two ends of the amplified target sequence and the amplicon.

In the invention, primer complementary pairing fragments containing the amplicon at two ends of the artificially synthesized differential target sequence are provided with binding sites of multiple PCR primers at two ends of the target sequence in an artificially synthesized mode, so that the primers can be amplified by the primers in a multiple PCR primer pool in the multiple PCR reaction process, the primers are bound to the nucleotide sequence of the artificially synthesized differential sequence, the chances of primer dimer formation and nonspecific amplification of the primers are reduced, and the enrichment and amplification of the multiple PCR primers are realized at the same time, and the library concentration is increased.

In the invention, each segment of artificially synthesized differential target sequence has difference with the theoretical amplification target sequence with the edit distance of more than 5bp, and the differential sequences are detected at least by more than 5bp in the sequencing read length range. The read length is preferably 35-600 bp, and more preferably 60-300 bp. For example, double-ended reads 60bp long sequencing are selected, and differences of 5bp can be read within 60 bp. The invention has no special requirements on the nucleotide sequence of the difference sequence, and mainly can distinguish artificially synthesized sequences and sample amplification sequences. The editing distance refers to the number of editing times required for completely aligning two nucleotide sequences when the two nucleotide sequences are aligned. For example, in the present embodiment, the nucleotide sequence of the difference sequence is a difference sequence with the length of aactggaagtcagaggtgag (SEQ ID No.1)20bp inserted compared with the theoretical amplicon sequence, but this is not to be construed as limiting the difference sequence.

Obtaining a plurality of different target sequences with different bands, and mixing not less than 10% of sequences in the total number of the plurality of different target sequences to construct and obtain an artificially synthesized different sequence set (IAC set).

In the IAC pool, the copy number of the target sequence with different sequences is not higher than 10⁸Copy/ml. To illustrate the case of the constructed IAC pool, in the examples of the present invention, the artificially synthesized variant target sequences are preferably 15 sequences shown in SEQ ID nos. 2 to 16 or 16 sequences shown in SEQ ID nos. 111 to 126 of the sequence listing, but are not to be construed as limiting the pool of multiple PCR artificially synthesized variant sequences involved in the methods of the present invention.

Taking a sample to be detected as a reaction template, adding the multiple PCR artificially synthesized sequence set with difference (IAC set) into a reaction system to carry out multiple PCR amplification reaction, thereby obtaining a high-quality multiple PCR amplification library.

A schematic representation of the amplification reaction in the IAC pool-fed PCR reaction system is shown in FIG. 2. Adding a nucleic acid to be detected, an enzyme, a multiple PCR primer mix and an IAC set when a PCR reaction system is prepared; the multiple PCR primers mix can simultaneously amplify the IAC set, and if the amplified target exists in the nucleic acid of the sample to be detected, the target can be amplified by the corresponding target in the multiple PCR primer set.

In the present invention, the system and procedure of the multiplex PCR amplification reaction vary according to the kind of different multiplex PCR primer sets that have amplification correspondence with different target sequences in the artificially synthesized set with differences. For example, the artificially synthesized variant sequence sets shown in SEQ ID Nos. 2 to 16 are described above, and the nucleotide sequences of the corresponding multiplex PCR primer sets are shown in SEQ ID Nos. 17 to 46. The procedures of the corresponding multiple PCR amplification reactions shown as SEQ ID No. 2-SEQ ID No.16 are pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 20 s; annealing at 63 deg.C for 2 min; extending for 72 ℃ for 2 min; 45 cycles; final extension 72 ℃ for 5 min. The system of the multiple PCR amplification reaction is a primer set of 4 mu l, ddH of the multiple PCR₂O6. mu.l, 3 × TENzyme mix 10. mu.l, synthetic set of sequence differences 2. mu.l, sample template 8. mu.l the concentration of each synthetic differential target sequence in the synthetic set of sequence differences is preferably not higher than 10⁸The copy/ml is preferably 500 to 4000 copies/ml.

In the invention, after obtaining the high-quality multiplex PCR amplification library, the method also comprises the steps of detecting a target fragment of the multiplex PCR amplification library, sequentially purifying an amplification product, connecting a sequencing joint, purifying the amplification product and sequencing on a computer, and identifying an artificial sequence and a real amplification sequence of a sample. The present invention is not particularly limited to the specific steps for detecting target fragments in the multiplex PCR amplification library, and may be performed by any method known in the art. Sequencing the purified multiplex PCR amplification library, and removing a sequencing result to obtain a target sequence containing the difference sequence; and screening and analyzing the amplified target sequence without the sequence difference. However, the method needs the sequencing length to cover the differential sequence on the IAC set so as to distinguish whether the detection result is from the IAC sequence or the real detection sequence; as shown in FIG. 3, reads at least one end of the sequencing overlay the difference sequence on the IAC. Comparing the screened reads which do not contain the differential fragment (tag) with the amplification target sequence of the amplicon, and judging whether the reads come from the target, thereby counting the number of the target targets in the detection sample and realizing the high-efficiency detection of the target pathogens.

The method for improving the quality of a library obtained by multiplex PCR amplification according to the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.