Library construction methods and methods for improving primer uniformity

Abstract

The application provides a library construction method and a method for improving primer uniformity, wherein the library construction method comprises the following steps: 1) providing DNA derived from a biological sample; 2) purifying the DNA by using magnetic beads; and 3) amplifying the purified DNA to form an amplicon library. By purifying the DNA (for example, the DNA after extraction) by using magnetic beads before amplification, the application removes residual organic reagents (for example, polysaccharides and salt ions) in the DNA, improves the library uniformity, thereby reducing the sequencing cost of products, expanding the throughput of amplicons, and improving the application value of clinical diagnosis.

Description

Technical Field

[0001] This invention relates to the field of gene sequencing, and more particularly to a library construction method and a method for improving primer uniformity. Background Technology

[0002] With the surge in information throughput in recent years, high-throughput sequencing technology has become increasingly widely used in life sciences and medicine, playing a particularly important role in disease diagnosis and prevention, mainly in prenatal screening, tumor diagnosis, prevention of major diseases, and health-related metagenomic analysis. Although whole-genome sequencing has made tremendous strides in terms of sequencing time and cost, the massive data analysis and extraction of genetic information are still time-consuming and labor-intensive. In comparison, whole-exome sequencing and gene panel sequencing can directly target most gene sequences that may cause diseases, while amplicon sequencing's advantages of high specificity, clear targeting, simple and rapid data analysis, and low cost will be more conducive to its widespread application in clinical disease detection.

[0003] Amplicon sequencing is a targeted capture sequencing technology that mainly uses multiplex PCR technology to specifically amplify and enrich sequences of multiple target regions simultaneously to obtain an amplicon library. Then, next-generation sequencing technology is used to sequence the amplicon library to obtain the sequence information of the target region.

[0004] Despite its numerous advantages, amplicon sequencing technology also has some drawbacks, the most prominent being poor amplicon library uniformity. This manifests as inconsistent sequencing depths, large standard deviations, and inconsistent sequencing data. Currently, when using FFPE DNA samples, the extracted FFPE DNA is directly used for amplicon library construction and sequencing. However, the preparation of FFPE DNA leaves behind some organic solvents, polysaccharides, or salt ions. These residual components can affect the activity of PCR enzymes, causing bias in the PCR amplification of amplicon primers and thus reducing library uniformity. Summary of the Invention

[0005] This invention provides a library construction method and a method for improving primer uniformity to solve the problem of residual components affecting PCR enzyme activity.

[0006] According to a first aspect of the present invention, a method for constructing a library to improve the uniformity of amplicon libraries is provided, the method comprising the following steps:

[0007] 1) Provide DNA derived from biological samples;

[0008] 2) The DNA was purified using magnetic beads; and

[0009] 3) The purified DNA is amplified to form an amplicon library.

[0010] The DNA of the biological sample can be any biological sample suitable for constructing a library in this field. The biological sample can be a body fluid sample or a tissue sample.

[0011] In a specific example, the biological sample can be a paraffin-embedded sample (e.g., an FFPE sample), and correspondingly, the DNA can specifically be FFPE DNA. FFPE specifically refers to Formalin Fixation and Paraffin Embedding, which can be understood as a sample fixed in formalin and embedded in paraffin.

[0012] DNA extraction can be performed on paraffin-embedded (FFPE) samples using the ReliaPrep FFPE gDNA Miniprep System (promega, Cat. NO: A2352) or the QIAamp DNA FFPE tissue kit (qiagen, 56404).

[0013] Optionally, the DNA mentioned is extracted DNA. If applied to amplification using PCR enzymes, then: when the extracted DNA is purified by magnetic beads after extraction, residual organic reagents, polysaccharides, and salt ions in the DNA can be removed, reducing their impact on PCR enzymes, improving library uniformity, thereby reducing the sequencing cost of the product, increasing the throughput of amplicon, and improving the application value in clinical diagnosis.

[0014] Optionally, the biological sample is a body fluid sample or a tissue sample.

[0015] Optionally, the biological sample is a paraffin-embedded sample.

[0016] Optionally, the biological sample is an FFPE sample.

[0017] Optionally, the ratio of magnetic beads used for extracting DNA can be in the range of 2.0x-4.5x, specifically 3.5x. Specifically, Agencourt AMPureXP beads with a magnetic bead ratio in the range of (2.5x-4.5x) (e.g., 3.5x) can be selected for purification, and the purified DNA can be quantified by Qubit for later use.

[0018] Optionally, the amplification includes:

[0019] i) Using the purified DNA as a template, perform the first round of PCR amplification using the first primer.

[0020] ii) The products of the first round of PCR amplification may be purified;

[0021] iii) Using the product from the first round of PCR amplification and purification as a template, a second round of PCR amplification was performed using a second primer; and

[0022] iv) The products of the second round of PCR amplification may be purified to obtain the target amplicon library.

[0023] Optionally, the first primer contains SEQ ID NO:1-94.

[0024] Optionally, the upstream primer of the first primer pair is Pool_FWD, a mixture of forward primers including the nucleotide sequences shown in SEQ ID NO: 1-47, and the downstream primer is Pool_REV, a mixture of reverse primers including the nucleotide sequences shown in SEQ ID NO: 48-94; the upstream primer of the second primer pair is a primer with an I5 index sequence, and the downstream primer is a primer with an I7 index sequence.

[0025] Optionally, both the first round of PCR amplification and the second round of PCR amplification are performed using the rhAmpSeq system.

[0026] Optionally, the ratio of magnetic beads used in the purification is 2.0x-4.5x.

[0027] According to a second aspect of the present invention, a method for improving primer uniformity based on amplicon sequencing is provided, comprising:

[0028] 1) Construct amplicon sequencing libraries using the methods described above;

[0029] 2) Sequencing and bioinformatics analysis were performed on the constructed library.

[0030] The library construction method and primer uniformity improvement method provided by this invention improve library uniformity by purifying DNA (e.g., extracted DNA) with magnetic beads before amplification, removing residual organic reagents (e.g., polysaccharides and salt ions) from the DNA, avoiding or reducing their impact on enzyme activity, thereby reducing the sequencing cost of the product, increasing the throughput of amplicon, and improving the application value of clinical diagnosis. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a flowchart illustrating the library construction method in an embodiment of the present invention;

[0033] Figure 2 This is a schematic diagram showing the recovery rate of DNA after purification with magnetic beads at different ratios in Test Example 1 of this invention;

[0034] Figure 3 This is a schematic diagram illustrating the effect of different proportions of magnetic beads purified on uniformity in Test Example 1 of this invention;

[0035] Figure 4 This is a flowchart illustrating step S13 in Embodiment 1 of the present invention;

[0036] Figure 5 This is a schematic diagram illustrating the principle of rhAmpSeq multiplex PCR amplification technology in Embodiment 1 of the present invention;

[0037] Figure 6 This is a schematic diagram illustrating the effect of magnetic bead purification on the uniformity of the library constructed from FFPE DNA extracted by Qiagen in Test Example 2 of this invention.

[0038] Figure 7 This is a schematic diagram illustrating the effect of magnetic bead purification on the uniformity of the library constructed from FFPE DNA extracted from Promega in Test Example 3 of this invention.

[0039] Figure 8 This is a schematic diagram illustrating the effect of magnetic bead purification on the uniformity of the library constructed from FFPE DNA extracted from Promega in Test Example 4 of this invention.

[0040] Figure 9 This is a flowchart illustrating the method for improving primer uniformity based on amplicon sequencing in Embodiment 2 of the present invention. Detailed Implementation

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] The technical solution of the present invention will be described in detail below with reference to specific embodiments. These specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some embodiments.

[0043] Example 1

[0044] Please refer to Figure 1This invention provides a library construction method for improving the uniformity of amplicon libraries, comprising the following steps:

[0045] S11: Provide DNA from biological samples. This DNA is extracted using the ReliaPrep FFPE gDNAMiniprep System (promega, Cat. NO: A2352) or QIAamp DNA FFPE tissue kit (qiagen, 56404) to extract DNA from paraffin-embedded (formalin fixed paraffin-embedded) samples. The ratio of magnetic beads used for the extracted DNA is 3.5x.

[0046] S12: The DNA is purified using magnetic beads at a ratio of 3.5x. The purified DNA is then quantified using Qubit for later use.

[0047] Because subsequent steps require the use of the inherent properties of RNase H2 enzyme, if step S12 is not performed and amplification is performed directly, the residual organic reagents in the DNA will affect the function of RNase H2 enzyme, thereby ultimately reducing the uniformity of the library. Therefore, by purifying the extracted DNA before amplification, the influence of residual organic reagents on RNase H2 enzyme (or other PCR enzymes) can be reduced, the uniformity of the library can be improved, thereby reducing the sequencing cost of the product, increasing the throughput of amplicon, and improving the application value of clinical diagnosis.

[0048] S13: Amplify the purified DNA to form an amplicon library. The PCR amplification method is described below. Figure 4 ,

[0049] include:

[0050] S131: Using the purified DNA as a template, perform the first round of PCR amplification;

[0051] Prepare a 20 μL reaction system according to Table 1 below:

[0052] Table 1

[0053]

[0054]

[0055] in:

[0056] Pool_FWD is a mixture of forward primers, including nucleotide sequences as shown in SEQ ID NO: 1-47; Pool_REV is a mixture of reverse primers, including nucleotide sequences as shown in SEQ ID NO: 48-94. Both were designed by the inventors and synthesized by IDT.

[0057] IDTE is IDTE buffer, a TE buffer from IDT Corporation, catalog number: 11-05-01-09.

[0058] The volume of DNA, Xul, is: If the amount of DNA added for library construction is 50ng, based on the concentration of DNA after purification by magnetic beads in step S12, the volume of DNA added (i.e., the input volume) can be determined as the ratio of the total amount of DNA to the DNA concentration. Therefore, X can be used to represent the input volume of different samples.

[0059] For the mixture obtained in the above reaction system, the target region is sequentially activated, amplified, and inactivated by enzyme to complete the first round of PCR amplification.

[0060] The first round of amplification targeting the region was performed using a ProFlex PCR System PCR instrument (Applied biosystems by life technologies, model: ProFlex) following the reaction procedure; refer to Table 3 below:

[0061] Table 3

[0062]

[0063] S132: The products of the first round of PCR amplification were optionally purified using Agencourt AMPure XP beads with 80% ethanol (prepared fresh for use);

[0064] S133: Using the product amplified and purified in the first round of PCR as a template, a second round of PCR amplification is performed using the second primer pair;

[0065] The reaction system used in step S133 is shown in Table 4 below:

[0066] Table 4

[0067]

[0068] in:

[0069] 4X rhAmpSeq LibraryMix1 and 4X rhAmpSeq LibraryMix2 in Tables 1 and 4 are two components from the same kit. Kit name: rhAmpSeq TMLibrary Kit; Product No.: 10000067; Manufacturer: IDT;

[0070] I5 index_primer is a primer with an I5 index sequence; I7 catalog number: 203029; manufacturer: IDT; I7index_primer is a primer with an I7 index sequence; I5 catalog number: 203037; manufacturer: IDT.

[0071] The mixture obtained from the above reaction system was subjected to enzyme activation, amplification, and inactivation in the target region in sequence to complete the second round of PCR amplification.

[0072] Specifically, the amplification process is shown in Table 5 below:

[0073] Table 5

[0074]

[0075] S134: The products of the second round of PCR amplification were optionally purified using Agencourt AMPure XP beads with 80% ethanol (prepared fresh for immediate use), and the purified library was subjected to quantitative quality control.

[0076] Both the first and second rounds of PCR amplification were performed using the rhAmpSeq system. For details on using rhAmpSeq multiplex PCR amplification technology, please refer to [link to relevant documentation]. Figure 5 This technology uses blocking primers containing RNA bases (rhAmp primers) and leverages the inherent properties of RNase H2 enzymes to recognize and cleave DNA:RNA duplexes. Specifically, RNase H2 enzymes target and cleave RNA bases within the DNA:RNA duplex, releasing the 3' end blocking sequence and activating the rhAmp primers. RNase H2 enzyme activity is highly specific; it only activates when the rhAmp primers bind correctly to the template DNA, cleaving RNA bases and activating the rhAmp primers. Only activated rhAmp primers can elongate to generate the amplicon target sequence, thus reducing non-specific hybridization and the amplification of primer dimers, while significantly simplifying the operational process.

[0077] Example 2

[0078] The difference from Example 1 is that the ratio of magnetic beads is 1.0x, while all other conditions remain unchanged.

[0079] Example 3

[0080] The difference from Example 1 is that the ratio of magnetic beads is 1.5x, while all other conditions remain unchanged.

[0081] Example 4

[0082] The difference from Example 1 is that the ratio of magnetic beads is 2.5x, while all other conditions remain unchanged.

[0083] Example 5

[0084] The difference from Example 1 is that the ratio of magnetic beads is 4.5x, while all other conditions remain unchanged.

[0085] Comparative Example 1

[0086] The difference from Example 1 is that step S12 was not performed, while all other conditions remained unchanged.

[0087] Test Example 1

[0088] The DNA recovery rate tests conducted in Examples 1-5 and Comparative Example 1 of this invention yielded the following results: Figure 2 , 3 As shown.

[0089] The optimal ratio of magnetic beads for purification was determined by the DNA recovery rate, and the results are as follows: Figure 2 As shown, Figure 2 The diagram illustrates the recovery rates of DNA purified by magnetic beads at different ratios. The recovery rates of 1.0x and 1.5x DNA are relatively low. Clinical samples are inherently precious and scarce, and a recovery rate of <70% does not meet QC standards. Therefore, magnetic bead ratios of 1.0x and 1.5x are not applicable, i.e., ratios below 1.5x are not suitable. The magnetic bead purification recovery rate in the range of 2.5x-4.5x is ≥80%, which is a more suitable range.

[0090] at the same time, Figure 3 The diagram illustrates the effect of different proportions of magnetic beads on homogeneity during purification. Figure 3 The results also clearly demonstrate that the library homogeneity is significantly improved after purification with 2.5x-4.5x magnetic beads.

[0091] Test Example 2

[0092] Fourteen FFPE DNA samples extracted using the QIAamp DNA FFPE tissue kit (qiagen, 56404) were selected. One sample underwent DNA magnetic bead purification, while the other did not (i.e., step S12 was not performed; this was the control group). The treated DNA was then tested according to the method described in this embodiment of the invention. Figure 6 The homogeneity of the library was significantly improved after DNA purification treatment.

[0093] Test Example 3

[0094] Thirty-nine FFPE DNA samples extracted using the ReliaPrep FFPE gDNA Miniprep System (promega, Cat. NO: A2352) were selected. DNA was then purified using magnetic beads, and unpurified DNA (i.e., step S12 was not performed, serving as a control group) was also treated. The treated DNA was then tested according to the method of this invention. Figure 7 The homogeneity of the library was significantly improved after DNA purification treatment.

[0095] Test Example 4

[0096] Another 24 FFPE DNA samples extracted using the ReliaPrep FFPE gDNA Miniprep System (promega, Cat. NO: A2352) were selected and subjected to DNA magnetic bead purification and unpurified DNA (i.e., step S12 was not performed, control group). The treated DNA was tested according to the method of this embodiment of the invention, such as... Figure 8 The homogeneity of the library was significantly improved after DNA purification treatment.

[0097] Example 6

[0098] like Figure 9 As shown, this embodiment of the invention also provides a method for improving primer uniformity based on amplicon sequencing, comprising:

[0099] S21: Construct an amplicon sequencing library using the method described in any of the embodiments 1-5 above;

[0100] S22: Sequencing and bioinformatics analysis of the constructed library.

[0101] In summary, this invention improves library uniformity by purifying DNA (e.g., extracted DNA) with magnetic beads before amplification, removing residual organic reagents (e.g., polysaccharides and salt ions), thereby reducing sequencing costs, increasing amplicon throughput, avoiding or minimizing their impact on enzyme activity, and enhancing its clinical diagnostic value. Furthermore, the invention offers advantages such as simple operation and low cost.

[0102] 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. sequence list <110> Shanghai 3D Biomedical Technology Co., Ltd. <120> Library construction methods and methods to improve primer uniformity <141> 2021-12-27 <160> 94 <170> SIPOSequenceListing 1.0 <210> 1 <211> 66 <212> DNA / RNA <213> Artificial sequence (none) <400> 1 acactctttc cctacacgac gctcttccga tctgaaattc tctgaagcaa acaaactacr 60 ctggtg 66 <210> 2 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 2 acactctttc cctacacgac gctcttccga tcttccacac ttaccctgat acatrcacag 60 c 61 <210> 3 <211> 67 <212> DNA / RNA <213> Artificial sequence (none) <400> 3 acactctttc cctacacgac gctcttccga tctcctgaat tttacctact gtttacaaac 60 rcaccat 67 <210> 4 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 4 acactctttc cctacacgac gctcttccga tctgttcctt gagtaaaagg tgctratgct 60 a 61 <210> 5 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 5 acactctttc cctacacgac gctcttccga tctcttggta ttgccctgtg tcrcagcta 59 <210> 6 <211> 65 <212> DNA / RNA <213> Artificial sequence (none) <400> 6 acactctttc cctacacgac gctcttccga tctgagaaca cgaaaaatat tcctactcrc 60 gcata 65 <210> 7 <211> 57 <212> DNA / RNA <213> Artificial sequence (none) <400> 7 acactctttc cctacacgac gctcttccga tctcctgggc ccagtcctat rutttta 57 <210> 8 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 8 acactctttc cctacacgac gctcttccga tctaagtctg cagttgaaaa gccrcaacga 60 <210> 9 <211> 63 <212> DNA / RNA <213> Artificial sequence (none) <400> 9 acactctttc cctacacgac gctcttccga tctggcatgg agatattttc cttctgrgtc 60 tta 63 <210> 10 <211> 65 <212> DNA / RNA <213> Artificial sequence (none) <400> 10 acactctttc cctacacgac gctcttccga tctcgttggt atataaaacc tgactactrc 60 atttg 65 <210> 11 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 11 acactctttc cctacacgac gctcttccga tcttccaaga atgtaagtgg gagtrgattc 60 a 61 <210> 12 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 12 acactctttc cctacacgac gctcttccga tcttattcac agagacttgg cagcrcagaa 60 t 61 <210> 13 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 13 acactctttc cctacacgac gctcttccga tcttccacct ttctccagct aattrcatct 60 c 61 <210> 14 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 14 acactctttc cctacacgac gctcttccga tcttgtgaaa attccagtgg ccatrcaaag 60 a 61 <210> 15 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 15 acactctttc cctacacgac gctcttccga tctcctttca tgctctcttc ccrcaggtt 59 <210> 16 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 16 acactctttc cctacacgac gctcttccga tctctatttt tcctcacagc tcgttrcatc 60 gc 62 <210> 17 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 17 acactctttc cctacacgac gctcttccga tctcctttgg aaaacctgca gatrcatcac 60 <210> 18 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 18 acactctttc cctacacgac gctcttccga tctcatgtac tggtccctca ttgrcactga 60 <210> 19 <211> 63 <212> DNA / RNA <213> Artificial sequence (none) <400> 19 acactctttc cctacacgac gctcttccga tcttgctaac caagttcttt cttttgrcac 60 agc 63 <210> 20 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 20 acactctttc cctacacgac gctcttccga tctctcagga acacggttaa tgtrcatagc 60 <210> twenty one <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> twenty one acactctttc cctacacgac gctcttccga tctaggatga ctgttaccac catarcaggc 60 c 61 <210> twenty two <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> twenty two acactctttc cctacacgac gctcttccga tctcaaaatc atctgtgccc agrcagtgg 59 <210> twenty three <211> 58 <212> DNA / RNA <213> Artificial sequence (none) <400> twenty three acactctttc cctacacgac gctcttccga tctaccgcac atcatctcgt arcatgag 58 <210> twenty four <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> twenty four acactctttc cctacacgac gctcttccga tctctgtctt tttcttccag tttgcrcaag 60 gg 62 <210> 25 <211> 63 <212> DNA / RNA <213> Artificial sequence (none) <400> 25 acactctttc cctacacgac gctcttccga tctttgatga cattgcatac attcgaraag 60 acg 63 <210> 26 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 26 acactctttc cctacacgac gctcttccga tctgtcaagg ttgctgattt tggtrcttgc 60 g 61 <210> 27 <211> 58 <212> DNA / RNA <213> Artificial sequence (none) <400> 27 acactctttc cctacacgac gctcttccga tcttgtggtc tcccataccc trctcagg 58 <210> 28 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 28 acactctttc cctacacgac gctcttccga tctgggaaaa tgacaaagaa cagctrcaaa 60 gg 62 <210> 29 <211> 69 <212> DNA / RNA <213> Artificial sequence (none) <400> 29 acactctttc cctacacgac gctcttccga tctgctcatc ttttctttat gttttcgaat 60 ttrctcgat 69 <210> 30 <211> 67 <212> DNA / RNA <213> Artificial sequence (none) <400> 30 acactctttc cctacacgac gctcttccga tctcagctac accatatatg aatggagaaa 60 rcatctt 67 <210> 31 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 31 acactctttc cctacacgac gctcttccga tctgggatta aagctggcta tggrcacctc 60 <210> 32 <211> 64 <212> DNA / RNA <213> Artificial sequence (none) <400> 32 acactctttc cctacacgac gctcttccga tctacctttt cttatgtgct tttagggrcc 60 cacg 64 <210> 33 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 33 acactctttc cctacacgac gctcttccga tcttcttatt ggctttggtc ttcaargtag 60 cg 62 <210> 34 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 34 acactctttc cctacacgac gctcttccga tctagaaacc gaggtatgaa attcgrctgg 60 ac 62 <210> 35 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 35 acactctttc cctacacgac gctcttccga tctgcaaata cagctttggt gcrcacctc 59 <210> 36 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 36 acactctttc cctacacgac gctcttccga tctaggagct agagcttgat gagrcagcac 60 <210> 37 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 37 acactctttc cctacacgac gctcttccga tcttgtattg gtctctcatg gcarctgtag 60 <210> 38 <211> 64 <212> DNA / RNA <213> Artificial sequence (none) <400> 38 acactctttc cctacacgac gctcttccga tctgtctgaa ctgaagataa tgactcarcc 60 tggc 64 <210> 39 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 39 acactctttc cctacacgac gctcttccga tctatgatac tcactgtcca tcagcrctcc 60 ac 62 <210> 40 <211> 57 <212> DNA / RNA <213> Artificial sequence (none) <400> 40 acactctttc cctacacgac gctcttccga tctacttcct cttctgccct rcccagc 57 <210> 41 <211> 65 <212> DNA / RNA <213> Artificial sequence (none) <400> 41 acactctttc cctacacgac gctcttccga tctcctctat tgttggatca tattcgtcrc 60 acaat 65 <210> 42 <211> 65 <212> DNA / RNA <213> Artificial sequence (none) <400> 42 acactctttc cctacacgac gctcttccga tctcaactgt gttagattga ctgtttctrc 60 atcca 65 <210> 43 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 43 acactctttc cctacacgac gctcttccga tctaggatcc agcacttcag taarggtctc 60 <210> 44 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 44 acactctttc cctacacgac gctcttccga tctatgatga cttcccagag ctrccagat 59 <210> 45 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 45 acactctttc cctacacgac gctcttccga tcttgaaagc tactccactg tttgrgcatg 60 t 61 <210> 46 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 46 acactctttc cctacacgac gctcttccga tctcctttct cctacacaca ggrcttcac 59 <210> 47 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 47 acactctttc cctacacgac gctcttccga tctaaacatt cccgcctcac atrgatccg 59 <210> 48 <211> 68 <212> DNA / RNA <213> Artificial sequence (none) <400> 48 gtgactggag ttcagacgtg tgctcttccg atcttcttca gtgataatgc aatatttgtt 60 grgctgta 68 <210> 49 <211> 63 <212> DNA / RNA <213> Artificial sequence (none) <400> 49 gtgactggag ttcagacgtg tgctcttccg atctgtttct gattgcacat tttggggct 60 tta 63 <210> 50 <211> 69 <212> DNA / RNA <213> Artificial sequence (none) <400> 50 gtgactggag ttcagacgtg tgctcttccg atctgtagga gagaaaagag tttaactaaga 60 aargtctta 69 <210> 51 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 51 gtgactggag ttcagacgtg tgctcttccg atctggacag ctagaagcag agatrctgaa 60 t 61 <210> 52 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 52 gtgactggag ttcagacgtg tgctcttccg atctgggatt ccctatactg agaccrattt 60 ta 62 <210> 53 <211> 68 <212> DNA / RNA <213> Artificial sequence (none) <400> 53 gtgactggag ttcagacgtg tgctcttccg atctttttat atttaaatgt atgtctcccc 60 trggccta 68 <210> 54 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 54 gtgactggag ttcagacgtg tgctcttccg atctattgtg ccattgcatt ccaarcctgg 60 c 61 <210> 55 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 55 gtgactggag ttcagacgtg tgctcttccg atcttgacca ataagcaagt cactgruggc 60 ta 62 <210> 56 <211> 66 <212> DNA / RNA <213> Artificial sequence (none) <400> 56 gtgactggag ttcagacgtg tgctcttccg atctgcatga atgtctagag cttcctatar 60 catcat 66 <210> 57 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 57 gtgactggag ttcagacgtg tgctcttccg atctcaagga acccacaggt aatgrctttt 60 a 61 <210> 58 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 58 gtgactggag ttcagacgtg tgctcttccg atctggctct aaaatgctct gttctrcaaa 60 at 62 <210> 59 <211> 63 <212> DNA / RNA <213> Artificial sequence (none) <400> 59 gtgactggag ttcagacgtg tgctcttccg atctgggtac tcacgtttcc tttaacrcac 60 att 63 <210> 60 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 60 gtgactggag ttcagacgtg tgctcttccg atcttagaac gcaccataga agccrcacga 60 a 61 <210> 61 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 61 gtgactggag ttcagacgtg tgctcttccg atctggtcct tcctgtcctc ctagrcagga 60 c 61 <210> 62 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 62 gtgactggag ttcagacgtg tgctcttccg atctcccttc gcacttctta carcttgcc 59 <210> 63 <211> 63 <212> DNA / RNA <213> Artificial sequence (none) <400> 63 gtgactggag ttcagacgtg tgctcttccg atctcctctt cataaacatc tcgggarcaa 60 gcg 63 <210> 64 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 64 gtgactggag ttcagacgtg tgctcttccg atctaccctg taaatttctc atgggrcagc 60 tg 62 <210> 65 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 65 gtgactggag ttcagacgtg tgctcttccg atctctccct tctcaggatt cctarcagga 60 t 61 <210> 66 <211> 65 <212> DNA / RNA <213> Artificial sequence (none) <400> 66 gtgactggag ttcagacgtg tgctcttccg atctacttac tgttcaagga tttcacagrc 60 acaga 65 <210> 67 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 67 gtgactggag ttcagacgtg tgctcttccg atctcagggc ctctctatgt catargttga 60 c 61 <210> 68 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 68 gtgactggag ttcagacgtg tgctcttccg atctctcctg tgatctgcaa tctagrcgcc 60 tc 62 <210> 69 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 69 gtgactggag ttcagacgtg tgctcttccg atctcatctt accaggcagt cgrctctcg 59 <210> 70 <211> 58 <212> DNA / RNA <213> Artificial sequence (none) <400> 70 gtgactggag ttcagacgtg tgctcttccg atctcttgat gccgagtcct grcccatg 58 <210> 71 <211> 64 <212> DNA / RNA <213> Artificial sequence (none) <400> 71 gtgactggag ttcagacgtg tgctcttccg atctccacct cacagttatt gaacatcrct 60 ctgc 64 <210> 72 <211> 65 <212> DNA / RNA <213> Artificial sequence (none) <400> 72 gtgactggag ttcagacgtg tgctcttccg atctaagatc caatccattt ttgttgtcrc 60 agcct 65 <210> 73 <211> 63 <212> DNA / RNA <213> Artificial sequence (none) <400> 73 gtgactggag ttcagacgtg tgctcttccg atctgagttt gcagactttc caaagcrcat 60 CCT 63 <210> 74 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 74 gtgactggag ttcagacgtg tgctcttccg atctggatgt caggcagatg ccrcagaac 59 <210> 75 <211> 63 <212> DNA / RNA <213> Artificial sequence (none) <400> 75 gtgactggag ttcagacgtg tgctcttccg atctccattt tagcacttac ctgtgarctc 60 caa 63 <210> 76 <211> 69 <212> DNA / RNA <213> Artificial sequence (none) <400> 76 gtgactggag ttcagacgtg tgctcttccg atctgtacac atgaagccat cgtatatatt 60 carcattta 69 <210> 77 <211> 69 <212> DNA / RNA <213> Artificial sequence (none) <400> 77 gtgactggag ttcagacgtg tgctcttccg atcttcagca tttgacttta ccttatcaat 60 gtrctcgat 69 <210> 78 <211> 57 <212> DNA / RNA <213> Artificial sequence (none) <400> 78 gtgactggag ttcagacgtg tgctcttccg atctctggga ggtggtggtg rgtccca 57 <210> 79 <211> 64 <212> DNA / RNA <213> Artificial sequence (none) <400> 79 gtgactggag ttcagacgtg tgctcttccg atctctgcat gatcttcctg ctttgaarca 60 aatt 64 <210> 80 <211> 63 <212> DNA / RNA <213> Artificial sequence (none) <400> 80 gtgactggag ttcagacgtg tgctcttccg atcttcactt aatttggatt gtggcarcag 60 agt 63 <210> 81 <211> 65 <212> DNA / RNA <213> Artificial sequence (none) <400> 81 gtgactggag ttcagacgtg tgctcttccg atctgggtcc acataaatat attcatgtrc 60 catca 65 <210> 82 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 82 gtgactggag ttcagacgtg tgctcttccg atctaagatg ggatactcca gggrctcagg 60 <210> 83 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 83 gtgactggag ttcagacgtg tgctcttccg atctgctcac tgatcttctc aaagtrcgtc 60 aa 62 <210> 84 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 84 gtgactggag ttcagacgtg tgctcttccg atctccccag gattcttaca gaaaarcaag 60 tc 62 <210> 85 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 85 gtgactggag ttcagacgtg tgctcttccg atctgtccag tgaaaatcct cactcrcagg 60 tg 62 <210> 86 <211> 62 <212> DNA / RNA <213> Artificial sequence (none) <400> 86 gtgactggag ttcagacgtg tgctcttccg atctactaca gaatcacatg ccacarcagt 60 gt 62 <210> 87 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 87 gtgactggag ttcagacgtg tgctcttccg atctcatcac acaccataac tccarcacat 60 g 61 <210> 88 <211> 67 <212> DNA / RNA <213> Artificial sequence (none) <400> 88 gtgactggag ttcagacgtg tgctcttccg atcttgactg aatataaact tgtggtagtt 60 rggagca 67 <210> 89 <211> 66 <212> DNA / RNA <213> Artificial sequence (none) <400> 89 gtgactggag ttcagacgtg tgctcttccg atcttgatcc acattgtatg gtttttaggr 60 caccaa 66 <210> 90 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 90 gtgactggag ttcagacgtg tgctcttccg atcttaccca agaagactct gtccrcagcc 60 t 61 <210> 91 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 91 gtgactggag ttcagacgtg tgctcttccg atctatacaa gagcttccgg agacrcagca 60 t 61 <210> 92 <211> 61 <212> DNA / RNA <213> Artificial sequence (none) <400> 92 gtgactggag ttcagacgtg tgctcttccg atctggtagt tactnagccc atgcrcatcg 60 a 61 <210> 93 <211> 60 <212> DNA / RNA <213> Artificial sequence (none) <400> 93 gtgactggag ttcagacgtg tgctcttccg atctgctaat gtgcgtttct ccarcactta 60 <210> 94 <211> 59 <212> DNA / RNA <213> Artificial sequence (none) <400> 94 gtgactggag ttcagacgtg tgctcttccg atcttatcta gcagagctgc ggrcagacg 59

Claims

1. A library construction method for improving the uniformity of amplicon libraries, characterized in that, The method includes the following steps: 1) Provide DNA derived from biological samples; 2) The DNA was purified using magnetic beads at a ratio of 2.5x to 4.5x. 3) The purified DNA is amplified to form an amplicon library; The amplification of the purified DNA specifically includes: i) Using the purified DNA as a template, perform the first round of PCR amplification using the first primer. ii) The products of the first round of PCR amplification may be purified; iii) Using the product from the first round of PCR amplification and purification as a template, a second round of PCR amplification was performed using a second primer; and iv) The products of the second round of PCR amplification may be purified to obtain the target amplicon library; The first primer is the nucleotide sequence shown in SEQ ID NO: 1-94; Both the first and second rounds of PCR amplification were performed using the rhAmpSeq system.

2. The method according to claim 1, characterized in that, The DNA mentioned therein is extracted DNA.

3. The method according to claim 1 or 2, characterized in that, The biological sample mentioned therein is a body fluid sample or a tissue sample.

4. The method according to claim 1 or 2, characterized in that, The biological sample mentioned above is a paraffin-embedded sample.

5. The method according to claim 1 or 2, characterized in that, The biological sample mentioned above is an FFPE sample.

6. A method for improving primer uniformity based on amplicon sequencing, characterized in that, include: 1) Construct an amplicon sequencing library using the method described in any one of claims 1-5; 2) Sequencing and bioinformatics analysis were performed on the constructed library.

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