A library construction method for improving amplicon library uniformity

Abstract

The present disclosure relates to a library construction method for improving amplicon library uniformity, the method comprising the steps of: 1) providing DNA; 2) performing enzymatic breakage on the DNA using a DNA fragmentation enzyme to form an enzymatically broken DNA; and 3) performing amplification on the enzymatically broken DNA to form an amplicon library.

Description

Technical Field

[0001] This invention belongs to the field of high-throughput gene sequencing. Specifically, this invention relates to a library construction method for improving the uniformity of amplicon libraries. 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 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 primarily utilizes multiplex PCR to simultaneously and specifically amplify and enrich sequences from multiple target regions, obtaining an amplicon library. Next-generation sequencing (NGS) is then used to sequence this library, thereby obtaining the sequence information of the target region. Amplicon sequencing offers advantages such as flexible panel design, cost-effectiveness, speed, good reproducibility, and high specificity, making it the preferred technology widely used in basic biological research and clinical disease diagnosis.

[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. Poor library uniformity significantly increases sequencing costs and limits amplicon throughput.

[0005] Numerous studies have shown that as amplicon throughput increases, primer dimers and nonspecific fragments also increase significantly, leading to a sharp decline in library homogeneity. Differences in amplification efficiency among different primers are a key factor contributing to poor library homogeneity. Furthermore, the multilevel spatial structure of DNA affects the complete binding of DNA and primers; insufficient binding between the DNA template and primers reduces primer amplification efficiency, thus resulting in poor amplicon library homogeneity.

[0006] Currently, methods to improve amplicon uniformity mainly involve adjusting the concentration of different primers to reduce the amplification efficiency differences between primers, thereby improving library uniformity. While this method can improve library uniformity to some extent, it cannot completely solve the problem. Another approach is to control the number of amplicones to enhance library uniformity, but this method somewhat limits the application of this technology in clinical diagnosis. In conclusion, improving library uniformity has become a pressing issue in the field of amplicon sequencing technology and a crucial hurdle that must be overcome to further reduce amplicon sequencing costs and increase throughput.

[0007] Therefore, there is an urgent need in this field for methods to solve the above problems in order to improve the uniformity of libraries. Summary of the Invention

[0008] Current amplicon sequencing technology uses extracted DNA samples directly for amplicon library construction and sequencing. The multi-level spatial structure of DNA can affect the full binding of DNA and primers. If the DNA template and primers cannot bind fully, primer-template binding bias will occur, which will amplify the differences in primer amplification efficiency, thus leading to poor uniformity of the amplicon library.

[0009] The inventors of this application unexpectedly discovered that digesting DNA samples with DNA fragmentase before amplicon library construction can significantly improve the homogeneity of the amplicon library. For example, the inventors of this application unexpectedly discovered that digesting FFPE DNA samples with DNA fragmentase before amplicon library construction can significantly improve the homogeneity of the amplicon library.

[0010] Based on this, the present invention provides: A library construction method for improving the uniformity of amplicon libraries, the method comprising digesting and breaking down a DNA sample with a DNA fragmentation enzyme before constructing the amplicon library; The use of a DNA fragmentation enzyme and amplification reagent in the preparation of a kit for the aforementioned method; and A kit for improving the uniformity of amplicon libraries, the kit comprising: a DNA fragmentation enzyme for digesting DNA prior to amplification; and an amplification reagent for amplifying the digested DNA.

[0011] Compared with the prior art, the present invention has at least the following advantages: i. Increasing the effective amount of DNA template allows primers to bind more fully to DNA; ii. Improve the uniformity of the document library; iii. Applicable to a wide variety of database construction technologies, including but not limited to rhAmpSeq; iv. Significantly improves the homogeneity of libraries constructed using rhAmpSeq multiplex PCR technology; v. The operation process is simple and the cost is low.

[0012] In a first aspect, this disclosure provides a library construction method for improving the uniformity of amplicon libraries, the method comprising the following steps: 1) Provide DNA derived from biological samples; 2) The DNA is cleaved using a DNA fragmentation enzyme to form fragmented DNA; and 3) The DNA fragmented by the enzyme digestion is amplified to form an amplicon library.

[0013] In some implementations, the DNA is extracted DNA.

[0014] In some implementations, the biological sample is a body fluid sample or a tissue sample.

[0015] In some embodiments, the biological sample is a paraffin-embedded sample, such as an FFPE sample.

[0016] In some implementations, the length of the DNA fragments after enzyme digestion is in the range of 500-4000 bp.

[0017] In some embodiments, the amplification includes i) performing a first round of PCR amplification using the enzyme-digested DNA as a template through a first primer; ii) optionally purifying the DNA; iii) performing a second round of PCR amplification using the product of the first round of PCR amplification as a template through a second primer; and iv) optionally purifying the DNA to obtain a target amplicon library.

[0018] In some implementations, the first primer contains SEQ ID NO: 1-94.

[0019] In some implementations, the first and second rounds of PCR amplification are performed using the rhAmpSeq system.

[0020] In a second aspect, this disclosure provides the use of DNA fragmentation enzymes and amplification reagents in the preparation of kits for use in the method described in any embodiment of the first aspect.

[0021] In some embodiments, the amplification reagent includes a first primer and a second primer, wherein the first primer contains SEQ ID NO: 1-94.

[0022] In a third aspect, this disclosure provides a kit for improving the uniformity of amplicon libraries, the kit comprising: a DNA fragmentation enzyme for digesting DNA prior to amplification; and an amplification reagent for amplifying the digested DNA.

[0023] In some embodiments, the amplification reagent includes a first primer and a second primer, wherein the first primer contains SEQ ID NO: 1-94.

[0024] The following description and examples illustrate embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein and therefore can be modified. Those skilled in the art will recognize that many variations and modifications exist in the present invention, all of which are included within its scope. Attached Figure Description

[0025] Figure 1 The results, as described in Example 2, show that the homogeneity of the amplicon libraries obtained by enzyme digestion of 22 different gDNA samples, including control and experimental groups, is significantly improved. Figure 1 In the graph, the horizontal axis represents the sample number, and the vertical axis represents the library homogeneity percentage. The highest homogeneity is 1 (100%), and the closer the value is to 100%, the higher the homogeneity. The squares in the graph represent samples that have been digested with DNA fragmentation enzymes, while the circles represent samples that have not been digested.

[0026] Figure 2 This diagram illustrates the homogeneity of amplicon libraries obtained from four different FFPE DNA samples subjected to different lengths of restriction enzyme digestion (500bp, 1000bp, 2000bp, 4000bp, 8000bp digestion groups and a control group), as described in Example 3. The horizontal axis represents the sample number, and the vertical axis represents the library homogeneity percentage. The highest homogeneity was 1 (100%), with values ​​closer to 100% indicating higher homogeneity. The results show that when the DNA fragments are in the 500-4000bp range, the library homogeneity is improved to nearly 1 (100%). Detailed Implementation

[0027] The inventors of this application unexpectedly discovered that digesting DNA samples with DNA fragmentation enzymes before amplicon library construction can significantly improve the homogeneity of the amplicon library. For example, digesting FFPE DNA samples with DNA fragmentation enzymes before amplicon library construction can significantly improve the homogeneity of the amplicon library.

[0028] More specifically, the inventors of this application unexpectedly discovered that digesting FFPE DNA samples with NEBNext® dsDNAFragmentase® before constructing amplicon libraries can significantly improve the uniformity of the amplicon libraries, as demonstrated in the examples of this application.

[0029] Compared with the prior art, the present invention has at least the following advantages: i. Increasing the effective amount of DNA template allows primers to bind more fully to DNA; ii. Improve the uniformity of the document library; iii. It is applicable to a wide variety of database construction technologies, including but not limited to rhAmpSeq and DamSeq; iv. Significantly improves the homogeneity of libraries constructed using rhAmpSeq multiplex PCR technology; v. The operation process is simple and the cost is low.

[0030] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” used herein are also intended to include the plural forms. Furthermore, the open-ended expressions “comprising” and “including” are to be interpreted as potentially containing structural components or method steps not mentioned, but it should be noted that these open-ended expressions also cover situations where the invention consists only of the stated components and method steps (i.e., they cover the closed-ended expressions “consisting of…”).

[0031] As used throughout, a range is used as a shorthand to describe each and all values ​​within that range. Any value within the range, such as an integer value, a value that increments by 10 (when the end of the range is a multiple of 10), or a value that increments by one-tenth (when the end of the range is one decimal place), can be chosen as the end of the range. For example, the range 150 bp-5 kb is used to describe all values ​​within that range, such as 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, 200 bp, 210 bp, 220 bp...4950 bp, 4960 bp, 4970 bp, 4980 bp, 4990 bp and 5000 bp (in increments of 10 bp), and includes all subranges, such as 200-4000 bp, 300-3000 bp, 400-2000 bp, 500-1000 bp, 600-1500 bp, etc.

[0032] All scientific and technical terms mentioned in this specification have the same meaning as commonly understood by those skilled in the art, and in case of conflict, the definitions in this specification shall prevail. To make the description of this invention easier to understand, some terms are explained below.

[0033] The term "high-throughput sequencing" used in this article, also known as next-generation sequencing (NGS) or massively parallel sequencing (MPS), refers to a sequencing technology that uses the principle of "sequencing while synthesis" to simultaneously perform parallel sequencing reactions on hundreds of thousands to millions of DNA molecules. The raw image data or electrochemical signals obtained are then analyzed through bioinformatics to ultimately obtain information such as the nucleic acid sequence or copy number of the sample. It is also called high-throughput sequencing, deep sequencing, or second-generation sequencing. The basic procedure of high-throughput sequencing involves randomly fragmenting the DNA to be tested into small fragments, constructing a library through steps such as end repair, ligation of adapter sequences, and PCR, and finally sequencing using sequencers such as Illumina or Ion Torrent.

[0034] The term "biological sample" as used herein refers to a sample of biological tissue or body fluid containing nucleic acids or peptides. Such samples are typically derived from humans, but include tissues isolated from non-human primates or rodents (e.g., mice and rats). Biological samples may also include tissue secretions, such as biopsy and autopsy samples, frozen sections obtained for histological purposes, cerebrospinal fluid, blood, plasma, serum, sputum, feces, tears, mucus, hair, skin, etc. Biological samples also include explants and / or primary and / or transformed cell cultures derived from patient tissues. "Biological sample" also refers to a single cell or cell population or a quantity of tissue or body fluid derived from an animal. Most commonly, a biological sample has been removed from the animal, but the term "biological sample" can also refer to cells or tissues analyzed in vivo, i.e., not removed from the animal. Typically, a "biological sample" will contain cells from an animal, but the term can also refer to non-cellular biological material that can be used to measure the expression levels of polynucleotides or peptides, such as non-cellular components of blood, serum, saliva, cerebrospinal fluid, or urine. Many types of biological samples can be used in this invention, including but not limited to tissue biopsies or blood samples.

[0035] The term "amplifier library construction" as used in this article refers to the amplicon library construction method, which uses multiplex PCR technology to enrich target DNA regions, thereby constructing libraries for next-generation sequencing.

[0036] In this article, the term "homogeneity" in amplicon libraries refers to the uniformity of sequencing depth.

[0037] 1. DNA sample The DNA used in this invention can be derived from various sources, such as direct extraction from biological samples or preparation by reverse transcription of RNA. Methods for extracting nucleic acids, such as DNA, from samples are well known in the art; for example, DNA extraction can be performed using phenol and chloroform, or using commercially available DNA extraction reagents.

[0038] In some embodiments, the DNA is genomic DNA (gDNA). In some embodiments, the DNA is extracted DNA. DNA extraction can be performed using any method known in the art, such as Qiagen reagent or Promega reagent.

[0039] In some embodiments, the biological sample is a body fluid sample or a tissue sample, preferably selected from biopsy samples, tumor tissue samples, cell cultures, solidified samples (such as paraffin-embedded samples, for example FFPE samples), whole blood, plasma, serum, saliva, cerebrospinal fluid, sweat, sputum, bronchoalveolar lavage fluid, urine, feces, secretions, milk, and peritoneal fluid, etc.

[0040] In some embodiments, the biological sample is a paraffin-embedded sample, such as an FFPE sample.

[0041] It should be understood that DNA can be purified using conventional purification methods in the art, such as the PrepSEQ™ kit (from Applied Biosystems) and the method in U.S. Patent No. 5,234,809, etc.

[0042] 2. DNA fragmentation by enzyme digestion After obtaining the DNA, it can be fragmented using a DNA fragmentation enzyme. In some embodiments, the DNA fragmentation enzyme is a fragmentase (NEBNext® dsDNA Fragmentase®) from NEB Corporation. In some embodiments, the DNA fragmentation enzyme includes, but is not limited to, Tn5 transposases, DNase I, and Endonuclease V used in high-throughput sequencing, as well as various other commercially available DNA fragmentation enzymes.

[0043] Although not bound by any theory, the inventors of this application have discovered that by enzymatically digesting and breaking down the extracted DNA, the multi-level spatial structure of the DNA is opened up, allowing primers to bind more fully to the DNA and improving the uniformity of the library.

[0044] 3. Amplification DNA can be amplified through various mechanisms, some of which include PCR. Samples can be amplified on an array. See, for example, U.S. Patent No. 6,300,070 and U.S. Patent Application Serial No. 09 / 513,300.

[0045] Other suitable amplification methods include ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4,560 (1989), Landegren et al., Science 241,1077 (1988), and Barringer et al., Gene 89:117 (1990)), transcriptional amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88 / 10315), self-sustaining sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87,1874 (1990) and WO90 / 06995), selective amplification of the target polynucleotide sequence (US Patent No. 6,410,276), consensual sequence-initiated polymerase chain reaction (CP-PCR) (US Patent No. 4,437,975), and arbitrary-initiated polymerase chain reaction (AP-PCR). (US Patent Nos. 5,413,909, 5,861,245) and nucleic acid-based sequence amplification (NABSA) (see US Patent Nos. 5,409,818, 5,554,517 and 6,063,603, each of which is incorporated herein by reference).

[0046] In some embodiments, amplification is performed using PCR. In some embodiments, the amplification includes i) performing a first round of PCR amplification using the enzyme-digested DNA as a template through a first primer; ii) optionally purifying the DNA; iii) performing a second round of PCR amplification using the product of the first round of PCR amplification as a template through a second primer; and iv) optionally purifying the DNA to obtain a target amplicon library.

[0047] In some implementations, the first primer contains or consists of SEQ ID NO: 1-94.

[0048] In some implementations, the first and second rounds of PCR amplification are performed using the rhAmpSeq system.

[0049] In some implementations, the reaction system used for the first round of PCR amplification includes rhAmpSeq LibraryMix1, forward and reverse primers, and template DNA. In some implementations, the forward and reverse primers are shown in Tables 2 and 3, respectively.

[0050] In some implementations, the reaction system used for the second round of PCR amplification includes rhAmpSeq LibraryMix2, i5 (index 5), and i7 (index 7), as well as template DNA (the product purified from the first round of PCR).

[0051] 4. Reagent kit This disclosure also provides a kit for improving the uniformity of amplicon libraries, the kit comprising: a DNA fragmentation enzyme for digesting DNA prior to amplification; and an amplification reagent for amplifying the digested DNA.

[0052] In some embodiments, the DNA fragmentation enzyme is a fragmentase (NEBNext® dsDNA Fragmentase®) from NEB Corporation. In some embodiments, the DNA fragmentation enzyme includes, but is not limited to, Tn5 transposases, DNase I, and Endonuclease V used for high-throughput sequencing, as well as various other commercially available DNA fragmentation enzymes.

[0053] In some embodiments, the amplification reagents include rhAmpSeq Library Mix 1 or rhAmpSeq Library Mix 2, forward and reverse primers, index 5 and index 7 primers, and template DNA, etc. In some embodiments, the forward and reverse primers are shown in Tables 2 and 3, respectively.

[0054] While various embodiments of the invention have been described above, it should be understood that they are provided by way of example only and not as limitations. Many changes to the disclosed embodiments may be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Therefore, the breadth and scope of the invention should not be limited by any of the embodiments described above.

[0055] All references mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated herein by reference for all purposes, and are cited as if they were individually cited. Example

[0056] Unless otherwise stated, all materials used in the embodiments herein are commercially available, and all specific experimental methods used to conduct the experiments are conventional experimental methods in the art or are performed according to the steps and conditions recommended by the manufacturer, and can be conventionally determined by those skilled in the art as needed.

[0057] Example 1: Construction of an amplicon library using DNA fragmentation via enzyme digestion 1) Sample preparation: DNA extraction was 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). Cell line DNA and blood gDNA were extracted using the QIAamp DNA Blood Mini Kit.

[0058] 2) The extracted DNA was digested using NEBNext® dsDNA Fragmentase® (M0348L) according to the enzyme digestion system prepared in Table 1. The DNA was incubated at 37°C for 20 min for digestion. The fragmented DNA was then quantified using Qubit for later use.

[0059] Table 1 Enzyme digestion interruption conditions 3) Target region amplification: Prepare a 20 μL reaction system according to the table below. The reaction system is as follows (Pool_FWD and Pool_REV refer to the forward primer mixture and reverse primer mixture, respectively, designed by the applicant and synthesized in IDT. Specific primer information is shown in Tables 2 and 3, where all primers have a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end (the nucleotide after the letter r) is a ribonucleotide.

[0060] Table 2. Forward primer sequences Table 3. Reverse primer sequences Table 4. Target Region Amplification Reaction System The volume of DNA, X ul: The amount of DNA added for library construction is 50 ng. Based on the DNA concentration, the volume of DNA added is determined as the ratio of the total amount of DNA to the DNA concentration. X represents the volume of DNA added for different samples.

[0061] The first round of amplification targeting the region was performed using a ProFlex PCR System PCR instrument (Applied biosystems by life technologies, model: ProFlex) according to the reaction procedure in Table 5; Table 5. Target Region Amplification Procedure 4) Purification of the target amplification product: Agencourt AMPure XP beads (1.5x) were used to purify the target product with 80% ethanol (prepared fresh for use).

[0062] 5) Prepare the amplification (Index PCR) reaction system for the target product according to Table 6: Table 6. Target product amplification reaction system Amplification was performed according to the reaction procedure in Table 7 below; Table 7. Indexing PCR Amplification Procedure 6) Purification of the target library: Agencourt AMPure XP beads (1.0x) were used to purify the target product with 80% ethanol (prepared fresh for use). The purified library was then subjected to quantitative quality control.

[0063] 7) Perform sequencing on the library and analyze the data.

[0064] Example 2: The effect of enzyme digestion disruption on library uniformity Twenty-two different gDNA samples (two blood gDNA samples: gB_1, gB_1; two cell line DNA samples: HCT-15, H460; and the remaining 18 FFPE gDNA samples) were subjected to enzyme digestion and fragmentation treatment (control group (untreated), enzyme-digested group), and the tests were performed according to the procedure in Example 1. (See attached...) Figure 1 As can be seen, the homogeneity of the library after enzyme digestion was improved to nearly 1 (100%). This indicates that the homogeneity of the library was significantly improved after DNA fragmentation enzyme digestion.

[0065] Example 3: The effect of different lengths of enzyme digestion fragments on library homogeneity Four different FFPE DNA samples were selected and subjected to enzyme digestion of different lengths (500bp, 1000bp, 2000bp, 4000bp, 8000bp digestion groups and a control group), and the tests were performed according to the procedure in Example 1. (See attached...) Figure 2It is evident that the homogeneity of the library is improved to nearly 1 (100%) for DNA fragments in the 500-4000 bp range after enzyme digestion, while the improvement in homogeneity is not significant for fragments >4000 bp (e.g., 8000 bp), although there is some improvement. This indicates that DNA fragmentation enzyme digestion significantly improves the homogeneity of the library in the 500-4000 bp range.

[0066] While various embodiments of the invention have been described above, it should be understood that they are provided by way of example only and not as limitations. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications will fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents. sequence list <110> Shanghai 3D Biomedical Technology Co., Ltd. <120> A library construction method for improving the uniformity of amplicon libraries <130> CPCH2162595N <160> 94 <170> PatentIn version 3.5 <210> 1 <211> 65 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 1 acactctttc cctacacgac gctcttccga tctgaaattc tctgaagcaa acaaactacc 60 tggtg 65 <210> 2 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 2 acactctttc cctacacgac gctcttccga tcttccacac ttaccctgat acatcacagc 60 <210> 3 <211> 66 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 3 acactctttc cctacacgac gctcttccga tctcctgaat tttacctact gtttacaaac 60 caccat 66 <210> 4 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 4 acactctttc cctacacgac gctcttccga tctgttcctt gagtaaaagg tgctatgcta 60 <210> 5 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 5 acactctttc cctacacgac gctcttccga tctcttggta ttgccctgtg tccagcta 58 <210> 6 <211> 64 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 6 acactctttc cctacacgac gctcttccga tctgagaaca cgaaaaatat tcctactccg 60 cata 64 <210> 7 <211> 56 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 7 acactctttc cctacacgac gctcttccga tctcctgggc ccagtcctat utttta 56 <210> 8 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 8 acactctttc cctacacgac gctcttccga tctaagtctg cagttgaaaa gcccaacga 59 <210> 9 <211> 62 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 9 acactctttc cctacacgac gctcttccga tctggcatgg agatattttc cttctggtct 60 ta 62 <210> 10 <211> 64 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 10 acactctttc cctacacgac gctcttccga tctcgttggt atataaaacc tgactactca 60 tttg 64 <210> 11 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 11 acactctttc cctacacgac gctcttccga tcttccaaga atgtaagtgg gagtgattca 60 <210> 12 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 12 acactctttc cctacacgac gctcttccga tcttattcac agagacttgg cagccagaat 60 <210> 13 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 13 acactctttc cctacacgac gctcttccga tcttccacct ttctccagct aattcatctc 60 <210> 14 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 14 acactctttc cctacacgac gctcttccga tcttgtgaaa attccagtgg ccatcaaaga 60 <210> 15 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 15 acactctttc cctacacgac gctcttccga tctcctttca tgctctcttc cccaggtt 58 <210> 16 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 16 acactctttc cctacacgac gctcttccga tctctatttt tcctcacagc tcgttcatcg 60 c 61 <210> 17 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 17 acactctttc cctacacgac gctcttccga tctcctttgg aaaacctgca gatcatcac 59 <210> 18 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 18 acactctttc cctacacgac gctcttccga tctcatgtac tggtccctca ttgcactga 59 <210> 19 <211> 62 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 19 acactctttc cctacacgac gctcttccga tcttgctaac caagttcttt cttttgcaca 60 gc 62 <210> 20 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 20 acactctttc cctacacgac gctcttccga tctctcagga acacggttaa tgtcatagc 59 <210> twenty one <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> twenty one acactctttc cctacacgac gctcttccga tctaggatga ctgttaccac catacaggcc 60 <210> twenty two <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> twenty two acactctttc cctacacgac gctcttccga tctcaaaatc atctgtgccc agcagtgg 58 <210> twenty three <211> 57 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> twenty three acactctttc cctacacgac gctcttccga tctaccgcac atcatctcgt acatgag 57 <210> twenty four <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> twenty four acactctttc cctacacgac gctcttccga tctctgtctt tttcttccag tttgccaagg 60 g 61 <210> 25 <211> 62 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 25 acactctttc cctacacgac gctcttccga tctttgatga cattgcatac attcgaaaga 60 cg 62 <210> 26 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 26 acactctttc cctacacgac gctcttccga tctgtcaagg ttgctgattt tggtcttgcg 60 <210> 27 <211> 57 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 27 acactctttc cctacacgac gctcttccga tcttgtggtc tcccataccc tctcagg 57 <210> 28 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 28 acactctttc cctacacgac gctcttccga tctgggaaaa tgacaaagaa cagctcaaag 60 g 61 <210> 29 <211> 68 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 29 acactctttc cctacacgac gctcttccga tctgctcatc ttttctttat gttttcgaat 60 ttctcgat 68 <210> 30 <211> 66 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 30 acactctttc cctacacgac gctcttccga tctcagctac accatatatg aatggagaaa 60 catctt 66 <210> 31 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 31 acactctttc cctacacgac gctcttccga tctgggatta aagctggcta tggcacctc 59 <210> 32 <211> 63 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 32 acactctttc cctacacgac gctcttccga tctacctttt cttatgtgct tttagggccc 60 acg 63 <210> 33 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 33 acactctttc cctacacgac gctcttccga tcttcttatt ggctttggtc ttcaagtagc 60 g 61 <210> 34 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 34 acactctttc cctacacgac gctcttccga tctagaaacc gaggtatgaa attcgctgga 60 c 61 <210> 35 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 35 acactctttc cctacacgac gctcttccga tctgcaaata cagctttggt gccacctc 58 <210> 36 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 36 acactctttc cctacacgac gctcttccga tctaggagct agagcttgat gagcagcac 59 <210> 37 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 37 acactctttc cctacacgac gctcttccga tcttgtattg gtctctcatg gcactgtag 59 <210> 38 <211> 63 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 38 acactctttc cctacacgac gctcttccga tctgtctgaa ctgaagataa tgactcacct 60 ggc 63 <210> 39 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 39 acactctttc cctacacgac gctcttccga tctatgatac tcactgtcca tcagcctcca 60 c 61 <210> 40 <211> 56 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 40 acactctttc cctacacgac gctcttccga tctacttcct cttctgccct cccagc 56 <210> 41 <211> 64 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 41 acactctttc cctacacgac gctcttccga tctcctctat tgttggatca tattcgtcca 60 caat 64 <210> 42 <211> 64 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 42 acactctttc cctacacgac gctcttccga tctcaactgt gttagattga ctgtttctca 60 tcca 64 <210> 43 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 43 acactctttc cctacacgac gctcttccga tctaggatcc agcacttcag taaggtctc 59 <210> 44 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 44 acactctttc cctacacgac gctcttccga tctatgatga cttcccagag ctccagat 58 <210> 45 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 45 acactctttc cctacacgac gctcttccga tcttgaaagc tactccactg tttggcatgt 60 <210> 46 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 46 acactctttc cctacacgac gctcttccga tctcctttct cctacacaca ggcttcac 58 <210> 47 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 47 acactctttc cctacacgac gctcttccga tctaaacatt cccgcctcac atgatccg 58 <210> 48 <211> 67 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 48 gtgactggag ttcagacgtg tgctcttccg atcttcttca gtgataatgc aatatttgtt 60 ggctgta 67 <210> 49 <211> 62 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 49 gtgactggag ttcagacgtg tgctcttccg atctgtttct gattgcacat tttggggctt 60 ta 62 <210> 50 <211> 68 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 50 gtgactggag ttcagacgtg tgctcttccg atctgtagga gagaaaagag tttaactaaga 60 aagtctta 68 <210> 51 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 51 gtgactggag ttcagacgtg tgctcttccg atctggacag ctagaagcag agatctgaat 60 <210> 52 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 52 gtgactggag ttcagacgtg tgctcttccg atctgggatt ccctatactg agaccatttt 60 a 61 <210> 53 <211> 67 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 53 gtgactggag ttcagacgtg tgctcttccg atctttttat atttaaatgt atgtctcccc 60 tggccta 67 <210> 54 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 54 gtgactggag ttcagacgtg tgctcttccg atctattgtg ccattgcatt ccaacctggc 60 <210> 55 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 55 gtgactggag ttcagacgtg tgctcttccg atcttgacca ataagcaagt cactguggct 60 a 61 <210> 56 <211> 65 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 56 gtgactggag ttcagacgtg tgctcttccg atctgcatga atgtctagag cttcctatac 60 atcat 65 <210> 57 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 57 gtgactggag ttcagacgtg tgctcttccg atctcaagga acccacaggt aatgctttta 60 <210> 58 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 58 gtgactggag ttcagacgtg tgctcttccg atctggctct aaaatgctct gttctcaaaa 60 t 61 <210> 59 <211> 62 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 59 gtgactggag ttcagacgtg tgctcttccg atctgggtac tcacgtttcc tttaaccaca 60 tt 62 <210> 60 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 60 gtgactggag ttcagacgtg tgctcttccg atcttagaac gcaccataga agcccacgaa 60 <210> 61 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 61 gtgactggag ttcagacgtg tgctcttccg atctggtcct tcctgtcctc ctagcaggac 60 <210> 62 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 62 gtgactggag ttcagacgtg tgctcttccg atctcccttc gcacttctta cacttgcc 58 <210> 63 <211> 62 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 63 gtgactggag ttcagacgtg tgctcttccg atctcctctt cataaacatc tcgggacaag 60 cg 62 <210> 64 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 64 gtgactggag ttcagacgtg tgctcttccg atctaccctg taaatttctc atgggcagct 60 g 61 <210> 65 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 65 gtgactggag ttcagacgtg tgctcttccg atctctccct tctcaggatt cctacaggat 60 <210> 66 <211> 64 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 66 gtgactggag ttcagacgtg tgctcttccg atctacttac tgttcaagga tttcacagca 60 caga 64 <210> 67 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 67 gtgactggag ttcagacgtg tgctcttccg atctcagggc ctctctatgt catagttgac 60 <210> 68 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 68 gtgactggag ttcagacgtg tgctcttccg atctctcctg tgatctgcaa tctagcgcct 60 c 61 <210> 69 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 69 gtgactggag ttcagacgtg tgctcttccg atctcatctt accaggcagt cgctctcg 58 <210> 70 <211> 57 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 70 gtgactggag ttcagacgtg tgctcttccg atctcttgat gccgagtcct gcccatg 57 <210> 71 <211> 63 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 71 gtgactggag ttcagacgtg tgctcttccg atctccacct cacagttatt gaacatcctc 60 tgc 63 <210> 72 <211> 64 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 72 gtgactggag ttcagacgtg tgctcttccg atctaagatc caatccattt ttgttgtcca 60 gcct 64 <210> 73 <211> 62 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 73 gtgactggag ttcagacgtg tgctcttccg atctgagttt gcagactttc caaagccatc 60 ct 62 <210> 74 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 74 gtgactggag ttcagacgtg tgctcttccg atctggatgt caggcagatg cccagaac 58 <210> 75 <211> 62 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 75 gtgactggag ttcagacgtg tgctcttccg atctccattt tagcacttac ctgtgactcc 60 aa 62 <210> 76 <211> 68 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 76 gtgactggag ttcagacgtg tgctcttccg atctgtacac atgaagccat cgtatatatt 60 cacattta 68 <210> 77 <211> 68 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 77 gtgactggag ttcagacgtg tgctcttccg atcttcagca tttgacttta ccttatcaat 60 gtctcgat 68 <210> 78 <211> 56 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 78 gtgactggag ttcagacgtg tgctcttccg atctctggga ggtggtggtg gtccca 56 <210> 79 <211> 63 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 79 gtgactggag ttcagacgtg tgctcttccg atctctgcat gatcttcctg ctttgaacaa 60 att 63 <210> 80 <211> 62 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 80 gtgactggag ttcagacgtg tgctcttccg atcttcactt aatttggatt gtggcacaga 60 gt 62 <210> 81 <211> 64 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 81 gtgactggag ttcagacgtg tgctcttccg atctgggtcc acataaatat attcatgtcc 60 atca 64 <210> 82 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 82 gtgactggag ttcagacgtg tgctcttccg atctaagatg ggatactcca gggctcagg 59 <210> 83 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 83 gtgactggag ttcagacgtg tgctcttccg atctgctcac tgatcttctc aaagtcgtca 60 a 61 <210> 84 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 84 gtgactggag ttcagacgtg tgctcttccg atctccccag gattcttaca gaaaacaagt 60 c 61 <210> 85 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 85 gtgactggag ttcagacgtg tgctcttccg atctgtccag tgaaaatcct cactccaggt 60 g 61 <210> 86 <211> 61 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 86 gtgactggag ttcagacgtg tgctcttccg atctactaca gaatcacatg ccacacagtg 60 t 61 <210> 87 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 87 gtgactggag ttcagacgtg tgctcttccg atctcatcac acaccataac tccacacatg 60 <210> 88 <211> 66 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 88 gtgactggag ttcagacgtg tgctcttccg atcttgactg aatataaact tgtggtagtt 60 ggagca 66 <210> 89 <211> 65 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 89 gtgactggag ttcagacgtg tgctcttccg atcttgatcc acattgtatg gtttttaggc 60 accaa 65 <210> 90 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 90 gtgactggag ttcagacgtg tgctcttccg atcttaccca agaagactct gtcccagcct 60 <210> 91 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 91 gtgactggag ttcagacgtg tgctcttccg atctatacaa gagcttccgg agaccagcat 60 <210> 92 <211> 60 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <220> <221> misc_feature <222> (45)..(45) <223> n is a, c, g, t, or u <400> 92 gtgactggag ttcagacgtg tgctcttccg atctggtagt tactnagccc atgccatcga 60 <210> 93 <211> 59 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 93 gtgactggag ttcagacgtg tgctcttccg atctgctaat gtgcgtttct ccacactta 59 <210> 94 <211> 58 <212> DNA <213> Artificial sequence <220> <223> It has a 3SpC3 modification at the 3' end, and the 6th nucleotide starting from the 3' end is a ribonucleotide. <400> 94 gtgactggag ttcagacgtg tgctcttccg atctttatcta gcagagctgc ggcagacg 58

Claims

1. A method for constructing a library to improve the uniformity of sequencing depth of an amplicon library, the method comprising the following steps: 1) Provide DNA derived from biological samples; 2) The DNA is cleaved using a DNA fragmentation enzyme to form fragmented DNA; and 3) The DNA fragmented by the enzyme digestion is amplified to form an amplicon library. The DNA mentioned is extracted DNA, the biological sample is an FFPE sample, and the length of the DNA fragments after enzyme digestion is in the range of 500-4000 bp. The amplification includes i) using the enzyme-digested DNA as a template, performing a first round of PCR amplification with a first primer; ii) optionally purifying the DNA; iii) using the product of the first round of PCR amplification as a template, performing a second round of PCR amplification with a second primer; and iv) optionally purifying the DNA to obtain a target amplicon library, wherein the first and second rounds of PCR amplification are performed using the rhAmpSeq system.

2. The method according to claim 1, characterized in that... The first primer consists of SEQ ID NO: 1-94.

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