Amplification-based library construction method

Abstract

The present disclosure relates to a method of amplicon-based library construction, characterized by comprising the steps of: 1) providing DNA, such as extracted DNA; 2) performing sonication of the DNA to form fragmented DNA; and 3) performing amplification of the fragmented 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 method for constructing a library based on amplicon sequencing. 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 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 and the molecular cross-linking of FFPE DNA can affect 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] This invention utilizes ultrasonic fragmentation of DNA, such as extracted DNA, to open up the multi-level spatial structure of DNA, allowing primers to bind more fully to the DNA and improving library homogeneity. The method is simple to operate and low in cost.

[0009] In a first aspect, this disclosure provides a method for constructing a library based on amplicon, characterized by the following steps: 1) Provide DNA, such as extracted DNA; 2) The DNA is ultrasonically fragmented to form fragmented DNA; and 3) The fragmented DNA is amplified to form an amplicon library.

[0010] In some embodiments, the length of the fragmented DNA ranges from 150 bp to 5 kb, preferably 200 bp to 2000 bp, for example 500 bp to 2000 bp, more preferably 400 bp to 1500 bp, and most preferably 500 bp to 1000 bp. In some embodiments, the peak length of the fragmented DNA is between 300 bp and 2000 bp, for example 500 bp to 2000 bp, preferably between 500 bp and 1000 bp.

[0011] In some embodiments, the amplification includes i) performing a first round of PCR amplification using the fragmented 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; preferably, the first and second rounds of PCR amplification are performed using an rhAmpSeq system.

[0012] In some embodiments, the ultrasonic disruption is performed using an ultrasonic breaker, such as a Covaris ultrasonic breaker, preferably a Covaris M series ultrasonic breaker, and more preferably a Covaris M220 ultrasonic breaker.

[0013] In some embodiments, the DNA is derived from a biological sample, preferably a body fluid sample or a tissue sample, and more preferably a sample 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.

[0014] In a first aspect, this disclosure provides a method for improving the uniformity of an amplicon library, characterized in that DNA is ultrasonically fragmented prior to amplification to form fragmented DNA.

[0015] In some embodiments, the length of the fragmented DNA ranges from 150 bp to 5 kb, preferably 200 bp to 2000 bp, for example 500 bp to 2000 bp, more preferably 400 bp to 1500 bp, and most preferably 500 bp to 1000 bp. In some embodiments, the peak length of the fragmented DNA is between 300 bp and 2000 bp, for example 500 bp to 2000 bp, preferably between 500 bp and 1000 bp.

[0016] In some embodiments, the amplification includes i) performing a first round of PCR amplification using the fragmented 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; preferably, the first and second rounds of PCR amplification are performed using an rhAmpSeq system.

[0017] In some embodiments, the ultrasonic disruption is performed using an ultrasonic breaker, such as a Covaris ultrasonic breaker, preferably a Covaris M series ultrasonic breaker, and more preferably a Covaris M220 ultrasonic breaker.

[0018] In some embodiments, the DNA is derived from a biological sample, preferably a body fluid sample or a tissue sample, and more preferably a sample 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.

[0019] 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

[0020] Figure 1 This shows the results obtained after ultrasonic fragmentation of different clinical FFPE DNA samples. Figure 1 In the graph, the horizontal axis represents the sample number, and the vertical axis represents the library homogeneity percentage. The highest homogeneity of the library is 1 (100%), and the closer the value is to 100%, the higher the homogeneity.

[0021] Figure 2 This shows the results obtained after sonicating different cell line samples. Figure 2 In the graph, the horizontal axis represents the sample number, and the vertical axis represents the library homogeneity percentage. The highest homogeneity of the library is 1 (100%), and the closer the value is to 100%, the higher the homogeneity.

[0022] Figure 3 This shows the results obtained after ultrasonic fragmentation of DNA samples from different cell lines and different blood DNA samples. Figure 3 In the graph, the horizontal axis represents the sample number, and the vertical axis represents the library homogeneity percentage. The highest homogeneity of the library is 1 (100%), and the closer the value is to 100%, the higher the homogeneity.

[0023] Figure 4 The results show the impact of different interruption conditions on different samples. Figure 4 In the graph, the horizontal axis represents the sample number, and the vertical axis represents the library homogeneity percentage. The highest homogeneity of the library is 1 (100%), and the closer the value is to 100%, the higher the homogeneity. Detailed Implementation

[0024] Several aspects of the invention are described below with reference to illustrative examples. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. However, those skilled in the art will readily recognize that the invention may be practiced without one or more of these specific details or may be practiced in other ways.

[0025] Current amplicon sequencing technology uses extracted DNA samples directly for amplicon library construction and sequencing. The multi-level spatial structure of DNA affects 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.

[0026] The inventors of this application have pioneered the use of ultrasonic fragmentation of DNA derived from biological samples, such as extracted DNA, to significantly improve library homogeneity. The inventors have unexpectedly discovered that ultrasonic fragmentation of DNA samples before amplicon library construction can significantly improve the homogeneity of the amplicon library. For example, the inventors have unexpectedly discovered that ultrasonic fragmentation of FFPE DNA samples before amplicon library construction can significantly improve the homogeneity of the amplicon library.

[0027] Compared with the prior art, the present invention has at least the following advantages: i. Unlocking the multilevel spatial structure of DNA; ii. Improve amplification efficiency and library homogeneity (especially libraries constructed using rhAmpSeq multiplex PCR technology); iii. Applicable to various sample sources and a wide range of library construction techniques, including but not limited to rhAmpSeq; iv. Simple operation process and low cost; and v. It can generate amplicon libraries suitable for NGS, for use in deep targeted sequencing, etc.

[0028] 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…”).

[0029] 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, 500 bp-2000 bp, 400-2000 bp, 500-1000 bp, 600-1500 bp, etc.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The terms "amplifier library construction" and "amplifier-based library construction" used in this article are interchangeable, referring to the amplicon library construction method, which uses multiplex PCR technology to enrich target DNA regions, thereby constructing libraries for next-generation sequencing.

[0034] In this article, the term "homogeneity" in amplicon libraries refers to the uniformity of sequencing depth.

[0035] In this article, the peak value also refers to the main segment distribution, which is the range in which the target segments are relatively concentrated. It is generally understood as the segment length with the largest proportion.

[0036] 1. Provide DNA 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.

[0037] 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.

[0038] 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.

[0039] In some embodiments, the biological sample is a paraffin-embedded sample, such as an FFPE sample.

[0040] 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.

[0041] 2. Ultrasonic interruption After obtaining the DNA, various techniques can be used to ultrasonically break it down. In some embodiments, the ultrasonic breaking is performed using an ultrasonic disruptor, such as a Covaris ultrasonic disruptor, preferably a Covaris M series ultrasonic disruptor, and more preferably a Covaris M220 ultrasonic disruptor. In some embodiments, the ultrasonic disruptor is used in conjunction with a matching disruption tube. In some embodiments, the ultrasonic disruptor is either a contact or non-contact type.

[0042] In some implementations, ultrasonic breaking is performed using an ultrasonic disruptor according to the manufacturer's instructions. In some implementations, ultrasonic breaking is performed using the following parameters: temperature 20-25°C, e.g., 20°C; target peak BP: 500-1000 bp, e.g., 500 bp or 1000 bp; peak incident power: 50 W; duty cycle: 2-10%, e.g., 10% or 2%; pulse cycles per pulse: 200; processing time 50-90 s, e.g., 50 s or 90 s.

[0043] In some embodiments, the length of the fragmented DNA ranges from 150 bp to 5 kb, preferably from 200 bp to 2000 bp, for example, 500 bp to 2000 bp, more preferably from 400 bp to 1500 bp, and most preferably from 500 bp to 1000 bp. In some embodiments, the peak length of the fragmented DNA is between 300 bp and 2000 bp, for example, 500 bp to 2000 bp, preferably between 500 bp and 1000 bp.

[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 fragmented 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 and second rounds of PCR amplification are performed using the rhAmpSeq system.

[0048] In some embodiments, the reaction system used for the first round of PCR amplification includes rhAmpSeq LibraryMix1, forward and reverse primers, and template DNA. In some embodiments, the first primer contains or consists of SEQ ID NO:1-94. In some embodiments, the forward and reverse primers are shown in Tables 3 and 4, respectively.

[0049] In some implementations, the second primer comprises i5 (index 5) and i7 (index 7) or is composed of them. In some implementations, the reaction system for the second round of PCR amplification includes rhAmpSeq Library Mix2, i5 (index 5) and i7 (index 7), and template DNA (the product purified from the first round of PCR).

[0050] 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.

[0051] 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

[0052] 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.

[0053] Example 1: Construction of an amplicon library using DNA fragmented by sonication 1) Sample preparation: DNA extraction was performed on paraffin-embedded (FFPE) samples using the ReliaPrep FFPE gDNA Miniprep System (promega, catalog number: A2352) or the QIAamp DNA FFPE tissue kit (qiagen, catalog number: 56404). Cell line DNA and blood gDNA were extracted using the QIAamp DNABlood Mini Kit.

[0054] 2) The extracted DNA was fragmented using a Covaris M220 and matching fragmentation tubes according to the conditions in Table 1. The fragmented DNA was then quantified using Qubit for later use.

[0055] Table 1. Conditions for Ultrasonic Interruption 3) Target region amplification: Prepare a 20 μL reaction system according to Table 2 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 at IDT. Specific primer information is shown in Tables 3 and 4 (where the 6th nucleotide starting from the 3' end (i.e., the nucleotide after r) is a ribonucleotide, and / 3SpC3 / is the C3 spacer arm modification at the 3' end)): Table 2. Target Region Amplification Reaction System Table 3. Forward primer sequences Table 4. Reverse primer sequences 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.

[0056] 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 were used to purify the target product with 80% ethanol (prepared fresh for use).

[0057] The specific purification steps are as follows: a. Add 30 μl of magnetic beads (1.5x magnetic beads) to the PCR tube containing the reaction solution, then vortex to mix, briefly centrifuge, and incubate at room temperature for 10 min; b. Place on a magnetic rack and let stand until the liquid becomes clear. Use a pipette to remove the supernatant (be careful not to touch the magnetic beads and keep the PCR tube on the magnetic rack). c. Add 200 μl of 80% ethanol to the PCR tube, let stand for 30 seconds, and discard the supernatant; d. Repeat c; e. Use a 10μl pipette tip to remove as much liquid as possible from the PCR tube, and let it stand at room temperature until the magnetic beads are dry; f. Add 22 μl of nuclease-free water, remove the PCR tube from the magnetic rack, vortex to mix, and centrifuge briefly; g. Incubate at room temperature for 5 min, then place the PCR tube on a magnetic rack until the liquid becomes clear, and add 20 μl of the supernatant to a new PCR tube; h. Add 30 μl of magnetic beads to the PCR tube containing the supernatant, then vortex to mix, briefly centrifuge, and incubate at room temperature for 10 min; i. Place on a magnetic rack and let stand until the liquid becomes clear. Use a pipette to remove the supernatant (be careful not to touch the magnetic beads and keep the PCR tube on the magnetic rack at all times). j. Add 200 μl of 80% ethanol to the PCR tube, let stand for 30 seconds, and discard the supernatant; k. Repeat g; 1. Use a 10μl pipette tip to remove as much liquid as possible from the PCR tube, and let it stand at room temperature until the magnetic beads are dry; m. Add 20 μL of the reaction liquid prepared according to Table 6 to the dried magnetic beads, vortex to mix, incubate at room temperature for 3 min, and then place on a PCR instrument to perform the PCR reaction.

[0058] 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.

[0059] 7) Perform sequencing on the library and conduct bioinformatics workflow analysis.

[0060] Example 2: The effect of ultrasonic disruption on the homogeneity of FFPE sample libraries 2a) Twelve different clinical FFPE DNA samples were selected for ultrasonic fragmentation (control group (unfractionated), ultrasonic fragmentation of 500 bp, ultrasonic fragmentation of 1000 bp). The specific procedure was performed as described in Example 1. The results are as follows. Figure 1 As shown, from Figure 1 It is evident that both 500bp and 1000bp ultrasound interruption conditions can significantly improve library homogeneity.

[0061] 2b) Three different cell line samples (NCIH2347, HCT-15, and NCIH1299) were selected for sonication fragmentation (control group (unfractionated), 500bp fragmentation by sonication, and 1000bp fragmentation by sonication). The specific procedure was as described in Example 1. The results are as follows. Figure 2 As shown, from Figure 2 It is evident that the uniformity of the library was significantly improved after cell line DNA was fragmented by ultrasound.

[0062] 2c) Five different cell line DNA samples (LS180, LS411N, H1975, H1650, H460) and two different blood DNA samples (SLB_1, SLB_2) were selected for ultrasonic fragmentation (control group (unfractionated), ultrasonic fragmentation of 500 bp). The specific procedure was as described in Example 1. The results are as follows. Figure 3 As shown, from Figure 3 It is evident that the homogeneity of the library was significantly improved after cell line DNA and blood DNA were fragmented by ultrasound.

[0063] 2d) Four different FFPE DNA samples were selected for ultrasonic fragmentation (control group (unfractionated), 500bp, 1000bp, 2000bp, 4000bp, and 8000bp groups). The specific procedure was as described in Example 1. The results are as follows. Figure 4 As shown, library uniformity is significantly improved when the target peak bp is in the range of 500bp-2000bp. Library uniformity is also improved when the target peak bp is 4000bp, but the improvement effect is slightly lower than that in the range of 500-2000bp. Library uniformity is not significantly improved when the target peak bp is 8000bp.

[0064] 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. 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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 based on amplicon, characterized in that... Includes the following steps: 1) Provide DNA; 2) The DNA is ultrasonically broken to form broken DNA, wherein the length of the broken DNA ranges from 150 bp to 3 kb, and the peak length of the broken DNA is between 500 bp and 2000 bp. and 3) Amplify the fragmented DNA to form an amplicon library, wherein the amplification includes i) performing a first round of PCR amplification using the fragmented 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 the target amplicon library.

2. The method as described in claim 1, characterized in that... The DNA mentioned is extracted DNA.

3. The method as described in claim 1 or 2, characterized in that... The length of the fragmented DNA ranges from 200 bp to 2000 bp.

4. The method as described in claim 3, characterized in that... The length of the fragmented DNA ranges from 500 bp to 2000 bp.

5. The method as described in claim 3, characterized in that... The length of the fragmented DNA ranges from 400 bp to 2000 bp.

6. The method as described in claim 1, characterized in that... The peak length of the fragmented DNA is between 500 bp and 1000 bp.

7. The method as described in claim 1 or 2, characterized in that... The first and second rounds of PCR amplification were performed using the rhAmpSeq system.

8. The method as described in claim 1 or 2, characterized in that... The ultrasonic disruption was performed using an ultrasonic disruptor.

9. The method as described in claim 8, characterized in that... The ultrasonic breaker mentioned is a Covaris ultrasonic breaker.

10. The method as described in claim 9, characterized in that... The Covaris ultrasonic breaker mentioned is the Covaris M series ultrasonic breaker.

11. The method as described in claim 10, characterized in that... The ultrasonic breaker mentioned in the Covaris M series is the Covaris M220 ultrasonic breaker.

12. The method as described in claim 1 or 2, characterized in that... The DNA was derived from a biological sample.

13. The method as described in claim 12, characterized in that... The sample is a body fluid sample or a tissue sample.

14. The method as described in claim 12, characterized in that The samples were selected from tumor tissue samples, cell cultures, whole blood, plasma, serum, saliva, cerebrospinal fluid, sweat, sputum, bronchoalveolar lavage fluid, urine, feces, milk, and peritoneal fluid.

15. The method as described in claim 12, characterized in that... The sample was a biopsy sample.

16. The method as described in claim 12, characterized in that... The sample was a secretion.

17. The method as described in claim 12, characterized in that The sample is a sample that has undergone curing treatment.

18. The method as described in claim 17, characterized in that... The solidified sample is a paraffin-embedded sample.

19. The method as described in claim 18, characterized in that... The paraffin-embedded sample was an FFPE sample.

20. A method for improving the uniformity of amplicon libraries, characterized in that... Prior to amplification, the DNA is sonicated to form fragmented DNA, wherein the length of the fragmented DNA ranges from 150 bp to 3 kb, and the peak length of the fragmented DNA is between 500 bp and 2000 bp. The amplification includes i) using the fragmented DNA as a template and 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 and performing a second round of PCR amplification with a second primer; and iv) optionally purifying the DNA to obtain a target amplicon library.

21. The method as described in claim 20, characterized in that... The length of the fragmented DNA ranges from 200 bp to 2000 bp.

22. The method as described in claim 21, characterized in that The length of the fragmented DNA ranges from 500 bp to 2000 bp.

23. The method as described in claim 21, characterized in that... The length of the fragmented DNA ranges from 400 bp to 2000 bp.

24. The method as described in claim 21, characterized in that... The peak length of the fragmented DNA is between 500 bp and 1000 bp.

25. The method as described in claim 20 or 21, characterized in that... The first and second rounds of PCR amplification were performed using the rhAmpSeq system.

26. The method according to claim 20 or 21, characterized in that... The first primer comprises the forward primer shown in SEQ ID NO: 1-47 and the corresponding reverse primer shown in SEQ ID NO: 48-94.

27. The method as described in claim 20 or 21, characterized in that... The ultrasonic disruption was performed using an ultrasonic disruptor; and / or The DNA was derived from a biological sample.

28. The method as described in claim 27, characterized in that... The ultrasonic breaker mentioned is a Covaris ultrasonic breaker.

29. The method as described in claim 28, characterized in that... The Covaris ultrasonic breaker mentioned is the Covaris M series ultrasonic breaker.

30. The method as described in claim 29, characterized in that... The ultrasonic breaker mentioned in the Covaris M series is the Covaris M220 ultrasonic breaker.

31. The method as described in claim 27, characterized in that... The sample is a body fluid sample or a tissue sample.

32. The method as described in claim 27, characterized in that The samples were selected from tumor tissue samples, cell cultures, whole blood, plasma, serum, saliva, cerebrospinal fluid, sweat, sputum, bronchoalveolar lavage fluid, urine, feces, milk, and peritoneal fluid.

33. The method as described in claim 27, characterized in that... The sample was a biopsy sample.

34. The method as described in claim 27, characterized in that The sample was a secretion.

35. The method as described in claim 27, characterized in that... The sample is a sample that has undergone curing treatment.

36. The method as described in claim 35, characterized in that... The solidified sample is a paraffin-embedded sample.

37. The method as described in claim 36, characterized in that... The paraffin-embedded sample was an FFPE sample.

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