Method for producing library of nucleic acids

Abstract

The present invention relates to a method for obtaining a nucleic acid library of a sample comprising a polynucleotide, the method comprising the steps of: a. Providing the polynucleotide with a plurality of modified primers wherein the modified primers are the starting point of a polymerase for nucleic acid amplification; b. Amplifying the polynucleotide using a polymerase; c. Fragmenting the amplified polynucleotide, thereby obtaining a mixture of fragmented and unfragmented polynucleotides comprising the modified primer; d. Linking a plurality of linker oligonucleotides to the mixture obtained in step c), thereby obtaining a mixture of polynucleotides comprising said linker oligonucleotides, said linker oligonucleotides comprising binding sites for amplification primers; e. Providing an amplification primer to the mixture obtained in step d), wherein the amplification primer is the starting point of the polymerase for nucleic acid amplification; f, nucleic acid amplification is started by providing polymerase; characterised in that the modified primer provided in step a) comprises a functional group wherein the functional group is a blocking group located at the 5'end of the modified primer, thereby preventing the ligation of linker oligonucleotides, or wherein the functional group is at least one nucleotide analogue, thereby preventing the ligation of linker oligonucleotides. And wherein the nucleotide analogue is resected after step d) by an endonuclease, thereby removing the primer binding site provided by the linker oligonucleotide, thereby preventing binding of the amplification primer provided in step f) and nucleic acid amplification of the fragmented polynucleotide comprising the modified primer.

Description

Technical Field

[0001] The present invention relates to the field of next generation sequencing and nucleic acid library preparation. Background Art

[0002] Next-generation sequencing is an emerging technology that is expanding into all areas of biomedical research and clinical diagnostics.

[0003] Instruments used for next-generation sequencing have a limited read length (the number of molecules downstream of the sequencing primer whose sequence can be accurately determined). In many applications, this read length is not sufficient to determine the sequence of the target complete DNA fragment. Therefore, a process called library preparation is an important part of sequencing applications. To this end, the DNA fragments to be sequenced are multiplied and multiple copies are subsequently shortened by a process called fragmentation. These fragments are sequenced and the obtained sequences are aligned with a reference sequence to determine the sequence of the complete fragment.

[0004] The ideal outcome of a fragmentation-based library preparation workflow is to obtain DNA library molecules that evenly cover the DNA molecules to be sequenced ( Figure 4a However, typical libraries from fragmentation-based library preparation workflows show a high proportion of unfragmented DNA (10x Genomics User Guide CG000208Rev E; ChromiumNext GEM Single Cell 5' Reagent Kits v2, Section 4.6, p. 57), which is a major drawback in the library preparation process (e.g. Figure 4b shown).

[0005] For libraries containing many unfragmented nucleic acids, it is shown that many of the reads obtained by sequencing originate from the most 3' end of the enriched target (e.g., libraries generated according to 10x Genomics User Guide CG000208Rev E / 10x Genomics ChromiumNextGEM Single Cell 5'ReagentKits v2). In order to ensure that enough reads are obtained from shorter fragments, the number of sequencing reads must be increased. This leads to higher costs because the number of reads of the sample must be increased, and therefore the number of different samples that can be sequenced in parallel must be reduced. If not compensated by increasing the number of reads of each sample, there is a high risk of insufficient coverage for the entire sequence of the assembled initial DNA molecule.

[0006] In addition, primer dimers from the initial targeted enrichment may be present in the library. This occurs especially in targeted enrichments where multiple targets are amplified simultaneously (in these multiplex reactions, it is not possible to design a large number of primers without any one that will form primer dimers). These primer dimers are also typically carried over during library preparation and will also be sequenced.

[0007] The presence of primer dimers (no information about the target) and lack of fragmentation (uneven coverage of the target) can only be compensated by increasing the number of reads. This results in increased costs or lower coverage.

[0008] Here we describe an improved or alternative method for nucleic acid library preparation to reduce the amount of unfragmented nucleic acids, thereby avoiding sequencing these molecules. This makes sequencing more accurate and saves costs because the number of sequencing reads can be reduced. This method can also be used to reduce the number of primer dimers. Summary of the invention

[0009] The object of the present invention is a method for obtaining a nucleic acid library of a sample containing polynucleotides, the method comprising the following steps:

[0010] a. providing a plurality of modified primers to the polynucleotide, wherein the modified primers are starting points for a polymerase for nucleic acid amplification;

[0011] b. amplifying the polynucleotide using a polymerase;

[0012] c. fragmenting the amplified polynucleotide to obtain a mixture of fragmented and unfragmented polynucleotides comprising the modified primer;

[0013] d. connecting a plurality of linker oligonucleotides to the mixture obtained in step c) to obtain a polynucleotide mixture comprising the linker oligonucleotides, wherein the linker oligonucleotides comprise a binding site for an amplification primer;

[0014] e. providing an amplification primer to the mixture obtained in step d), wherein the amplification primer is a starting point for a polymerase for nucleic acid amplification;

[0015] f. Initiate nucleic acid amplification by providing a polymerase.

[0016] The key factor is the modified primer. The modified primer provided in step a) comprises a functional group. The functional group is a blocking group (Fig. 2) at the 5' end of the modified primer, thereby preventing the connection of the adapter oligonucleotide, or the functional group is at least one nucleotide analog, and wherein the nucleotide analog is excised by an endonuclease after step d), thereby removing the primer binding site (Fig. 3) provided by the adapter oligonucleotide. Thus, the combination of the amplification primer provided in step f) and the nucleic acid amplification of the fragmented polynucleotide comprising the modified primer are prevented.

[0017] In addition, amplification of primer dimers is prevented.

[0018] The method of the present invention can be combined with the statistical fragmentation technique disclosed in (PCT / EP / 2020 / 081731). In this method, a nucleotide analog is incorporated into the nucleic acid during amplification. The nucleotide analog is removed by an endonuclease to produce a fragmented nucleic acid library.

[0019] The target nucleic acid library obtained by the method of the present invention can be used for sequencing. For sequencing, any method known in the art can be used.

[0020] definition

[0021] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0022] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods and their respective components, which components are essential to the method or composition, but may still include unspecified elements, whether essential or not.

[0023] The words "bind" and "hybridize" and their grammatical equivalents are used interchangeably. If two nucleic acid strands are complementary to each other, they will hybridize. Hybridization can occur under conditions known in the art.

[0024] As used herein, the term "complementary" refers to the ability to precisely pair between two nucleotides by Watson and Crick base pairing. To explain, if a nucleotide at a given position of a nucleic acid strand is able to form a hydrogen bond with a nucleotide of the other nucleic acid strand, then the two nucleic acids are considered complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," where only some of the nucleotides bind, or it can be complete when there is total complementarity between the single-stranded molecules.

[0025] "Primer" used herein is a single-stranded oligonucleotide made of nucleotides, which can bind to a complementary nucleic acid sequence. It should be understood that all primers described in the present invention can be used as the starting point for nucleic acid synthesis / amplification. According to the present invention, modified primers can be used. This modified primer contains a functional group and can prevent the connection of an adapter molecule or can be removed from a polynucleotide. According to the present invention, the functional group is a blocking group located at the 5' end of the modified primer, or the functional group is at least one nucleotide analog contained in the modified primer. The modified primer can also be referred to as a "modified targeted enrichment primer". In addition, unmodified primers can also be used in the disclosed method. These primers do not carry a functional group that can prevent the connection of an adapter molecule or can be removed from a polynucleotide. The amplification primers used herein also have the characteristics of unmodified primers.

[0026] The terms "nucleic acid synthesis" and nucleic acid amplification used herein can be used interchangeably. The process of nucleic acid synthesis is well known in the art. In short: for nucleic acid synthesis, a template nucleic acid is provided, which can be single-stranded or double-stranded. In the case of initial use of double-stranded nucleic acid, the first step is to denature into a single nucleic acid chain (complementary and reverse complementary) using techniques known in the art. Single-stranded nucleic acid does not require a denaturation step. In the next step, a primer that binds to the complementary region of the nucleic acid chain is provided. The 3' end of the primer is then extended using a polymerase, and a complementary chain is produced by filling complementary nucleotides. As a result, a complementary nucleic acid chain is formed. For further amplification, double-stranded nucleic acid denaturation is required, and then another round of nucleic acid synthesis can be initiated. According to the present invention, nucleic acid synthesis can be symmetrical or asymmetrical. In the symmetrical nucleic acid synthesis process, only one primer is used. Therefore, only one chain is synthesized. In the symmetrical reaction process, a pair of two primers (forward primer and reverse primer) can be provided. One primer binds to the complementary nucleic acid chain, and the other primer binds to the reverse complementary nucleic acid chain. Therefore, nucleic acid synthesis can be initiated on two chains. Unless otherwise indicated, the term "primer" as used herein may include a forward primer, a reverse primer or both. In a specific embodiment, the term "primers having the same specificity" relates to all forward primers used or all reverse primers used.

[0027] The term "adaptor" or "adapter" as used herein refers to an oligonucleotide that can be connected to a polynucleotide. They contain primer binding sequences to facilitate amplification or sequencing of the associated nucleic acid. The primer binding sequences of the adapter molecule can be the same (identical) or different sequences. Thus, for example, the 5' adapter can contain the same or different primer binding sequences, while the 3' adapter can contain the same or different primer binding sequences. The same primer binding sequence that may be present in different members of a variety of nucleic acid molecules can allow a single universal amplification primer complementary to the universal / identical primer binding sequence to amplify a variety of different sequences. The adapter molecule can also include the sequence of one or more sample tags and molecular tags (barcodes). The adapter can be double-stranded (symmetrical), partially double-stranded (asymmetric) or single-stranded. One or more adapter molecules can be located at the 5' or 3' end of the nucleic acid. The adapters at the 5' end and 3' end of the adapter-target-adapter can be the same or different.

[0028] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a biopolymer composed of nucleotide monomers covalently bonded in a chain. The amplified nucleic acid can be named "amplicon". The nucleic acid can be DNA or RNA. It can contain one or more nucleotide analogs. Some non-limiting examples of analogs include: 8-oxo-7,8-dihydroguanine (8-oxoG), uridine (U), inosine (I), 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil, 3-methyladenine, 7-methylguanine, 1,N6-vinylidene adenine and hypoxanthine and its derivatives, such as deoxy-8-oxo-7,8-dihydroguanine (d8-oxoG), deoxyuridine (dU), deoxyinosine (dI). These nucleotide analogs can be removed by endonucleases, especially structure-specific endonucleases.

[0029] As used herein, the term "plurality" of something means two or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] All steps used in the drawings are based on the steps mentioned in claim 1.

[0031] Figure 1: Example of a prior art library preparation workflow. The workflow and its desired results are shown in Figure 1. Figure 1a As shown. First, one or more molecules are amplified using a forward primer (black box) and a reverse primer (bold box). In this example, the one or more molecules amplified are barcoded (striped box). Multiple molecules are generated in this amplification step; for simplicity, only a single amplicon is described in this figure. Next, the amplicon is fragmented. For simplicity, only the 5' fragment is shown. According to the method for fragmentation, the resulting fragment can be blunted using a polymerase with 3' to 5' polymerase activity and 3' to 5' exonuclease activity. Subsequently, a DNA ligase is used to connect the adapter molecule containing the primer binding site. In order to facilitate the connection, a polynucleotide kinase can be added in this step or the previous step to phosphorylate the 5' end of the DNA fragment. In addition, in the case of using an adapter with a T protruding end, an enzyme with A tailing activity can be added to this step or the previous step. Finally, the library fragment can be amplified. In this example, an amplification primer binding site is present in the original molecule, and the second amplification primer binds to the adapter present in the connection. Therefore, the desired fragmented molecules containing these primers will be amplified.

[0032] Using the prior art library preparation workflow, the library generated from the unfragmented amplicons will be amplified in the final amplification reaction ( Figure 1b). This figure shows an example of an unfragmented amplicon. The position of the reverse primer is indicated by a bold box. Adapters can be attached to such fragments, and the resulting library molecules will be amplified in the final amplification reaction, so the unfragmented amplicon will be present in the final library.

[0033] Figure 1c : With the prior art library preparation workflow, a library generated from primer dimer molecules will be amplified in the final amplification reaction. The figure shows an example of primer dimers that may be generated in an amplification reaction. The location of the reverse primer is indicated by a bold box. Adapters can be attached to such fragments, and the resulting library molecules will be amplified in the final amplification reaction, so that unfragmented amplicons will be present in the final library.

[0034] Figure 2: Example of library preparation workflow using modified primers comprising blocking groups. The library preparation workflow used in this example is the same as Example 1, except that a modified reverse primer with a blocking group is used.

[0035] Figure 2a The effect of modified primers with blocking groups on the desired outcome of library preparation is shown. The presence of the blocking group has no effect on the desired outcome: if the amplicon is fragmented in step c), the resulting 5' fragments will not contain the modified primers, so the adapter can be connected in step d). The resulting library molecules can be amplified in the final amplification reaction (step f)). Please note that in this example an adapter oligonucleotide (called MIL-Adapter) is used, in which the complete primer binding site of the adapter is only present in the bottom strand.

[0036] Figure 2b : When using modified primers with blocking groups, library molecules generated by unfragmented amplicons will not be amplified in the final amplification reaction (step f). The figure shows an example of unfragmented amplicons in step d). The positions of the primers are indicated by bold boxes. During the adapter connection process, the blocking groups prevent the connection of the adapter to the unfragmented amplicon. Therefore, the adapter containing the binding site of the amplification primer will not connect to such an amplicon, so exponential amplification will not occur in the final amplification reaction (step f).

[0037] Figure 2c : When using modified primers with blocking groups, the library molecules generated by primer dimers will not be amplified in the final amplification reaction (step f)). The figure shows an example of primer dimers generated in step b). The position of the primers is indicated by a thick frame. During the adapter connection process, the blocking group prevents the connection of the adapter to the primer dimer. Therefore, the adapter containing the binding site of the amplification primer (step f)) will not be connected to such primer dimers, so exponential amplification will not occur in the final amplification reaction (step f)).

[0038] Figure 3: Example of a library preparation workflow using modified primers containing nucleotide analogs that can be cleaved off in a subsequent step.

[0039] Figure 3a : Effect of modified primers containing nucleotide analogs on the desired result of library preparation. The presence of nucleotide analogs has no effect on the desired result: if the amplicon is fragmented in step c), the resulting 5' fragments will not contain modified primers, so the adapter can be connected in step d), and subsequent treatment with endonucleases will not have any effect on these fragments. Therefore, the resulting library molecules can be amplified in the final amplification reaction (step f)). Please note that in this example, an adapter oligonucleotide (called MIL-adapter) is used, in which the complete primer binding site of the adapter is only present in the bottom strand.

[0040] Figure 3b : When using modified primers containing nucleotide analogs, library molecules generated from unfragmented amplicons will not be amplified in the final amplification reaction (step f)). The figure shows an example of unfragmented amplicons in step c). The positions of the primers are indicated by bold boxes, and the nucleotide analogs are indicated by "X". Adapters will be connected to unfragmented amplicons containing amplification primers, but these molecules containing reverse amplification primers will be cut during subsequent endonuclease treatment. Therefore, the library from unfragmented amplicons containing reverse amplification primers will not be exponentially amplified in step f).

[0041] Figure 3c : When using modified primers containing nucleotide analogs, library molecules generated by primer dimers will not be amplified in the final amplification reaction (step f)). The figure shows an example of primer dimers generated in step b). The positions of the primers are indicated by bold boxes, and the nucleotide analogs are indicated by "X". The adapter will be attached to the primer dimers, but these molecules containing reverse amplification primers will be cut during the subsequent nuclease treatment. Therefore, the library derived from primer dimers containing reverse amplification primers will not be exponentially amplified in step f).

[0042] Figure 4 shows the impact of unfragmented amplicons and primer dimers on NGS libraries. Figure 4aAn example of an RNA-Seq application is shown, in which a target cDNA molecule is amplified using specific primers (targeted enrichment). In this example, the cDNA molecules are previously barcoded at the 5' end before amplification (indicated by gray dotted lines at the bottom of the figure), and the amplification primers are selected so that the 5' barcode remains in the amplicon after targeted enrichment. During targeted enrichment, multiple copies of the same molecule (same 5' barcode) will be generated. The top two figures describe the size of the DNA fragments before (left) and after (right) library preparation (represented by electropherograms). The lower schematic describes how the sequencing primers will bind to the library fragments (black arrows) and which regions of the fragments will be determined by sequencing (dotted arrows; note that the read length of the sequencer is not enough to cover the entire fragment). Upper left: After targeted enrichment, the specific fragment exists together with a smaller primer dimer. This specific fragment is too large to be sequenced with a single read. Upper right: The fragment is statistically shortened (while maintaining the 5' barcode), and a linker with a sequencing primer is added to the shortened fragment. Inserts (indicated by solid grey lines) are determined by reverse reads, and barcodes are determined by forward reads. After sequencing, all reads with the same barcode (forward reads) will be aligned to the reference sequence. When enough reads covering the entire sequence (full length) are obtained, the sequence of the starting molecule can be determined.

[0043] Figure 4b Shows Figure 4a Example of prior art library preparation results for the same RNA-Seq application described in . Prior art methods do not result in complete fragmentation of the targeted enrichment product. Therefore, such a library will contain many unfragmented polynucleotides. Therefore, the reads covering the 3' end of the target will be over-represented (bottom right). In order to achieve complete coverage of the fragments, the number of sequencing reads must be increased.

[0044] FIG5 shows a workflow for evaluating different blocking groups and non-specific fragmentation, as described in Example 1 of the present invention. Figure 5a An exemplary workflow is shown, and Figure 5b -f displays the results obtained.

[0045] Figure 6 shows a workflow for evaluating different blocking groups and fragmentation based on uracil incorporation and excision, as described in Example 2 of the present invention. Figure 6a An exemplary workflow is shown, and Figure 6b -f displays the results obtained.

[0046] FIG. 7 shows a workflow for evaluating modified primers comprising nucleotide analogs and non-specific fragmentation, as described in Example 3 of the present invention. Figure 7a An exemplary workflow is shown, and Figure 7b-c shows the results obtained.

[0047] FIG. 8 shows a workflow for evaluating modified primers comprising nucleotide analogs and fragmentation based on uracil incorporation and excision, as described in Example 4 of the present invention. Figure 8a An exemplary workflow is shown, and Figure 8b The results obtained are shown.

[0048] Fig. 9 Additional information for the sequence analysis of Examples 3 and 4 is shown.

[0049] Fig.10a and 10b Additional information from the sequence analysis of Example 5 is shown.

[0050] Fig.11a and 11b Additional information from the sequence analysis of Example 6 is shown. DETAILED DESCRIPTION

[0051] The present invention provides a method to overcome the limitations of current nucleic acid library preparation. The main purpose is to reduce the number of unfragmented nucleic acids that can be sequenced. This is achieved by using modified primers for initial nucleic acid amplification during library preparation.

[0052] The method of the present invention is used to prepare a nucleic acid library. The nucleic acid library can be used in a variety of downstream applications, such as next generation sequencing or polymerase chain reaction.

[0053] Samples that can be used for nucleic acid library preparation as described herein can be derived from any specimen, such as whole animals, organs, tissue sections, cell aggregates or single cells of invertebrates (e.g., Caenorhabditis elegans, Drosophila melanogaster), vertebrates (e.g., zebrafish, African clawed frogs) and mammals (e.g., mice, Homo sapiens). Biological samples can be in the form of tissue sections, cell aggregates, suspended cells, adherent cells or body fluids.

[0054] The nucleic acid used for initial library preparation can be a polynucleotide chain made of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can have a linear or circular conformation.

[0055] In the first step (a) of the method of the present invention, a modified primer is provided, and then a nucleic acid is amplified using a polymerase (step b). The nucleic acid provided by the sample is used as a template for nucleic acid amplification. Techniques and conditions for nucleic acid synthesis and amplification, such as polymerase chain reaction, are well known in the art.

[0056] Standard polymerases that can be used for these reactions are, for example, Taq polymerase, proofreading polymerases such as Pfu polymerase or Kod polymerase, fusion polymerases such as KAPA HiFi polymerase (Roche Diagnostics), T7 DNA polymerase, Klenow fragment, T9 DNA polymerase, Phi29 polymerase. The polymerase can be an optimized variant capable of incorporating dUTP nucleotides, such as KAPA HiFi uracil + polymerase (Roche Diagnostics).

[0057] After nucleic acid amplification, step c) fragmentation of polynucleotides follows. In this step, the polynucleotides can be double-stranded. Several techniques for polynucleotide fragmentation are known in the art, such as physical shearing using a Covaris sonicator, enzymatic fragmentation, and Tagmentation (examples are disclosed in PCT / EP / 2020 / 081731). Fragmentation of polynucleotides produces a mixture of fragmented and unfragmented polynucleotides. The mixture contains: fragmented polynucleotides comprising modified primers, fragmented polynucleotides not comprising modified primers, and unfragmented polynucleotides comprising the modified primers. It is assumed that the unfragmented polynucleotides contain modified primers.

[0058] The resulting fragments may be blunt ended, or may have 3' or 5' single stranded overhangs. Prior to adapter ligation, the fragmented nucleic acids may be treated with an enzyme or enzyme mixture having 5'→3' polymerase activity and 3'→5' exonuclease activity to generate blunt ends.

[0059] At fragments with 5' protruding ends, a 5'→3' polymerase activity is used to synthesize the reverse complementary second strand ("filling in"); at fragments with 3' protruding ends, a 3'→5' exonuclease activity is used to remove the protrusion. After this treatment, all fragments have blunt ends. Typical enzymes used for such reactions are, for example, the Klenow fragment of E. coli DNA polymerase or T4 DNA polymerase. The filling reaction usually also contains a polynucleotide kinase to add a phosphate group to the 5' end of the treated fragment. Optionally, an enzyme with A-tailing activity may also be present in step c). After the nucleic acid is fragmented in step c), a plurality of adapter oligonucleotides are then connected to the mixture obtained in step c) in step d). The result is a mixture of polynucleotides containing the adapter oligonucleotides, wherein the adapter oligonucleotides contain binding sites for amplification primers. The adapter oligonucleotide used for connection contains at least one amplification primer binding sequence. It may also contain other primer sequences suitable for amplification and sequencing. In addition, the adapter molecule may also contain a barcode sequence. By adding specific barcodes, individual molecules can be labeled (e.g., by oligonucleotide barcodes, such as unique molecular identifiers), allowing multiple independent DNA molecules to be sequenced in parallel.

[0060] The adapter molecule may be a double-stranded molecule with a blunt end, or at least partially double-stranded molecule comprising a single-stranded nucleic acid chain overhang (e.g., a T overhang of the sample in the case of an enzyme having A-tailing activity in step c). Examples of adapters include Figure 1a shown.

[0061] The joint submolecule can be connected to any side of the polynucleotide, i.e. adjacent to one side of the forward primer or adjacent to one side of the reverse primer (as exemplarily shown in Fig. 1-3) or connected to both sides of the polynucleotide contained in the mixture obtained in step c). If the joint molecule is connected to both sides of the polynucleotide, the joint molecule on each side can be identical or different. The side connected depends on the primer of the modification used. If the reverse primer modified is used, the joint on one side of the reverse primer will be prevented from connecting in the unfragmented polynucleotide, and the joint will be connected to the fragmented polynucleotide. If the forward primer modified is used, the joint on one side of the reverse primer will be prevented from connecting in the unfragmented polynucleotide, and the joint will be connected to the fragmented polynucleotide. If the mixture of the forward primer modified and the reverse primer modified is used, the joint will be prevented from connecting at the polynucleotide still comprising the forward or reverse primer.

[0062] The connection of the linker molecule is carried out by ligase. Common examples of this type of ligase are, for example, T4 DNA ligase, Taq ligase or an equivalent enzyme. Before step d, the fragment can also be made single-stranded, and a single-stranded linker, for example, a heat-stabilized 5'App DNA / RNA ligase or an equivalent enzyme can be connected. The result is a polynucleotide that produces the linker.

[0063] After the connection of the adapter molecule, an amplification step follows, wherein an amplification primer is provided to the mixture obtained in step d), wherein the amplification primer is the starting point for a polymerase for nucleic acid amplification. In order to initiate nucleic acid amplification, a polymerase is provided. An amplification primer can also be used to initiate a sequencing reaction.

[0064] In one embodiment of the invention, no washing steps may be required after fragmentation (step c) and / or adaptor ligation (step d).

[0065] The next section lists a number of preferred variations and embodiments of the first aspect of the invention.

[0066] Modified and unmodified primers

[0067] The key element of the present invention is the use of a modified primer in step a). The modified primer comprises a functional group, which is a blocking group located at the 5' end of the modified primer, which prevents the connection of the adapter oligonucleotide; or the functional group is at least one nucleotide analog contained in the modified primer, wherein the nucleotide analog is excised by an endonuclease after step d), thereby removing the primer binding site provided by the adapter oligonucleotide. In both cases, the modification results in the absence of the adapter oligonucleotide in the polynucleotide and fragment comprising the modified primer. Therefore, due to the absence of the primer binding sequence, the amplification primer added in step f) cannot bind to such polynucleotides and amplification cannot be performed.

[0068] The modified primer provided in step a) may be selected from forward and / or reverse primers. The forward and reverse primers may be the same or different.

[0069] In a specific embodiment, further multiple unmodified primers can be provided in step a). The unmodified primers do not include functional groups that cause the absence of linker oligonucleotides in the polynucleotides and fragments comprising the unmodified primers. According to the stoichiometry between the unmodified primers provided in step a) of the invented method and the modified primers, the amount of the potential unfragmented polynucleotides that will be amplified in step f) can be adjusted. In another embodiment, the further multiple unmodified primers provided in step a) can have the same specificity as the modified primers, for example, unmodified forward primers and modified forward primers or unmodified reverse primers and modified reverse primers.

[0070] Unmodified primers can be selected from forward and / or reverse primers. The molar ratio of multiple modified and unmodified primers can be 99: 1, 95: 5 or 90: 10. In another embodiment, the molar ratio of multiple modified and unmodified primers with the same specificity can be 99: 1, 95: 5 or 90: 10.

[0071] In another embodiment, at least 50%, 75%, 90% or 99% of the primers provided in step a) are modified primers. In a preferred embodiment, at least 50%, 75%, 90% or 99% of the primers with the same specificity provided in step a) are modified primers.

[0072] In a preferred embodiment, all reverse primers provided in step a) may be unmodified primers, and all forward primers may be modified primers, or vice versa. In yet another embodiment, 90% of the reverse primers and 90% of the forward primers may be modified.

[0073] In another embodiment of the present invention, 25% of the reverse primers can be modified, and 75% of the reverse primers and all forward primers can be unmodified. In another embodiment of the present invention, 50% of the reverse primers can be modified, and 50% of the reverse primers and all forward primers can be unmodified. In another embodiment of the present invention, 75% of the reverse primers can be modified, and 25% of the reverse primers and all forward primers can be unmodified.

[0074] In another embodiment of the present invention, 25% of the forward primers can be modified, and 75% of the forward primers and all reverse primers can be unmodified. In another embodiment of the present invention, 50% of the forward primers can be modified, and 50% of the forward primers and all reverse primers can be unmodified. In another embodiment of the present invention, 75% of the forward primers can be modified, and 25% of the forward primers and all reverse primers can be unmodified.

[0075] Modified primer-blocking group

[0076] In one variant of the invention, the modified primer provided in step a) comprises a functional group at the 5' end, which is a blocking group (Fig. 2). The blocking group prevents the connection of the adapter oligonucleotide to the polynucleotide (step d). Connection cannot occur. The modification of the primer prevents phosphorylation of the 5' group of the polynucleotide comprising the modified primer, which is necessary for the connection of the adapter molecule.

[0077] The blocking group is selected from blocking groups known in the art. Commonly used blocking groups are, for example, fluorophores or quenchers coupled to the 5' end of the nucleotide, 5' amino modifiers (including C6 amino and C12 amino), 5' biotin (including bibiotin and biotin-TEG), 5' thiol modifiers (including C6 thiol), carbon spacers (including spacers C3, C6, C12), oligoethylene glycol spacers (including spacers 9, 12, 18, HEG spacers), digoxin, Acrydite, C3-Azid, DBCO, DBCO-TEG, cholesterol-TEG, PC-amino modifiers. In one embodiment, the blocking group can be selected from biotin, oligoethylene glycol spacers with 1 to 25 glycol units, and carbon spacers with 3 to 12 carbon atoms. Commonly used oligoethylene glycol spacers include spacers 9, 12, 18, and HEG spacers. In a preferred embodiment, the blocking group may be selected from the group consisting of a spacer group C3 and a HEG spacer group.

[0078] In another embodiment of the method of the present invention, step b is performed by providing natural nucleotides (N) a, t, g, c and a nucleotide analog (A). The preferred molar ratio of N and A is between 150:1 and 10:1, more preferably between 150:1 and 25:1. Based on this, the fragmentation of the amplified polynucleotide in step c) is performed by excision of the A nucleotide by an endonuclease. The nucleotide analog is incorporated into the newly synthesized nucleic acid instead of its natural counterpart (step b). The newly synthesized nucleic acid can then be treated with an endonuclease (step c). These enzymes excise the nucleotide analog and create a gap in the newly synthesized nucleic acid. This then leads to fragmentation. The newly synthesized nucleic acid can then be treated with an endonuclease such as listed in Table 1. Examples of such enzyme mixtures capable of excising the nucleotide analog uracil are uracil-DNA glycosylase (UDG) and endonuclease III, or UDG and endonuclease VIII (Melamade et al., 1994; Jiang et al., 1007). Alternatively, commercial enzymes or enzyme cocktails such as USER enzyme or thermolabile USER enzyme from New England Biolabs (Cat. Nos. M5508 and M5507, New England Biolabs, Ipswich, MA, USA) can be used.

[0079] Fragment length can be controlled by adjusting the ratio between N and A, as previously disclosed in (PCT / EP / 2020 / 081731).

[0080] The nucleotide analogue can be selected from 8-oxo-7,8-dihydroguanine (8-oxoG), uridine (U), inosine (I), 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil, 3-methyladenine, 7-methylguanine, 1,N6-vinylideneadenine and hypoxanthine and derivatives thereof, such as deoxy-8-oxo-7,8-dihydroguanine (d8-oxoG), deoxyuridine (dU), deoxyinosine (dI). In a preferred embodiment, the nucleotide analogue is deoxyuridine (dUTP).

[0081] Modified primers – nucleotide analogs

[0082] In another variant of the present invention, the modified primer provided in step a) comprises at least one nucleotide analog (Fig. 3). The nucleotide analog is excised by endonuclease after step d). By doing so, the primer binding site provided by the linker oligonucleotide is removed. This makes it impossible to start the nucleic acid synthesis reaction of this chain of the primer incorporating the modification.

[0083] In one embodiment, the modified primer provided in step a) comprises a nucleotide analog. In the nucleic acid amplification process, this primer is incorporated into the newly synthesized nucleic acid. It is followed by fragmentation and connection of the adapter molecule. The nucleotide analog is then excised by an endonuclease, thereby creating a gap in the double-stranded nucleic acid structure. This then results in the generation of a polynucleotide lacking an adapter molecule comprising a primer binding site. Due to the lack of a connector, and therefore due to the lack of an amplification primer binding site, the polynucleotide lacking an adapter molecule cannot be amplified.

[0084] The nucleotide analogue can be selected from 8-oxo-7,8-dihydroguanine (8-oxoG), uridine (U), inosine (I), 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil, 3-methyladenine, 7-methylguanine, 1,N6-vinylideneadenine and hypoxanthine and its derivatives, such as deoxy-8-oxo-7,8-dihydroguanine (d8-oxoG), deoxyuridine (dU), deoxyinosine (dI). In a preferred embodiment, the nucleotide analogue is 8-oxo-7,8-dihydroguanine (8-oxoG). The treatment after step d) is performed using Fpg (formamidopyrimidine [fapy]-DNA glycosylase).

[0085] In another embodiment of the method of the present invention, additional step b) is performed by providing natural nucleotides (N) a, t, g, c and a nucleotide analog (A). The preferred molar ratio of N and A is between 150:1 and 10:1, more preferably between 150:1 and 25:1. Based on this, the fragmentation of the amplified polynucleotide in step c) is performed by removing the A nucleotides with a nuclease endonuclease. Nucleotide analogs are incorporated into the newly synthesized nucleic acid instead of their natural counterparts. The nucleotide analog used as a functional group in the primer provided in step a) is different from the nucleotide analog provided in step b).

[0086] The newly synthesized nucleic acid can then be treated with the endonucleases listed in, for example, Table 1. These enzymes excise nucleotide analogs and create gaps in the newly synthesized nucleic acid. This then results in fragmentation. Examples of such enzyme mixtures that create gaps at uracil sites are uracil-DNA glycosylase (UDG) and endonuclease III, or UDG and endonuclease VIII (Melamade et al., 1994; Jiang et al., 1007). Alternatively, commercial enzymes or enzyme mixtures can be used, such as USER enzymes from New England Biolabs or heat-labile USER enzymes (Cat. Nos. M5508 and M5507, New England Biolabs, Ipswich, MA, USA).

[0087] Fragment length can be controlled by adjusting the ratio between N and A, as previously disclosed in (PCT / EP / 2020 / 081731).

[0088] The nucleotide analogue may be selected from 8-oxo-7,8-dihydroguanine (8-oxoG), uridine (U), inosine (I), 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil, 3-methyladenine, 7-methylguanine, 1,N6-vinylideneadenine and hypoxanthine and derivatives thereof, such as deoxy-8-oxo-7,8-dihydroguanine (d8-oxoG), deoxyuridine (dU), deoxyinosine (dI). In a preferred embodiment, the nucleotide analogue used as a functional group in the primer provided in step a) is 8-oxo-7,8-dihydroguanine, and the nucleotide analogue provided in step b) is deoxyuridine.

[0089] Table 1: List of modified bases and enzymes that can be used in the method (option: modified base and subsequent cleavage of the modified base after linker ligation):

[0090]

[0091]

[0092] All definitions, features and embodiments, variants specified herein with respect to the first aspect of the invention disclosed herein also apply mutatis mutandis in the context of the other variants and embodiments of the invention disclosed herein.

[0093] Example

[0094] The following examples are intended to explain the present invention in more detail, but the present invention is not limited to these examples.

[0095] The method was evaluated in a single cell targeted RNA sequencing application (targeted RNA-Seq). To achieve this goal, we used cDNA generated from human T cells using the Chromium Single Cell 5'Library&Gel BeadKit (PN-1000014, 10x Genomics, Pleasanton, CA, USA) according to the instructions given in the ChromiumNext GEM Single Cell V(D)J ReagentKits v1.1 User Guide (CG000208 Rev E). Aliquots of the same cDNA were used in the following examples.

[0096] To amplify specific targets followed by library preparation (RNA-Seq), we used targeted enrichment primers from the Single Cell V(D)J Enrichment Kit, Human T Cell (PN-1000005, 10x Genomics, Pleasanton, CA, USA) following the instructions given in the ChromiumNext GEMSingle Cell V(D)J Reagent Kits v1.1 User Guide (CG000208 Rev E, steps 4 and 5).

[0097] For all the following examples, we used the ChromiumNext GEM Single Cell V(D)J Reagent Kits v1.1 User Guide (CG000208 Rev E) as the baseline protocol and modified specific steps in the protocol. The exact modifications are described in the corresponding examples.

[0098] In each example, we generated a reference library (labeled "10x G" or "10x Genomics") in parallel exactly following the described protocol (ChromiumNext GEM Single Cell V(D)J Reagent Kits v1.1 User Guide, CG000208 Rev E).

[0099] Example 1: Evaluation of methods for using modified primers containing blocking groups in RNA-Seq experiments

[0100] To evaluate this approach, we amplified only the alpha chain cDNA of the T cell receptor.

[0101] SEQ ID NO: 3 and SEQ ID NO: 4 were used for targeted enrichment 1 (Step 4.1 in the ChromiumNext GEM Single Cell V(D)J Reagent Kits v1.1 User Guide, CG000208 Rev E), and SEQ ID NO: 5 and SEQ ID NO: 6 were used for targeted enrichment 2 (Step 4.3 in the ChromiumNext GEM Single Cell V(D)J Reagent Kits v1.1 User Guide, CG000208 Rev E).

[0102] All primers were either unmodified (labeled "10x Genomics Control" or "10x G") or modified as described in Table 2. For adapter ligation (Step 5.2 in the ChromiumNext GEM Single Cell V(D)J Reagent Kits v1.1 User Guide, CG000208 Rev E), we used adapters provided in the 10x Genomics kit ("10xGenomics" adapters) or used adapters generated by annealing oligonucleotides of sequence SEQ ID NO: 1 and SEQ ID NO: 2 ("Adapter-Mil").

[0103] Table 2: Evaluation of different blocking groups and nonspecific fragmentation

[0104]

[0105] The differences between the proposed method and the standard 10x Genomics workflow are as follows Figure 5a Table 2 lists the modifications evaluated in this example.

[0106] The impact of the proposed method was evaluated by assessing the size distribution of the libraries using D5000 or High Sensitivity D5000 Screen Tapes (Cat. Nos. 5067-5588 and 5067-5592, Agilent, Santa Clara, CA, USA) using an Agilent 4200 TapeStation system after a final sample index post-PCR cleanup (step 5.5 in the ChromiumNext GEM Single Cell V(D)J Reagent Kits v1.1 User Guide, CG000208 Rev E) using SPRIselect beads (Cat. No. B23317, Beckman Coulter, Brea, CA, USA).

[0107] The final library for each modified primer set (solid line) is plotted against the 10x Genomics control (dotted line). Figure 5b shown.

[0108] The results are shown in Figures 5c to 5e (Note that the experiment was split into two rounds, so two different controls were generated. Pairwise comparisons of libraries generated with modified primers and controls generated in the same round are depicted).

[0109] Most modified primers did show the expected results: a reduction in peaks for the unfragmented libraries compared to the 10x Genomics control baseline. Only the library generated with primers containing O-ME modified nucleotides did not show a significant reduction.

[0110] We were also unable to observe an effect of modifications to Spacer C3 and Spacer C9 in the forward primer. This was expected since adapter ligation of the unfragmented amplicon occurs on the reverse primer side. However, modifications in the forward primer still reduce primer-dimer derived library molecules and are therefore of interest.

[0111] We next sequenced subsets of representative libraries. As expected, we did not see significant differences in the number of reads, valid barcodes, and the number of cells with T cell receptor α chain-producing contigs (analyzed using 10xGenomics Cell Ranger software).

[0112] To evaluate the coverage of each library, we generated merged coverage maps. These coverage maps were generated by combining the mapping position of each read to its reference sequence into a single map where 0 corresponds to the first base and 1 corresponds to the last base.

[0113] These coverage plots show a peak of reads aligned to the 3' end of the reference sequence ( Figure 5f ; 10x genomics control; coverage plot). This peak originates from the unfragmented amplicon. As expected, this peak is significantly reduced in both libraries generated with 5' modified targeted enrichment primers (biotin and HEG spacer groups, Figure 5f ).

[0114] Example 2: Evaluation of methods using targeted modified primers containing blocking groups in RNA-Seq experiments based on dUTP incorporation / USER enzyme fragmentation

[0115] We also evaluated methods using an alternative fragmentation method disclosed in PCT / EP / 2020 / 081731. Briefly, this fragmentation method statistically incorporates dUTP nucleotides during the amplification reaction and then excises the uracil base using a uracil-N-glycosylase / endonuclease cocktail.

[0116] like Figure 6aLibraries were generated as shown: During targeted enrichment, we statistically incorporated uracil bases by using KAPA HiFi HS Uracil Master Mix (KK2801, Roche Diagnostics, Rotkreuz, Switzerland) supplemented with 1 μl of 1 mM dUTP (total reaction volume: 100 μl).

[0117] After the second targeted enrichment, we used USER II enzyme (Cat. No. M5508, New England Biolabs, Ipswich, MA, USA) to remove uracil bases.

[0118] Next, the samples were Ultra TM II EndRepair / dA-TailingModule (Cat. No. E7546, New England Biolabs, Ipswich, MA, USA), and then use Ultra TM II Ligation Module (Cat. No. E7595, New England Biolabs, Ipswich, MA, USA) was used for connector connection.

[0119] The adapters used in the adapter ligation process were generated by annealing oligonucleotides with the sequences specified by SEQ ID NO: 1 and SEQ ID NO: 2; as a control, we used 10x genomics AdaptorMix (PN 220026, 10xGenomics, Pleasanton, CA, USA).

[0120] The exact combinations of oligonucleotide modifications and linkers are specified in Table 3.

[0121] Figure 6b Results for the different conditions evaluated in the experiment are shown (Agilent Tapestation results after sample index PCR and cleanup). Libraries generated using standard primers and standard 10x Genomics adapters (dotted lines) show characteristic peaks for unfragmented amplicons ( Figure 6b Libraries generated using modified primers and standard 10x Genomics adapters (dashed line) show a reduced peak of unfragmented amplicons: although long fragments were ligated to the top strand, which does not contain the modification, the presence of the modification in the second strand reduced ligation efficiency, most likely through steric effects ( Figure 6bMiddle). Library generated using modified primers and "Adapter-Mil". The peak of the unfragmented amplicon can no longer be detected. Therefore, the modification completely prevents the adapter from ligating to the fragment containing the amplification primer, so the unfragmented amplicon is no longer subjected to sequencing ( Figure 6b bottom).

[0122] The results of each library preparation are as follows Figures 6c to 6d As shown: for all modifications evaluated (biotin, HEG spacer, spacer C3, spacer C9), we could successfully demonstrate that the peak of the unfragmented library could be removed by the proposed method.

[0123] Next, we sequenced all libraries using the modified primers in combination with the Mil-adapter. As expected, we did not see significant differences in the number of reads, valid barcodes, and the number of cells with T cell receptor α chain producing contigs (analyzed using 10x Genomics Cell Ranger software).

[0124] Table 3: Evaluation of different blocking groups, fragmentation based on uracil incorporation and excision

[0125]

[0126] We next generated the coverage plot described in Example 1. As expected, we observed a peak of reads aligned to the 3' end of the reference sequence ( Figure 6e continued Coverage plot), which is derived from the unfragmented amplicons.

[0127] Surprisingly, this peak derived from the unfragmented amplicon completely disappeared in the library generated with the 5' modified targeted enrichment primer (reverse primer). This data strongly suggests that the combination of the 5' modified targeted enrichment primer and fragmentation by statistical incorporation of uracil nucleotides followed by USER enzyme treatment is able to completely remove the unfragmented amplicon.

[0128] Example 3: Evaluation of methods using modified primers containing nucleotide analogs in RNA-Seq experiments

[0129] We next evaluated methods that utilize modified nucleotides followed by cleavage of these modified nucleotides after adaptor ligation.

[0130] Samples were processed according to the protocol provided in the ChromiumNext GEM Single Cell V(D)J Reagent Kits v1.1 User Guide (CG000208 Rev E) with the following modifications (see also Figure 7a ):

[0131] Modified targeted enrichment primers (reverse) (SEQ ID NO: 7 and SEQ ID NO: 8) were used. For certain conditions, "Adapter-Mil" (Example 2; SEQ ID NO: 1 and SEQ ID NO: 2) was used.

[0132] After adaptor ligation, samples with modified primer (8-oxo-G) were treated with formamidopyrimidine DNA glycosylase (Fpg) for the indicated times (Cat. No. M0240, New England Biolabs, Ipswich, MA, USA).

[0133] Figure 7b and 7c Results are shown for the different conditions evaluated in the experiment (Agilent Tapestation results after sample index PCR and cleanup). For both amounts of Fpg, the peak of the library from the unfragmented amplicon was significantly reduced. This reduction was already observable after 15 minutes of incubation.

[0134] Example 4: Evaluation of methods for using modified primers containing nucleotide analogs in dUTP incorporation / USER enzyme fragmentation-based RNA-Seq experiments

[0135] We also evaluated methods utilizing modified nucleotides followed by excision of these modified nucleotides after linker ligation by an alternative fragmentation method utilizing statistical incorporation of uracil bases followed by excision of these uracil bases as disclosed in PCT / EP / 2020 / 081731.

[0136] The protocol was the same as Example 2, except that primers containing 8-oxo-G modifications (SEQ ID NO: 7 and SEQ ID NO: 8) and subsequent Fpg digestion (Example 3) were used; for an overview of the workflow steps, see Figure 8a As observed in Example 2, this method is also able to completely remove the peak of the unfragmented amplicon (see Figure 8b ).

[0137] Sequence Analysis of Examples 3 and 4

[0138] Next, using the same method as in Examples 1 and 2, we sequenced a subset of the libraries from Examples 3 and 4 and compared the results with the libraries generated using the 10x Genomics protocol ( Fig. 9 ) (All libraries had comparable read numbers, valid barcodes, and number of cells with T-cell receptor α chain-producing contigs; analyzed using 10x Genomics Cell Ranger software).

[0139] As expected, we observed a significant reduction in unfragmented amplicons in libraries generated using modified nucleotides in modified target enrichment primers treated with Fpg after adapter ligation.

[0140] Strikingly, for libraries generated using the 10x genomics fragmentation module, we still observed small amounts of unfragmented amplicons; libraries generated using the fragmentation method of dUTP incorporation followed by USER enzyme treatment no longer showed reads from unfragmented amplicons.

[0141] This observation is very important because it will allow the amount of unfragmented amplicon to be fine-tuned from zero to any desired amount by mixing unmodified and modified primers.

[0142] Example 5: Different ratios between modified and unmodified primers allow preventing adapter oligonucleotide ligation on a subset of input DNA

[0143] The proposed method allows preventing adapter oligonucleotide ligation on only a subset of input DNA by carefully selecting which primers are modified and which are not modified. Furthermore, it is even possible to prevent adapter oligonucleotide ligation on only a subset of amplicons.

[0144] To show this advantage, we used the ligation step, using "Adapter-Mil", using the same setup as in Example 3. Instead of using only modified reverse primers, we used different ratios of modified and unmodified primers. Two replicates were generated for each ratio. After library preparation, the sequences were analyzed using the "Sequence Analysis of Examples 3 and 4" section and Fig. 9 The samples were sequenced and analyzed using the same method described in .

[0145] Fig.10a and Fig.10b The results in clearly show that the peak of the unfragmented amplicon decreases as the ratio of modified primers increases.

[0146] This observation has great advantages for methods like the one shown in this example. In single-cell TCR RNA-Seq using 5' barcoded TCR cDNA targeted enrichment products, the target region that requires high coverage is located in the 5' 2 / 3 of the cDNA. However, the constant region at the 3' end is important for determining the subtype of the T cell receptor. Since only a few reads are required in the non-diversified constant region, this method allows focusing on the 5' 2 / 3 of the cDNA; by incorporating a small portion of unmodified primers, it can still ensure that the 3' end is covered by a small number of reads.

[0147] Example 6: Different ratios between modified and unmodified primers can block ligation to a subset of amplicons in a PCR reaction

[0148] In this example, we also evaluated whether we could block ligation to a subset of amplicons in a PCR reaction. To do this, we used the same approach as in Example 5, except that we used different combinations of modified and unmodified amplification primers for TRA (T cell receptor alpha) and TRB (T cell receptor alpha).

[0149] like Fig.11a and Fig.11b As can be seen, this approach allows to specifically prevent ligation to only a subset of amplicons (note that the coverage plots for TRA and TRB are shown in Fig.11a and Fig.11b are separated in ).

[0150] This result is again very important because it greatly reduces the number of workflow steps: the alternative approach requires separate reactions (i.e., one reaction with the template to which the unfragmented amplicons should not be ligated, and a second reaction with the template to which the unfragmented amplicons should be ligated), and it would even reduce sensitivity because the individual template molecules would be randomly distributed into the two different reactions.

Claims

1. A method for obtaining a nucleic acid library of a sample containing polynucleotides, the method comprising the following steps: a. providing a plurality of modified primers to the polynucleotide, wherein the modified primers are starting points for a polymerase for nucleic acid amplification; b. amplifying the polynucleotide using a polymerase; c. fragmenting the amplified polynucleotide to obtain a mixture of fragmented and unfragmented polynucleotides comprising the modified primer; d. connecting a plurality of linker oligonucleotides to the mixture obtained in step c) to obtain a mixture of polynucleotides comprising the linker oligonucleotides, wherein the linker oligonucleotides comprise a binding site for an amplification primer; e. providing an amplification primer to the mixture obtained in step d), wherein the amplification primer is a starting point for a polymerase for nucleic acid amplification; f. Initiate nucleic acid amplification by providing a polymerase; Characterized in that the modified primer provided in step a) comprises a functional group, wherein the functional group is a blocking group located at the 5' end of the modified primer, thereby preventing the attachment of an adaptor oligonucleotide, or wherein the functional group is at least one nucleotide analogue, and wherein the nucleotide analogue is cleaved off by an endonuclease after step d), thereby removing the primer binding site provided by the adaptor oligonucleotide, Thereby, binding of the amplification primers provided in step f) and nucleic acid amplification of the fragmented polynucleotide comprising the modified primers are prevented.

2. The method according to claim 1, characterized in that In step a) a plurality of unmodified primers comprising no functional group are further provided.

3. The method according to claim 2, characterized in that In step a) a plurality of modified and unmodified primers are provided in a molar ratio of 90:

10.

4. The method according to claim 2, characterized in that: At least 50% of the primers provided in step a) are modified primers.

5. The method according to any one of claims 1 to 4, characterized in that The blocking group is selected from biotin, oligoethylene glycol having 1 to 25 glycol units, and a carbon spacer group having 3 to 12 carbon atoms.

6. The method according to any one of claims 1 to 5, characterized in that The modified primer provided in step a) comprises a functional group at the 5' end, which is a blocking group; Step b) is performed by providing natural nucleotides (N) a, t, g, c and a nucleotide analogue (A), wherein the molar ratio of N to A is between 150:1 and 10:1; and - Step c) is performed by cleaving the A nucleotide with an endonuclease.

7. The method according to any one of claims 1 to 4, characterized in that The nucleotide analog used as a functional group in the primer provided in step a) is selected from 8-oxo-7,8-dihydroguanine, deoxyuridine, deoxyinosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil, 3-methyladenine, 7-methylguanine, 1,N6-vinylidene adenine, hypoxanthine, deoxy-8-oxo-7,8-dihydroguanine, deoxyuridine and deoxyinosine.

8. The method according to any one of claims 1 to 4 and 7, characterized in that Step b) is performed by providing natural nucleotides (N) a, t, g, c and a nucleotide analog (A), wherein the molar ratio of N to A is between 150:1 and 25:1, and Step c) is performed by excising the A nucleotide with an endonuclease, wherein the nucleotide analogue used as the functional group in the primer provided in step a) is different from the nucleotide analogue provided in step b).

9. The method according to any one of claims 1-4 and 7-8, characterized in that The nucleotide analogue used as the functional group in the primer provided in step a) is 8-oxo-7,8-dihydroguanine, and the nucleotide analogue provided in step b) is deoxyuridine.

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