Method for producing nucleic acid library

Abstract

The present invention relates to a method for obtaining a nucleic acid library of a sample containing polynucleotides, a. providing a plurality of modified primers to the polynucleotide, wherein the modified primer serves as a starting point for nucleic acid amplification for a polymerase, b. amplifying the polynucleotide using a polymerase, c. obtaining a mixture of fragmented polynucleotides containing the modified primer and non-fragmented polynucleotides by fragmenting the amplified polynucleotide, d. obtaining a mixture of polynucleotides containing the adapter oligonucleotide by ligating a plurality of adapter oligonucleotides to the mixture obtained in step c), wherein the adapter oligonucleotide contains a binding site for an amplification primer, e. providing an amplification primer to the mixture obtained in step d), wherein the amplification primer serves as a starting point for nucleic acid amplification for a polymerase, f. initiating nucleic acid amplification by providing a polymerase comprising a method, wherein the modified primer provided in step a) contains a functional group, wherein · the functional group is a blocking group at the 5'-end of the modified primer that prevents ligation of the adapter oligonucleotide, or · the functional group is at least one nucleotide analog, in which case the nucleotide analog is excised by an endonuclease after step d) to remove the primer binding site provided by the adapter oligonucleotide, thereby preventing binding of the amplification primer provided in step f) and nucleic acid amplification of the fragmented polynucleotide containing the modified primer, characterized in that it is directed to a method.

Description

Technical Field

[0001] The present invention relates to the fields of next-generation sequencing and nucleic acid library preparation.

Background Art

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

[0003] Instruments for next-generation sequencing have a limited read length (the number of molecules downstream of the sequencing primer whose sequences can be accurately determined). In many applications, this read length is insufficient to determine the sequence of an entire DNA fragment of interest. Therefore, a process called library preparation is an essential part of sequencing applications. For this purpose, the DNA fragments to be sequenced are amplified, and then these numerous copies are shortened by a process called fragmentation. These fragments are sequenced, and the resulting sequences are aligned to a reference to determine the sequence of the entire fragment.

[0004] The ideal result 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 exhibit a significant proportion of non-fragmented DNA (10x Genomics User Guide CG000208 Rev E; Chromium Next GEM Single Cell 5’ Reagent Kits v2, Section 4.6, page 57). This is a significant shortcoming during library preparation (a typical example is shown in Figure 4b).

[0005] In the case of libraries containing many non-fragmented nucleic acids, many reads obtained by sequencing have been shown to be derived from the most 3’ end of the enriched target (e.g., libraries prepared according to the 10x Genomics User Guide CG000208 Rev E / the 10x Genomics Chromium Next GEM Single Cell 5’ Reagent Kits v2). To ensure that sufficient reads are obtained from shorter fragments, it is necessary to increase the number of sequencing reads. This leads to an increase in cost. This is because it is necessary to increase the number of reads for a given sample and, therefore, reduce the number of different samples that can be sequenced in parallel. Without compensating by increasing the number of reads per sample, there is a high risk of insufficient coverage to assemble the entire sequence of the original DNA molecule.

[0006] Furthermore, primer dimers from the initial target enrichment may be present in the library. This is particularly the case in target enrichment where multiple targets are amplified simultaneously (in these multiplex reactions, it is impossible to design a large number of primers that do not form primer dimers). These primer dimers are often carried over during library preparation and are similarly sequenced.

[0007] The presence of primer dimers (without information about the target) and non-fragmented species (unequal coverage of the target) can only be compensated for by increasing the number of reads. This leads to either an increase in cost or a decrease in coverage.

[0008] Here, the inventors describe an improved or alternative nucleic acid library preparation method for reducing the number of non-fragmented nucleic acids in order to avoid sequencing these molecules. This makes the sequencing more accurate and reduces costs because the number of sequencing reads can be reduced. This method can also be used to reduce the number of primer dimers.

[0009] Summary of the Invention An object of the present invention is a method for obtaining a nucleic acid library of a sample containing a polynucleotide, comprising: a. providing a plurality of modified primers to the polynucleotide, wherein the modified primer serves as a starting point for nucleic acid amplification for a polymerase; b. amplifying the polynucleotide using a polymerase; c. obtaining a mixture of fragmented polynucleotides containing the modified primer and non-fragmented polynucleotides by fragmenting the amplified polynucleotide; d. obtaining a mixture of polynucleotides containing the adapter oligonucleotide by ligating a plurality of adapter oligonucleotides to the mixture obtained in step c), wherein the adapter oligonucleotide contains a binding site for an amplification primer; e. providing an amplification primer to the mixture obtained in step d), wherein the amplification primer serves as a starting point for nucleic acid amplification for a polymerase; f. starting nucleic acid amplification by providing a polymerase The key element is the modified primer. The modified primer provided in step a) contains a functional group. The functional group is a blocking group at the 5' end of the modified primer that prevents ligation of the adapter oligonucleotide (Figure 2), or the functional group is at least one nucleotide analog, in which case the nucleotide analog is excised by an endonuclease after step d) to remove the primer binding site provided by the adapter oligonucleotide (Figure 3). This prevents the binding of the amplification primer provided in step f) and the nucleic acid amplification of the fragmented polynucleotide containing the modified primer.

[0010] In addition, amplification of primer dimers is prevented.

[0011]

[0012] ​The method of the present invention can be combined with the statistical fragmentation technique disclosed in (PCT / EP / 2020 / 081731). In this method, one type of nucleotide analog is incorporated into the nucleic acid during amplification. The nucleotide analog is excised by an endonuclease, thus generating a fragmented nucleic acid library.

[0013] The target nucleic acid library obtained by the method of the present invention can be used for sequencing. Any method known in the art can be used for sequencing.

[0014] Definitions Unless otherwise defined, all 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.

[0015] As used herein, the term "comprising" (or "comprises") is used with respect to compositions, methods, and their respective components, and they include elements not specified that are not essential to the method or composition, whether or not they are essential.

[0016] The words "bind" and "hybridize" and their grammatical equivalents can be used interchangeably. Hybridization occurs when two nucleic acid strands are complementary to each other. Hybridization can occur under conditions known in the art.

[0017] As used herein, the term "complementary" refers to the ability of two nucleotides to form exact pairs by Watson-Crick base pairing. By way of illustration, two nucleic acids are considered to be complementary to each other at a given position if the nucleotide at that position in one nucleic acid strand can form a hydrogen bond with a nucleotide in another nucleic acid strand. Complementarity between two single-stranded nucleic acid molecules may be "partial" (in which case only a portion of the nucleotides bind) or "complete" (in which case there is complete complementarity between the single-stranded molecules). As used herein, a "primer" is a single-stranded oligonucleotide composed of nucleotides that can bind to a complementary nucleic acid sequence. It is understood that any of the primers described in the present invention can serve as a starting point for nucleic acid synthesis / amplification. According to the present invention, modified primers can be used. Such modified primers contain a functional group and can prevent ligation of adapter molecules or can be removed from the polynucleotide. According to the present invention, the functional group is a blocking group at the 5' end of the modified primer or the functional group is at least one nucleotide analog contained in the modified primer. Modified primers may also be referred to as "modified target enrichment primers". In addition, in the methods of the present disclosure, unmodified primers can also be used. These primers do not retain a functional group that can prevent ligation of adapter molecules or a functional group that can be removed from the polynucleotide. Amplification primers as used herein also have the characteristics of unmodified primers.

[0018] As used herein, the terms "nucleic acid synthesis" and nucleic acid amplification can be used interchangeably. The process of nucleic acid synthesis is well known in the art. Briefly, a template nucleic acid is provided for nucleic acid synthesis. This can be single-stranded or double-stranded. If double-stranded nucleic acid is used first, the first step is denaturation to single nucleic acid strands (complementary strand and reverse complementary strand) using techniques known in the art. The denaturation step is not required for single-stranded nucleic acid. In the next step, a primer that binds to the complementary region of the nucleic acid strand is provided. Next, a polymerase is used to extend the 3' end of the primer and fill it with complementary nucleotides to generate a complementary strand. As a result, a complementary nucleic acid strand is formed. For further amplification, denaturation of the double-stranded nucleic acid is required, without which a new round of nucleic acid synthesis cannot be initiated. Based on the present invention, nucleic acid synthesis can be symmetric or asymmetric. In symmetric nucleic acid synthesis, only one primer is used. Thus, only one strand is synthesized. In a symmetric reaction, a pair of two primers can be provided (forward primer and reverse primer). One primer binds to the complementary nucleic acid strand and the other binds to the reverse complementary nucleic acid strand. Thereby, nucleic acid synthesis can be initiated on both strands. Unless otherwise stated, the expression "primer" as used herein can include a forward primer, a reverse primer, or both. The expression "primers of the same specificity" refers to all of the forward primers used or all of the reverse primers used in a particular embodiment.

[0019] As used herein, the term "adaptor" (either "adaptor" or "adapter") refers to an oligonucleotide that can be ligated to a polynucleotide. These contain primer binding sequences to facilitate amplification or sequencing of the associated nucleic acid. The primer binding sequences of the adaptor molecules can be the same (identical) or different sequences. Thus, for example, a 5' adaptor can contain the same or different primer binding sequences, and a 3' adaptor can contain the same or different primer binding sequences. Identical primer binding sequences that can be present in different members of a plurality of nucleic acid molecules can enable amplification of a number of different sequences using a single universal amplification primer that is complementary to the universal / identical primer binding sequence. The adaptor molecule can further contain sequences for one or more sample labels and molecular labels (barcodes). The adaptor can be double-stranded (symmetric), partially double-stranded (asymmetric) or single-stranded. One or more adaptor molecules can be located at the 5' or 3' end of the nucleic acid. The adaptors at the 5' and 3' ends of the adaptor-target-adaptor can be the same or different.

[0020] The terms "polynucleotide" and "nucleic acid" can be used interchangeably and refer to biopolymers composed of covalently linked nucleotide monomers in a chain. An amplified nucleic acid can be referred to as an "amplicon". The nucleic acid can be DNA or RNA. It can include 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-ethenoadenine, and hypoxanthine, as well as their derivatives, such as deoxy-8-oxo-7,8-dihydroguanine (d8-oxoG), deoxyuridine (dU), deoxyinosine (dI). These nucleotide analogs can be excised by endonucleases, particularly structure-specific endonucleases.

[0021] As used herein, the term "a plurality of" means two or more.

[0022] All of the steps used in the drawings are based on the steps recited in claim 1.

Brief Description of the Drawings

[0023]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0024] The present invention provides a method for overcoming the current limitations in nucleic acid library preparation. The main objective is to reduce the number of non-fragmented nucleic acids that can be sequenced. This is achieved by using modified primers for the first nucleic acid amplification during library preparation.

[0025] The method of the present invention is a method for preparing a nucleic acid library. This nucleic acid library can be used for several downstream applications such as next-generation sequencing or polymerase chain reaction.

[0026] Samples that can be used for preparing the nucleic acid library 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., Danio rerio, Xenopus laevis) and mammals (e.g., Mus musculus, Homo sapiens). Biological samples can take the form of tissue sections, cell aggregates, floating cells, adherent cells or body fluids.

[0027] The nucleic acid used for the first library preparation can be a polynucleotide chain composed of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can have a linear or circular structure.

[0028] In the first step (a) of the method of the present invention, a modified primer is provided, followed by amplification of the nucleic acid using a polymerase (step b). The nucleic acid provided by the sample functions as a template for nucleic acid amplification. Techniques and conditions such as polymerase chain reaction for synthesizing and amplifying nucleic acids are well known in the art.

[0029] Standard polymerases that can be used for these reactions are proofreading polymerases such as Taq polymerase, 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 that can incorporate dUTP nucleotides such as KAPA HiFi Uracil+ polymerase (Roche Diagnostics).

[0030] Subsequently, after nucleic acid amplification, step c) fragmentation of the polynucleotide is performed. During this step, the polynucleotide can be double-stranded. Techniques for polynucleotide fragmentation are known in the art, such as physical shearing using a Covaris sonicator, enzymatic fragmentation, and tagmentation (an example is disclosed in PCT / EP / 2020 / 081731). Fragmentation of the polynucleotide results in a mixture of fragmented polynucleotides and unfragmented polynucleotides. This mixture contains fragmented polynucleotides with modified primers, fragmented polynucleotides without modified primers, and unfragmented polynucleotides containing said modified primers. The unfragmented polynucleotides are considered to contain modified primers.

[0031] The resulting fragments can have blunt ends or 3' or 5' single-stranded overhangs. Blunt ends can be generated by treating the fragmented nucleic acid with an enzyme or enzyme mixture that exhibits 5'→3' polymerase activity and 3'→5' exonuclease activity prior to adapter ligation.

[0032] For fragments with 5' overhangs, the reverse complementary second strand is synthesized using 5'→3' polymerase activity (the "fill-in"), and for fragments with 3' overhangs, the overhangs are removed using 3'→5' exonuclease activity. 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 fill-in reaction typically also contains polynucleotide kinase to add phosphate groups to the 5' ends of the processed fragments. Optionally, step c) may also contain an enzyme with A-tailing activity. Subsequently, after fragmentation of the nucleic acid in step c), in step d), ligation of multiple adapter oligonucleotides to the mixture obtained in step c) is performed. As a result, a mixture of polynucleotides containing the adapter oligonucleotides is obtained, where the adapter oligonucleotides contain binding sites for amplification primers. The adapter oligonucleotides used for ligation contain at least one amplification primer binding sequence. It may further contain additional 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 tagged (e.g., by oligonucleotide barcodes such as unique molecular identifiers), thereby enabling parallel sequencing of a large number of independent DNA molecules.

[0033] The adapter molecule may be a double-stranded with blunt ends or at least partially double-stranded including a single-stranded nucleic acid strand overhang (e.g., a T overhang in a sample using an enzyme with A-tailing activity in step c). An example of an adapter is shown in Figure 1a.

[0034] The adapter molecule is ligated to one side of the polynucleotide contained in the mixture obtained in step c), i.e., adjacent to the site of the forward primer or adjacent to the reverse primer side (as in the typical examples shown in FIGS. 1 to 3), or can be ligated to both sides of the polynucleotide. When the adapter molecule is ligated to both sites of the polynucleotide, the adapter molecules on each side may be the same or different. The side on which ligation occurs depends on the modified primer used. When a modified reverse primer is used, adapter ligation on the reverse primer side is prevented in non-fragmented polynucleotides, but the adapter ligates to fragmented polynucleotides. When a modified forward primer is used, adapter ligation on the reverse primer side is prevented in non-fragmented polynucleotides, but the adapter ligates to fragmented polynucleotides. When a mixture of a modified forward primer and a modified reverse primer is used, adapter ligation in polynucleotides still containing the forward primer or the reverse primer is prevented.

[0035] Ligation of the adapter molecule is performed by a ligase. General examples of such ligases are, for example, T4 DNA ligase, Taq ligase or equivalent enzymes. Prior to step d), the fragment can be made single-stranded and a single-stranded adapter can be ligated. For example, thermostable 5’ App DNA / RNA ligase or equivalent enzymes. As a result, a polynucleotide containing the adapter is generated.

[0036] Subsequently, after ligation of the adapter molecule, an amplification step is performed. In this step, an amplification primer is provided to the mixture obtained in step d). Here, the amplification primer serves as a starting point for nucleic acid amplification for the polymerase. A polymerase is provided to initiate nucleic acid amplification. The amplification primer can also be used to initiate a sequencing reaction.

[0037] In one embodiment of the present invention, a washing step may not be required after fragmentation (step c) and / or adapter ligation (step d).

[0038] In the following section, numerous preferred variations and embodiments of the first aspect of the present invention are enumerated.

[0039] Modified primers and unmodified primers The key element of the present invention is the use of a modified primer in step a). The modified primer contains a functional group. This functional group is a blocking group that is located at the 5' end of the modified primer and prevents the ligation of the adapter oligonucleotide, or the functional group is at least one nucleotide analog contained in the modified primer. In this case, the nucleotide analog is excised by an endonuclease after step d) to remove the primer binding site provided by the adapter oligonucleotide. In either scenario, this modification results in the absence of the adapter oligonucleotide in the polynucleotide and fragment containing the modified primer. As a result, the amplification primer added in step f) cannot bind to such a polynucleotide due to the absence of the primer binding sequence, and amplification is hindered.

[0040] The modified primer provided in step a) can be selected from a forward primer and / or a reverse primer. The forward primer and the reverse primer may be the same or different.

[0041] In certain embodiments, in step a), a further plurality of unmodified primers may be provided. The unmodified primers do not contain a functional group, whereby adapter oligonucleotides are absent from the polynucleotides and fragments containing the unmodified primers. Depending on the stoichiometry between the unmodified primers and the modified primers provided in step a) of the method of the invention, the amount of potential non-fragmented polynucleotides amplified in step f) can be adjusted. In yet another embodiment, the further plurality of unmodified primers provided in step a) may have the same specificity as the modified primers. For example, the unmodified forward primer and the modified forward primer or the unmodified reverse primer and the modified reverse primer may have the same specificity.

[0042] The unmodified primers may be selected from forward primers and / or reverse primers. The molar ratio of the plurality of modified primers to the plurality of unmodified primers can be 99 to 1, 95 to 5, or 90 to 10. In another embodiment, the molar ratio of the plurality of modified primers to the plurality of unmodified primers having the same specificity can be 99 to 1, 95 to 5, or 90 to 10.

[0043] 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 having the same specificity provided in step a) are modified primers.

[0044] In a preferred embodiment, all of the reverse primers provided in step a) are unmodified primers and all of the 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.

[0045] In another embodiment of the present invention, 25% of the reverse primers are modified, and 75% of the reverse primers and all forward primers can be unmodified. In yet another embodiment of the present invention, 50% of the reverse primers are 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 are modified, and 25% of the reverse primers and all forward primers can be unmodified.

[0046] In another embodiment of the present invention, 25% of the forward primers are modified, and 75% of the forward primers and all reverse primers can be unmodified. In yet another embodiment of the present invention, 50% of the forward primers are 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 are modified, and 25% of the forward primers and all reverse primers can be unmodified.

[0047] Modified primer - blocking group In a variant of the present invention, the modified primer provided in step a) contains a functional group that is a blocking group at the 5'-end (Figure 2). This blocking group prevents the ligation of the adapter oligonucleotide to the polynucleotide (step d). Ligation cannot occur. This modification of the primer prevents the phosphorylation of the 5'-group of the polynucleotide containing the modified primer that is required for the ligation of the adapter molecule.

[0048] The blocking group is selected from blocking groups known in the art. Commonly used blocking groups include, for example, a fluorophore or quencher coupled to the 5'-end of a nucleotide, a 5'-amino modification group (including C6 amino and C12 amino), 5'-biotin (including bis-biotin and biotin-TEG), a 5'-thiol modification group (including C6 thiol), a carbon spacer (including spacer C3, spacer C6, spacer C12), an oligoethylene glycol spacer (including spacer 9, spacer 12, spacer 18, HEG spacer), digoxigenin, acrydite, C3-azide, DBCO, DBCO-TEG, cholesteryl-TEG, and a PC-amino modification group. In certain embodiments, the blocking group can be selected from the group consisting of biotin, an oligoethylene glycol spacer having 1 to 25 glycol units, and a carbon spacer having 3 to 12 carbon atoms. Commonly used oligoethylene glycol spacers include spacer 9, spacer 12, spacer 18, and HEG spacer. In a preferred embodiment, the blocking group can be selected from the group consisting of spacer C3 and HEG spacer.

[0049] In another embodiment of the present invention, further, step b) is performed by providing natural nucleotides (N) a, t, g, c and one type of nucleotide analog (A). The preferred molar ratio of N to A is 150:1 to 10:1, more preferably 150:1 to 25:1. Based on this, the fragmentation of the amplified polynucleotide in step c) is performed by excision of nucleotide A by an endonuclease. The nucleotide analog is incorporated into the newly synthesized nucleic acid in place of its natural counterpart (step b). Next, the newly synthesized nucleic acid can be treated using an endonuclease (step c). These enzymes excise the nucleotide analog and nick the newly synthesized nucleic acid. Next, this results in fragmentation. Next, the newly synthesized nucleic acid can be treated using an endonuclease as listed in Table 1. Examples of such enzyme mixtures that can excise 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, commercially available enzymes or enzyme mixtures such as New England Biolabs' USER enzyme or thermolabile USER enzyme may be used (catalog numbers M5508 and M5507, New England Biolabs, Ipswich, Massachusetts, USA).

[0050] The fragment length can be controlled by adjusting the ratio between N and A, as previously disclosed in (PCT / EP / 2020 / 081731).

[0051] Nucleotide analogs can be selected from the list of 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-ethenoadenine, and hypoxanthine, and their derivatives, such as deoxy-8-oxo-7,8-dihydroguanine (d8-oxoG), deoxyuridine (dU), deoxyinosine (dI). In a preferred embodiment, the nucleotide analog is deoxyuridine (dUTP).

[0052] Modified primer - nucleotide analog In another variation of the present invention, the modified primer provided in step a) comprises at least one nucleotide analog (Figure 3). This nucleotide analog is excised by an endonuclease after step d). By doing so, the primer binding site provided by the adapter oligonucleotide is removed. This makes it impossible to initiate the nucleic acid synthesis reaction of this strand incorporating the modified primer.

[0053] In one embodiment, the modified primer provided in step a) comprises a nucleotide analog. Such a primer is incorporated into the newly synthesized nucleic acid during nucleic acid amplification. Subsequently, fragmentation and ligation of adapter molecules are then performed. Next, the nucleotide analog is excised by an endonuclease, thereby creating a nick in the double-stranded nucleic acid structure. This then results in the generation of a polynucleotide lacking the adapter molecule containing the primer binding site. The polynucleotide lacking the adapter molecule cannot be amplified because it lacks the adapter and thus lacks the amplification primer binding site.

[0054] Nucleotide analogs can be selected from the list of 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-ethenoadenine, and hypoxanthine, and their derivatives, such as deoxy-8-oxo-7,8-dihydroguanine (d8-oxoG), deoxyuridine (dU), deoxyinosine (dI). In a preferred embodiment, the nucleotide analog is 8-oxo-7,8-dihydroguanine (8-oxoG). The treatment after step d) is performed using Fpg (formamidopyrimidine [fapy]-DNA glycosylase).

[0055] In another embodiment of the present invention, further, step b) is performed by providing natural nucleotides (N) a, t, g, c and one type of nucleotide analog (A). The preferred molar ratio of N to A is 150:1 to 10 / 1, more preferably 150:1 to 25:1. Based on this, the fragmentation of the amplified polynucleotide in step c) is performed by excision of nucleotide A by an endonuclease. The nucleotide analog is incorporated into the newly synthesized nucleic acid instead of its natural counterpart. The nucleotide analog serving as a functional group in the primer provided in step a) is different from the nucleotide analog provided in step b).

[0056] Next, the newly synthesized nucleic acid can be treated with endonucleases as listed in Table 1. These enzymes excise nucleotide analogs and nick the newly synthesized nucleic acid, which then leads to fragmentation. Examples of such enzyme mixtures that nick 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, commercially available enzymes or enzyme mixtures such as New England Biolabs' USER enzyme or thermolabile USER enzyme (catalog numbers M5508 and M5507, New England Biolabs, Ipswich, Massachusetts, USA) may be used.

[0057] The fragment length can be controlled by adjusting the ratio between N and A as previously disclosed (PCT / EP / 2020 / 081731).

[0058] The nucleotide analogs can be selected from the list of 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-ethenoadenine, and hypoxanthine, and their derivatives, such as deoxy-8-oxo-7,8-dihydroguanine (d8-oxoG), deoxyuridine (dU), deoxyinosine (dI). In a preferred embodiment, the nucleotide analog that serves as a functional group in the primer provided in step a) is 8-oxo-7,8-dihydroguanine, and the nucleotide analog provided in step b) is deoxyuridine. Table 1: List of modified bases and enzymes that can be used in the method (options: modified bases and subsequent excision of modified bases after adapter ligation)

Table 1

[0059] Regarding the first aspect of the invention disclosed in this specification, all the definitions, features, embodiments, and modifications defined in this specification are also applicable mutatis mutandis to other modifications and embodiments of the invention disclosed in this specification.

Examples

[0060] The following examples are intended to explain the present invention in more detail, but the present invention is not limited to these examples.

[0061] The method was evaluated in a single-cell targeted RNA sequencing application (targeted RNA-Seq). Towards this goal, the inventors used cDNA prepared from human T cells by using the Chromium Single Cell 5’ Library&Gel Bead Kit (PN-1000014, 10x Genomics, Pleasanton, CA, USA) according to the instructions described in the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide (CG000208 Rev E). The same cDNA aliquots were used in the following examples.

[0062] To amplify specific targets and subsequently perform library preparation (RNA-Seq), the inventors used the target enrichment primers of the single Cell V(D)J Enrichment Kit, Human T Cell (PN-1000005, 10x Genomics, Pleasanton, CA, USA) according to the instructions described in the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide (CG000208 Rev E, Steps 4 and 5).

[0063] For each of the following examples, the inventors used the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide (CG000208 Rev E) as the baseline protocol and made changes to specific steps in this protocol. Details of the changes are described in each example.

[0064] In each example, the inventors strictly followed the protocol (Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide, CG000208 Rev E) to prepare a reference library (labeled "10x G" or "10x Genomics") in parallel.

[0065] Example 1: Evaluation of the method using modified primers containing blocking groups in RNA-Seq experiments To evaluate this method, the inventors amplified only the cDNA of the alpha chain of the T cell receptor.

[0066] SEQ ID NO: 3 and SEQ ID NO: 4 were used for target enrichment 1 (step 4.1 of the Chromium Next 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 target enrichment 2 (step 4.3 of the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide, CG000208 Rev E).

[0067] All primers were either unmodified (labeled as "10x Genomic Control" or "10x G") or modified as described in Table 2. For adapter ligation (Step 5.2 of Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide, CG000208 Rev E), either the adapter included in the 10x Genomics kit ("10x Genomics" adapter) or an adapter prepared by annealing oligonucleotides with the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 ("Adapter-Mil") was used. Table 2: Evaluation of Various Blocking Groups and Nonspecific Fragmentation [Table 2]

[0068] The differences between the method proposed herein and the standard 10x Genomics workflow are shown in Fig. 5a. The modifications evaluated in this example are listed in Table 2.

[0069] After final Post Sample Index PCR Cleanup using SPRIselect beads (Catalog No. B23317, Beckman Coulter, Brea, CA, USA) (Step 5.5 of Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide, CG000208 Rev E), the Agilent 4200 TapeStation system was used with D5000 or High Sensitivity D5000 Screen Tape (Catalog Nos. 5067-5588 and 5067-5592, Agilent, Santa Clara, CA, USA) to evaluate the size distribution of the library, thereby evaluating the impact of the method proposed herein.

[0070] As shown in Fig. 5b, the final libraries for each modified primer set (solid lines) were plotted pairwise against the 10× Genomics control (dotted lines).

[0071] The results are shown in Figs. 5c - 5e. (Note that since the experiment was divided into two rounds, two different controls were created. The pairwise comparison of the libraries made using modified primers is shown against the control made in the same round.)

[0072] Most of the modified primers showed the expected results. That is, the peak of the unfragmented library decreased compared to the 10x Genomics control baseline. Only the libraries made with primers containing O - ME modified nucleotides did not show a substantial reduction.

[0073] The inventors were also unable to observe the effect of spacer C3 and spacer C9 modifications in the forward primer. Since adapter ligation to the unfragmented amplicon occurs on the reverse primer side, this was expected. Nevertheless, the modification in the forward primer is still interesting as it reduces library molecules derived from primer dimers.

[0074] Next, the inventors sequenced a subset of representative libraries. As expected, there was no substantial difference in the number of reads, the number of valid barcodes, and the number of cells with productive contigs regarding the T - cell receptor alpha chain (analyzed using the 10x Genomics Cell Ranger software).

[0075] To evaluate the coverage of each library, consolidated coverage plots were created. These coverage plots were created by integrating the mapping positions of each read to the reference into a single figure where 0 corresponds to the first base and 1 corresponds to the last base.

[0076] These coverage plots show a high peak of reads that align to the 3’ end of the reference (Figure 5f; 10x genomics control; coverage plot). This peak is derived from non-fragmented amplicons. As expected, this peak is significantly reduced in the two libraries made using 5’-modified target enrichment primers (biotin and HEG spacer, Figure 5f).

[0077] Example 2: Evaluation of a method using target-modifying primers containing a blocking group in an RNA-Seq experiment based on dUTP spike-in / USER enzyme fragmentation The inventors evaluated this method using the alternative fragmentation method disclosed in PCT / EP / 2020 / 081731. Briefly, this fragmentation method uses the statistical incorporation of dUTP nucleotides during the amplification reaction, followed by excision of uracil bases using a uracil-N-glycosylase / endonuclease mixture.

[0078] Libraries were prepared as shown in Figure 6a. That is, during target enrichment, the inventors statistically incorporated uracil bases using 1 μl of KAPA HiFi HS uracil master mix (KK2801, Roche Diagnostics, Rotkreuz, Switzerland) supplemented with 1 mM dUTP (total reaction volume: 100 μl).

[0079] After a second target enrichment, uracil bases were excised using USER II enzyme (catalog number M5508, New England Biolabs, Ipswich, Massachusetts, USA).

[0080] Next, the sample was subjected to the NEBNext® Ultra™ II End Repair / dA-Tailing Module (Catalog No. E7546, New England Biolabs, Ipswich, MA, USA), followed by adapter ligation using the NEBNext® Ultra™ II Ligation Module (Catalog No. E7595, New England Biolabs, Ipswich, MA, USA).

[0081] The adapter used for adapter ligation was prepared by annealing oligonucleotides having the sequences specified in SEQ ID NO: 1 and SEQ ID NO: 2. As a control, the inventors used the 10x genomics adapter mix (PN 220026, 10x Genomics, Pleasanton, CA, USA).

[0082] The details of the oligonucleotide modifications and adapter combinations are shown in Table 3.

[0083] Figure 6b shows the results for different conditions evaluated in the experiment (Agilent Tapestation results after sample index PCR and cleanup). Libraries prepared using standard primers and standard 10x Genomics adapters (dotted line) show characteristic peaks of non-fragmented amplicons (Figure 6b, top). Libraries prepared using modified primers and standard 10x Genomics adapters (dashed line) show a reduction in the peaks of non-fragmented amplicons. That is, long fragments in the top strand without modification are ligated, but the presence of modification in the second strand reduces the ligation efficiency, probably due to steric effects (center of Figure 6b). Libraries prepared with modified primers and "Adapter-Mil". Non-fragmented amplicons can no longer be detected. Therefore, this modification completely prevents adapter ligation to fragments containing amplification primers, and thus non-fragmented amplicons are no longer subjected to sequencing (Figure 6b, bottom).

[0084] The results for each library preparation are shown in FIGS. 6c-6d. For all of the modifications evaluated (biotin, HEG spacer, spacer C3, spacer C9), we were able to successfully demonstrate that the peaks of the unfragmented library can be removed by the method proposed herein.

[0085] Next, we used the modified primers in combination with the Mil-adapter to sequence all libraries. As expected, no substantial differences were seen in the number of reads, the number of valid barcodes, and the number of cells with productive contigs with respect to the T cell receptor alpha chain (analyzed using 10x Genomics Cell Ranger software). Table 3: Evaluation of fragmentation based on different blocking groups, uracil incorporation and excision [Table 3]

[0086] Next, we created the coverage plots described in Example 1. As expected, we observed a high peak of reads that aligned to the 3' end of the reference, originating from unfragmented amplicons (coverage plots in FIGS. 6e and later).

[0087] Surprisingly, this peak originating from unfragmented amplicons completely disappeared in libraries made using 5'-modified target enrichment primers (reverse primers). This data strongly indicates that unfragmented amplicons can be completely removed by the combination of 5'-modified target enrichment primers and the statistical incorporation of uracil nucleotides followed by fragmentation with USER enzyme treatment.

[0088] Example 3: Evaluation of the method using modified primers containing nucleotide analogs in RNA-Seq experiments Next, the inventors evaluated a method of utilizing modified nucleotides and subsequently excising these modified nucleotides after adapter ligation.

[0089] Samples were processed according to the protocol provided in the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide (CG000208 Rev E) with the following modifications (see also Figure 7a).

[0090] Modified target enrichment primers (reverse) were used (SEQ ID NO: 7 and SEQ ID NO: 8). Under some conditions, "Adapter-Mil" (Example 2; SEQ ID NO: 1 and SEQ ID NO: 2) was used.

[0091] After adapter ligation, samples with modified primers (8-oxo-G) were treated with formamidopyrimidine DNA glycosylase (Fpg) for the indicated time (Catalog No. M0240, New England Biolabs, Ipswich, Massachusetts, USA).

[0092] Figures 7b and 7c show the results for the different conditions evaluated in this experiment (Agilent Tapestation results after sample index PCR and cleanup). For both amounts of Fpg, the peak of the library derived from non-fragmented amplicons was significantly reduced. This reduction could already be observed after 15 minutes of incubation.

[0093] Example 4: Evaluation of a method using modified primers containing nucleotide analogs in RNA-Seq experiments based on dUTP spike-in / USER enzyme fragmentation The inventors also evaluated a method of utilizing modified nucleotides and subsequently excising these modified nucleotides after adapter ligation with an alternative fragmentation method that utilizes the statistical incorporation of uracil bases and subsequent excision of these uracil bases, as disclosed in PCT / EP / 2020 / 081731.

[0094] The protocol was the same as in Example 2, except for the use of primers containing 8-oxo-G modifications (SEQ ID NO: 7 and SEQ ID NO: 8) and subsequent Fpg digestion (Example 3). See Figure 8a for an overview of the workflow steps. As observed in Example 2, this method can also completely remove the peaks of non-fragmented amplicons (see Figure 8b).

[0095] Sequence analysis of Example 3 and Example 4 Next, the inventors sequenced a subset of the libraries from Example 3 and Example 4 and compared the results to libraries generated using the same approach as in Examples 1 and 2 using the 10x Genomics protocol (Figure 9) (all libraries had an equivalent number of reads, valid barcodes, and number of cells with productive contigs for the T cell receptor alpha chain; analyzed using 10x Genomics Cell Ranger software).

[0096] As expected, the inventors observed a significant reduction in non-fragmented amplicons in libraries generated by using modified nucleotides in the modified target enrichment primers and treating them with Fpg after adapter ligation.

[0097] Notably, for libraries generated using the 10x genomics fragmentation module, a small amount of non-fragmented amplicons was still observed. Libraries generated by a fragmentation approach using dUTP spike-in followed by USER enzyme treatment no longer showed reads from non-fragmented amplicons.

[0098] This observation is very interesting because it allows for fine-tuning the amount of non-fragmented amplicons from zero to any desired amount by mixing unmodified and modified primers.

[0099] Example 5: By varying the ratio of modified primers to unmodified primers, it becomes possible to prevent adapter oligonucleotide ligation in a subset of input DNA. In the method proposed herein, by carefully selecting which primer to modify and which primer to leave unmodified, it becomes possible to prevent only adapter oligonucleotide ligation in a subset of input DNA. Additionally, it is even possible to prevent adapter oligonucleotide ligation only to a subset of amplicons.

[0100] To demonstrate this advantage, the inventors used the same setup as in Example 3, using "Adapter-Mil" and a ligation step. Instead of using only modified reverse primers, the inventors used modified and unmodified primers in various ratios. Two replicates were made for each ratio. After library preparation, the samples were sequenced and analyzed in the same way as described in the section "Sequence analysis of Examples 3 and 4" and in FIG. 9.

[0101] The results in FIGS. 10a and 10b clearly show that the peak of non-fragmented amplicons decreases with an increase in the proportion of modified primers.

[0102] This observation is very advantageous for the method as shown in this example. In single-cell TCR RNA-Seq using 5'-barcoded TCR cDNA target enrichment products, the regions of interest that require high coverage are located within the 2 / 3 of the 5'-end of the cDNA. Nevertheless, the constant region at the 3'-end is also important for determining the subtype of the T cell receptor. Since only a few reads are needed for the non-diverse constant region, this method allows focusing on the 2 / 3 of the 5'-side of the cDNA, and still, by spiking in a small number of unmodified primers, it is possible to ensure that the 3'-end is covered by a small number of reads.

[0103] Example 6: By changing the ratio of modified primers to unmodified primers, ligation to a subset of amplicons in a PCR reaction can be blocked. In this example, it was also evaluated whether ligation to a subset of amplicons in a PCR reaction could be blocked. For this purpose, the inventors used the same approach as in Example 5, except that modified amplification primers and unmodified amplification primers for TRA (T cell receptor alpha) and TRB (T cell receptor beta) were used in various combinations.

[0104] As can be seen from FIGS. 11a and 11b, this approach makes it possible to specifically prevent only ligation of a subset of amplicons (note that in FIGS. 11a and 11b, the coverage plots for TRA and TRB are separated).

[0105] This result is also very interesting because it dramatically reduces the number of workflow steps. In an alternative approach, it would be necessary to separate the reactions (i.e., one reaction with a template to which non-fragmented amplicons should not be ligated and another reaction with a template to which non-fragmented amplicons should be ligated), and each template molecule would be randomly distributed among two different reactions, which could even reduce sensitivity.

Claims

1. To obtain a nucleic acid library of samples containing polynucleotides, a. A step of providing a plurality of modified primers to the polynucleotide, where the modified primers serve as a starting point for nucleic acid amplification for polymerase. b. Amplification of the polynucleotide using polymerase, c. A step of obtaining a mixture of fragmented polynucleotides containing the modified primers and unfragmented polynucleotides by fragmentation of the amplified polynucleotides. d. A step of obtaining a mixture of polynucleotides containing the adapter oligonucleotides by ligating a plurality of adapter oligonucleotides to the mixture obtained in step c), wherein the adapter oligonucleotides include binding sites for amplification primers. e. A step of providing an amplification primer to the mixture obtained in step d), where the amplification primer serves as a starting point for nucleic acid amplification for the polymerase. f. A step of initiating nucleic acid amplification by providing polymerase. A method including, The modified primer provided in step a) contains a functional group, where, - The functional group is a blocking group located at the 5' end of the modified primer that prevents ligation of the adapter oligonucleotide, or - The functional group is at least one nucleotide analog, in which case the nucleotide analog is excised by an endonuclease after step d) to remove the primer binding site provided by the adapter oligonucleotide. This prevents the binding of the amplification primer provided in step f) and the nucleic acid amplification of the fragmented polynucleotide containing the modified primer. A method characterized by the following:

2. The method according to claim 1, characterized in that, in step a), a plurality of unmodified primers that do not contain functional groups are further provided.

3. The method according to claim 2, characterized in that in step a), a plurality of modified primers and a plurality of unmodified primers are provided in a molar ratio of 90 to 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 claim 1 or 2, characterized in that the blocking group is selected from the group consisting of biotin, oligoethylene glycol having 1 to 25 glycol units, and carbon spacers having 3 to 12 carbon atoms.

6. - The modified primer provided in step a) contains a functional group that is a blocking group at its 5' end. Step b) is carried out by providing natural nucleotides (N) a, t, g, c and one type of nucleotide analog (A), wherein the molar ratio of N to A is 150:1 to 10:1, and - Step c) is performed by excision of the A nucleotide by an endonuclease. The method according to claim 1 or 2, characterized by the above.

7. The method according to claim 1 or 2, characterized in that the nucleotide analog that plays a role as a functional group in the modified primer provided in step a) is selected from the group consisting of 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-ethenoadenine, hypoxanthine, deoxy-8-oxo-7,8-dihydroguanine, deoxyuridine, and deoxyinosine.

8. Step b) is carried out by providing natural nucleotides (N) a, t, g, c and one type of nucleotide analog (A), where the molar ratio of N to A is 150:1 to 25:

1. Step c) is performed by excision of the A nucleotide by an endonuclease, Here, the nucleotide analog that plays a role as a functional group in the modified primer provided in step a) is different from the nucleotide analog provided in step b). The method according to claim 1 or 2, characterized in that

9. The method according to claim 1 or 2, characterized in that the nucleotide analog that plays a role as a functional group in the modified primer provided in step a) is 8-oxo-7,8-dihydroguanine, and the nucleotide analog provided in step b) is deoxyuridine.

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