WO2016135300A1

WO2016135300A1 - Efficiency improving methods for gene library generation

Info

Publication number: WO2016135300A1
Application number: PCT/EP2016/054103
Authority: WO
Inventors: Katja HEITZ; Nan Fang
Original assignee: Qiagen Gmbh
Priority date: 2015-02-26
Filing date: 2016-02-26
Publication date: 2016-09-01

Abstract

The present invention provides new methods and kits to improve the efficiency of next generation sequencing (NGS) library construction. In particular, methods and kits are provided, where multiple subsequent enzymatic steps are performed in the same reaction container, and where one or several enzymes of one step is inactivated before the subsequent step is initiated. We have demonstrated that adding specific enzyme inhibitors to the reaction tube improves the reduction of undesirable enzyme activity from a preceding enzymatic step and improves the efficiency of the subsequent reaction, thereby increasing library yield and specificity when compared to available inactivation techniques.

Description

EFFICIENCY IMPROVING METHODS FOR GENE LIBRARY GENERATION

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Next-generation sequencing (NGS), also known as high-throughput sequencing allows to acquire genome-wide data using highly parallel sequencing approaches for molecular biology applications, in vitro clinical diagnostics, or for forensics. Such applications include e.g. de novo genome sequencing, DNA sequencing, transcriptome sequencing and epigenomics, as well as genetic screening for the identification of rare genetic variants and for efficient detection of either inherited or somatic mutations in cancer genes.

Hence, several sequencing platforms have been developed, which allow for low-cost, high-throughput sequencing. Such platforms include lllumina® (Solexa), GS FLX by Roche 454, Ion torrent: Proton / PGM by Life Technologies, and SOLiD™. NGS technologies, NGS platforms and common applications/fields for NGS technologies are e.g. reviewed in Voelkerding et al. (Clinical Chemistry 55:4 641-658, 2009) and Metzker (Nature Reviews/Genetics Volume 11 , January 2010, pages 31-46). Three main steps exist in NGS on most current platforms: preparation of the sample for high-throughput sequencing, immobilization on a suitable surface, and the actual sequencing. The preparation step involves random fragmentation of the genomic DNA and addition of adapter sequences to the fragment ends. The commonly used method to generate platform-specific NGS libraries uses multi-step enzymatic reaction protocols to ligate adapters to the DNA fragments to be analyzed.

First, DNA fragments are generated with mechanical, chemical, or enzymatic

fragmentation or by target-specific PCR. Subsequently, the DNA fragments are end- repaired. The end-repair step requires at least two enzymes: (a) a polynucleotide kinase, normally the T4 Polynucleotide Kinase (PNK) that phosphorylates the 5'-terminus of the double stranded DNA fragments; and (b) an enzyme or enzymes with polymerase and exonuclease activities that make the ends of the DNA fragments blunt by either fill-in or trimming reactions, such as e.g. T4 DNA Polymerase. After the end-repair step, for sequencing on platforms, such as those provided by lllumina®, a so-called A-addition step is required, which generates a terminal adenine as a docking site for the

sequencing adapters that have an overhang formed by thymidine nucleotides, i.e. a T- overhang. In this step, an A-overhang is added to the 3'-terminus of the end-repaired PCR product, e.g. by Klenow Fragment exo-, the large fragment of the DNA polymerase I having 5'- 3' polymerase activity, but lacking both 3'- 5' exonuclease activity and 5'- 3' exonuclease activity. Alternatively, the A-addition step can also be facilitated with enzymes having terminal nucleotide transferase activity, such as the Taq polymerase. Following the A-addition step, the sequencing adapter can be ligated to the DNA by a ligase, such as the T4 DNA Ligase. For other sequencing platforms, such as Ion Torrent PGM/ Proton by Life Technologies®, the A-addition step is not required and blunt-ended adapters are ligated by a T4 ligase directly to the end-repaired DNA fragments.

Following each enzymatic reaction step, either reaction clean-up or heat-inactivation can be used to remove or inactivate the enzymes so that they do not interfere with subsequent reaction steps.

The methods using reaction clean-up are tedious, time-consuming, and cause a loss of DNA material. Compared to the clean-up method, the method using heat-inactivation has the advantage of saving both time and handling steps. Additionally, it is therefore more suited for analyzing small amounts of input DNA, for example lower input amount than 1 ng. However, if the enzymes are not completely inactivated through heating, they potentially post negative impact on a/the subsequent reaction step/s and hence the total library construction efficiency. In particular, the low efficiency can be a draw-back if a sequencing library needs to be constructed from a small amount of input DNA. Thus, there is a need in the art for simpler sample preparation methods for a NGS library protocol generation, especially when small amounts of DNA are to be analyzed.

SUMMARY OF THE INVENTION

The present invention relates to methods and kits for specific inhibition of enzymes involved in the DNA preparation for NGS library construction protocols. These specific inhibitors are preferably directed to enzymes of preceding reaction steps in a multi-step sequencing library construction protocol without, however, affecting the enzymatic activity of the enzyme used in a present reaction step or any subsequent step of a protocol. For example, such enzyme inhibitors may be used for specific inhibition of end- repair enzymes (e.g. polynucleotide kinase, such as the T4 Polynucleotide Kinase or T4 DNA Polymerase) in an A-addition step. Alternatively, they may be used for specific inhibition of A-addition enzymes (e.g. Taq polymerase or Klenow Fragment (3^'→5^' exo-) in a ligation step. Suitable inhibitors include, but are not restricted to chemical compounds and antibodies that inhibit said enzymes competitively, allosterically, or both. By using such specific inhibitors, a significant increase of the library yield is achieved. Hence, the present methods and kits are particularly suited for generating libraries with low-input DNA samples, which are as low as 1 pg or higher, preferably 10pg or higher.

In particular, the invention provides a method of generating a sequencing library, wherein the method comprises the steps of:

(i) providing DNA fragments; (ii) end-repairing the DNA fragments by a polynucleotide kinase enzyme and an enzyme with polymerase and exonuclease activities to obtain blunt-ended, 5' phosphorylated DNA fragments;

(iii) optionally adding a terminal adenine to the end of the end-repaired DNA fragments by a deoxynucleotidyl transferase enzyme; and

(iv) ligating the DNA fragments, optionally having the terminal adenine base, with sequencing adaptors by a DNA ligase;

whereby after completion of step (ii) and/or the optional step (iii) the enzyme or enzymes used in that/those step/s is/are inactivated by the addition of (a) specific inhibitor(s).

None of the inhibitors of the above method inhibits the enzyme activity of a/the subsequent step(s).

In some embodiments, the steps of the above method, which comprise enzyme inactivation by a specific inhibitor, do not comprise heat-inactivation of said enzyme(s). In some embodiments, the steps of the above method, which comprise enzyme inactivation by a specific inhibitor, do not comprise a subsequent purification step from said enzyme(s). In some alternative embodiments, the steps of the above method, which comprise addition of (a) specific inhibitor(s), comprise an upstream heat-inactivation of said enzyme(s), but do not comprise a purification step from said enzyme(s).

In some embodiments, the above method may further comprise step (v), wherein the ligated fragments of step (iv) are purified and size-selected for sequencing. These fragments may be amplified prior to sequencing.

In some embodiments, the polynucleotide kinase enzyme of step (ii) is the T4

Polynucleotide Kinase (PNK) and the enzyme with polymerase and exonuclease activities is the T4 DNA Polymerase; the deoxynucleotidyl transferase enzyme of step (iii) is a Taq polymerase or a Klenow Fragment exo-; and/or the DNA ligase enzyme of step (iv) is a T4 ligase. In some embodiments, the method further comprises DNA library sequencing on a sequencing platform selected from lllumina®, GS-FLX Roche 454, SOLiD™, and Ion torrent: Proton / PGM sequencing. Another aspect of the invention refers to a kit for generating a multi-step sequencing library, wherein the kit comprises:

(i) a polynucleotide kinase and a DNA polymerase for catalyzing the end-repair reaction;

(ii) optionally a deoxynucleotidyl transferase for catalyzing the terminal adenine addition;

(iii) a DNA ligase for ligating the DNA fragments and the adaptors; and

one or more of the following:

a specific polynucleotide kinase inhibitor and a specific DNA polymerase inhibitor; an inhibitor of the exonuclease activity of the enzyme with polymerase and exonuclease activities; optionally a deoxynucleotidyl transferase-specific inhibitor; and a specific DNA ligase inhibitor.

In some embodiments, the polynucleotide kinase enzyme of the above kit is the T4 Polynucleotide Kinase (PNK) and the enzyme with polymerase and exonuclease activity is the T4 DNA Polymerase; the deoxynucleotidyl transferase enzyme is a Taq polymerase or a Klenow Fragment exo-; and/or the DNA ligase enzyme is a T4 DNA ligase.

In any of the above methods or the above kit, the inhibitor is an organic molecule or an antibody. None of the inhibitors of the above kits inhibits the enzyme activity of a/the subsequent step(s).

In some embodiments, the enzyme selected for catalyzing the terminal adenine addition in the kits and methods referred to above is a thermostable polymerase, preferably a thermostable Taq polymerase. In preferred embodiments, said enzyme is inhibited by a Taq-specific inhibitor, wherein the inhibitor of Taq polymerase is a Taq antibody. BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 :

Absorption spectra of the library yield for a sample that was generated using a modified NGS library construction protocol comprising a Taq antibody (blue line), which was added before the ligation step, and a sample without a Taq antibody before initiating the ligation step (control, red line). Said antibody is used for the inactivation of Taq polymerase^'s enzyme activity, whereas in the sample without the Taq antibody, only heat inactivation of the Taq polymerase was applied. All other parameters and conditions for library generation were identical.

FIGURE 2:

Figure 2 illustrates corresponding qPCR results of the samples of Figure 1.

DETAILED DESCRIPTION OF THE INVENTION Definitions The terms "next generation sequencing" and "high-throughput sequencing" are used as synonyms.

The term "library" refers to a large number of nucleic acid fragments, here the collection of DNA fragments for sequencing analysis. The libraries referred to herein are generated by fragmentation of a sample to be analyzed, end-repairing, optionally addition of a terminal adenine, and ligation of fragments into adaptors. Optionally, the purified DNA fragments are amplified or enriched before they are sequenced.

The term "inhibitor" refers to a substance that decreases the rate of, or prevents, an enzymatic reaction. Inhibitors of the current invention refer to antibodies, fragment antigen binding (Fab) fragments or chemical compounds, which specifically inhibit the target enzyme^'s activity by binding to the enzyme^'s active site or by binding allosterically to the enzyme. Said inhibitors include, but are not restricted to the following: specific T4 polynucleotide kinase inhibitors, specific T4 DNA Polymerase inhibitors of its exonuclease activity, specific Taq polymerase activity inhibitors, specific Klenow

Fragment (3^'→5^' exo-) inhibitors, and specific T4-ligase inhibitors. Said inhibitors may be competitive, allosteric, or both. A "specific inhibitor" refers to any of the above inhibitors, which inhibit the activity of the enzyme or enzymes from one step, without affecting the activity of any enzyme of any subsequent step, preferably the next step.

As used herein, the term "antibody" refers to an immunoglobulin, which may be derived from natural sources or which may be synthetically produced. The terms "antibody" and "immunoglobulin" are used synonymously throughout the document, unless indicated otherwise.

An "antibody fragment" is a portion of a whole antibody which retains the ability to exhibit antigen binding activity, e.g. a Fab fragment.

The term "restriction endonuclease" is used herein in its commonly accepted sense as a site specific endodeoxyribonuclease and isoschizomers thereof. Restriction

endonucleases are well-known compounds as is the method of their preparation; see for example Roberts, Critical Reviews in Biochemistry, November 1976, pages 123-164. Representative restriction endonucleases which may be employed in the method of the invention include, but are not restricted to: Alu I, Ava I, Ava II, Bal I, Bam HI, Bel I, Bgl I, Bst E II, Eco R I, Hae II, Hae III, Hinc II, Hind II, Hind III, Hinf I, Hha I, Hpa I, Hpa II, Hph I, Hin 389I, Kpn II, Pst I, Rru I, Sau 3A, Sal I, Sma I, Sst I, Sst II, Tac I, Taq I, Xba I, Xho I and the like, many of which are commercially available (e.g. NEB, Promega, Life Technologies, and Thermo Scientific). Other restriction endonucleases which may be employed and their preparation are listed in Roberts, pages 127-130.

The term "median fragment size" means that half of the fragments have a longer length and half of the fragments have a shorter length.

As used herein, the term "about" when used together with a numerical value (e.g., a percentage value) is intended to encompass a deviation of 20%, preferably 10%, more preferably 5%, even more preferably of 2%, and most preferably of 1 % from that value. When used together with a numerical value it is at the same time to be understood as individually disclosing that exact numerical value as a preferred embodiment in accordance with the present invention. The terms "bp" and "bps" are abbreviations of nucleotide base pairs.

"T4 Polynucleotide Kinase" refers to an enzyme that catalyzes the transfer and exchange of P, from the γ position of ATP to the 5^'-hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3^'-monophosphates.

"T4 DNA Polymerase" refers to an enzyme that catalyzes the synthesis of DNA in the 5^'→3^' direction and requires the presence of template and primer. This enzyme has a 3^'→5^' exonuclease activity which is much more active than that found in DNA

Polymerase I (E. coli). T4 DNA Polymerase does not exhibit 5^'→3^' exonuclease activity.

"Klenow fragment exo-" or "Klenow fragment (3^'→5^' exo-)" refers to an N-terminal truncation of DNA Polymerase I, which retains polymerase activity, but has lost the 5^'→3^' exonuclease activity and the 3^'→5^' exonuclease activity.

"Taq polymerase" refers to a highly thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus. The enzyme catalyzes 5'→3' synthesis of DNA, has no detectable 3'→5' exonuclease (proofreading) activity and possesses low 5'→3' exonuclease activity. In addition, Taq DNA Polymerase exhibits deoxynucleotidyl transferase activity, which is often applied in the addition of additional adenines at the 3'- end of PCR products to generate 3^' adenine overhangs.

The terms "deoxynucleotidyl transfer", "terminal nucleotide addition" and "terminal nucleotide transfer" are used herein as synonyms.

"T4 DNA Ligase" refers to an enzyme that catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA or RNA. This enzyme joins both blunt end and cohesive (sticky) ends. The term "small organic molecule" refers to a chemical compound that has a low molecular weight of about 1000 daltons (Da). Small organic molecules are used for high- throughput screening analysis of the gene function, protein interaction, cellular processing, biochemical pathways, or other chemical interactions, such as the inhibition of the enzymes that are used for the generation of next generation sequencing libraries.

The term "PCR" refers to polymerase chain reaction, which is a standard method in molecular biology for DNA amplification. The term "qPCR" refers to quantitative real-time PCR, a method used to amplify and simultaneously detect the amount of amplified target DNA molecule fragments. The process involves PCR to amplify one or more specific sequences in a DNA sample. At the same time, a detectable probe, typically a fluorescent probe, is included in the reaction mixture to provide real-time quantification. Two commonly used fluorescent probes for quantification of real-time PCR products are: (1 ) non-sequence-specific fluorescent dyes (e.g., SYBR® Green) that intercalate into double-stranded DNA molecules in a sequence non-specific manner, and (2) sequence-specific DNA probes (e.g., oligonucleotides labeled with fluorescent reporters) that permit detection only after hybridization with the DNA targets or after incorporation into PCR products.

The term "DNA" in the present invention relates to any one of viral DNA, prokaryotic DNA, archaeal DNA, and eukaryotic DNA. The DNA may also be obtained from any one of viral RNA, and mRNA from prokaryotes, archaea, and eukaryotes by generating complementary DNA (cDNA) by using a reverse transcriptase.

Methods

The present invention refers to methods of generating multi-step sequencing libraries, which comprise multiple enzymatic steps. In particular, the method referred herein is characterized in that after completion of one step, the enzyme of this step is inactivated with an inhibitor, before the following step is initiated. In preferred embodiments, the inactivation by an inhibitor is optionally combined with heat-inactivation, whereby the heat-inactivation is preferably carried out before inactivation by the respective enzyme inhibitor. In some embodiments, no additional inactivation and/or purification steps are required before proceeding to the following step. Said inhibitor is specific to an enzyme of one step only and it does not adversely affect subsequent steps as the inhibitor does not affect the activity of any of the enzymes of the subsequent protocol steps. In summary, by using the methods referred to herein below, a more rapid and efficient preparation of NGS libraries is achieved. Said multi-step sequencing protocols may involve one, two, or more steps, which involve addition of a specific inhibitor.

For DNA analysis by NGS sequencing methods, first, DNA fragments are generated in the suitable size range with mechanical, chemical, enzymatic methods, or by PCR-based target enrichment. In this context, the DNA fragments provided in the methods of this invention originate from DNA samples. Alternatively, the DNA fragments may derive from mRNA, from which complementary DNA (cDNA) is generated by using the reverse transcriptase enzyme in protocols known to the skilled person.

In one embodiment, the invention provides a method of generating a sequencing library, wherein the library comprises the steps of:

(i) providing DNA fragments by any of the above DNA fragment generation methods;

(ii) end-repairing the DNA fragments by a polynucleotide kinase enzyme and an enzyme with polymerase and exonuclease activities to obtain blunt-ended, 5' phosphorylated

DNA fragments;

(iv) ligating the DNA fragments by a DNA ligase, optionally having the terminal adenine base, with sequencing adaptors;

whereby after completion of step (ii) and/or the optional step (iii), the enzyme or enzymes used in that/those steps is/are inactivated by the addition of (a) specific inhibitor(s). Most notably, none of the above inhibitors impedes the activity of any one of the enzymes used in any one of the subsequent steps. In preferred embodiments, said enzyme(s) do(es) not impede the activity of the enzyme or enzymes of a next step. In some embodiments, the steps of the above method, which comprise enzyme inactivation by a specific inhibitor, do not comprise heat-inactivation of said enzyme(s). In some embodiments, the steps of the above method, which comprise enzyme inactivation by a specific inhibitor, do not comprise a subsequent purification step from said enzyme(s).

Alternatively, in some embodiments, the steps of the above method, which comprise addition of (a) specific inhibitor(s), comprise an upstream heat-inactivation of said enzyme(s), but do not comprise a purification step from said enzyme(s), whereby the inhibition by addition of (a) specific inhibitor(s) inactivates the residual enzyme activity after the heat-inactivation step.

This is dual-inactivation approach is particularly applicable in cases, where the heat- inactivation of (an) enzyme(s) requires long heat-inactivation times at high temperatures, and where less time-consuming methods are desired. For example, the Taq polymerase requires inactivation for 40 minutes at 95°C.

In some embodiments, the method may further comprise step (v), wherein step (v) comprises purification and size-selection of the ligated fragments of step (iv). The ligated fragments may be amplified prior to sequencing. The purification and size selection may be carried out by a suitable kit, such as the GeneRead™ Size Selection Kit. The library fragments are subsequently sequenced by using sequencing platforms known to the person skilled in the art, such as lllumina® (Solexa), GS FLX by Roche 454, Ion torrent: Proton / PGM by Life Technologies, and SOLiD™. The size of the DNA fragment length is a key factor for library construction and for sequencing. Typical median lengths of DNA fragments for NGS libraries are between about 150 bps and about 1000 bps, preferably between about 150 bps and about 600 bps, more preferably between about 200 bps and about 500 bps. Most preferably, the median length is about 200 bps, about 300 bps, or about 500 bps.

The preferred amount of DNA starting material for generating a NGS sequencing library and for subsequent sequence analysis ranges from about 1 pg to about 1 μg, preferably from about 10 pg to about 1 μg, and more preferably 10 pg-1 ng. For genomic DNA analysis, the amount of starting material is preferably about 1 pg-1 μg, preferably from about 10 pg to about ^g, and more preferably 10 pg- 1 ng.

In some embodiments, the fragmentation step is mechanical. Preferably, the mechanical fragmentation is among others achieved by ultrasonic acoustic shearing, nebulization forces, sonication, hydrodynamic shearing (e.g. in French pressure cells or by needle shearing). More preferably, specific median fragment length sizes of DNA can be prepared e.g. by ultrasonic acoustic shearing, such as Adaptive Focused Acoustics (AFA)™ by using a Covaris® instrument, according to the manufacturer's instructions.

In some embodiments, the fragmentation of DNA step is chemical. Chemical shear may also be employed for the breakup of long RNA fragments. This is typically performed through heat digestion of RNA with a divalent metal cation (magnesium or zinc). The length of the RNA (1 15 bp-350 bp) can be adjusted by increasing or decreasing the time of incubation.

In some embodiments, the fragmentation step is enzymatic. Preferably, said enzymatic fragmentation is achieved by digestion of DNA by an endonuclease. Such

endonucleases are described in more detail in the Definitions section. Preferably, the fragmentation may also be carried out by employing a transposase known to the person skilled in the art. When applying enzymes for the fragmentation reaction, said fragmentation step may be inactivated by heat. Alternatively, the fragmentation may be inactivated by an inhibitor, which specifically inhibits the activity of the enzyme used for fragmentation. Such inhibitors include a specific antibody or a specific organic molecule, preferably a small organic molecule. Preferably, said inhibitor does not affect the activity of any one of the subsequent library generation steps, preferably the enzyme(s) of the next step, i.e. the enzymes of the end-repairing step. In preferred embodiments, the steps (ii)-(iv) of the library generation method are carried out in a single reaction container, such as a reaction tube. In some embodiments, after completion of step (ii) or (iii) and before initiating the subsequent step, respectively, an enzyme-specific antibody or an organic molecule, preferably a small organic molecule, inhibitor, is applied to the reaction container for inactivating the enzyme of step (ii) or step (iii), respectively. In other embodiments, such an inhibition is optionally preceded by a heat-inactivation, whereby the enzyme inhibitor inactivates residual enzyme activity after heat inactivation, before the subsequent step is initiated. In steps that do not comprise an inactivation step by a specific inhibitor, such as a specific antibody or a specific organic molecule, preferably a small organic molecule, inhibitor, the enzyme or enzymes of such a step is/are heat-inactivated or a subsequent purification removes said enzyme or enzymes. An inactivation step by heat comprises an at least 10-minute to an at least 20-minute period of an elevated temperature of 65°C or higher, depending on the respective enzyme^'s thermal stability and e.g. the buffer conditions. For example, the enzymes in a GeneRead™ DNA Library Prep I Kit, the T4 Polynucleotide Kinase (PNK) and the T4 DNA Polymerase are inactivated for 20 minutes at 75°C. The Klenow fragment exo- of the kit is inactivated at 75°C for 10 minutes. Taq polymerase is a highly thermostable enzyme, its enzyme activity having a half-life of about 40 minutes at 95°C.

Step (ii), the end-repair step, is carried out by an enzyme or two enzymes with (a) polynucleotide kinase activity (PNK) and (b) an enzyme with polymerase and

exonuclease activities, whereby the exonuclease activity makes the ends of the DNA blunt by fill-in or trimming reactions. Preferably, the enzymes of step (ii) comprise a T4 Polynucleotide Kinase (PNK) and a T4 DNA Polymerase.

T4 Polynucleotide Kinase (PNK) may be inhibited by specific antibodies or by specific organic molecules, preferably small organic molecules, e.g. described in WO

2010/139069 A1.

T4 DNA Polymerase inhibitors include, but are not restricted to antibodies or organic molecules, preferably small organic molecules, e.g. butylphenyl nucleotides, such as N2- (p-n-butylphenyl)dGTP (BuPdGTP), aphidicolin and pyrophosphate analogs.

Step (iii), the A-addition step, is carried out by an enzyme, which generates an adenine docking site for adapters that have a thymidine overhang (T-overhang). Preferably, the enzyme of step (iii) is a Taq polymerase or Klenow Fragment exo-, the large fragment of the DNA polymerase I having 5'- 3' polymerase activity but lacking both 3'- 5' exonuclease activity and 5'- 3' exonuclease activity. In some preferred embodiments, the enzyme of step (iii) is a thermostable polymerase, preferably a Taq polymerase.

In some embodiments, the Klenow fragment exo- is inhibited by an organic molecule, preferably a small organic molecule, or an antibody. Organic molecules inhibiting Klenow Fragment exo- include, but are not restricted to 5R-5,6-dihydro-5-hydroxythymidine and structural analogues, such as 5,6-dihydro-5-methylthymidine.

Specific inhibitors of Taq polymerase include, but are not restricted to antibodies, and organic molecules, preferably, small organic molecules, which inhibit the Taq activity of the terminal nucleotide, such as terminal adenine, transfer. In some embodiments, said inhibitors inhibit the terminal nucleotide transfer and the polymerase activity of a Taq polymerase. Antibodies that are known to the person skilled in the art to inhibit the Taq polymerase in hot start PCR reactions include, but are not restricted to e.g. Platinum® Taq Antibody (Life Technologies), JumpStart™ Taq Antibody (Sigma-Aldrich), and QIAGEN proprietary Taq antibody. In a preferred embodiment, the Taq inhibitor is QIAGEN proprietary Taq antibody. The concentration range of such an antibody inhibitor is about 0.001 to about 100 μg/μL, preferably about 0.005 to about 50 μg/μL, more preferably, about 0.005 to about 50 μg/μL, and most preferably about 0.01 to about 10 μg/μL.

Suitable organic molecules include, but are not restricted to, anionic polymers such as HotMaster Inhibitor (as in HotMaster Taq, 5 PRIME GmbH), 2-amino-5-(2'-deoxy-3-d- ribofuranosyl) pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy-3-d-ribofuranosyl)-3- methyl-2-pyridone-5'-triphosphate (d*TTP) (Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group, Mao-Jun Guo and Michael J. Waring, Nucleic Acids Research, Volume 26, Issue 8, p. 1863-1869), or aptamer (see, for example, Patent Nos. US 5,475,096, US 5,670,637, US 5,696,249, US 5,874,557, and US 5,693,502).

Step (iv), the ligation step, joins either blunt or cohesive (sticky) ends of DNA fragments with either blunt or cohesive (sticky) ends of adapter molecules. Successful ligation of cohesive (sticky) ends requires complementary sequences. In preferred embodiments, a fragment comprising terminal, i.e. 3' adenine overhangs serves as a docking site for the sequencing adapters, which comprise a complementary terminal, i.e. 3' thymidine overhang. By using such TA cloning, it is not necessary to design a specific pair of primers for each DNA fragment to be analyzed. The same primers can be used for amplification of different templates provided that each template is modified by addition of the same universal primer-binding sequences to its 5' and 3' ends. The adapter sequence can therefore be any DNA fragment of interest, as long as it has a 3' thymidine overhang.

Preferably, the enzyme of step (iv) is a T4 DNA ligase or an E. coli DNA ligase, whereby the E. coli DNA ligase only ligates cohesive (sticky) DNA. Therefore, more preferably, step (iv) comprises T4 DNA ligase.

The ligation step is carried out at 4-37°C, preferably 4-25°C, depending on the optimal temperature for the ligase ^'s activity.

After the generation of the ligated fragments, said fragments are purified and size- selected on e.g. silicon containing surface of a binding matrix in the presence of a salt, preferably a chaotropic salt. The size of DNA molecules that bind to the binding matrix can be controlled e.g. by the salt concentration or the pH value of the binding mixture. Such purification is e.g. described in WO 2014/122288 A1. Suitable columns applying such a size selection method include the GeneRead™ Size Selection Kit. A further DNA size selection method includes agarose gel electrophoresis. The purified fragments may be used directly for subsequent sequencing. Alternatively, prior to the sequencing step, the purified fragments may be amplified for library enrichment by PCR-based methods known to the person skilled in the art, or by capture-by-hybridization, i.e. on-array or in- solution hybrid capture; or by capture-by-circularization, i.e. molecular inversion probe- based methods. Preferably, library enrichment is carried out by PCR amplification.

Typically, the amplification is followed by the quantification. Alternatively, the

amplification and quantification may be performed simultaneously.

Furthermore, in order to obtain optimal sequencing results, the amount of the amplified or non-amplified DNA fragments is preferably determined. If the amount of the DNA on a platform is too high, DNA fragment clusters will overlap and therefore, adversely affect the sequencing data.

Quantification of the libraries may be carried out by using quantitative real-time PCR methods. qPCR for the rapid quantification of DNA libraries has been demonstrated inter alia by Buehler (Buehler et al., Methods 50 (2010), S15-S18). As shown in this publication, qPCR can provide accurate quantitative measurements of DNA libraries. In addition, as only those fragments containing the next-generation library adapters are amplified, qPCR can minimize overestimation of the DNA concentrations in such libraries - fragments with no or only one adapter will not be amplified. For optical readout, said method may employ non-specific fluorophores or hybridization probes, e.g. by using GeneRead™ Library Quant Kit.

In preferred embodiments, said methods comprise an antibody, more preferably a Taq antibody, which allows for a rapid inactivation of Taq polymerase, whereas heat- inactivation requires long inactivation steps at high temperatures, which are not favorable to the standard library construction procedures: the Taq polymerase is a highly thermostable enzyme. Its enzyme activity has a half-life of about 40 minutes at 95°C. After the NGS library generation, said library is sequenced on a sequencing platform. Suitable sequencing platforms include, but are not restricted to lllumina®, Roche 454 SOLiD™, and Ion torrent: Proton / PGM sequencing by Life Technologies.

Kits

The present invention also provides kits for performing the methods of this invention. Such kits comprise enzymes and inhibitors used in the steps (ii-iv) of the above described method.

Preferably, such kits include:

(i) a polynucleotide kinase and a DNA polymerase with polymerase and exonuclease activities for catalyzing the end-repair reaction;

(ii) optionally a deoxynucleotidyl transferase for catalyzing the terminal adenine addition; (iii) a DNA ligase for ligating the DNA fragments and the adaptors; and one or more of the following:

a specific polynucleotide kinase inhibitor and a specific DNA polymerase inhibitor; optionally a deoxynucleotidyl transferase-specific inhibitor; and a specific DNA ligase inhibitor.

In more preferred embodiments, the polynucleotide kinase enzyme is the T4

Polynucleotide Kinase (PNK) and the enzyme with polymerase and exonuclease activity is the T4 DNA Polymerase; the deoxynucleotidyl transferase enzyme is a Taq polymerase or a Klenow Fragment exo-; and/or the DNA ligase enzyme is a T4 ligase.

In more preferred embodiments, the inhibitors referred to above include specific antibodies or organic molecules, preferably, small organic molecules. Such specific antibodies include antibodies directed against the T4 Polynucleotide Kinase, T4 DNA Polymerase, Klenow Fragment exo-, Taq Polymerase, or the T4 DNA Ligase. None of the inhibitors inhibits the enzyme activity of (a) subsequent step(s).

T4 Polynucleotide Kinase (PNK) may be inhibited by specific antibodies or by specific small organic molecules, e.g. described in WO 2010/139069 A1 .

T4 DNA Polymerase inhibitors include, but are not restricted to antibodies or small organic molecules, e.g. butylphenyl nucleotides, such as N2-(p-n-butylphenyl)dGTP (BuPdGTP), aphidicolin and pyrophosphate analogs.

In some embodiments, the Klenow fragment exo- is inhibited by a small organic molecule or an antibody. Small organic molecules inhibiting Klenow Fragment exo- include, but are not restricted to 5R-5,6-dihydro-5-hydroxythymidine and structural analogues, such as 5,6-dihydro-5-methylthymidine. Specific inhibitors of Taq polymerase include, but are not restricted to antibodies, and organic molecules, preferably small organic molecules, which inhibit the Taq activity of the terminal nucleotide, such as a terminal adenine, transfer,. In some embodiments, said inhibitors inhibit the terminal nucleotide transfer and the polymerase activity of a Taq polymerase. Antibodies that are known to the person skilled in the art to inhibit the Taq polymerase in hot start PCR reactions include, but are not restricted to e.g.

Platinum® Taq Antibody (Life Technologies), JumpStart™ Taq Antibody (Sigma- Aldrich), and QIAGEN proprietary Taq antibody. In a preferred embodiment, the Taq inhibitor is QIAGEN proprietary Taq antibody. Such Taq antibodies inhibit the Taq polymerase^'s terminal nucleotide transfer activity, or the Taq polymerase^'s terminal nucleotide transfer activity and polymerase activity. The concentration range of such an antibody inhibitor is about 0.001 to about 100 μg/μL, preferably about 0.005 to about 50 μg/μL, more preferably, about 0.005 to about 50 μg/μL, and most preferably about 0.01 to about 10 μg/μL.

Suitable organic molecules include, but are not restricted to, anionic polymers such as HotMaster Inhibitor (as in HotMaster Taq, 5 PRIME GmbH), 2-amino-5-(2'-deoxy-3-d- ribofuranosyl) pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy-3-d-ribofuranosyl)-3- methyl-2-pyridone-5'-triphosphate (d*TTP) (Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group, Mao-Jun Guo and Michael J. Waring, Nucleic Acids Research, Volume 26, Issue 8, p. 1863-1869)., or aptamer (see, for example, Patent Nos. US 5,475,096, US 5,670,637, US 5,696,249, US 5,874,557, and US 5,693,502).

In preferred embodiments, the terminal adenine addition is catalyzed by a thermostable polymerase, preferably a Taq polymerase. Preferably, said inhibitor is an antibody, more preferably, a Taq inhibiting antibody, and even more preferably, an antibody that specifically inhibits both the polymerase and terminal transferase activities of Taq. EXAMPLES gDNA from Ecoli DH10B is sheared to an average fragment size of 300 bp (Covaris S220 Focused-ultrasonicator, Covaris), and 10 pg of sheared DNA is used for each library construction test. GeneRead™ DNA Library Prep I Core Kit, GeneRead™ DNA I Amp Kit, GeneRead™ Adapter I Set 12-Plex (72), and GeneRead™ Size Selection Kit (all from QIAGEN) are used according to manufacturer's instructions with the following modifications: 0.5 U of the Taq polymerase (QIAGEN) and 0.5 mM of dATP (QIAGEN) are added to the end-repair reaction; the temperature profile for end-repair reaction is 30 minutes at 25°C, and 30 minutes at 72°C, where the 72°C step was used to both inactivate end-repair enzymes and utilize the terminal transferase activity of the Taq enzyme to add an adenine to the 3' of the DNA fragments. The separate A-addition step using Klenow fragment (3'- 5' exo-) is therefore removed from the protocol.

0.05 μΜ of sequencing adaptor was used in the ligation steps at 25 °C. Before the ligation step, 0.25μg/μl of Taq antibody (Propriety of QIAGEN) was added. Following the ligation steps, the library was first purified with the GeneRead™ Size Selection Kit (QIAGEN), amplified for 22 cycles with adaptor-specific primers by PCR using the GeneRead™ DNA I Amp Kit( QIAGEN) and purified again with GeneRead™ Size Selection Kit (QIAGEN). For determining the amount of amplified DNA, the purified fragments were analyzed by qPCR with the Standards and Primers from GeneRead Library Quantification Kit (QIAGEN) in combination with QuantiFast Sybr Green PCR Mix (QIAGEN) following manufacturer's instructions.

Example 1 :

The above amplified product of the test and control samples was qualitatively analyzed by using Agilent Bioanalyzer and High Sensitivity DNA Analysis Kit (Agilent). Example 2:

The above amplified product of the test and control samples was quantitatively analyzed by using qPCR method (QuantiFast Sybr Green Kit, QIAGEN).

Claims

1. A method of generating a sequencing library, wherein the method comprises the steps of:

(i) providing DNA fragments;

(ii) end-repairing the DNA fragments by a polynucleotide kinase enzyme and an enzyme with polymerase and exonuclease activities to obtain end-repaired DNA fragments;

whereby after completion of step (ii) and/or the optional step (iii), the enzyme or enzymes used in that/those steps is/are inactivated by the addition of (a) specific inhibitor(s).

2. The method of claim 1 , wherein the inactivation of one or more enzymes by a/the specific inhibitor(s) is not achieved by purification from said enzyme(s).

3. The method of claim 1 , wherein the inactivation of one or more enzymes by a/the specific inhibitor(s) is not achieved by heat-inactivation of said enzyme(s).

4. The method of claim 1 , wherein the steps of the above method, which comprise inhibition by addition of (a) specific inhibitor(s), comprise an upstream heat- inactivation of said enzyme(s), but do not comprise a purification step from said enzyme(s).

5. The method of claims 1-4, further comprising step (v), wherein the ligated

fragments of step (iv) are purified and size-selected for sequencing.

6. The method of claim 5, wherein the adaptor-ligated fragments are amplified prior to sequencing. The method of claims 5 or 6, wherein the method further comprises DNA library sequencing on a sequencing platform selected from lllumina®, Roche 454, SOLiD™, and Ion Torrent: Proton / PGM sequencing.

A kit for generating a multi-step sequencing library, wherein the kit comprises:

(i) a polynucleotide kinase and an enzyme with polymerase and exonuclease activities for catalyzing the end-repair reaction;

(iii) a DNA ligase for ligating the DNA fragments and the adaptors; and one or more of the following:

a specific polynucleotide kinase inhibitor and a specific DNA polymerase inhibitor; an inhibitor of the exonuclease activity of the enzyme with polymerase and exonuclease activities, optionally a deoxynucleotidyl transferase-specific inhibitor; and a specific DNA ligase inhibitor.

The method of any one of claims 1-7, or the kit of claim 8,

wherein the polynucleotide kinase enzyme is the T4 Polynucleotide Kinase (PNK) and the enzyme with polymerase and exonuclease activity is the T4 DNA

Polymerase;

wherein the deoxynucleotidyl transferase enzyme is a Taq polymerase or a

Klenow Fragment exo-; and/or

wherein the DNA ligase enzyme is a T4 ligase.

The method of any one of claims 1-7 and 9, or the kit of any one of claims 8-9, wherein the inhibitor is an organic molecule, aptamer, or an antibody.

The method any one of claims 1 -7 and 9-10, or the kit of any one of claims 8-10, wherein none of the inhibitors inhibits the enzyme activity of an enzyme or enzymes of a/the subsequent step(s).

12. The method of any one of claims 1-7 and 9-1 1 , or the kit of any one of claims 8- 1 1 , wherein the enzyme catalyzing the terminal adenine addition is a

thermostable polymerase.

13. The method of any one of claims 1-7 and 9-12, or the kit of any one of claims 8- 12, wherein the enzyme catalyzing the terminal adenine addition is a thermostable polymerase Taq.

14. The method or the kit of claim 13, wherein the inhibitor is a Taq polymerase antibody.