Methods for testing nucleic acid amplification products

Abstract

The present invention relates to a method for testing a nucleic acid amplification product comprising the steps of: ai) providing an amplification product comprising upstream and downstream primer regions, ii) an amplified target region between the upstream and downstream primer regions, b) testing the amplification product by amplifying the amplification product in a PCR reaction, wherein i) the upstream and downstream PCR primers in the PCR reaction bind in the upstream and downstream primer regions of the amplification product, and ii) first and second oligonucleotide probes are positioned downstream of the first and second PCR primers and within the upstream and downstream primer regions, c) performing a PCR reaction and testing the newly formed amplification product, wherein the upstream and downstream primer regions of step ai) have a length of less than 35 nucleotides and more than 20 nucleotides.

Description

[Background technology]

[0001] background Next generation sequencing techniques (e.g., pyrosequencing, sequencing by synthesis, and sequencing by oligonucleotide ligation and detection) overcome the major limitations of the first generation approach. Sequencing reactions can be performed in parallel with many different samples (templates) immobilized in arrays in the same flow cell. The sample density per unit area can be very high, and the total number of samples can be increased by enlarging the array. The samples can be exposed to a series of sequencing reagents in parallel in a shared fluid volume inside the flow cell. Also, the samples in the array can be monitored with a camera to record the sequencing data from all of the samples in real time as the sequencing reaction proceeds in parallel with the periodic exposure to reagents passing through the flow cell; see WO 2013 / 019751.

[0002] Next-generation technologies currently on the market rely on in vitro libraries with a specific configuration. The various fragments to be sequenced are each flanked by adapters to form library members. The adapters provide primer binding sites for clonal amplification of each library member on a support (e.g., a flat surface or beads). The adapters introduce binding sites that allow amplification of all members of the library with the same primer or a pair of adapter-specific primers. Also, one or both of the adapters may provide binding sites for sequencing primers. In addition, the adapters may introduce library-specific index sequences that allow members of different libraries to be pooled and sequenced together in the same flow cell without losing track of the original library for each member. Library sets can be constructed from different nucleic acid samples in parallel, for example, in different wells of a multi-well plate. However, despite best efforts to achieve uniform reaction conditions between wells, the concentration and quality of the libraries may vary widely. As the sequencing capacity of NGS instrumentation continues to increase, researchers can pool more samples, or libraries, into one sequencing run, greatly reducing the per-sample cost of sequencing. However, NGS library concentrations can vary widely based on the quantity and quality of the input nucleic acid sample, as well as the target enrichment method used. To ensure that each pooled library is sequenced to the desired depth, NGS libraries must be carefully quantified and normalized so that each sample achieves the required number of reads. Common library quantification methods include fluorescence spectrophotometry and quantitative PCR (qPCR). Both methods provide relatively accurate measurements of library concentration, but there are assay-specific considerations associated with these techniques. In this paper, we compare two library quantification techniques: Invitrogen TM Qubit TMUsing the Invitrogen Fluorometer or qPCR, respectively TM Qubit TM dsDNA HS Assay Kit and Invitrogen TM Collibri TM Provide a comparison of Library Quantification Kits. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2013 / 019751 Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention The present disclosure provides methods for characterizing a nucleic acid library by using a digital amplification assay. The present invention therefore relates to a method for testing a nucleic acid amplification product, said method comprising the steps of: a. Below: i. upstream and downstream primer regions, and upstream and downstream primers which bind to said upstream and downstream primer regions, respectively, and optionally and preferably ii. a target region to be amplified between the upstream and downstream primer regions; Providing an amplification product resulting from linear or exponential amplification, b. testing the amplified product by amplifying the amplified product in a PCR reaction, i. the upstream and downstream PCR primers in the PCR reaction bind within the upstream and downstream primer regions of the amplification product; ii. a first and a second oligonucleotide probe are located downstream of the first and the second PCR primers and within the upstream and the downstream primer regions; process, c. performing a PCR reaction and testing the newly formed amplification products, wherein the upstream and downstream primer regions of step ai) have a length of less than 35 nucleotides and more than 20 nucleotides; The present invention relates to a method comprising the steps of:

[0005] Preferably, the amplification is exponential.

[0006] An exemplary method of library characterization is provided. However, the invention can be applied to various types of amplification products. In the method, a nucleic acid library can first be obtained. The library can include members each having a first adapter region and a second adapter region. Characterization of these libraries has been difficult to standardize because the adapters are often of different lengths or very short, making assay development very difficult.

[0007] The testing step is preferably i) quantifying the amount of amplification product, or ii) checking whether said amplification product contains the desired insert, or iii) both.

[0008] At least a subset of the members may have an insert disposed between the first and second adaptor regions. At least a portion of the library may be divided into partitions. A digital assay may be performed with adaptor region probes on the partitions to generate data indicative of whether a library member is present in each partition. A property of the library may be determined based on the data. Another exemplary method of library characterization is provided. In the method, a nucleic acid library may be obtained. The library may include members each having a first constant region and a second constant region. At least a subset of the members may have a variable region disposed between the first and second constant regions. Partitions may be formed that include members of the library upon limiting dilution. Members of the library may be amplified within the partitions using primers for each constant region. Amplification data may be collected from constant region probes within the partitions. A level of the library members may be determined based on the amplification data.

[0009] Library characterization prior to sequencing can be problematic. Only properly formed library members containing both adapters in the correct relative orientation generate a clonal population that can be reliably interrogated by sequencing. Malformed members in the library (e.g., members flanked by two copies of only one of the adapters) can be difficult to distinguish from fully formed ones. However, the malformed members generally cannot be amplified on the support (a prerequisite for sequence acquisition), or do not have a binding site for the sequencing primer, or both. As a result, the malformed members can occupy space, consume reagents, and reduce the amount of useful sequence information generated by a next-generation sequencing run in direct proportion to the proportion of malformed members in the library; see WO 2013 / 019751.

[0010] The method of the present invention has solved these problems. Figure 1 shows the main setup. If the amplification step b) is a digital PCR amplification, it is preferred in the method of the present invention. In this preferred embodiment, the template is subdivided into compartments or droplets. Digital assays can be performed on the compartments with adapter region probes to generate data indicating whether a library member is present in each compartment. The properties of the library or template can be determined based on this data.

[0011] Digital polymerase chain reaction (digital PCR, digital PCR, dPCR, or dePCR) is a biotechnological improvement of the conventional polymerase chain reaction method that can be used to directly quantify and clonally amplify nucleic acid strands, including DNA, cDNA, or RNA. The key difference between dPCR and traditional PCR is in the way the amount of nucleic acid is measured. dPCR is a more precise method than PCR, but is prone to error in the hands of an inexperienced user. A "digital" measurement quantitatively and discontinuously measures a certain variable, whereas an "analog" measurement extrapolates a certain measurement based on the measured pattern. PCR performs one reaction per single sample. dPCR also performs one reaction within a sample, but the sample is separated into many compartments, and the reaction is performed individually within each compartment. This separation allows for more reliable collection and sensitive measurement of the amount of nucleic acid. The above method has been clearly shown to be useful for testing variations in gene sequences, such as copy number variants and point mutations, and is routinely used for clonal amplification of samples for next generation sequencing.

[0012] The present invention is characterized by the fact that the primers are very short when compared to standard primers. Similarly, the probes used are very short when compared to standard probes. This leads to problems with melting temperatures and melting curves.

[0013] Current methods for NGS library quantification are electrophoresis, quantitative real-time PCR, and more recently, next-generation sequencing and digital PCR. Among them, SYBR Green-based assays, which use two primers each specific to target one of the two adapters, are used by most customers. A more recent development is the TaqMan probe-based assay, which uses two primers and two probes, each specific to one of the two ligated adapters in the library. In contrast to SYBR Green-based assays, they are more accurate because non-specific amplicons do not produce a fluorescent signal. The probes of the Taqman probe-based assays currently on the market are intended and only work for the quantification of NGS libraries from a specific preparation kit. This indicates a probe design that targets the variable part of the adapter. Also, it requires the use of multiple assays for the quantification of different Illumina libraries.

[0014] In accordance with a preferred embodiment of the present invention, the method of the present invention overcomes this limitation, where the probes are in the conserved adapter regions P5 and P7. In a further preferred embodiment, this is combined with nanoplate-based digital PCR on the QIAcuity platform.

[0015] Preferably, both probes are designed to target either the same strand or opposite strands, and they preferably have different fluorophores or different labeling systems. Even more preferably, the labeling system comprises a rare earth cryptate or rare earth chelator in combination with a fluorescent or chemiluminescent dye, in particular a cyanine type dye. In the context of the present invention, fluorophores include the use of dyes, such as, for example, FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, fluorescein isothiocyanate (FITC), IRD-700 / 800, cyanine dyes (e.g., CY3, CY5, CY3.5, CY5.5, Cy7), xanthene, 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), TET, 6-carboxy-4',5'-dichloro-2',7'-diamine, 5-carboxy-2',5 ... 6-Carboxy-4',5'-dichloro-2',7'-dimethodyfluorescein (JOE), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine-6G (R6G5), 6-carboxyrhodamine-6G (RG6), rhodamine, rhodamine green, rhodamine red, rhodamine 110, BODIPY dyes (e.g., BODIPY The dye may be selected from the group including: TMR), Oregon Green, coumarins (e.g., umbelliferone), benzimides (e.g., Hoechst 33258); phenanthridines (e.g., Texas Red), Yakima Yellow, Alexa Fluor, PET, ethidium bromide, acridinium dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, and the like.

[0016] Preferably, the oligonucleotide probes of the present invention are hydrolysis probes.

[0017] Hydrolysis probes are a common detection chemistry for monitoring sequence-specific amplification in PCR or digital PCR (dPCR). Just as with SYBR Green dye, signal detection is achieved via monitoring the increase in fluorescence as the reaction proceeds. However, TaqMan TM The fluorescent signal in the chemistry depends on probe hydrolysis rather than hybridization, hence the name "hydrolysis probe." In the hydrolysis probe design, there are two primers and a probe. The probe is also designed to be complementary to the target and contains a fluorophore and a quencher at either end; see also FIG. 1.

[0018] During the amplification process, the probe binds to a specific target sequence during the annealing step. Due to the proximity between the donor (fluorophore) and acceptor (quencher) on the probe, there is no fluorescence. During the extension step, the 5'-3' exonuclease activity of the polymerase hydrolyzes the probe, releasing the fluorophore from the quenching effect, and the fluorescence is read by a detector.

[0019] With this design, one target DNA ligated to both adaptors produces dual fluorescent signals, which can be detected and quantified in digital PCR after end-point amplification.Since the target region can in principle be relatively short, the primers and probes preferably contain LNA to compensate for the reduced binding affinity.

[0020] Thus, for the first time, the present invention provides one assay that can simultaneously quantify different types of libraries in one reaction. The combination of the above assay with the QIAcuity platform and QIAcuity probe master mix chemistry makes it possible to harness the absolute quantification precision and accuracy of digital PCR for NGS library quantification. The above assay design also works in combination with qPCR.

[0021] The present invention also provides a nucleic acid amplification composition, comprising: a. at least two primers having a length between 10 and 22 nucleotides, and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that increase template binding strength; and b. at least two oligonucleotide probes having a length between 8 and 17 nucleotides and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that enhance template binding strength; The present invention relates to a nucleic acid amplification composition comprising:

[0022] The present invention also relates to the use of the compositions of the invention for the analysis of nucleic acid libraries.

[0023] The present invention also provides a. at least two primers having a length between 10 and 22 nucleotides, and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that increase template binding strength; and b. at least two oligonucleotide probes having a length between 8 and 17 nucleotides and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that enhance template binding strength; The present invention relates to a kit comprising:

[0024] Analysis or testing herein refers to determining whether amplification has occurred, determining whether the target sequence is between the primer regions and, if necessary, has the correct length, and determining the amount of amplification product having the correct target sequence.Preferably, in the method of the present invention, accurate quantification of the amplification product is desired.

[0025] As used herein, a "primer region" (or priming region) is a region in a nucleic acid molecule that allows the binding of an oligonucleotide primer. This region is typically between 15 and 40 nucleotides in length, more preferably between 20 and 35 nucleotides in length, and most preferably between 15 and 25 nucleotides in length. The sequence therein is the reverse complement of the sequence of the primer that binds said "primer region". Of course, said sequence must not be a 100% reverse complement. It may differ slightly, provided that the respective primer can bind.

[0026] As used herein, an "amplification product" is either a double-stranded or single-stranded nucleic acid product resulting from a nucleic acid amplification reaction. Such reactions can be isothermal or non-isothermal amplifications. It typically contains sequences derived from one or more primers used to amplify the target nucleic acid. Preferably, as used herein, it is double-stranded and derived from PCR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Invention Depending on the sequencing target and the output amount from different types of sequencing equipment, the library used for sequencing consists of either a single library or a pool of partial libraries. In both cases, accurate quantification is required to achieve optimal cluster density for sequencing run and prevent oversaturation or undersaturation of flow cells. Equimolar pooling of partial libraries before sequencing requires an additional step of accurate quantification.

[0028] The quantity and quality of the prepared NGS DNA library is determined by the use of different methods. These include UV absorption (e.g., Nanodrop); intercalating dyes (e.g., QuBit; Invitrogen, SYBR Green); electrophoresis coupled with intercalating dyes (Agilent Bioanalyzer), 5' hydrolysis probes (e.g., TaqMan®) coupled with real-time quantitative PCR (qPCR; Kapa Biosystem), NGS library quantification (MiSeq) or droplet digital PCR (Bio-Rad). The main drawback of DNA quantification using intercalating dyes is the non-specific binding of the dye to any double-stranded DNA. If there are non-target DNA fragments in the library to which the dye binds, this will result in over-quantification of the target library DNA.

[0029] Digital PCR offers certain advantages over other commonly used DNA quantification strategies (e.g., QuBit and qPCR). With absolute quantification of single molecules, digital PCR uses less input DNA and does not require back-calculation of the library to the average size determined by Bioanalyzer assay. This still provides similar sensitivity and accuracy as qPCR, while dPCR-based quantification consumes less time and reagents. It is also more sensitive compared to quantification using QuBit and PicoGreen. Another advantage of digital PCR is that amplification occurs in separate compartments. Even if amplification efficiency varies from amplicon to amplicon or from extraction to extraction, enough amplicons are generated in a dPCR run to determine whether the target was present or not. Thus, digital PCR provides a binary value for the template of interest (present (even if poorly amplified); or absent), which uses Poisson statistics to correct for the possibility of having one or more template molecules per compartment, revealing the absolute titer of molecules / μl.

[0030] Compared to droplet digital PCR (ddPCR) (as provided by BioRad), dPCR on the QIAcuity system uses 96-well and 24-well nanoplates in a standard format suitable for use in a fully automated workflow. TM The Library Quantification Kit is limited to quantification of Illumina TruSeq libraries. Similarly, Thermo Fisher Scientific's qPCR NGS library quantification assays are limited to individual Illumina library types (e.g., Illumina® Nextera Library Quantification Assay or Illumina® TruSeq DNA / RNA Library Quantification Assay).

[0031] The present invention relates to a method for testing a nucleic acid amplification product, the method comprising: a. Below: i. an upstream and a downstream primer region, and an upstream primer and a downstream primer respectively binding said upstream and downstream primer regions, and optionally and preferably ii. a target region to be amplified between the upstream and downstream primer regions; Providing an amplification product derived from a linear or exponential amplification comprising: b. testing the amplified product by amplifying the amplified product in a PCR reaction, i. the upstream and downstream PCR primers in the PCR reaction bind within the upstream and downstream primer regions of the amplification product; ii. a first and a second oligonucleotide probe are located downstream of the first and the second PCR primers and within the upstream and the downstream primer regions; process, c. performing a PCR reaction, preferably a digital PCR reaction, to test the newly formed amplification product, wherein the upstream and downstream primer regions of step ai) have a length of less than 35 nucleotides and more than 20 nucleotides; The present invention relates to a method comprising the steps of:

[0032] In preferred embodiments, the target region is between 15-35 nucleotides in length, between 16-34 nucleotides in length, between 17-33 nucleotides in length, between 18-32 nucleotides in length, between 19-31 nucleotides in length, between 20-30 nucleotides in length, between 21-29 nucleotides in length, or between about 20-28 nucleotides in length.

[0033] The nucleic acid amplification products to be analyzed according to the present invention may be derived from the amplification of any suitable source of genomic DNA, mitochondrial DNA, chloroplast DNA, cDNA, etc. The fragments (targets) may have any suitable length (e.g., about 10-10,000, or 20-2,000 nucleotides, among others). The fragments may or may not be size-selected prior to attachment to the adaptors (primer regions). The fragments may be generated from the source nucleic acid material by any suitable approach (e.g., shearing, chemical digestion, enzymatic digestion, amplification with one or more primers, reverse transcription, end-polishing, or any combination thereof, among others). The fragments may have blunt or overhanging ends and may be at least predominantly double-stranded or single-stranded. The target nucleic acid may be RNA or DNA. DNA is preferred.

[0034] In a preferred embodiment, the upstream and downstream primer regions are derived from the amplification of library adapters. Thus, libraries are generated by ligating adapters to inserts, which then serve, in turn, as upstream and downstream primer regions. Each adapter (or adapter region) may have any suitable structure before and / or after binding to an insert. The adapters before binding may comprise a nucleic acid or a nucleic acid analog. Each adapter may be formed by one or more oligonucleotide strands, each having any suitable length (e.g., at least about 6, 8, 10, 15, 20, 30, or 40 nucleotides, among others, and / or less than about 200, 100, 75, or 50 nucleotides, among others). The adapters may be provided by one or more oligonucleotides chemically synthesized in vitro. The adapters may be configured to be bound to an insert at only one of its two ends. In some cases, the adapters may be partially or completely single-stranded before binding to an insert, such as when the adapters are provided by primers that bind to the insert via primer extension. The adapters of the library primarily serve as upstream and downstream primer regions. The target region of the present invention herein is preferably the amplified library insert.

[0035] Ideally, in a method according to the invention, the primers are between 10 and 22 nucleotides in length and / or the probes are between 8 and 17 nucleotides in length.

[0036] Preferably, the primers are between 11 and 17 nucleotides in length, more preferably they are between 12 and 16 nucleotides in length. They do not have to have the same length and can be different.

[0037] Preferably, the probes are between 9 and 16 nucleotides in length, more preferably they are between 11 and 14 nucleotides in length. They do not have to have the same length and can be different.

[0038] Preferably, the combined length of the primer plus probe is between 18 and 35 nucleotides, more preferably between 23 and 30 nucleotides in length.

[0039] Preferably, in the method of the present invention, there are two primers, a first upstream and a second downstream primer.The first probe is adjacent to and downstream of the first upstream primer.The first primer and the first probe may have the same 5'-3' orientation, or in an alternative embodiment, the first probe binds to the opposite strand and has a 3'-5' orientation, while the first primer is naturally oriented in a 5'-3' manner.

[0040] The second probe is adjacent to and downstream of a second, so-called downstream primer, and may bind the same strand and have the same 3'-5' orientation, or may bind the opposite strand.

[0041] Preferably, in the method of the invention, two double-stranded scorpions are used as probes.

[0042] Four alternative embodiments are shown in FIG. 2. All four options are feasible. In variants (A) and (B), the two probes bind specifically to opposite strands of the library fragment. In variant (A), both probes bind closely downstream of each primer. Due to the short distance between both primers and the probe, variant (A) has the advantage of a very efficient emission of a fluorescent signal by the 5'-3' exonuclease activity of Taq polymerase during strand synthesis. In variant (B), both probes bind to a distant second adapter sequence downstream of the primer binding site. Variant (B) has the advantage that each fluorescent signal indicates the polymerization of the library fragments spanning from one adapter to the other. The disadvantage of variant (B) is that for longer fragments (>500 bp), the amplification efficiency decreases, resulting in a smaller emission of a fluorescent signal. In variants (C) and (D), the two probes specifically bind to the same strand of the library fragment. In both variants, one probe binds closely downstream of one primer, and the second probe binds to a second adaptor sequence downstream of the same primer. This has the advantage that a single extension step gives rise to a double signal indicating polymerization of the library fragments spanning from one adaptor to the other.

[0043] In the method of the invention, preferably at least one of the primers contains one or more locked nucleic acid nucleotides (LNA) or another nucleotide analogue that enhances template binding strength. LNA was the first nucleotide analogue to be synthesized with its sugar locked into a C3'-endo conformation. This analogue shows a significantly increased target binding affinity (ΔTm / modification = approximately +5°C compared to native DNA). Other modified nucleotides are also available; see Chem Commun (Camb). 2017 Aug 14; 53(63): 8910-8913, Published online 2017 Jul 27. doi: 10.1039 / c7cc05159j, PMCID: PMC5708354, PMID: 28748236. Locked nucleic acid (LNA) enhances binding affinity of triazole-linked DNA towards RNA†.

[0044] In the methods of the invention, preferably at least one of the probes contains one or more locked nucleic acid nucleotides (LNA) or other nucleotide analogues that increase template binding strength.

[0045] In a preferred embodiment, all primers and probes comprise LNA. One skilled in the art can determine the ideal amount of LNA nucleotides per probe or primer.

[0046] In a preferred embodiment, the one or more primers comprise between 1 and 8 LNA nucleotides, between 2 and 7 LNA nucleotides, between 3 and 6 LNA nucleotides or between 4 and 5 LNA nucleotides, the amount also depending on the length of the primer.

[0047] Ideally, the one or more probes comprise between 2 and 12 LNA nucleotides, between 3 and 11 LNA nucleotides, between 4 and 10 LNA nucleotides, between 5 and 9 LNA nucleotides, between 6 and 8 LNA nucleotides or about 7 LNA nucleotides, the amount also depending on the length of the probe.

[0048] In a preferred embodiment, the amplification products from step a) result from a step of amplifying a nucleic acid library. In a further preferred embodiment, said library is a sequencing library.

[0049] Thermophiles, like other bacteria, contain five types of DNA polymerases, referred to as polymerases I, II, III, IV, and V. Given the nature of the habitat of thermophiles, these enzymes typically exhibit heat stability and are generally referred to as thermostable DNA polymerases. DNA polymerase I ("Pol I") is the most abundant polymerase and is generally responsible for certain types of DNA repair, including repair-like reactions that allow ligation of Okazaki fragments during DNA replication. Pol I is essential for repairing DNA damage induced by UV irradiation and radiomimetic drugs. DNA polymerase II is thought to play a role in repairing DNA damage that induces the SOS response. In mutants lacking both Pol I and DNA polymerase III, DNA polymerase II repairs UV-induced damage. DNA polymerase III is a multisubunit replicase.

[0050] Thermostable DNA polymerases have proven very useful in several applications of molecular biology. One such application is the polymerase chain reaction (PCR). The PCR process is described, for example, in U.S. Pat. Nos. 4,683,195 and 4,683,202, the disclosures of which are incorporated herein by reference. In a PCR reaction, primers, templates, and nucleoside triphosphates are combined in a suitable buffer with DNA polymerase for the basic steps of heat denaturation of target DNA, hybridization of the primers to the template with cooling of the reaction mixture, and primer extension to generate extension products complementary to the template sequence. The heat denaturation is repeated, and the primers are annealed to the extension products with cooling of the reaction mixture, and the previously generated extension products serve as templates for subsequent primer extension reactions. This cycle is repeated many times, resulting in exponential amplification of the desired nucleic acid sequence. The use of thermostable DNA polymerases provides for repeated heating / cooling cycles without loss of enzyme activity. Preferred polymerases herein are Taq polymerase, Vent polymerase, Deep Vent polymerase, Bst polymerase, Pfu polymerase, Tth polymerase, etc. Ideally and preferably, the polymerase has displacement activity and / or 5'-3' exonuclease activity to displace the probe and release the label. The probe is preferably a TaqMan probe that includes a label and a quencher.

[0051] The present invention also relates to the following nucleic acids as expressly claimed herein (see SEQ ID NOs: 1-24). The kits of the present invention may use the following primers and probes as claimed herein:

[0052] In a preferred embodiment, the primers and probes bind at the p5 and p7 regions of the Illumina library adapter; see FIG. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0053] The present invention also relates to a nucleic acid selected from the following group: [Table 2-1] [Table 2-2] The + indicates that the nucleotide following the + is an LNA (locked nucleic acid).

[0054] The method according to the invention, wherein said primers and / or probes are located in the underlined regions: [Table 3-1] [Table 3-2]

[0055] The present invention also provides a. at least two primers having a length between 10 and 22 nucleotides, and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that increase template binding strength; and b. at least two oligonucleotide probes having a length between 8 and 17 nucleotides and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that enhance template binding strength; The present invention relates to a nucleic acid amplification composition comprising:

[0056] The present invention also relates to the use of a composition according to the invention for the analysis of a nucleic acid library.

[0057] Preferably, the library comprises adaptors, the adaptors comprising conserved regions for amplification, and preferably, the target regions are between 15-35 nucleotides in length, between 16-34 nucleotides in length, between 17-33 nucleotides in length, between 18-32 nucleotides in length, between 19-31 nucleotides in length, between 20-30 nucleotides in length, between 21-29 nucleotides in length, or between about 20-28 nucleotides in length.

[0058] The present invention also provides a. at least two primers having a length between 10 and 22 nucleotides, and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that increase template binding strength; and b. at least two oligonucleotide probes having a length between 8 and 17 nucleotides and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that enhance template binding strength; The present invention relates to a kit comprising:

[0059] Preferably, a method, composition or kit according to the invention has a probe which is a hydrolysis probe and said oligonucleotide probe carries a label, wherein the labels on said two probes are different.

[0060] Preferably, the method, composition or kit according to the present invention has a nucleic acid amplification product derived from a step of amplifying a nucleic acid library, wherein said library is a sequencing library. Preferably, said sequencing library is an ILLUMINA library.

[0061] Sequencing libraries are made to perform various analyses (e.g., whole genome sequencing, whole exome sequencing, targeted DNA sequencing, whole transcriptome sequencing, targeted RNA sequencing, ChIP-seq, RIP-seq, epigenetic studies, etc.). ILLUMINA libraries are intended to be run on ILLUMINA sequencing platforms, such as MiSeq system, NextSeq system or NovaSeq system. All ILLUMINA library types use two types of sequencing adapters, each of which shares one of two adapter sequences P5 and P7 at its distal end.

[0062] Depending on the intended analysis, different library preparation kits are used. This results in ILLUMINA libraries that all share the P5 and P7 adapter regions, but the composition of the remainder of the adapter sequence can vary substantially. Figure 1 illustrates examples of ILLUMINA library preparation kits with different adapter sequences.

[0063] Preferably, in the method according to the invention, the amplification step b) is a digital PCR assay and the template is subdivided into partitions or droplets. EXAMPLES

[0064] Working Example Example 1 QIAcuity Systems and Workflows QIAcuity is designed as a walk-away instrument that consolidates and automates all plate processing steps. Only plate preparation has to be done manually before starting the run. This includes pipetting the target, reagents, and master mix in the input wells of the plate and closing the wells with a nanoplate seal. Once the preparation is done and the experiment is set up, the plate is placed into an open plate slot in the instrument tray. By reading the plate barcode, the instrument associates the plate with the experiment predefined in the software. After pressing the play button, all further steps are performed by the instrument fully automated.

[0065] Partitioning In a first step, the microchannels and partitions of the plate are filled with the input volume in the wells. This is done by piercing 24 / 96 pins in the elastic top seal and input wells. This creates a peristaltic pressure that pumps the liquid in the input wells into the microchannels and partitions. Subsequently, the connecting channels between the partitions are closed by a pressure controlled rolling process; see FIG. 8.

[0066] Thermocycling The second step is a high-precision plate thermocycler that performs the polymerase chain reaction. Cycling profiles can be set in the QIAcuity software suite or in the instrument's software. The QIAcuity thermal cycler is a plate thermocycler with fast and precise temperature control of the various cycling steps. Several Peltier elements are used for temperature generation and control. For optimal thermal contact between the plate and the thermocycler, the plate is clamped onto a heated surface during cycling.

[0067] Imaging The final step is image acquisition of all wells. The user can select the detection channel in the experimental setup. Compartments with target molecules inside fluoresce and are brighter than those without targets.

[0068] Calculation of the concentration of the target library fragment in the analyzed samples After end-point PCR, individual wells of the nanoplate are imaged in both channels, green and yellow, by the imaging module of the dPCR instrument. Images are analyzed by the QIAcuity software suite. In this analysis, each partition of the well is defined as valid or invalid based on distinct signal criteria. For each partition of the well, relative fluorescence values ​​are calculated for the yellow and green channels. To distinguish positive signals from negative signals, the software suite sets an automatic threshold in both channels, green and yellow. This results in distinct signal populations of partitions that are double negative (0), show only green (G) or yellow (Y) signals, and are positive for both channels (GY) (illustrated in a two-dimensional scatter plot in Figure 9). The QIAcuity software suite provides an export table that lists the total number of these four types of partitions, the volume cycled, and the total number of valid partitions for each well.

[0069] Using these numbers, the concentration of the target fragment in the library can be calculated using Poisson statistics. The statistical calculations were adapted from Regan JF, Kamitaki N, Legler T, Cooper S, Klitgord N, et al. (2015) A Rapid Molecular Approach for Chromosomal Phasing.

[0070] In it, the ligation of both signals for one target fragment is taken into account for the calculation. The target fragment is a DNA fragment that is ligated to both adapters, generating two signals, green and yellow (GY). Since Illumina NGS libraries may also contain non-target fragments that only produce one of the two signals, partitions with only green or yellow signals are predicted. This may result in partitions with double positive signals arising from the coincidence of one signal fragment being located simultaneously in one partition. The number of these coincidental double positive partitions (Nch) is calculated by: Equation 1: Nch = NG*NY / N0

[0071] Here, N indicates the number of partitions, so that NY and NG are the counts of single positive partitions, N0 is the count of double negative partitions that show no fluorescent signal, and Nch is the count of chance double positive partitions that show both fluorescent signals.

[0072] In the presence of target fragments with linked fluorophore signals (GY) and fragments with single signals (G and Y), there are additional double-positive partitions. Double-positive signals can result from five different combinations of fragments in one partition: G+Y, G+GY, Y+GY, GY, and GY+G+Y. The combination of G+Y is taken into account in Equation 1. For the calculation of the total number of GY, the combination of G and Y in the partition can be ignored, because in all of these cases, GY is present.

[0073] The total number of partitions that do not contain a target fragment with an attached fluorophore signal is calculated using: Equation 2: NnotGY = N0+NG+NY+Nch

[0074] From this, the concentration λ (average number of copies / partition) of the target fragment with the attached fluorophore is calculated using: Equation 3: λGY = ln(Ntot)-ln(NnotGY) where Ntot is the total number of valid partitions. The concentration (in copies / μl) of the target fragment with the fluorophore signal linked in the reaction is calculated using: Equation 4: c(GY) = λGY*Ntot / volume cycled

[0075] An example of a multi occupancy count output table for one well is shown below. The table summarizes the well E5 counts (counts) of four different partitions based on their assigned fluorescent signatures (groups), the total number of valid partitions (total) and the summed and cycled volumes (in μl) of all valid partitions (volume). The partition groups ++ (double positive), +- (positive in the first category), -+ (positive in the second category) and - (double negative) correspond to the signal order shown in the category column. [Table 4]

[0076] Example 2 During NGS library preparation, target DNA is ligated to DNA adapters. Each target DNA is ligated to two different adapters that form library fragments ready for sequencing. Accurate quantification of these full-length library fragments in NGS libraries is a critical step in the QC of next-generation sequencing. The present invention allows for the quantification of these fragments in Illumina NGS libraries using digital PCR. Both adapters in Illumina NGS libraries have a conserved region and a variable region. The variable region differs in length and sequence composition between different library types, whereas the conserved regions P5 and P7 are identical for all Illumina library types (see figure below). The above assay captures all Illumina library types in one assay. Thus, the design of both duplex Scorpions specifically targets these two regions. This requires the design of short oligos, since P5 is only 29 bp and P7 is only 24 bp long (see figure below). To accommodate the oligos required for successful PCR and binding of the Scorpion fluorophore sequence to the extended Scorpion primer sequence, the assay design uses Locked Nucleic Acids (LNAs). A detailed summary of the sequence composition of the oligos is provided in the Materials and Methods section below.

[0077] The product assay consists of two duplex scorpions, each targeting the opposite strand to one of the two conserved adapter sequences as shown in FIG. 10. Each duplex scorpion consists of two oligos, a fluorophore primer and a quencher oligo capable of forming a duplex. The fluorophore primer consists of three parts, a fluorophore domain, a HEG (hexethylene glycol) spacer and an annealing primer domain. The fluorophore domain contains a fluorophore at the 5' end and a sequence that is reverse complementary to the extension sequence downstream of the binding site of the primer domain. The HEG spacer allows the fluorophore domain flexibility to flip and bind the extended primer during amplification. The HEG further prevents extension by polymerase. The primer domain consists of a primer sequence that specifically binds to a conserved adapter sequence. The quencher oligo contains a quencher at the 3' end and specifically hybridizes with the fluorophore domain of the fluorophore primer. There is one possible design variant with two double-stranded scorpions, each specific for one of the two adapters. The oligo parameters are listed in the table below. Assay design framework overview [Table 5-1] [Table 5-2]

[0078] To generate a signal for a complete fragment, both Scorpion primers must bind to the target sequence on opposite strands. An Illumina NGS library fragment with both adapters will bind both Scorpion primers and emit two fluorescent signals.

[0079] At the start of PCR, the fluorophore primer and quencher oligo are in a duplex state (Figure 11). In this bound form, the fluorophore of the fluorophore primer and the quencher of the quencher oligo are in close proximity and fluorescence is quenched, so no signal can be detected. In the presence of an NGS library fragment with both adapters, both fluorophore primers bind their specific target sequences. The primers are extended. During denaturation, the quencher oligo separates from the fluorophore primer, releasing the fluorescent signal of the fluorophore. When cooled for annealing, the fluorophore domain of the extended fluorophore primer binds / hybridizes intramolecularly to the fragment downstream of the primer. Back hybridization events to the quencher oligo are very rare, since intramolecular binding is kinetically much more favorable. Only the unextended fluorophore primers rebind to the quencher and their fluorescence is quenched again. Extension of the reverse primer leads to hydrolysis of the fluorophore of the intramolecularly bound fluorophore domain due to the exonuclease activity of the polymerase. With each cycle, more and more intramolecularly bound fluorophore domains are generated, causing an intense fluorescent signal at the end of the PCR reaction.

[0080] This causes a dual fluorescent signal in each partition that contains NGS fragments with both adaptors, P5 and P7. In digital PCR, the fragments are randomly distributed into thousands of partitions, where end-point PCR causes a binary value of presence or absence of the template of interest for each partition. After correcting using Poisson statistics for the possibility of having one or more template molecules per partition, these binary values ​​reveal the absolute quantification of molecules / μl. For the product assay design, the binary signal is based on the presence and absence of a dual positive signal for both fluorophores of the two probes.

[0081] Our invention allows for the quantification of Illumina NGS library fragments using digital PCR (e.g., with the QIAcuity Nanoplate dPCR System). During end-point PCR within the partitions of the dPCR reaction, the amplification of each complete NGS library fragment produces two fluorescent signals (each specific for one of the two conserved adapter sequences, P5 and P7). For example, the QIAcuity software automatically detects and quantifies partitions with double-positive fluorescent signals and calculates the absolute number of double-positive library fragments in the template and their corresponding concentration in the library based on Poisson statistics.

[0082] In a preferred embodiment of the invention, we provide a kit that contains one assay in one tube, plus two tubes of 1 ml HO and one tube of 1 ml QIAcuity probe master mix. The assay consists of two double-stranded Scorpion primers (two fluorophore primers and two quencher oligos) premixed in one tube.

[0083] dPCR settings In the first step, when setting up a dPCR reaction, two dilutions of the Illumina library to be quantified are prepared. The degree of dilution depends on the expected concentration of each library. For the dPCR reaction, a defined amount of the mix of assay components (primer and probe mix), master mix and water are placed in a reaction tube. A defined amount of pre-diluted library is added to the above mix and mixed thoroughly. The final mix is ​​then transferred to the wells of a dPCR 8.5K nanoplate, which is then sealed and loaded into the QIAcuity dPCR instrument.

[0084] dPCR reaction To initiate a dPCR run, appropriate protocols for partitioning, cycling and imaging must be specified by the user in the QIAcuity software suite. The assay requires specific cycling and imaging conditions according to the assay design and the fluorophores of the probes used. Protocols for the dPCR reactions are provided with the developed kits.

[0085] Data analysis After the imaging step, the acquired data is analyzed using the QIAcuity software suite. The assay product is designed in such a way that the end-point PCR of the dPCR reaction reveals a signal intensity of the positive partition that is strong enough for the software to distinguish it from the negative partition. The automatic threshold function of the software performs this discrimination in both signal channels. It indicates whether one or both signals are detected for each partition in the well. Partitions with positive signals for both channels, so-called multi-occupied partitions, represent target partitions. The figures of multi-occupied partitions, effective partitions, cycled volumes and other parameters for each well are compiled in a table that must be exported from the software suite for further analysis. Using an external Excel data sheet, the concentration of NGS library fragments with double positive signals is calculated from the data in the exported table. The calculation applies Poisson statistics.

[0086] Data Output and Interpretation The duplex Scorpion assay design applied to NGS library fragments in digital PCR on QIAcuity produces data that allows the same readouts and data interpretation as using the previous design based on two probes and two primers. We are currently testing whether we see the same differences in relative fluorescence unit (RFU) signal intensity for short and long NGS library fragments. First test results show a similar correlation between RFU signal intensity and library fragment length (Figure 13).

[0087] material and method Protocols and Procedures One reaction in a 96-well 8.5K nanoplate contains the following reagents at the indicated concentrations: [Table 6]

[0088] The above dPCR reactions are run on a QIAcuity dPCR system using the following cycling and system settings: [Table 7]

[0089] An example of a double-stranded Scorpion design The present invention also relates to the following nucleic acids (see SEQ ID NOs: 25-44) which are expressly claimed herein. Various designs were tested. The nucleotide after + is LNA. HEG is hexethylene glycol. FAM and HEX are dyes. Q denotes a quencher. According to WIPO Standard 26, the sequences separated by the HEG spacer are assigned two SEQ ID NOs representing each part separately, with the sequence of the second SEQ ID NO being underlined. [Table 8-1] [Table 8-2]

[0090] The oligonucleotide combinations used in our feasibility studies were: Duplex Scorpion combinations tested (green if functional) [Table 9]

[0091] Diagram Descriptions and Examples [Brief description of the drawings]

[0092] [Figure 1-1] Figure 1 illustrates the polylobal nature of Illumina library adapter sequences. Illumina libraries contain conserved P5 and P7 regions, as well as variable regions that vary in length and composition between Illumina library types. [Figure 1-2] Same as above.

[0093] [Diagram 2] FIG. 2 shows that preferably the assay consists of a set of two primers, forward and reverse, and two 5'-hydrolysis probes labeled with different fluorophores. The two probe sets target the same strand in either the forward or reverse orientation, or opposite strands, e.g., primer-probe pairs P5 and P7 (each on the same strand). The probes are modified with respective quenchers at the 3' end. Darker colors indicate conserved adapter regions, while lighter colored regions represent adapter regions that vary in sequence, e.g., between different Illumina library types.

[0094] [Diagram 3]Figure 3 shows an exemplary target sequence for the novel assay design. Four representative Illumina libraries were selected for proof-of-concept testing of the initial assay design. The libraries were prepared using different library preparation kits and differ in the length of the inserted target DNA; see below. The fragment lengths correspond to the library fragments including their ligated adaptors. [Table 10]

[0095] [Figure 4] Figure 4 shows an exemplary assay design consisting of two primers and two probes. Each probe is labeled with a different fluorophore (FAM and HEX) at the 5' end and a quencher at the 3' end. The primer and probe designs used for proof-of-concept studies are shown as aligned sequences. The oligo sequences are aligned to the dual-indexed Illumina TrueSeq target library sequence with adapter regions P5 and P7 highlighted. Primers F4, F5 and F6 and the probe target the same strand. Reverse primer R5 targets the opposite strand. The locked nucleic acid (LNA) in the oligo is highlighted.

[0096] [Figure 5-1]Figure 5 shows two examples of P5 / P7 targeted assay designs. In dPCR, both assays produce clear signal-to-noise separation in both channels that can be seen in the 2D scatter plots. Furthermore, both assays precisely quantitate three dilutions of library test template, which are shown by the average concentration in copies / μl reaction in the bar graphs. The 2D scatter plots and quantification bar graphs of the two assay designs are shown. The points in the 2D scatter plots show the relative fluorescence in green and yellow of individual partitions of dPCR reactions with negative signals highlighted in grey and double positive partitions highlighted in dark blue. Each bar in the bar graph shows the average concentration in copies / μl of each of the three replicate reactions. The predicted concentration of the analyzed test library template is shown below the bar. NTC: No template control. [Figure 5-2] Same as above.

[0097] [Figure 6-1] FIG. 6 shows that an experiment was performed that clearly shows the advantages over the most sophisticated product concepts currently on the market; a comparison to BioRad's dual probe-based assay. In total, four Illumina test libraries of different types and lengths of target DNA were used for quantification. The four test libraries were quantified on both digital PCR systems, using one inventive assay design on QIAcuity and BioRad's competitive assay on a QX200 ddPCR instrument. The assay on the QX200 was run according to the manufacturer's instructions and using instrument-specific chemistry. In contrast to the Biorad assay, which cannot capture the QIAseq library, the inventive assay design captures both TruSeq and QIAseq test libraries. 2D scatter plots of both dPCR and ddPCR runs are shown, highlighting the RFU values ​​of the double positive cutoff in blue (dPCR) and orange (ddPCR). [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0098] [Figure 7] Figure 7 Materials and Methods a) Equipment and Plastics The assay of the present invention was tested on QIAcuity 1, 4 and 8 using 96-well nanoplates (96LV). b) Chemistry Digital PCR on QIAcuity used the standard QIAcuity dPCR probe master mix. A detailed protocol for setting up the dPCR reaction is shown in Figure 7. Oligos were ordered from Biomers and IDT.

[0099] c) Protocol An overview of the protocol applied is shown in FIG. dPCR reaction setup protocol 1. Thaw QIAcuity Probe PCR Master Mix, template DNA, primers, probe, and RNase-free water. Mix QIAcuity Probe PCR Master Mix and each individual reaction vigorously. Centrifuge briefly to collect liquid at the bottom of the tube. 2. Prepare the reaction mix for the number of reactions required according to Table 2. Due to the hot start, it is not necessary to keep samples on ice during reaction setup or while programming the QIAcuity instrument. 3. Vortex the above reaction mix. 4. Dispense an appropriate volume of the reaction mix (which contains all components except the template) into the wells of a standard PCR plate. Template DNA or cDNA is then added to each well containing the reaction mix. 5. Transfer the contents of each well from the standard PCR plate to a well of the nanoplate. 6. Seal the nanoplate properly using the QIAcuity Nanoplate Seal provided in the QIAcuity Nanoplate Kits. 7. The sealed plate was placed into the QIAcuity dPCR instrument which performed all subsequent steps (priming, rolling, cycling and imaging) automatically. 8. Image analysis was performed using the QIAcuity software suite.

[0100] [Figure 8] Figure 8. Device organization; see detailed explanation above.

[0101] [Figure 9] Figure 9. Schematic two-dimensional scatter plot of different signal populations. After setting thresholds for the green and yellow channels, the partitions that are double negative (0), that show only green (G) or yellow (Y) signals, and that are positive for both channels (GY) are located in four distinct regions in the 2D scatter plot.

[0102] [Figure 10] FIG. 10 This figure shows a preferred embodiment of an assay design based on two duplex Scorpions, each consisting of one fluorophore-primer and one quencher oligo.

[0103] [Figure 11] Figure 11 This figure shows a preferred embodiment of duplex-scorpion-based detection of an exemplary NGS library fragment with respect to one of two adapters. Two duplex scorpions, each specific to one of two adapters P5 and P7 (in the ILLUMINA NGS library), are preferred. The specificity for the adapters can be adjusted according to the library to be tested.

[0104] [Figure 12] Figure 12 shows the workflow as developed by the inventors. It shows the preferred workflow for NGS library quantification on the QIAcuity dPCR system.

[0105] [Figure 13] FIG. 13 FIG. 13 shows fragment length dependent signal, intensity after end point dPCR.

Claims

1. A method for testing a nucleic acid amplification product, the method comprising: a. The following: i. An upstream and a downstream primer region, and an upstream primer and a downstream primer that bind to the upstream and the downstream primer regions respectively, and optionally and preferably, ii. A target region to be amplified between the upstream and the downstream primer regions, Providing an amplification product comprising; b. Testing the amplification product by amplifying the amplification product in a PCR reaction, wherein i. The upstream and downstream PCR primers in the PCR reaction bind within the upstream and downstream primer regions of the amplification product, ii. The first and second oligonucleotide probes are located downstream of the first and second PCR primers and within the upstream and the downstream primer regions Step; c. Performing a PCR reaction, preferably a digital PCR reaction, and testing the newly formed amplification product, wherein in step a.i.) the upstream and downstream primer regions have a length of less than 35 nucleotides and greater than 20 nucleotides. A method comprising the steps.

2. The method according to claim 1, wherein the primer has a length between 10 nucleotides and 22 nucleotides and / or the probe has a length between 8 and 17 nucleotides.

3. The method according to claim 1 or 2, wherein at least one of the primers comprises one or more locked nucleic acid nucleotides (LNA) or another nucleotide analog that increases template binding strength.

4. The method according to claim 1, wherein at least one of the probes comprises one or more locked nucleic acid nucleotides (LNA) or another nucleotide analog that increases template binding strength.

5. The method according to claim 1, wherein all primers and probes comprise LNA.

6. The method according to claim 1, wherein the one or more primers comprise between 1 and 8 LNA nucleotides.

7. The method according to claim 1, wherein the one or more probes comprise between 2 and 12 LNA nucleotides.

8. The method according to claim 1, wherein the amplification product from step 1a) is derived from the step of amplifying a nucleic acid library.

9. The method according to claim 8, wherein the library is a sequencing library and / or the probe is two duplex scorpion probes.

10. A nucleic acid amplification composition comprising: a. At least two primers having a length between 10 and 22 nucleotides and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that increase template binding strength, and b. At least two oligonucleotide probes having a length between 8 and 17 nucleotides and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that increase template binding strength, wherein the probes are preferably two duplex scorpion probes. A nucleic acid amplification composition comprising the same.

11. Use of the composition according to claim 10 for the analysis of a nucleic acid library.

12. a. At least two primers having a length between 10 and 22 nucleotides and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that increase template binding strength, and b. At least two oligonucleotide probes having a length between 8 and 17 nucleotides and optionally containing one or more locked nucleic acid nucleotide analogs or other nucleotide analogs that increase template binding strength. A kit comprising the same.

13. The method according to claim 1, the composition according to claim 10, or the kit according to claim 12, wherein the probe is a hydrolysis probe, the oligonucleotide probe has a label, and the labels on the two probes are different.

14. The method, composition, or kit according to claim 13, wherein the nucleic acid amplification product is derived from the step of amplifying a nucleic acid library, and the library is a sequencing library.

15. The method according to claim 1, wherein the amplification step b) is a digital PCR assay, and the template is subdivided into partitions or droplets.

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