DETERMINATION OF THE QUANTITY AND QUALITY OF A DNA LIBRARY

Abstract

The invention relates to an adapter dimer-specific probe (33) for detecting adapter dimers (3) in a DNA library, the probe (33) being labelled, particularly by a fluorophore (35), and being designed to bind specifically to a first adapter nucleotide sequence of a first adapter molecule (2a) and to a second adapter nucleotide sequence of a second adapter molecule (2b).

Description

[0001] Description

[0002] title

[0003] Determining the quantity and quality of a DNA library

[0004] The present invention relates to an adapter dimer-specific probe, a method for detecting adapter dimers in a deoxyribonucleic acid (DNA) library, and a cartridge designed to carry out the method, according to the preamble of the independent claims.

[0005] State of the art

[0006] The publication by Hardigan et al., "CRISPR / Cas9-targeted Removal of Unwanted Sequences From small-RNA Sequencing Libraries. Nucleic Acids Research 2019," concerns the removal of adapter dimers from nucleic acid libraries. Removal occurs through specific cleavage of the adapter dimers using the CRISPR / Cas9 system. To verify successful removal of the adapter dimers, quantification of the adapter dimers using quantitative PCR is described. This process uses adapter-overlapping primers.

[0007] The publication by Shore et al., "Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation," published in PLoS One 2016, concerns adapter molecules for creating sequenceable nucleic acid libraries while preventing the formation of adapter dimers. WO 2014 / 144979 A1 discloses a reduction or inhibition of adapter dimer formation during nucleic acid library preparation.

[0008] WO 2019 / 222706 A1 relates to the sequencing of genomic DNA. Methods relating to polynucleotide libraries are provided.

[0009] WO 2009 / 106308 A2 describes the processing of nucleic acids to create sequenceable nucleic acid libraries.

[0010] WO 2015 / 044412 A1 discloses adapter molecules for creating sequenceable nucleic acid libraries and avoiding the formation of adapter dimers.

[0011] Today, next-generation sequencing (NGS) methods are used almost exclusively to sequence DNA samples. The term NGS refers to a wide range of different sequencing techniques, all of which are essentially characterized by the automated ability to perform a large number of tests in parallel and in a short period of time. For example, NGS makes it possible to discover new disease-relevant genes and disease-causing genetic mutations, i.e., changes in the genome. Such insights help to understand fundamental biological processes and gain information about the development of hereditary diseases.

[0012] One of the NGS methods is amplicon sequencing (also called targeted sequencing), in which the fragment lengths of the DNA fragments to be sequenced are already known before the sequencing run due to appropriate sample preparation.

[0013] In NGS, precise quantification and quality analysis of the DNA library are performed before each sequencing run to ensure a successful sequencing run. Quantification determines the concentration of fragments in the DNA library. Quality analysis includes, for example, determining the fragment lengths of the DNA fragments to be sequenced and identifying the formation of unwanted adapter dimers or other artifacts. Quantification and quality analysis are performed independently of each other using different methods and technologies.

[0014] Disclosure of the invention

[0015] According to the invention, an adapter dimer-specific probe, a method for detecting adapter dimers in a DNA library, and a cartridge designed to carry out the method are provided with the characterizing features of the independent claims.

[0016] While the available NGS methods differ in the reagents and protocols used, the essential procedural steps are the same for all NGS methods. First, the DNA to be sequenced must be prepared for sequencing. This involves cutting the DNA sample to be sequenced into small fragments, either enzymatically or mechanically. The length of the DNA fragments depends on the respective method. Short, artificially produced pieces of DNA known as adapter molecules are then attached to the two free ends of the DNA fragments, usually through a ligation step. The adapter molecules are constructed in such a way that they have a complementary nucleotide sequence to the primers used. Primers are oligonucleotides with a known nucleotide sequence that serve as a starting point for DNA-replicating enzymes such as DNA polymerase.

[0017] In order to provide a sufficient number of copies of DNA fragments for sequencing, the DNA fragments flanked by adapter molecules are amplified using a polymerase chain reaction (PCR)-based process, for example. If the DNA is fragmented, provided with adapter molecules, and specifically enriched, it is referred to as a DNA library. Real-time quantitative PCR (qPCR), for example, is particularly suitable for quantifying the DNA library. In this method, the amplified DNA fragments are quantified in real time during a PCR cycle. Quantification is carried out, for example, using fluorescence measurements. Fluorescent dyes are incorporated into the DNA fragments, whereby the fluorescence increases proportionally with the amount of amplified DNA fragments.

[0018] During DNA library preparation, adapter molecules not only bind to the free ends of the DNA fragments to be amplified, but can also combine with each other, forming adapter dimers. These are unwanted and distort the results both during quantification of the fragments in the DNA library and during subsequent sequencing. During quantification, the number of DNA fragments flanked by adapter molecules appears higher than it actually is if adapter dimers are present, because the adapter dimer fragments are also quantified.

[0019] In order to evaluate the quantification of fragments in a DNA library and to control the formation of artifacts such as adapter dimers, additional quality control methods must be performed in addition to qPCR.

[0020] According to the invention, an adapter dimer-specific probe for detecting adapter dimers, in particular in a DNA library, is disclosed.

[0021] The adapter dimer-specific probe comprises a label that makes it detectable. This label is, in particular, a fluorophore, so that fluorescence radiation is detected when the fluorophore is excited with light of a specific wavelength. Alternatively, the label of the adapter dimer-specific probe is a chemiluminescent label, by means of which light emission is detected as a result of a chemical reaction (chemiluminescence) or an enzymatic reaction (bioluminescence). Furthermore, the label of the adapter dimer-specific probe is alternatively a radioactive label, in which the radioactive radiation is detected, or a redox-active component whose activity is detected by electrochemical methods.

[0022] The adapter dimer-specific probe is, for example, a so-called Taqman probe, which contains a signal quencher. In the case of a fluorophore as a label, for example, the signal quencher quenches the fluorescence signal of the fluorophore by transferring at least part of its energy to the signal quencher when excited by a light source.

[0023] Suppression only occurs when the signal suppressor and the fluorophore are in close spatial proximity to each other. During DNA amplification, particularly of the complementary DNA strand, for example, by a polymerase, the probe covalently bound to the DNA strand is degraded. This causes the signal suppressor and the fluorophore to move away from each other, and an increasing fluorescence of the fluorophore can be measured.

[0024] The adapter dimer-specific probe is designed to specifically bind to a first adapter nucleotide sequence of a first adapter molecule and to a second adapter nucleotide sequence of a second adapter molecule.

[0025] An advantage of the adapter dimer-specific probe according to the invention is that adapter dimers are directly and specifically bound by the adapter dimer-specific probe and can thus be detected quickly and easily. Thus, no additional time-consuming and / or costly methods for detecting adapter dimers are necessary.

[0026] Furthermore, a method for detecting adapter dimers in a DNA library during qPCR is disclosed, wherein the DNA library comprises DNA fragments flanked at both free ends by adapter molecules and adapter dimers.

[0027] The method comprises the following steps: a) Addition of an adapter dimer-specific probe to a qPCR mixture comprising the DNA library b) Determination of the number of adapter dimers contained in the DNA library using the signal emitted by the adapter dimer-specific probe c) Calculation of the actual number of DNA fragments in the DNA library taking into account the determined number of adapter dimers. In general, qPCR is a very precise method for determining the concentration of DNA fragments in the DNA library, since only specifically adapter-bound fragments are amplified and thus quantified. However, if adapter dimers have also formed, the primers in qPCR also bind to them, so that the adapter dimers are also quantified. In this case, the measured DNA concentration is higher than it actually is.

[0028] Thus, for accurate quantification, it is traditionally necessary to determine the fragment lengths of the fragments contained in the DNA library using another method, such as gel electrophoresis or, if necessary, capillary gel electrophoresis, in order to calculate the actual number of DNA fragments in the DNA library. In this case, the number of adapter dimers is subtracted from the number of DNA fragments.

[0029] An advantage of the method according to the invention is that the number of adapter dimers in the DNA library is determined in real time directly during qPCR using the adapter dimer-specific probe. This eliminates the need for time-consuming and labor-intensive methods, such as determining the fragment lengths of the DNA fragments and the adapter dimers in the DNA library, especially when the fragment lengths of the DNA fragments are known, as is the case, for example, with a DNA library for amplicon sequencing. If the fragment lengths of the DNA fragments are known, the number of DNA fragments can be calculated from the corrected quantification without the need for further analytical methods and procedures following the qPCR. The advantage here is that the probe according to the invention and the method according to the invention thus save work steps as well as time, financial, and material resources.

[0030] The determined number of adapter dimers can also be a decisive quality indicator for whether the DNA library can be used for subsequent sequencing.

[0031] Further advantageous embodiments emerge from the subclaims.

[0032] In a particularly advantageous embodiment of the method, at least one adapter-specific probe for a first adapter molecule is further added to the qPCR mixture in step a) and in step b) the number of first adapter molecules contained is additionally determined by means of the signal emitted by the adapter-specific probe and in step c) the actual number of DNA fragments in the DNA library is calculated taking into account the determined number of adapter dimers and the determined number of first adapter molecules.

[0033] The advantage here is that both the number of adapter dimers in the DNA library is detected in real time directly during qPCR using the adapter dimer-specific probe, as is the number of first adapter molecules using the adapter-specific probe. The adapter-specific probe binds both to first adapter molecules flanking DNA fragments and to first adapter molecules of adapter dimers. The adapter-specific probe thus detects the total number of first adapter molecules. To calculate the actual number of DNA fragments in the DNA library, the number of detected adapter dimers is subtracted from the number of detected first adapter molecules. This eliminates the need for time-consuming and labor-intensive methods to determine the actual number of DNA fragments, such as determining the fragment lengths of the DNA fragments and the adapter dimers in the DNA library.

[0034] During qPCR, the fragments contained in the DNA library are quantified using the signal from the adapter-specific probe, and the quality of the DNA library is determined using the signal from the adapter dimer-specific probe. This saves work steps, as well as time, financial resources, and material.

[0035] The determined number of adapter dimers can also be a decisive quality indicator for whether the DNA library can be used for subsequent sequencing.

[0036] In a further advantageous embodiment of the method, the amount of DNA to be used for subsequent sequencing, in particular NGS sequencing, is determined based on the calculation of the actual number of DNA fragments in the DNA library.

[0037] Determining the exact number of DNA fragments in the DNA library for DNA sequencing is of utmost importance. If the DNA load on the sequencer is too low, its full potential will not be utilized, making it uneconomical. If the load is too high, the signals for sequencing can no longer be read correctly. The advantage is that the calculated actual number of DNA fragments in the DNA library allows the optimal amount of DNA to be used to be calculated, thus ensuring optimal loading of the sequencer.

[0038] Furthermore, the determined number of adapter dimers is crucial for whether the DNA library can be used for sequencing. If the proportion of adapter dimers is too high, it should not be used.

[0039] It is advantageous if the method is amplicon sequencing, where the length of the DNA fragments is known.

[0040] The advantage here is that the length of the DNA fragments is known due to the specific sample preparation. Depending on the qPCR approach, this parameter is required to calculate the quantity and quality of the DNA library and determine the optimal amount of DNA for amplicon sequencing. Thus, choosing amplicon sequencing may eliminate the need for additional procedures, particularly for determining the length of the DNA fragments, thus saving time, costs, and fewer work steps.

[0041] The invention further relates to a cartridge, in particular a microfluidic cartridge, as described for example in DE102016222072A1 or DE102016222075A1, wherein the cartridge is designed to carry out the method according to the invention.

[0042] The described process steps for the real-time detection of adapter dimers from a DNA library during qPCR, or for the real-time detection of adapter dimers and initial adapter molecules from a DNA library during qPCR, are then all performed within the cartridge. Subsequent sequencing, for example, also takes place within the cartridge. Preparatory steps for qPCR also take place within the cartridge.

[0043] Combined quantity and quality control is particularly advantageous in a microfluidic system, as no additional methods need to be implemented. Alternatively, all described steps can be performed manually.

[0044] Short description of the drawing

[0045] Embodiments of the present invention are illustrated in the drawing and explained in more detail in the following description of the figures. It shows:

[0046] Fig. 1: the schematic representation of an adapter dimer-specific probe according to the invention for the detection of adapter dimers,

[0047] Fig. 2: the schematic representation of an inventive

[0048] Method for detecting adapter dimers in a DNA library during qPCR in a first embodiment,

[0049] Fig. 3: the schematic representation of the method according to the invention for detecting adapter dimers in a DNA library during a qPCR in a second embodiment, and

[0050] Fig. 4: the schematic representation of an inventive

[0051] Cartridge designed to carry out the method according to Figure 3.

[0052] Embodiments of the invention

[0053] Figure 1 shows an adapter dimer-specific probe 33 according to the invention for detecting adapter dimers 3, in particular in a DNA library.

[0054] The adapter dimer-specific probe 33 comprises a first DNA nucleotide sequence 8a, which is complementary to a portion of the adapter nucleotide sequence of a first adapter molecule 2a, and a second DNA nucleotide sequence 8b, which is complementary to a portion of the adapter nucleotide sequence of a second adapter molecule 2b. By means of the first and second DNA nucleotide sequences 8a, 8b, the adapter dimer-specific probe 33 thus binds specifically to adapter dimers 3. The adapter dimer-specific probe 33 comprises a fluorophore 35 as a label. If the fluorophore 35 is excited with light of a specific wavelength, it emits detectable fluorescent radiation. In an alternative embodiment not shown, the label of the adapter dimer-specific probe 33 is a chemiluminescent label, a radioactive label, or a redox-active component.

[0055] The adapter dimer-specific probe 33 is, for example, a Taqman probe comprising a signal suppressor 37. The signal suppressor 37 quenches the fluorescence signal of the fluorophore 35 by the fluorophore 35 transferring at least part of its energy to the signal suppressor 37 when excited by the light source.

[0056] Suppression only occurs when the signal suppressor 37 and the fluorophore 35 are in close proximity to each other. During DNA amplification, for example, by a DNA polymerase, the adapter dimer-specific probe 33 covalently bound to the DNA strand is degraded. As a result, the signal suppressor 37 and the fluorophore 35 move away from each other, and an increasing fluorescence of the fluorophore 35 can be measured.

[0057] In an alternative embodiment, the adapter dimer-specific probe 33 is a different type of probe.

[0058] Figure 2 shows a method according to the invention for detecting adapter dimers 3 in a DNA library during qPCR in a first embodiment.

[0059] First, and not shown in Figure 2, a DNA sample is prepared in such a way that DNA fragments 1 are present. For this purpose, the DNA sample is enzymatically or mechanically broken down into DNA fragments 1, or alternatively, specific DNA fragments 1 are enriched, for example, using amplicon PCR (targeted PCR). The fragment lengths of the DNA fragments 1 depend on the respective method.

[0060] Subsequently, and as shown in step A, first adapter molecules 2a and second adapter molecules 2b are added to the DNA fragments 1.

[0061] The adapter molecules 2a, 2b are, for example, pseudo-double-stranded adapter molecules 2a, 2b and are asymmetrically attached to the two free ends 1a, 1b of the DNA fragments 1, for example, by ligation. The first adapter molecules 2a thus bind specifically to the first free end 1a of the DNA fragments 1, and the second adapter molecules 2b thus bind specifically to the second free end 1b of the DNA fragments 1. In this way, DNA fragments 21 flanked by adapter molecules 2a, 2b are formed. The first adapter molecule 2a has a complementary nucleotide sequence to a first primer. The second adapter molecule 2b has a complementary nucleotide sequence to a second primer.

[0062] The adapter molecules 2a and 2b not only bind to the free ends of DNA fragments 1, but can also combine with each other, albeit less efficiently, to form adapter dimers 3. These are unwanted and, among other things, distort the results of subsequent quantification of DNA fragments 1.

[0063] In a further step, not shown in the figure, the flanked DNA fragments 21 and the adapter dimers 3 are amplified, for example by means of a PCR method.

[0064] This results in a DNA library comprising DNA fragments 1 flanked at both free ends by adapter molecules 2a, 2b, and adapter dimers 3.

[0065] In order to determine the amount of DNA fragments 1 and adapter dimers 3, for example for analysis or for sequencing the DNA fragments 1, a qPCR is carried out. In preparation for this and described in step B, a first primer 4a and a second primer 4b are added to the mixture, which serve as starting points for DNA-replicating enzymes, such as DNA polymerase. The first primer 4a binds specifically to the complementary nucleotide sequence of the first adapter molecule 2a and the second primer 4b binds specifically to the complementary nucleotide sequence of the second adapter molecule 2b. In this case, the nucleotide sequence of the adapter molecules 2a, 2b that is complementary to the primers 4a, 4b is, for example, only a portion of the nucleotide sequence of the adapter molecules 2a, 2b at the respective unbound ends of the adapter molecules 2a, 2b.Furthermore, an adapter dimer-specific probe 33, as described for example in Figure 1, is added to the qPCR approach.

[0066] The adapter dimer-specific probe 33 binds specifically to a first adapter nucleotide sequence of a first adapter molecule 2a and to a second adapter nucleotide sequence of a second adapter molecule 2b. Thus, the adapter dimer-specific probe 33 is designed to bind specifically to adapter dimers 3. In the subsequent qPCR, the flanked DNA fragments 21 and the adapter dimers 3 are amplified. During DNA amplification, the fluorescence emitted by the adapter dimer-specific probe 33 is measured after each cycle, and an adapter dimer-specific fluorescence curve 333 is recorded as shown in step C. The PCR cycles are shown on the x-axis, and the measured fluorescence is shown on the y-axis. Using a standard curve, a molecule number is assigned to the fluorescence signals. This allows the number of adapter dimers 3 to be calculated.

[0067] In addition, the amplified DNA fragments 1 are also quantified in real time after each PCR cycle. In step D above, a DNA fragment 1 is shown, as well as a DNA-intercalating marker 5. The DNA-intercalating marker 5, for example a fluorescent dye, is incorporated into the DNA fragments 1, as shown in step 4 below, whereby the signal 6 of the marker 5, in particular the fluorescence signal, increases proportionally with the amount of amplified DNA fragments 1. In addition, and not shown in the figures, the DNA-intercalating marker 5 is also incorporated into the adapter dimers 3. Thus, the total concentration of DNA fragments 1 and adapter dimers 3 is detected here. From this, a corrected quantification can be calculated by subtracting the concentration of adapter dimers 3 from the concentration of detected total DNA.If the fragment lengths of the DNA fragments 1 are known, such as in sample preparation for amplicon sequencing, the actual number of DNA fragments 1 in the DNA library can be calculated.

[0068] The determined number of adapter dimers 3 can also be a decisive quality indicator for whether the DNA library can be used for subsequent sequencing.

[0069] Figure 3 shows a method according to the invention for detecting adapter dimers 3 in a DNA library during qPCR in a second embodiment.

[0070] In contrast to the method described for Figure 2 in the first embodiment, in addition to the adapter dimer-specific probe 33, an adapter-specific probe 22 is also added to the qPCR mixture in step B'. The adapter-specific probe 22 binds specifically to a first adapter nucleotide sequence of a first adapter molecule 2a. Thus, the adapter-specific probe 22 binds both to a first adapter molecule 2a flanking a DNA fragment 1 and to a first adapter molecule 2a of an adapter dimer 3. The adapter-specific probe 22 thus makes it possible to detect the total number of first adapter molecules 2a, which is representative of the total number of flanked DNA fragments 21.

[0071] In the subsequent qPCR, the flanked DNA fragments 21 and the adapter dimers 3 are amplified. During DNA amplification, the fluorescence emitted by the adapter dimer-specific probe 33 is detected after each cycle, and an adapter dimer-specific fluorescence curve 333 is recorded as shown in step C'.

[0072] In addition, after each amplification cycle, the fluorescence emitted by the adapter-specific probe 22 is detected, and an adapter-specific fluorescence curve 222 is recorded as shown in step C'. The PCR cycles are shown on the x-axis, and the measured fluorescence is shown on the y-axis.

[0073] Using a respective standard curve, a molecule number is assigned to each adapter dimer-related fluorescence signal and each adapter-related fluorescence signal. This occurs in parallel with the acquisition of fluorescence curves 222 and 333. To calculate the actual number of DNA fragments 1 in the DNA library, the determined number of adapter dimers 3 is subtracted from the number of first adapter molecules 2a.

[0074] Alternatively, and not shown in Figure 3, the adapter-specific probe 22 binds specifically to an adapter nucleotide sequence of a second adapter molecule 2b. The actual number of DNA fragments 1 in the DNA library is relevant, for example, for determining the amount of DNA molecules to be used for subsequent sequencing, in particular for NGS sequencing.

[0075] The determined number of adapter dimers 3 can also be a decisive quality indicator for whether the DNA library can be used for subsequent sequencing.

[0076] Depending on the number of different DNA fragments 1 used simultaneously in the methods described for Figures 2 and 3, for example, and thus also the number of different adapter molecules 2a, 2b used, different artifacts, in particular adapter dimers 3, can form. Of course, a plurality of different chimeric sequence-specific probes for detecting artifacts, such as the adapter dimer-specific probe 33 for detecting adapter dimers 3, can then be used in the methods. For example, several adapter-specific probes 22 can be used to detect the adapter molecules 2a, 2b.

[0077] Figure 4 shows a cartridge 100 according to the invention, in particular a microfluidic cartridge. The cartridge 100 is designed to carry out a method according to the second embodiment as described above with reference to Figure 3.

[0078] The steps described in Figure 3 then all take place, for example, within the cartridge 100. Subsequent sequencing also takes place, for example, within the cartridge 100.

[0079] Alternatively, and not shown in Figure 4, the cartridge 100 is designed to carry out a method according to the first embodiment as described in Figure 2, in particular with subsequent sequencing.

[0080] In an alternative embodiment, all described steps are performed manually.

Claims

Claims 1. Adapter dimer-specific probe (33) for the detection of adapter dimers (3), in particular in a DNA library, wherein the probe (33) has a label, in particular a fluorophore (35), and is configured to bind specifically to a first adapter nucleotide sequence of a first adapter molecule (2a) and to a second adapter nucleotide sequence of a second adapter molecule (2b).

2. A method for detecting adapter dimers (3) in a DNA library during qPCR, wherein the DNA library comprises DNA fragments (1) flanked at both free ends by adapter molecules (2a, 2b) and adapter dimers (3) comprising the following steps: a) adding an adapter dimer-specific probe (33) according to claim 1 to a qPCR reaction comprising the DNA library; b) determining the number of adapter dimers (3) contained in the DNA library by means of the signal emitted by the adapter dimer-specific probe (33); c) calculating the actual number of DNA fragments (1) in the DNA library taking into account the determined number of adapter dimers (3).

3. Method according to claim 2, characterized in that in step a) at least one adapter-specific probe (22) for a first adapter molecule (2a) is added to the qPCR approach and in step b) the number of contained first adapter molecules (2a) is additionally determined by means of the signal output by the adapter-specific probe (22) and in step c) the calculation of the actual number of DNA fragments (1) in the DNA library is carried out taking into account the determined number of adapter dimers (3) and the determined number of first adapter molecules (2a).

4. Method according to one of claims 2 or 3, characterized in that, based on the calculation of the actual number of DNA fragments (1) in the DNA library, the amount of DNA to be used for subsequent sequencing, in particular NGS sequencing, is determined.

5. Method according to claim 4, characterized in that it is an amplicon sequencing in which the length of the DNA fragments (1) is known.

6. Cartridge (100), in particular microfluidic cartridge (100), wherein the cartridge (100) is configured to carry out a method according to one of claims 2-5.