Method and kit for manual and automated sample preparation for long-read sequencing

Abstract

The invention relates to a method and kit for extraction of high molecular weight DNA from a biological sample and / or clean-up of a DNA library, wherein after optional lysis of the biological sample and / or after preparation of the DNA library the mixture is contacted with a combination of a polyether, preferably polyethylene glycol, and at least one salt, preferably magnesium chloride, in the absence of monohydric alcohols, wherein subsequently the DNA is bound to a solid phase and subsequently the DNA is purified without the use of monohydric alcohols. The method may be performed manually or automatically.

Description

METHOD AND KIT FOR MANUAL AND AUTOMATED PREPARATION OF LONG-READ SEQUENCES State of the art

[0001] New Generation Sequencing (NGS) technology is gaining increasing importance in the natural sciences, medicine, and related fields. The goal of comprehensive genome sequencing is not only to identify specific target sequences (pathogen detection, mutation detection, etc.) but also to elucidate the entire genome sequence of an organism. Logically, when addressing such questions using these new sequencing technologies (e.g., nanopore sequencing; Oxford Nanopore Technologies), the length of the sequenced genome segments is paramount: the larger the sequencing read lengths, the simpler and more resource-efficient the bioinformatic analysis of the generated sequences and their assignment to the whole genome.

[0002] Currently, a number of sequencing platforms with varying capabilities for detecting longer DNA fragments are available on the market. For example, the Nanopore sequencing platform from Oxford Nanopore Technologies can perform sequencing of megabase-length fragments without pre-amplification, provided high-quality, high-molecular-weight DNA is used (Ultra-Long DNA Sequencing Kit SQK-ULK001).

[0003] However, the theoretical possibility of sequencing longer fragments is insufficient for the success of the entire sequencing process. The DNA sample must initially contain the required DNA fragment length, and this DNA must also be recoverable in the necessary quantity after tagmentation (the creation of a DNA library for subsequent nanopore sequencing). Oxford Nanopore Technologies recommends the Monarch HMW DNA Extraction Kits (New England Biolabs) for DNA sample preparation. The protocol estimates the total time required at 100 minutes. However, this method is not suitable for DNA reprocessing after tagmentation. The kits are based on binding high-molecular-weight DNA to glass beads. This process requires alcoholic components (isopropanol and ethanol) both for DNA binding and for the necessary washing steps.The problem lies in the fact that sequencing ultra-long DNA requires so-called transposase fragmentation-based DNA. DNA libraries are produced. After fragmentation of the DNA with transposases, accompanied by the simultaneous insertion of transposon sequences, the adaptors coupled to the so-called motor protein are attached. After the generation of this DNA library, the unattached adaptors must be removed from the library to prevent them from unnecessarily overloading the sequencing pores. This purification must be performed without isopropanol or ethanol, etc., to avoid damaging the motor protein. This means that it is not possible to use known classical purification methods, which are known to be highly efficient and fast (e.g., binding the DNA to solid phases using ethanol or isopropanol and washing buffers containing isopropanol or ethanol).

[0004] For purifying a DNA library for subsequent nanopore sequencing, ONP recommends performing spermine precipitation (approximately 12 hours). They recommend preparing a 16.8 mmol spermine solution and mixing it 1:1 with the prepared DNA library. After a rotator-mixer incubation, the sample is centrifuged to generate the DNA pellet. The pellet should be dissolved in an elution buffer for at least 12 hours. The problem with this method lies in the ratio between the spermine and DNA concentrations. The lower the DNA concentration, the higher the spermine concentration should be (E. Raspaud et al. Precipitation of DNA by Polyamines: A Poly electrolyte Behavior, Biophysical Journal 1998). Thus, even the slightest deviation from the specified DNA concentration leads to an inefficient precipitation reaction and consequently to DNA loss.An increase in spermine concentration then leads to the risk of potentially "blocking" the sequencing pores. Furthermore, a resuspension of the pelleted DNA lasting over 12 hours makes it impossible to prepare the experiment in a single day.

[0005] Alternative methods for purifying a DNA library for nanopore sequencing are described. A kit from Circulomics is commercially available (Matthew W. Mitchell et al. High molecular weight DNA extraction and long-read nextgeneration sequencing of human genomic reference standards. ASHG Virtual Meeting 2020). A Circulomix Nanobind disc is added to the prepared library and bound to the disc using an alcohol-free binding buffer, washed alcohol-free, and then eluted. Here too, a waiting period of at least 12 hours is required before the DNA can be used for sequencing. This kit is very expensive. Furthermore, it cannot It is used for the prior extraction of high-molecular-weight DNA. This requires an additional kit. Furthermore, the process is not automated.

[0006] Another published method for purifying the DNA library is based on the use of hexamminecobalt(III) chloride and glass beads (Inswasti Cahyani et al. FindingNemo in OneDay: Ultra-Long ONT Library Preparation from Cell to Flowcell in One Day, www.protocol.s.io).

[0007] The prior art also includes the two German patent applications DE 10 2017 204 267 Al and DE 10 2015 216 558 Al, as well as the corresponding patent applications derived therefrom WO2018 / 167138 Al, EP3596215 Al, US20200255820 Al, German patent no. DE 10 2017 204 267 B4 on the one hand, and on the other hand, publications WO2016 / 169678 Al and patents US 10,934,540 B2 and EP 3 286 325 AL. DE 10 2017 204 267 Al describes the removal of DNA bound to glass particles using a washing solution. DE 10 2015 216 558 Al discloses the use of a rough surface for binding nucleic acids.

[0008] None of these listed methods, however, provides a comprehensive solution for sample preparation for nanopore sequencing (extraction of high-molecular-weight DNA and purification of the DNA library after tagmentation). This applies to both manual implementation and automated protocols. Object of the invention

[0009] The invention was based on the objective of providing a simple and, above all, rapid method for purifying generated libraries for nanopore sequencing, or more generally, a method and a kit that enable the entire sample preparation for nanopore sequencing (extraction of high-molecular-weight DNA and purification of the DNA library). The method should be capable of being performed both manually and automatically. Solution to the task

[0010] Surprisingly, the problem can be solved ideally using the present invention in accordance with the features of the patent claims.

[0011] The invention preferably utilizes the method of DNA adsorption known from patent documents (US 10,851,368 B2; US 10,704,039 B2; US 10,936,540 B2). on rough or structured surfaces. These patent specifications describe the adsorption of DNA onto rough or structured surfaces of nucleic acid released after lysis in the presence of (preferably) ethanol or isopropanol, the subsequent washing of the nucleic acids with ethanol-containing buffers, and the final detachment of the nucleic acids using a low-salt buffer or water. However, there is no mention of purifying DNA for the production of a library for subsequent nanopore sequencing using the Ultra-Long DNA Sequencing Kit (SQK-ULK001; Oxford Nanopore Technologies). The difficulty here lies in the fact that alcoholic components such as ethanol or isopropanol cannot be used, as these components would lead to the denaturation of the motor protein coupled to the adaptors.Therefore, a method was needed that would allow efficient adsorption of the generated long-chain DNA fragments from the synthesized DNA library without the use of ethanol or isopropanol. The patent specifications cited above always use ethanol or isopropanol for DNA adsorption. Furthermore, the wash buffers must not contain these components. Surprisingly, a combination of polyethylene glycol (a polyether) with a monovalent or multivalent salt is exceptionally well-suited for adsorbing the DNA fragments onto known rough or structured surfaces. Using the method according to the invention, the adsorbed fragments are then washed without the previously used alcohol-containing wash buffers. It turns out that a wash buffer using a Tris solution is entirely sufficient for this purpose. This wash buffer also has the advantage that alcohol removal is no longer necessary.The process, based on the new components according to the invention, is very simple and fast. The same materials known from the cited patents can be used as rough or structured surfaces. For manual operation, magnetic plastic materials of various shapes and sizes with rough surfaces are preferably used. For automated operation, roughened plastic materials are used, which are typically employed in commercially available extraction machines for the separation of magnetic beads. These machines are so-called walk-away platforms that use plastic combs for separating magnetic beads. A globally known system of this kind is the so-called Magnetic Particle Processor "KingFisher" from Thermo. Numerous similar machines exist worldwide, all utilizing the same principle.The inventive method does not require magnetic beads. The plastic combs are sufficient. They are slightly roughened. This then enables the direct adsorption of the DNA fragments onto the plastic combs using the method according to the invention.

[0012] For manual purification of the libraries, the reaction mix is ​​placed in a reaction vessel containing polyethylene glycol and a monovalent or divalent salt. The mixture is then brought into contact with a material having a rough or structured surface, preferably magnetic or paramagnetic. The vessel is carefully inverted several times to avoid shearing the long-chain DNA fragments. The DNA fragments adsorb to the surface. The vessel is placed in a magnetic rack. The rough material is fixed in place, and the supernatant is discarded. A wash buffer without ethanol or isopropanol, preferably a Tris buffer, is then added. The vessel remains in the magnetic rack and is carefully inverted several times. The supernatant is discarded. The washing step is repeated once more. After the final wash, ddEEO (double-distilled water) is added.The vessel containing the rough materials is then incubated for a period of time with gentle shaking to allow the bound DNA fragments to dissolve. After this incubation, the DNA library can be stored or used immediately for nanopore sequencing.

[0013] The entire process thus takes less than an hour and is therefore much faster than previously used methods. In the automated method using walk-away platforms, which utilize plastic combs as described above, purification is also carried out using the reagents described. The adsorption of the DNA fragments to the plastic combs occurs through a slow vertical movement of the comb into the reaction cavity containing the reaction mixture. After the adsorption step, the comb moves into the cavity containing the wash buffer. Washing is performed by briefly immersing the comb in the buffer. The washing step is then repeated. In the final step, the DNA is dissolved in water again. This is achieved through a slow immersion and re-emergence of the comb.The dissolved DNA can then be used immediately for nanopore sequencing or stored in a refrigerator for later use. Automated purification is also completed in less than an hour. The inventive method and composition thus provide a very simple, efficient, and rapid means of purifying DNA fragments from a generated DNA library for subsequent nanopore sequencing. The process can be carried out automatically or manually. Surprisingly, another advantage was revealed when a mixture of polyethylene glycol and monovalent or multivalent salts was used for the adsorption of DNA onto rough surfaces.

[0014] This method is suitable not only for purifying the DNA library but also for extracting high-molecular-weight DNA, which is required for library production. Commercially available products for extracting high-molecular-weight DNA are offered by companies such as NEB and Circulomics. However, the purification of high-molecular-weight DNA is generally labor-intensive and time-consuming. Automated applications are scarcely available commercially. Another significant disadvantage is that the available methods and reagents or kits for extracting high-molecular-weight DNA and for the necessary purification of libraries prepared for sequencing differ. There is no single kit that allows both processes to be performed.

[0015] Based on the present invention, it is possible to isolate high-molecular-weight DNA from a biological sample and purify the resulting library using a single kit. Such a product or process does not yet exist worldwide. Those skilled in the art would be aware of the advantage of having such a solution available. Such a kit requires only additional reagents for sample lysis (lysis buffer and proteolytic enzyme) and, optionally, a wash buffer, which may also contain an ethanol-based component. According to the invention, such a kit for the manual extraction of high-molecular-weight DNA from a biological sample and the subsequent purification of the resulting library would contain the following components: 1. Lysis buffer and proteinase K 2. Polyethylene glycol solution 3. Magnesium chloride solution (as a multivalent salt) 4. optionally an ethanol-based washing buffer 5. Tris solution as a washing buffer without alcoholic components 6. Material with a rough plastic surface that is magnetic (granules or spheres) 7. ddH2O The extraction process is briefly outlined below: Extraction of high molecular weight DNA 1. Lysis of the initial sample (cells, tissue, bacteria, etc.) by adding a lysis buffer and proteinase K. 2. After lysis of the initial sample, a defined amount of polyethylene glycol and magnesium chloride is added, along with the rough material. 3. Carefully invert the reaction vessel several times. 4. Place the vessel in a magnetic rack. Fix the rough material with the adsorbed DNA and pour off the supernatant. 5. Add an ethanol-containing wash buffer and invert the vessel three times, discarding the supernatant. Repeat the step. 6. Add a Tris solution and invert the vessel three times, then pour off the supernatant. 7. Add water and dissolve the DNA by incubation in a thermoshaker at 300 rpm and 37°C for approximately 30 minutes.

[0016] After this time, the high-molecular-weight DNA can be used directly to create a DNA library for nanopore sequencing. It is also possible, of course, to store the DNA properly and use it at a later date.

[0017] For nanopore sequencing using the Ultra-Long DNA Sequencing Kit SQK-ULK001 (Oxford Nanopore Technologies), a defined amount of DNA is used to generate the DNA library. After this step is completed, the library is purified using the inventive method and kit as follows: 1. Addition of a defined amount of polyethylene glycol and magnesium chloride, as well as addition of the rough material to the reaction mixture with the generated library. 2. Carefully invert the reaction vessel several times. 3. Place the vessel in a magnetic rack. Fix the rough material with the adsorbed DNA and pour off the supernatant. 4. Add a Tris solution to wash the adsorbed DNA and invert the vessel three times, discarding the supernatant. 5. Add water and dissolve the DNA by incubation in a thermoshaker at 300 rpm and 37°C for approximately 30 minutes.

[0018] The DNA can now be applied directly to the flow cell for sequencing, but it can also be stored and used at a later time.

[0019] Similarly, the combined extraction of high-molecular-weight DNA and the subsequent purification of the library can be automated. This is achieved using walk-away platforms with rough plastic combs, as previously described. The method is simple and very fast. Furthermore, nanopore sequencing allows for the sequencing of very long fragments, which is a crucial quality characteristic.

[0020] The invention is described below with reference to exemplary embodiments. Examples of implementation Example 1: Manual extraction of high molecular weight DNA from blood for subsequent nanopore sequencing using the Ultra-Long DNA Sequencing Kit SQK-ULK001 (Oxford Nanopore Technologies), including purification of the generated DNA library.

[0021] Three milliliters of a whole blood sample were used. After lysis of the erythrocytes, the resulting cell pellet of nucleated blood cells was resuspended with 130 µl of 1x PBS buffer and transferred to a 2.0 ml reaction vessel. Then, 200 µl of a lysis buffer (Lysis Solution CBV; IST Innuscreen GmbH) and 30 µl of proteinase K were added. Cell lysis was performed at 55°C in a thermomixer for 15 minutes. After successful lysis, the mixture was coated with three paramagnetic plastic spheres with a roughened surface (diameter 3 mm). mm; polypropylene). Then, 200 µl of a 40% polyethylene glycol solution (PEG solution) and 40 µl of magnesium chloride (400 mM) were added. The vessel was then carefully inverted 30 times by 180°. During this step, the high-molecular-weight DNA adsorbed to the rough surface of the paramagnetic beads. The vessel was then placed in a magnetic rack to fix the beads, and the lysis mixture was poured out. Next, a wash buffer (Washing Solution MS; IST Innuscreen GmbH) was added. The vessel remained on the magnetic rack and was inverted three times, and the wash buffer was poured out. The washing step was repeated once more. Then, 800 µl of a Tris solution (100 mM Tris-HCl; pH 8) was added. The vessel was inverted twice, and the Tris solution was poured out.This final washing step with a non-ethanolic solution eliminates the need for a final drying step to completely remove the ethanol. The DNA on the beads was dissolved after adding 200 pl of ddH₂O, with the vessel being incubated for approximately 30 min at 300 rpm and a temperature of 37°C. The high-molecular-weight DNA was transferred to a new vessel using a wide-bore pipette tip, with careful up-and-down pipetting several times. The DNA was analyzed spectrophotometrically, and a quantity of 45 pg of DNA in a volume of 400 pl was used for further nanopore sequencing using the Ultra-Long DNA Sequencing Kit SQK-ULK001 (Oxford Nanopore Technologies). After DNA library preparation, it is necessary to remove excess adaptors. This was subsequently carried out using the method according to the invention.The mixture was again treated with a PEG solution (40%) and magnesium chloride solution (400 mM), as well as with roughened paramagnetic plastic beads. The vessel was then carefully inverted 30 times by 180° to allow the high-molecular-weight DNA to adsorb onto the rough surface of the paramagnetic beads. The vessel was then placed in a magnetic rack to fix the beads, and the reaction mixture was poured out. Next, 800 µl of a Tris solution (100 mM Tris-HCl; pH 8) was added. The vessel was inverted twice, and the Tris solution was poured off. The washing step was repeated once more. It was important during the purification of the DNA library that no alcoholic components were used as wash or binding buffers, as this would destroy the library. The DNA on the beads was dissolved after the addition of 225 µl ddH₂O, for which the vessel was held in place for approximately...The sample was incubated for 45 minutes at 300 rpm and a temperature of 37°C. After DNA extraction, DNA library preparation, and purification, the sample was transferred to the [process / system - context needed]. The Minion device (Oxford Nanopore Technologies) is used to perform long-read sequencing. Using this technology, it has been demonstrated that very high read lengths (up to > 300,000 base pairs) can be achieved using the inventive method, which combines extraction of high-molecular-weight DNA with purification of the DNA library. The entire process is completed in approximately 2 hours, allowing sequencing to begin on the same working day. Furthermore, the method is very easy to perform. Example 2: Automated extraction of high molecular weight DNA from blood for subsequent nanopore sequencing using the Ultra-Long DNA Sequencing Kit SQK-ULK001 (Oxford Nanopore Technologies) including purification of the generated DNA library using a walk-away platform (KingFisher Flex; Thermo)

[0022] Three milliliters of a whole blood sample were used. After lysis of the erythrocytes, the resulting cell pellet of nucleated blood cells was resuspended with 130 µL 1x PBS buffer and transferred to a well of a 96-well deep well plate for processing according to the automated protocol on the KingFisher Flex instrument. In the automated method using walk-away platforms (as in this case with the KingFisher Flex), the high-molecular-weight DNA is adsorbed onto the previously described plastic combs used in these instrument systems. Extraction was also performed using the reagents described for the manual process. After automated sample lysis on the instrument, the DNA fragments were adsorbed onto the plastic combs by a slow vertical movement of the comb within the reaction cavity containing the reaction mixture.After the adsorption step of the high-molecular-weight DNA, the comb successively moved into the deep-well plate cavities containing the wash buffers. During the extraction of the high-molecular-weight DNA, an ethanol-based wash buffer was used. The final wash step was then performed with a Tris solution. The high-molecular-weight DNA was transferred from the deep-well plate cavity to a new tube using a wide-bore pipette tip, with several careful pipetting operations. The DNA was analyzed spectrophotometrically, and a quantity of 45 pg of DNA in a volume of 400 pl was used for further nanopore sequencing using the Ultra-Long DNA Sequencing Kit SQK-ULK001 (Oxford Nanopore Technologies). After successful DNA sequencing... Library preparation was then carried out using the reagents already listed in Example 1, along with automated purification of the DNA library to remove excess adaptors. During automated purification of the DNA library, washing is performed only with the Tris solution. Washing is carried out by briefly immersing the comb in the respective buffer. In the final step, the DNA is then reconstituted in water. This is done by a slow immersion and resuspension motion of the comb. After the automated extraction of high-molecular-weight DNA, the preparation of the DNA library, and the automated purification of the DNA library, the sample is transferred to the Minion instrument (Oxford Nanopore Technologies) for long-read sequencing.Using this technology, it has been demonstrated that very high read lengths (up to > 300,000 base pairs) can be achieved by combining automated extraction of high-molecular-weight DNA with purification of the DNA library according to the invented method. The entire process is completed in approximately 2 hours, allowing sequencing to begin on the same working day. Furthermore, the method is very easy to perform. Definitions: Monohydric alcohol: Alcohols with only one OH group, such as ethanol or isopropanol Polyether: Long-chain compounds of the type hot polyethers (also polyalkylene glycols, Polyether polyols, polyalkylene oxides). Examples of this group of polymeric ethers are polyethylene glycol and polypropylene glycol. PEG 6000 or PEG 8000 means that n = 6000 or 8000, respectively. TRIS: Tris(hydroxymethyl)aminomethane (abbreviated TRIS or THAM), also called tromethamine, trometamol (INN), and TRIS buffer. Chemically, it is a primary amine with three alcoholic hydroxyl groups. Preferably, its salt (hydrochloride) is used with a pH of 7–9, preferably pH 8. High molecular weight DNA: DNA with a length greater than or equal to 100,000 bp.

Claims

Patent claims 1. A method for extracting high molecular weight DNA from a biological sample and / or purifying a DNA library, characterized in that, after optional lysis of the biological sample and / or after preparation of the DNA library, the mixture is brought into contact with a combination of a polyether and at least one salt in the absence of monohydric alcohols, wherein the DNA is subsequently bound to a solid phase and the DNA is then purified without the use of monohydric alcohols.

2. Method according to claim 1, characterized in that polyethylene glycol (PEG), preferably PEG 6000 or PEG 8000, is used as the polyether.

3. Method according to claim 1 or 2, characterized in that MgCh is used as the salt, preferably in a concentration between 1 and 100 mM, in particular between 5 and 50 mM.

4. Method according to one of claims 1 to 3, characterized in that polyether and salt are present together in a buffer in corresponding concentrations.

5. Method according to one of claims 1 to 4, characterized in that a material with a rough or structured surface is used as the solid phase.

6. Method according to any one of claims 1 to 5, characterized in that a washing buffer without monohydric alcohol is used for the purification of the bound DNA, an amino alcohol, preferably TRIS.

7. Method for extracting high molecular weight DNA from a biological sample, characterized by the following steps: a) Lysis of the biological sample b) Addition of polyethylene glycol and magnesium chloride as well as addition of the rough material c) Fixation of the rough material with the adsorbed DNA and casting off of the supernatant d) Addition of a wash buffer e) Addition of water and dissolution of the DNA. Method for purifying a DNA library, characterized by the following steps: a) Addition of polyethylene glycol and magnesium chloride, and addition of the rough Materials for the reaction with the generated library b) Fixation of the rough material with the adsorbed DNA and pouring off the supernatant c) Addition of a wash buffer without monohydric alcohol d) Addition of water and dissolution of the DNA Automated method according to one of claims 1 to 8, wherein the lysis reaction or the reaction with the generated library is located in a walk-away extraction device which uses plastic rods having a rough or structured surface on which the DNA is adsorbed by vertical movement of the plastic combs within the sample upon addition of polyethylene glycol and magnesium chloride and these plastic combs move according to the walk-away principle into vessels with the wash buffer and finally into the aqueous solution.Kit for manually performing the method according to any one of claims 1 to 8, comprising: a) lysis buffer b) polyethylene glycol solution c) magnesium chloride solution d) Tris solution as a wash buffer e) material with a rough surface made of plastic that is magnetic (granules or spheres) f) ddH2O. Use of the method according to any one of claims 1 to 9 for subsequent nanopore sequencing.

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