SEMI-SOLID STATE NUCLEIC ACID MANIPULATION

Abstract

The invention pertains to a method for isolating a nucleic acid, wherein the nucleic acid is stabilized in a hydrogel. The hydrogel can be dissolved to release the nucleic acid without breaking the molecule. A preferred hydrogel is alginate. The invention further concerns a method for sequencing the nucleic acid and a composition comprising the hydrogel and the nucleic acid.

Claims

1. A method for obtaining a hydrogel comprising a long manipulated nucleic acid, the method comprising: (a) combining a nucleic acid with an aqueous polymer solution; (b) gelling the polymer solution to form a hydrogel comprising the nucleic acid; and (c) manipulating the nucleic acid in the hydrogel, wherein the hydrogel can be dissolved at a temperature below 45° C.

2. The method according to claim 1, wherein the nucleic acid is provided in a carrier.

3. The method according to claim 2, wherein the carrier is at least one of an organelle and a cell.

4. The method according to claim 3, further comprising (c) lysing the organelle or to release the nucleic acid.

5. The method according to claim 1, wherein the polymers in the aqueous solution are at least one of: (i) ionic polymers, preferably anionic polymers having carboxylic pendant groups; and (ii) polysaccharides or derivatives thereof.

6. The method according to claim 5, wherein the polysaccharides or derivatives thereof comprise uronic acid.

7. The method according to claim 5, wherein the polysaccharides or the derivatives thereof are an alginate or a derivative thereof.

8. The method according to claim 2, wherein the carrier is a mitochondrion, a chloroplast, a nucleus, a plant cell, or a protoplast.

9. The method according to claim 1, wherein the nucleic acid is a DNA molecule.

10. The method according to claim 1, wherein the long manipulated nucleic acid is an isolated ultra-high molecular weight (uHMW) nucleic acid

11. The method according to claim 1, wherein the long manipulated nucleic acid is stabilized in a hydrogel microsphere.

12. A method for preparing a sequencing library, comprising: (a) obtaining a hydrogel comprising a long manipulated nucleic acid according to claim 1; and (b) modifying the nucleic acid in the hydrogel to obtain a sequencing library.

13. A sequencing method, comprising: (a) obtaining a sequencing library according to claim 12; (b) dissolving the hydrogel, preferably at a temperature below 45° C.; and (c) sequencing the library.

14. The method according to claim 13, wherein the hydrogel is dissolved at a temperature below 45° C.

15. The method according to claim 14, wherein the sequencing library is loaded on a sequencer flow cell before dissolving the hydrogel.

16. The method according to claim 13, wherein the hydrogel is dissolved by at least one of: (i) addition of a sequencing buffer; (ii) addition of a buffer comprising monovalent cations; (iii) lowering the temperature from about 20° C.-40° C. to about 2° C.-10° C.; and (iv) adjusting the pH from about 5-6 to about 7-8, or from about 7-8 to about 5-6.

17. The method according to claim 16, wherein the monovalent cations are sodium cations.

18. A method for obtaining a hydrogel comprising a nucleic acid-comprising carrier, the method comprising: (a) combining the nucleic acid-comprising carrier with an aqueous polymer solution; and (b) gelling the polymer solution to form the hydrogel comprising the nucleic acid-comprising carrier, wherein the hydrogel is a hydrogel according to claim 1.

19. The method according to claim 18, wherein the nucleic acid-comprising carrier is an organelle and wherein the method further comprises lysing a cell to obtain the organelle.

20. A hydrogel comprising at least one of a nucleic acid-comprising carrier, and an organelle and a long manipulated nucleic acid, wherein the hydrogel can be dissolved at a temperature below 45° C.

21. A kit of parts for obtaining a hydrogel comprising a long manipulated nucleic acid, wherein the kit comprises: (i) a polymer for forming a hydrogel according to claim 20; (ii) a cell and/or organelle lysis buffer; and (iii) optionally, one or more components for preparing a sequencing library.

Description

FIGURE LEGEND

[0253] FIG. 1. Exemplary embodiments of the invention. (FIG. 1A) Embodiment depicting encapsulation of nuclei in hydrogel beads followed by nuclei lysis and purification of the nucleic acids after gelling of the hydrogel. Step 1) Nucleic acids in a biological carrier, such as cells or nuclei, 2) Encapsulation of the biological carrier in a hydrogel, 3) Reversible gelling of the hydrogel and lysis of the biological carrier, 4) Purification and manipulation (e.g. sequence library preparation) of nucleic acids, 5) De-gelling and recovery of nucleic acids, 6) Further processing of nucleic acids (e.g. sequencing) (FIG. 1B) In another exemplary embodiment, nucleic acids (1) can be directly encapsulated in the hydrogel (2) followed by reversible gelling of the polymer and manipulation of the nucleic acids (3). Step 4) is the de-gelling and recovery of nucleic acids and 5) further processing of the nucleic acids. (FIG. 1C) Nucleic acids may also be associated in or to a carrier other than cells or organelles, for example artificial beads or scaffolds. Step 1) Presence of nucleic acids in or to a carrier other than biological cells or organelles, 2) encapsulation of nucleic acid-carrier complex in a hydrogel, 3) reversible gelling of the hydrogel and manipulation of the nucleic acids, 4) de-gelling and recovery of nucleic acids and 5) further processing of nucleic acids. In the shown exemplary embodiments, the trapped nucleic acids can be semi-solid state-based (enzymatically) manipulated (for example, library preparation) followed by further processing upon or after de-gelling of the hydrogel. Microspheres containing the sequence library can be “loaded” directly in the flow cell followed by dissolving the particles. The released library DNA molecules become accessible to the sequencing process

[0254] FIG. 2. Percentage of genomic DNA loss by diffusion from the hydrogel beads in the different buffers used during semi-solid state Nanopore library preparation of Example 1 and 2. Indicated is the amount of relative DNA recovered from each discarded buffer used during Nanopore library preparation incubation, i.e. buffer solution used for washing prior to DNA repair and end-preparation (1), buffer comprising enzymes for DNA repair and end preparation (2), MQ for washing after DNA repair and end preparation (3), ligation buffer for washing prior to ligation (4), ligation buffer comprising ligase and adapters for adapter ligation (5) and elution buffer for equilibration (6). The amount of input DNA used for encapsulation is set at 100%. In all fractions and for all Examples, almost no DNA was lost in the aqueous solutions indicating no or a very low level of diffusion of encapsulated DNA from the hydrogel in the solution throughout library preparation, with the highest percentage of loss during the step of adapter ligation (see, inset graph), however, size determination showed that this was unligated adapter DNA (data not shown).

[0255] FIG. 3. Read length distribution of all reads that mapped against a reference genome. On the x-axis, all individual reads that map against the reference are given. The y-axis shows the read length in bases.

[0256] FIG. 4. Schematic presentation of microfluidic-based synthesis of hydrogel microspheres comprising nucleic acid-comprising carriers, wherein the first reactant comprises nucleic acid-comprising carriers and the second reactant comprises the aqueous polymer solution. An optional third reactant may comprise a gelation inducer. 1A) Dispersed phase, e.g. cells, organelles, nucleic acids, biomolecules, 1B) Dispersed phase, aqueous polymer solution, 2) Continuous phase, e.g. oil-surfactant emulsion, 3) Collection phase.

[0257] FIG. 5. Pulse-field gel electrophoresis of uHMW genomic plant DNA. FIG. 5A.) Lane 1: Nuclear DNA isolated after lysis of sugar beet nuclei and further purification in an aqueous environment. Lane 2: Saccharomyces cerevisiae chromosomal pulse-field marker with the size of the chromosomes given in kb. Molecular weights of individual fragments are indicated next to the gels. FIG. 5B.) Lane 1: Saccharomyces cerevisiae chromosomal pulse-field marker. Lanes 2, 4 and 5: Genomic DNA derived from sugar beet, nuclei that were encapsulated and lysed in alginate spheres. Lane 3: Genomic DNA derived from Impatiens, nuclei that were encapsulated and lysed in alginate spheres. In lane 2 and 3, alginate spheres containing uHMW DNA were loaded directly in the pulse-field gel prior electrophoresis. In lane 4 and 5, the uHMW DNA was released from the alginate spheres by addition of sodium citrate to the wells.

Examples

[0258] Example 1a and b: Encapsulation of high molecular weight bacteriophage Lambda DNA in alginate, followed by semi-solid state library preparation and Nanopore sequencing

Example 1a

[0259] 10 μL Escherichia virus Lambda (λ) DNA (total amount of DNA: 883 ng) with a median size of 45 Kb and a concentration of 82.4 ng/μL was mixed overnight with 10 μL 1.5% Pronova® UP LVG alginate (NovaMatrix™) on a rotary platform at 4° C. After incubation, the alginate solution was pipetted into a 2 ml BD Plastipak™ syringe (BD) using a 200 μL wide bore pipette tip to avoid shearing of the DNA. An 0.45×13 mm microlance (BD) was attached to the syringe and the alginate solution was slowly dripped into a 100 mL beaker glass containing 20 mL of a 200 mM CaCl.sub.2 solution. To obtain round-shaped, millimetre-sized beads, the CaCl.sub.2 solution was stirred vigorously by placing the beaker glass with a magnetic rod on a magnetic stirrer plate prior dripping of the alginate solution. With this dripping method, about one to two beads could be generated using 20 μL 0.7% alginate-DNA mixture.

[0260] After gelling, the beads containing the encapsulated A DNA were transferred to a 2-mL Eppendorf tube and were incubated for 30 minutes in 60 μL DNA repair and end-preparation mixture (without enzymes) containing 17.1× diluted NEBNext® FFPE repair and Ultra II End-prep reaction buffers (NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing, New England Biolabs Inc.) for washing prior to DNA repair and end-preparation. The NEBNext® buffer solution was discarded by pipetting.

[0261] Subsequently, for DNA-repair and end preparation, the beads were incubated for 30 minutes at 20° C. and 10 minutes at 65° C. in 60 μL NEBNext® FFPE repair and Ultra II End-prep reaction mixture containing 17.1× diluted NEBNext® FFPE repair and Ultra II End-prep reaction buffers, 30× diluted NEBNext® FFPE DNA Repair Mix, and 20× diluted NEBNext® Ultra II End-prep Enzyme Mix (NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing, New England Biolabs Inc.).

[0262] After DNA repair and end preparation, the beads were washed in 500 μL nuclease-free water followed by a 30 minutes incubation at 20° C. in a fresh batch of 500 μL nuclease-free water. The water was discarded by pipetting and the beads were incubated for 30 minutes in 100 μL adapter ligation mixture (without enzymes and without adapters) containing 4× diluted Ligation Buffer (SQK-LSK109 Ligation Sequencing Kit, Oxford Nanopore Technologies) prior to adapter ligation. The ligation buffer solution was discarded by pipetting and the beads were incubated for 2 hours at 4° C. in 100 μL adapter ligation mixture containing 4× diluted Ligation Buffer (SQK-LSK109 Ligation Sequencing Kit, Oxford Nanopore Technologies), 10× diluted NEBNext® Quick T4 DNA Ligase (NEBNext® Companion Module for Oxford Nanopore Technologies Ligation Sequencing, New England Biolabs Inc.) and 20× diluted Adapter Mix (SQK-LSK109 Ligation Sequencing Kit, Oxford Nanopore Technologies) for adapter ligation.

[0263] After adapter ligation, the beads were equilibrated for 30 minutes at room temperature in 50 μL Elution Buffer (SQK-LSK109 Ligation Sequencing Kit, Oxford Nanopore Technologies). Meanwhile, a R9.4.1 flow cell (Oxford Nanopore Technologies) was primed according the manufacturer's instructions (Nanopore protocol Genomic DNA by Ligation, version GDE_9063_v109_revN_14Aug2019, Oxford Nanopore Technologies). Following equilibration of the beads, the Elution Buffer was discarded and the beads were incubated for 30 minutes on ice in 75 μL 2× diluted Sequencing Buffer (SQK-LSK109 Ligation Sequencing Kit, Oxford Nanopore Technologies).

[0264] Incubation of the alginate beads in Sequencing Buffer caused de-gelling of the alginate bead. The alginate slurry containing the λ DNA 1D sequencing library was loaded via the SpotON sample port in the R9.4.1 flow cell (FAL16833) and the sequencing run was started on a GridION sequencer (MinKNOW version 3.4.8). The raw sequence data was base called with Guppy version 3.0.6 (Oxford Nanopore Technologies®) and the base called sequence data was further processed with NanoPack tools (De Coster et al., 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34-15).

Example 1b

[0265] In Example 1 b, the semi-solid state library was prepared as described above, with the exception that 200 mM sodium citrate was added to the sequencing buffer, i.e. in the last step, the beads were incubated in 75 μL 2× diluted Sequencing Buffer (SQK-LSK109 Ligation Sequencing Kit, Oxford Nanopore Technologies) and 200 mM sodium citrate. The de-gelled alginate slurry with the A DNA library was loaded in a new R9.4.1 flow cell (FAL02990) and the sequencing was performed on a GridION sequencer.

[0266] Summary statistics of the two 42 hours λ DNA sequence runs are presented in Table 1.

TABLE-US-00001 TABLE 1 NanoPlot analysis of MinION sequencing data. Example 1a Example 1b Mean read length (bp) 13,654.4 8,128.9 Mean read quality 7.4 7 Median read length (bp) 3,292 702 Median read quality 7.3 3.8 Number of reads 97,693 39,999 Read length N50 (bp) 45,303 34,117 Total bases 1,333,941,793 325,149,673
Sequencing of the semi-solid state prepared Example 1a λ DNA library has produced more than one gigabase of data represented by about 98,000 reads. The median read length is 3.3 Kb with a median read quality of 7.3. In contrast, the sequence yield of the λ DNA sequence run of Example 1b is more than fourfold lower (about 325 megabases) and median read length is even smaller than 1 kb (702 bases). The differences in sequence metrics between both examples is due to the differences in the post-library preparation treatment of the large beads; i.e. the de-gelling and loading of the alginate in sequence buffer (Example 1a) or sequence buffer with 200 mM sodium citrate added (Example 1b).

[0267] In order to investigate that the lambda sequence reads originated from entrapped DNA and not from DNA that has been diffused out the beads into the aqueous solutions during the library preparation steps, DNA quantity was measured after each incubation step. FIG. 2 shows the relative DNA loss by diffusion in the different washing and enzymatic steps during library preparation. DNA was isolated from the used buffers and enzyme mixes with Ampure XP beads and the quantity was determined with a Qubit fluorometer (High Sensitive dsDNA Assay kit, ThermoFisher Scientific). As is shown in FIG. 2, no DNA was detectable in the different aqueous solutions throughout the entire Nanopore library preparation procedure. After incubation in the ligation enzyme mixture, containing also the sequence adapters, a low amount of low molecular weight (˜200 bp) DNA could be observed. However, based on the size of the fragments and the composition of the ligation mixture, we conclude that the recovered DNA are non-ligated Nanopore adapters.

[0268] In summary, the results show that the present invention provides for a useful platform for preparation of long read sequence libraries starting from encapsulated HMW DNA in semi-solid state.

Example 2: Encapsulation of High Molecular Weight Plant DNA in Alginate, Followed by Semi-Solid State Library Preparation and Nanopore Sequencing

[0269] For example 2, the same experimental procedure was followed as described for example 1a, with the difference of mixing 993 ng (10 μL) genomic plant DNA with a median peak size of about 80 kb and a size range of 10 to 128 kb with the alginate. The alginate slurry containing the plant DNA 1D sequencing library was loaded via the SpotON sample port in the R9.4.1 flow cell (FAK94960). Sequencing was performed on a GridION (MinKNOW version 3.4.8) and the raw sequence data was base called with Guppy version 3.0.6 (Oxford Nanopore Technologies®). Further analyses of the base called sequence data was done with NanoPack tools (De Coster et al., 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34-15).

Summary statistics of the 42 hours sequence run are presented in Table 2.

TABLE-US-00002 TABLE 2 NanoPlot analysis of MinION sequencing data Example 2 Mean read length (bp) 4,025 Mean read quality 4.7 Median read length (bp) 577 Median read quality 3.4 Number of reads 55,165 Read length N50 (bp) 26,126 Total bases 222,040,158

[0270] Sequencing of the semi-solid state prepared plant DNA library resulted in slightly more than 222 Mb of data represented by 55,165 reads. These results demonstrate the possibility for preparation and sequencing of long read sequence libraries starting from encapsulated ultra HMW DNA in semi-solid state.

[0271] Quantification of the amount of lettuce DNA in the different aqueous solutions during library preparation showed that, like for the lambda DNA examples, no diffusion has occurred during incubation of the hydrogel beads. Therefore, we conclude that the sequence reads obtained were derived from encapsulated DNA and were generated by means of semi-solid state library preparation. As was the case for the lambda Example, the small amount of DNA that was recovered in step 5 was adapter DNA.

Example 3: Encapsulation and Lysis of Plant Nuclei in Alginate, Followed by Semi-Solid State Library Preparation and Nanopore Sequencing of the Embedded DNA

[0272] Nuclei were isolated from young leaf tissue essentially following the instructions described for plant, algal or fungal tissues in Zhang et al., 2012 (Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research, Nature Protocols, vol. 7 (3): 467-478). After the final wash step, the nuclei were resuspended in a 1% alginate (A0682 alginic acid sodium salt from brown algae, Sigma-Aldrich) solution containing 0.5× phosphate buffered saline (PBS; 69 mM NaCl, 5 mM phosphate, 1.4 mM KCl, pH 7.4), 1 mM CaCl.sub.2, and 5 mM MgCl.sub.2.

[0273] After complete resuspension of the nuclei, the suspension was pipetted into a 2 ml BD Plastipak™ syringe (BD) with an 0.45×13 mm microlance (BD) attached using a 200 μL wide bore pipette tip to avoid damage of the nuclei. Subsequently, the alginate solution was slowly dripped into a 100 mL beaker glass containing 20 mL of a 200 mM CaCl.sub.2 solution under stirring conditions. The resulting millimetre-sized alginate beads containing plant nuclei were further incubated for 30 minutes in the 200 mM CaCl.sub.2 solution without stirring to facilitate further gelling of the alginate. After incubation of the bead in propidium iodide, a large number of fluorescent nuclei could be observed.

[0274] After complete gelling of the alginate, the beads were collected using a 500 μm Pluristrainer (PluriSelect Life Science) and transferred to a new 50 mL polypropylene tube containing 20 mL of lysis solution containing TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0), 50 mM CaCl.sub.2 and 300 μg/mL proteinase K and the nuclei were lysed overnight at 50° C. in a shaking incubator with an orbital agitation set at 75 rpm.

[0275] Following lysis, the beads containing the entrapped genomic DNA were washed twice for 30 minutes in fresh 20 mL wash solutions (TE buffer and 50 mM CaCl.sub.2). Complete deactivation of proteinase K was established by overnight incubation at 37° C. in a 20 mL solution containing TE buffer and 2 mM Pefabloc® SC (Sigma-Aldrich). The Pefabloc® SC was removed by incubating the beads twice in 20 mL TE buffer and 50 mM CaCl.sub.2 solution and the beads containing the encapsulated genomic DNA were stored in the same solution at 4° C. until use.

[0276] For nanopore library preparation, 4 beads were incubated for 30 minutes in 30 μL DNA repair and end-preparation mixture containing 8.6× diluted NEBNext® Ultra II End-prep reaction buffer (NEBNext® Ultra™ II End Repair/dA-Tailing Module, New England Biolabs Inc.), 0.83× NAD+ (PreCR® Repair Mix, New England Biolabs Inc.) and 5 mM CaCl.sub.2. The solution was discarded by pipetting and the beads were incubated for one hour at 20° C. and 20 minutes at 65° C. in 60 μL NEBNext® FFPE repair and Ultra II End-prep reaction mixture containing 8.6× diluted NEBNext® Ultra II End-prep reaction buffer (NEBNext® Ultra™ II End Repair/dA-Tailing Module, New England Biolabs Inc.), 0.83× NAD+ (PreCR® Repair Mix, New England Biolabs Inc.), 5 mM CaCl.sub.2, 20× diluted NEBNext® FFPE DNA Repair Mix (New England Biolabs Inc.), and 1.3× diluted NEBNext® Ultra II End-prep Enzyme Mix (NEBNext® Ultra™ II End Repair/dA-Tailing Module, New England Biolabs Inc.).

[0277] Following DNA repair and end preparation, the beads were washed with 1 mL 10 mM Tris-HCl and 5 mM CaCl.sub.2 solution and subsequently incubated for 15 minutes incubation at 20° C. in a fresh batch of 10 mM Tris-HCl and 5 mM CaCl.sub.2 solution. This incubation step was repeated once. After the second incubation step, the solution was discarded and the beads were incubated for 30 minutes at 20° C. in 50 μL adapter ligation mixture containing 2× diluted NEBNext® Ultra II Ligation Master Mix (NEBNext® Ultra™ II Ligation Module, New England Biolabs Inc.), 50× diluted NEBNext® Ligation Enhancer (NEBNext® Ultra™ II Ligation Module, New England Biolabs Inc.), 10 μL Oxford Nanopore Technologies Adaptor Mix (SQK-LSK108 Ligation Sequencing Kit 1D, Oxford Nanopore Technologies), and 5 mM CaCl.sub.2. After incubation, the ligation mixture was discarded and the ligation step was repeated with a fresh ligation mixture.

[0278] Following adapter ligation, the beads were equilibrated for two times 30 minutes at room temperature in 25 μL and 50 μL Elution Buffer (SQK-LSK108 Ligation Sequencing Kit 1D, Oxford Nanopore Technologies), respectively. Meanwhile, a R9.4 flow cell (ID FAH05684, Oxford Nanopore Technologies) was primed according the manufacturer's instructions (1D gDNA selecting for long reads, SQK-LSK108, Oxford Nanopore Technologies). After the equilibrations in the elution buffers, the beads were incubated for 30 minutes at 20° C. in 75 μL pre-sequencing mix containing 35 μL Running Buffer Fuel (RBF; SQK-LSK108 Ligation Sequencing Kit 1D, Oxford Nanopore Technologies), 4 μL Elution Buffer (SQK-LSK108 Ligation Sequencing Kit 1D, Oxford Nanopore Technologies), 25.5 μL Library Loading Beads (EXP-LLB001 Library Loading Beads Kit, Oxford Nanopore Technologies), and 10 μL 1.5 M sodium citrate. The beads were incubated for 30 minutes at 20° C. and the slurry was loaded using a wide-bore pipet via the SpotON sample port in the R9.4 flow cell and the sequencing run was started on a MinION MK 1 b sequencer.

[0279] Sequencing of the semi-solid state prepared plant DNA library resulted in 1,448 reads that could be mapped (Minimap2) against a reference whole genome sequence (FIG. 3). More than ten percent of the reads mapped with a similarity equal to or greater than 90%. These results show the possibility for preparation and sequencing of long read sequence libraries in a semi-solid state fashion starting from encapsulated plant nuclei.

Example 4: Effect of Encapsulation and Lysis of Plant Nuclei in Alginate on DNA Size

[0280] The effect of protecting nuclear DNA in alginate spheres was further analysed. To this end, genomic DNA was obtained after lysis of sugar beet nuclei and further purification in an aqueous environment. The fragment size of this conventionally isolated DNA was compared with the fragment size of genomic DNA isolated from alginate-embedded nuclei.

[0281] For the alginate-embedded samples, sugar beet and impatiens nuclei were encapsulated and lysed in alginate spheres according to the procedure described in Example 3. The alginate-embedded μHMW DNA was either loaded directly on a gel, or was first released from the alginate spheres and subsequently loaded on a gel.

[0282] As shown in FIG. 5, a gentle isolation procedure in solution typically results in genomic DNA with a size range between 50 to 300 kb. In stark contrast, lysing sugar beet or impatiens nuclei in alginate spheres results in DNA ranges between 200 kb and megabase-size with the majority of fragments between 200 and 800 kb. Lysing the nuclei in alginate thus significantly protects the genomic DNA from break down.