WO2006125458A1

WO2006125458A1 - Parallel sequencing of transformed nucleic acids in encapsulated cells

Info

Publication number: WO2006125458A1
Application number: PCT/EP2005/005768
Authority: WO
Inventors: Martin Held; Ilia Leviev; Josef P. Magyar; Sven Panke; Rene Pellaux; Marta Maria Reinoso-Garcia; Eve Silverberg; Marcel Walser
Original assignee: ETH Zürich
Priority date: 2005-05-27
Filing date: 2005-05-27
Publication date: 2006-11-30

Abstract

The present invention relates to a method for sequencing nucleic acids, comprising the provision of one or more capsules, each comprising one or more identical transformed cell(s) with at least one copy of a transformed nucleic acid therein, lysis of the cell(s) and subsequent sequencing of the transformed nucleic acid(s) in the capsule. A further aspect is directed to the use of these methods for sequencing transformed nucleic acids.

Description

Parallel sequencing of transformed nucleic acids in encapsulated cells

Background of the invention

The sequencing of large nucleic acid fragments such as genomic DNA is very labour intensive and time consuming because the size of these nucleic acid sequences commonly requires fragmentation, multiplication of the fragments, sequencing of the fragments and later assignment of the_. fragments to the sequence. In a typical approach (Sanger sequencing) genomic DNA is fragmented, for example, by restriction enzymes, subsequently cloned into vectors such as plasmids, cosmids, or artificial chromosomes (YACs, BACs, PACs) and said vectors are transformed into cells. From these transformed cells clones are then selected, propagated, harvested and the isolated nucleic acid is sequenced after cell lysis and purification.

For large nucleic acid sequences such as genomic DNA thousands up to many millions of fragments are generated and subjected to the above procedure. Before the final sequencing step the cell clones are either propagated or the DNA is isolated and amplified to provide sufficient starting material for the sequencing reaction.

Due to the high sample throughput in the bio- and life science industries microencapsulation is a frequently employed technology, especially for screening purposes and highly parallelized syntheses. Applications are as diverse as isolating substances or microbial isolates from nature (K. Zengler et al. (2002) Cultivating the uncultured, Proceedings of the National Academy of Sciences of the United States of America (99), 15681-15686), screening for new drug candidates (US 2003 027221), or enzyme activities (US 2002 150949). Depending on the specific application agarose, alginate, carrageenan, acrylamides or other materials that can be reacted to form gel-like structures are frequently found appropriate for enclosing the matter of interest such as cells, enzymes, particles and the like. Said technologies are sometimes also referred to as gel microdroplet encapsulation.

In the life and biosciences it is especially applications in the field of genomic research that frequently suffer from extremely high sample loads. Currently, most genomic applications rely on microtiter (MT) plates as reaction compartments. The standard format used by large nucleic acid sequencing facilities such as the Joint Genome Institute or the US Department of Energy (DOE) are MT-plates with 384 wells. Although these plates are appropriate for handling sample volumes of only a few microlitres, there still remains plenty of room for the improvement of genomic approaches by further downscaling of the reaction volumes.

For instance, US patent application 2003/0207260 discloses a method for nucleic acid analysis, wherein large biological entities such as singular cells, viruses and chromosomes are microencapsulated and the nucleic acids thereof are subsequently analyzed or detected by hybridisation in a highly parallelized fashion. Hence, this is a powerful technology for resequencing applications. A hybridized probe identifies the encapsulated nucleic acids directly by attaching a label and facilitates the selection of microcapsules with nucleic acids of interest. This method allows for the simultaneous analysis of very large numbers of cells, viruses or chromosomes and can detect altered genotypes within large populations. An additional benefit is that a permeable matrix of the capsule permits hybridisation in free solution and, thus, improves reaction rates. The encapsulated nucleic acids can optionally be fragmented and/or amplified within the capsule microcompartment. Libraries of encapsulated cells, viruses and chromosomes can be stored, assayed by hybridisation, or the nucleic acid content can be released from the capsule by digestion and be further processed, for example, by sequencing them after cloning into vectors such as YACs, BACs or PACs followed by sequencing or genome mapping.

Similarly, microcapsules were suggested as vessels for parallel nucleic acid amplification in EP1488006. For that purpose purified nucleic acids are encapsulated together with the compounds required for amplification, e.g. enzymes, dNTPs, etc. If the capsule allows for permeation, then these or other compounds can be added after the encapsulation and also be removed during the subsequent purification. Supposedly, this method produces individual microcapsules with amplified nucleic acids therein. Upon selection and degradation of a capsule the nucleic acid content may be analysed.

Even though the before mentioned microcapsule amplification technology can be employed for genomic analysis and genome assembly the system has a number of drawbacks. First of all, for providing one capsule with one nucleic acid type only, nucleic acids have to be efficiently diluted prior to gel formation. Depending on the requirements, a large number of empty microdroplets will have to be produced. Secondly, the sequence fidelity for parallel amplification with universal primers crucially depends on the concentration of the number of nucleic acids of interest within a single microdroplet. If the concentration of the nucleic acid species of interest in a microdroplet is one (1) nucleic acid species only, amplification and/or subsequent sequencing of said molecule is hardly possible when enzyme kinetics are considered, unless extremely small microdroplets are produced.

At present, there is a need for more efficient techniques that overcome the limits posed by enzyme kinetics. In addition, there is also a need in the art for methods for the convenient, economical and simultaneous sequencing of numerous, even millions of nucleic acids.

The above objects are solved by a method for sequencing nucleic acids, comprising:

(a) providing one or more capsules, each comprising one or more identical transformed cell(s) with at least one copy of a transformed nucleic acid therein, (b) lysis of the cell(s) in the capsule, and

(c) sequencing the transformed nucleic acid(s) after cell lysis in the capsule.

In preferred embodiments the present invention allows for the large scale, simultaneous sequencing of large numbers of nucleic acids in one suspension batch by concomitantly making efficient use of enzyme-catalyst resources and minimizing aberrant reactions of nucleic acids. For example, the starting material can be nucleic acid fragments obtained by the isolation of genomic nucleic acids and optional fragmentation thereof. The origin and type of the nucleic acids is uncritical. The size of the nucleic acid or fragment thereof should be selected to suit the needs of the cell and the needs of the later sequencing techniques. However, the size may be optimized later within the capsule where nucleic acids may be processed after encapsulation and cell lysis. To allow a one batch approach and also to avoid any aberrant sequencing of cellular nucleic acids it is preferred that the transformed nucleic acids of each capsule share identical sequence information that allows for the specific attachment of the primers used for sequencing and/or optional amplification.

The term "transformed nucleic acid" as used in the context of the present invention relates to the heterologous nucleic acid(s) of interest within the cell that is to be sequenced within the microcapsule. Hence, a transformed cell is one comprising one or more transformed nucleic acid(s). Transformed cell(s) within the same capsule should be identical in genetic make-up, quality and quantity of transformed nucleic acids.

The term "capsule" as used in the context of the present invention is meant to refer to a globular gel-like particle capable of encapsulating compounds such as nucleic acids, cells, reactants, etc, thereby providing a microenvironment from which some or all of the reactants may migrate or which they may enter by diffusion while cells and polynucleotides are preferably retained until lysis of the capsule. Preferably, said capsules have a homogeneous structure with respect to the same component(s) they are comprised of.

The presence of one transformed cell with at least one copy of a transformed nucleic acid in the capsule is sufficient to practice the present invention.

It is preferred that the transformed cell comprises at least two copies of a nucleic acid. The transformed nucleic acids in the transformed cell(s) may be the same or different.

Preferably, the transformed nucleic acid for sequencing is already present in the microcapsule in sufficient numbers to allow efficient sequencing and/or amplification.

More preferably, the transformed nucleic acid is already present in the cell in sufficient numbers to allow sequencing after cell lysis. For example, multiple copies may be present in the genome or plasmid(s) of the encapsulated cell.

If the copy number of the transformed nucleic acid is low, e.g. too low to reliably produce sufficient amounts of product to allow its analysis after linear enzymatic reactions, such as sequencing reactions, it is preferred that an additional step of multiplying the transformed nucleic acid(s) prior and/or after the lysis of the cell(s) in the capsule is performed.

In another preferred embodiment, the transformed nucleic acid(s) is (are) multiplied by the propagation of the transformed cell within the microcapsule before cell lysis. Alternatively, multiplication can be accomplished by the propagation of phages or viruses. The term "propagation of a cell", as used herein, is meant to encompass the propagation of the cell itself or cellular components within the cell such as phages or viruses. For multiplication by cell propagation care must be taken that either the substances for propagation are already located within the capsule or that the capsule is permeable and allows permeation of those substances necessary for propagation such as growth factors, nutrients, etc. Permeable capsules are generally preferred because they do not only allow the addition of substances to the microcompartment of the capsule but also purification as well as separation and disposal of catabolites. Of course, propagation must either be halted before the cell mass endangers the integrity of the capsule or disrupted capsules are sorted out during one of the subsequent steps.

If the cell is already dead or no longer fit to propagate or if it is simply considered more effective the transformed nucleic acid(s) can be multiplied by amplification after cell lysis to provide suitable numbers of nucleic acids for sequencing. For amplification the cells are lysed and optionally the capsules are purified to dispose of cellular debris. The substances for amplification may already by present in the capsule or be provided through permeation and diffusion from the outside environment.

It is preferred that the amplification is effected by enzymatically catalyzed processes, preferably by PCR (polymerase chain reaction) or by rolling cycle amplification (RCA).

In a more preferred embodiment the multiplication of the transformed nucleic acid(s) is effected by propagation of the transformed cell before lysis and subsequent amplification of the transformed nucleic acids after lysis.

The specific propagation, lysis, amplification and purification methods for practicing the present invention will depend on the cell, the capsule material, and the nucleic acids. Any of those methods known in the art may be employed and the adaptation of these methods to the specific needs of the invention is available through routine experimentation. For example, cells can be grown in chemically defined media. A non- restrictive example is Tris-HCI-buffered, phosphate-limited M9-medium supplemented with glucose or another readily metabolized carbon and energy source. Alternatively, cells can be grown on complex media such as LB, SOB or GYT (J. Sambrook, D. W. Russell / J. Sambrook (2001 ) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor). In principle, any medium can be used provided that the cells replicate. According to the present invention cell lysis may be affected by any method that leads to the release of nucleic acids from the embedded cell in such a fashion that said nucleic acids can be sequenced and capsule integrity is maintained. Non-restrictive examples are cell lysis by physical methods such as pressured drops, freezing/thawing-cycles, heating, osmotic shock, ultrasonication, the addition of chemical agents such as antibiotics, detergents, hydrogen peroxide, acids or bases, or by biological methods such as phage-induced lysis, digestion by e.g. lysozyme, or induced cell lysis by e.g. pTOD.

One independent aspect of the present invention relates to the use of peroxides, preferably hydrogen peroxide, for the lysis of cells within capsules, preferably within the capsules prepared from the hydrogel-forming polymers described below, for separating empty capsules from capsules filled with cells by their difference in density. Capsules that comprise cells that react with peroxides enzymatically, e.g. by catalase, will provide a gas as reaction product and lower the density of that peroxide-treated capsule, thus, resulting in less dense particles that preferably demonstrate an increased buoyancy and depending on the medium surrounding the capsules may float on top. In this manner, capsules comprising cells can be efficiently separated from empty capsules. It is preferred to add 0,1 to 3 % (w/v) peroxide, more preferably 1 to 2 % peroxide to the capsules.

For practicing the method of the present invention the capsule can be provided with reactant(s) during and/or after encapsulation of cell(s). Alternatively, said reactants can be provided by the cell itself. Preferably, said reactant(s) is (are) suitable for (i) the multiplication of the cell(s), (ii) the multiplication of nucleic acid(s), (iii) the sequencing and/or labelling of nucleic acids, or (iv) for general reasons of capsule handling.

In a preferred embodiment, high molecular weight structures such as nucleic acids and enzymes can be brought into the capsule by using cell(s) as a carrier-vesicle(s) for said high molecular weight structures. High molecular weight compounds that otherwise can not be fed to the capsules due to their high molecular weight are brought into the capsule by lysis of the embedded cells whereupon said high molecular weight compounds are released into the capsule.

In a preferred embodiment the capsule is provided with reactants and catalysts that are produced by the cell, preferably macromolecular compounds, more preferably nucleic acids or enzyme-catalysts.

More preferably, the reactant(s) is (are) selected from the group consisting of growth factors, nutrients, antibiotics, buffers, enzymes, primers, labels, nucleotides, proteins, cell preservative agents, particulates for separation by gravimetric methods, or magnetic particles. Particulates for separation by gravimetric methods will influence the density of the capsule and preferably these particulates may be changed in size and mass inside the capsule by controlled degradation or formation such as aggregation and/or build-up.

The capsule for use in the present invention comprises either a living or a dead transformed cell, or a transformed resistant dormant body such as spores, cysts, or sclerotic structures. The advantage of the cell is that (i) it provides a multitude of copies of the nucleic acid(s) of interest as genomic, plasmid or phage copies, (ii) that it protects the nucleic acid(s) from environmental conditions once encapsulated, (iii) that it provides a large physical entity that is advantageous for being encapsulated singularly, (iv) that nucleic acid fragments' approximate size can be preselected upon transformation of said nucleic acid, and (v) that it can be guaranteed that nucleic acid fragments form an integral unity with an appropriate primer sequence. Hence, the method of the present invention does not require performing elaborate in vitro steps for nucleic acid purification and reactions prior to encapsulation but takes full advantage of the synthetic power of the cell.

The transformed cell may be selected from procaryotes or eucaryotes.

Procaryotic cells are preferred. Bacterial cells are more preferred, of these Gram-positive or Gram-negative strains are even more preferred. Most preferably, the prokaryotic cell for practising the present invention is selected from eubacteria, preferably from the group consisting of Salmonella typhimurium, Serratia marcescens, Streptomyces lividans, Pseudomonas sp, Bacillus sp, and Escherichia sp, most preferably Escherichia coli and Bacillus subtilis. Alternatively, the transformed cell is a eucaryotic cell, preferably a cell selected from the group consisting of mammalian cells, preferably immortalized mammalian cells, CHO cells, 293 cells, hybridoma cells, HELA cells, BHK cells, NIH3T3 cells and COS cells; insect cells, preferably Drosophila Schneider cells, Drosophila Kc, and Sf9 cells; fungal cells, preferably Candida albicans, Candida methylica, Candida utilis, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe, Saccharomyces cerevisia, Pichia pastoris, and Aspergillus niger, more preferably Saccharomyces cerevisia, Pichia pastoris, and Aspergillus niger.

Materials and technologies for preparing capsules for the use in the method of the present invention are well known to those trained in the art. In principle, all technologies can be used that a) produce appropriately sized capsules, and that b) produce capsules with high monodispersity. Furthermore, in preferred embodiments materials may be used that a) result in capsules with the desired permeability for all or some reactants used in their presence, and that b) result in capsules that are stable for all reaction conditions employed in their presence, and that c) show low or no inhibitory effects for the enzymatic reactions performed within the capsule structure. It is self-evident that the cell(s) and/or transformed and/or amplified and/or sequenced nucleic acid(s) should not be able to leave the capsule until lysis of the capsule except for the growth phase where some cell outgrowth can be tolerated.

More preferably, the capsules for practising the present invention are produced by a method for the production of single droplets (C. Heinzen et al., (2004) Use of vibration technology for jet break-up for encapsulation of cells and liquids in monodisperse microcapsules, in Nedovic, Viktor; Willaert, Ronnie (Eds.) Fundamentals of Cell

Immobilisation Biotechnology. Focus on Biotechnology, Springer, Heidelberg, Ch. 8A, 257-275).).

For practicing the method of the present invention the capsule for incorporating the cell should be appropriately sized. In a preferred embodiment the capsules provided have an average diameter in the range of 10 to 1000 μm, more preferably in the range of 20 to 700 μm, and even more preferably in the range of 50 to 400 μm.

For the capsule for practising the present invention it is preferred that the standard deviation of the capsule diameter of at least 1000 beads produced in one batch is ± 50 %, more preferably ± 25 %, and most preferably ± 5 %. In a further preferred embodiment the capsules provided for practising the present invention are produced in a suspension polymerisation process.

The capsules that are produced by suspension polymerisation have a size-distribution in the range of 0.1 to 1000 μm, preferably in the range of 1 to 400 μm, and more preferably 2 to 200 μm.

Preferably, capsules for use in the invention have a pore size that is permeable for all or part of the reactants employed. For practicing the method of the present invention, it is advantageous to employ a capsule material that is permeable to a number of reactants that are needed for, e.g. cell propagation, removal of catabolites, amplification, labelling and/or sequencing and that assist purification. In a preferred embodiment capsule materials with appropriate permeability are hydrogels made from crosslinked natural or synthetic polymers or crosslinked acrylic polymers. Further preferred are alginate hydrogels crosslinked by bi- ortrivalent metal cations.

In a preferred embodiment, the material(s) that is (are) used for production of the capsule for practising the present invention comprises one or more material(s) selected from the group consisting of hydrogel-forming polymers, preferably poly(diallyldimethylammonium chloride), poly(ethyleneimine), polylysine, cellulose derivatives, preferably carboxymethylcellulose and cellulose esters, agaroses, alginates, carrageenans, polyacrylamides, acrylic acids, and polysaccharides, more preferably alginates, pectinates, chitosans, and carrageenans.

More preferably, the alginate capsule material for practising the present invention is calcium, barium and/or strontium alginate, most preferably barium or strontium alginate.

It is noted that any capsule diameter can be chosen provided that the concentration of the nucleic acids within the volume of said capsule is sufficiently high to allow enzymatically reacting said nucleic in such a fashion that sequencing becomes feasible.

Preferred technologies to produce capsules with diameters in the indicated range are microdroplet production technologies. A further preferred technology is the laminar jet break up technology (C. Heinzen et al., supra) . It is desirable to employ a capsule material that is stable against all reaction conditions employed. Preferably, the capsules are not disintegrated by chemical, physical and mechanical stress induced by, for instance, growth of cells, cell lysis, in vitro amplification, or DNA sequencing. Preferred capsule materials are those listed above.

For practicing the method of the present invention, it is necessary to employ capsule materials that show low or no inhibitory effects for the enzymatic reactions such as amplification or sequencing reactions performed within the capsule structure. Capsule materials with appropriate low inhibitory effects on amplification and sequencing reactions are those listed above. Many capsule materials allow enclosed cells to proliferate.

The most preferred capsule materials are alginate hydrogels crosslinked by bi- or trivalent metal ions. Preferred cations of these are the bivalent cations calcium, strontium and barium. It was surprisingly found that barium alginate and strontium alginate microcapsules have a significantly reduced inhibitory effect on polymerase enzymes needed for PCR reactions. Hence, barium and strontium alginate will allow for a much better amplification than calcium alginate.

Therefore, a further independent aspect of the present invention is directed to barium and also strontium alginates in general and the use of these new alginates for any methods that comprise polymerase activity, preferably for PCR, RCA, RACE or sequencing.

As mentioned before, the copy number of transformed nucleic acid(s) in the capsule after cell lysis should exceed one copy to provide suitable enzyme kinetics.

It is also preferred that the capsule for practicing the method of the present invention has a copy number of the transformed nucleic acid(s) after cell lysis that lies in the range of 2 to 2 x 10⁸.

In yet another preferred embodiment the number of copies of the transformed nucleic acid(s) in a capsule after cell lysis is selected from the group consisting of at least 2, 20, 100, 1000, 10⁵, 10⁶, and 10⁷. The transformed nucleic acid is either a DNA or an RNA, preferably a DNA. If desired, an RNA can be reverse-transcribed within the capsule by in vitro techniques which are well known by those skilled in the art.

To ensure primer-selective amplification and/or sequencing and to avoid aberrant amplification and/or sequencing of cellular nucleic acids the transformed nucleic acid(s) preferably comprise(s) one or more primer specific region(s) for primer-selective amplification and/or sequencing, preferably one or two flanking primer-specific regions.

In a preferred embodiment the nucleic acids produced in the sequencing reaction are labelled, preferably labelled with a light absorbing label, more preferably with a fluorescent label. For a comprehensive review on the art of fluorescent compounds for DNA labelling M. Neumann et al., (2001 ) New techniques for DNA sequencing based on diode laser excitation and time-resolved fluorescence detection, in B. Valeur and J. -C. Brochon (Eds.) New Trends in Fluorescence Spectrosocopy. Applications to Chemical and Life Science Springer, Berlin, Ch. 16, 303-329) is referenced.

The nucleic acids multiplied and/or sequenced according to the invention can easily be purified while still present in the capsule. A preferred purification method for the microcapsules is the extraction of the capsule. More preferably, the capsule(s) is (are) separated from a liquid or solid medium by centrifugation, sieving, flotation, sedimentation, filtration, or magnetic binding. In one embodiment, the capsules are then contacted with a solute, preferably an aqueous buffer, and more preferably a buffer that contains organic compounds. Non-restrictive examples of preferred organic compounds are polyethyleneglycol or polyvinylpyrilidone and alcohols, esters, ethers, alkanes, or derivatives of BTXS (benzene, toluene, xylene, or styrene).

Although not a requirement capsules can be classified and physically separated by an appropriate method. For example, one class of capsules may contain a colony or cell and the second class is empty. One class of capsules may contain nucleic acids and another class does not. One class of capsules may contain fluorescent-labelled nucleic acids while another does not. One class may be disrupted or physically altered while another class is not. Examples for methods employed for class separation are flotation, sedimentation, density centrifugation, electro-osmotic hydrodynamic counter flow, diaelectrophoresis, FACS (fluorescence-activated cell-sorting), or COPAS (complex object parametric analyzer and sorter). Before amplifying and/or sequencing the nucleic acids in the capsule lysis of the cell(s) is required to allow free access to said nucleic acids.

As demonstrated above the sequencing method of the present invention uses standard propagation, amplification and/or sequencing techniques but is surprisingly effective for sequencing large numbers of nucleic acids and can be used to sequence thousands and even millions of nucleic acids simultaneously in a single batch approach with high fidelity due to the encapsulation of the sequencing components in a microcompartiment.

A further aspect of the present invention is directed to the use of a method according to the present invention for sequencing transformed nucleic acids.

Description of the Figures

Fig. 1 illustrates at the centre a Barium alginate bead with a diameter of -350 μm containing an E. coli colony (-100 μm diameter; -1 million cells). Initially the bead contained a single cell which was proliferated upon incubation in 50 % SOB medium (24 hours, 37°C).

Fig. 2 illustrates the results of a PCR (16 ng plasmid DNA, 3.8 kb) performed in the presence of different bivalent metal-ion salts in order to determine concentrations at which PCR is inhibited. In Fig. 2B BaCI₂ and CaCI₂ are compared on a gel: Lane 1: DNA standard; Lane 2: 5 mM BaCI₂; Lane 3; 2.5 mM BaCI₂; Lane 4: 1 mM BaCI₂; Lane 5: 0.5 mM BaCI₂; Lane 6: positive control; Lane 7: 5 mM CaCI₂; Lane 8: 2.5 mM CaCI₂; Lane 9: 1 mM CaCI2; Lane 10: 0.5 mM CaCI₂; Lane 11: positive control. In Fig. 2A the inhibition concentration for SrCI₂ are shown on a gel: Lane 1: DNA standard: Lane 2: 20 mM SrCI₂; Lane 3: 10 mM SrCI₂; Lane 4: 6 mM SrCI₂; Lane 5: 4 mM SrCI₂; Lane 6: 2 mM SrCI₂; Lane 7: 1 mM SrCI₂; Lane 8: 0.5 mM SrCI₂; Lane 9: positive control. The results demonstrate that CaCI₂ inhibits PCR at concentrations of > 1 mM, < 2.5 mM; BaCI₂ at concentrations of > 2.5 mM, < 5 mM; and SrCI₂ at concentrations > 4 mM, < 6 mM, thereby indicating that PCR is less strongly inhibited by Barium and Strontium than by Calcium ions. Fig. 3 shows a crude cell lysate of E. coli that has been embedded into Ba-alginate beads (400 μm in diameter). The beads were then subjected to PCR. After cycling, the beads were recovered from the PCR tube, briefly washed with water, stained with SYBRGreen (Invitrogen, Carlsbad, CA, US), and then placed on a UV-screen. As a result approximately half of the bead population is PCR positive (shiny spots) whereas the other half remains dark thereby indicating that either no PCR took place or that PCR yields were very low (dark spots). The distance indicated by the arrow heads is 10 cm.

Fig. 4 shows the result of a PCR performed within barium alginate beads. Barium alginate beads containing a partially purified plasmid DNA as well as empty barium alginate beads were prepared. Always six beads of either batch were than subjected to

PCR, were briefly washed with water in the PCR tube and then stained by SYBRGreen.

Both tubes were then illuminated by a UV-screen. The six empty alginate beads without

DNA (left side) did not yield a signal whereas the six beads with plasmids are fluorescent (right side). This demonstrates that the PCR reaction took place on the embedded plasmid.

Fig. 5 Two beads of 700 μm in diameter were incubated in the same PCR well. One bead contained partially purified plasmid DNA with an insert expected to result in a 250 bp PCR product while the other one contained a fragment expected to result in a 480 bp PCR product. The flanking regions of both inserts were identical and hybridize to the same primers. Upon cycling 250 bp fragments were formed in one bead (left side) and a 480 bp fragment in the other bead (right side). This result demonstrates the absence of any bead-to-bead crosstalk during PCR amplification.

Figs. 6A, 6B and 6C show the same calcium alginate bead with a diameter of 350 μm containing an E. coli colony with a diameter of roughly 30 μm incubated in 0.5 % SDS and 0.1 M NaOH for 60 minutes after 1 minute (Fig. 6A), 10 minutes (Fig. 6B), and 60 minutes (Fig. 6C). The colony in focus (upper colony in the bead in the centre) in Fig. 6A is dark at t=1 minute and microstructured. The edges of the colony became transparent after 30 minutes thereby indicating that the colony lysis is initiated from the outside. After 60 minutes the colony is clear and all microstructures vanished which indicates complete colony lysis. In the following the invention will be further explained by specific examples which are provided for the purpose of illustration only and are not to be construed as limiting the scope of the claims.

Examples

Example 1 : Encapsulation of cells

Escherichia coli cells (XL-10 Gold, Stratagene, La JoIIa, California, US) containing DNA plasmid pUC18 were diluted to a final concentration of 2.3x10³ cells/ml in a solution of 26 mM sodium chloride, 20 mM Tris-HCI, pH 7.0 and 0.01 g Dextrane blue. This solution was mixed 1 :1 with sodium alginate (4%) and droplets of approximately 450 μm in diameter were produced using mechanical vibration-induced laminar-jet technology (flow 6.5 ml/min; frequency 2.3 kHz; nozzle diameter 200 μm). Droplets were solidified in 20 mM barium chloride for 1 hour.

Solid spheres (400 μm) were rinsed in Luria broth (LB) and subsequently mixed with semi-solid Luria broth (containing 0.8% Agar) with X-gal (45 μg/ml), IPTG (0.02%) and Ampicillin (50 μg/l). After overnight incubation at 37°C the number of spheres containing recombinant E. coli were compared to those without bacteria (1000 spheres counted): 94.7 % (947) of spheres contained no bacteria, 5.2 % (52) contained one bacterial colony and 0.1 % (1) contained more than one bacterial colony.

Ten (10) beads each containing a single colony were taken and incubated in 500 μl of bead-lysis buffer (20 mM (NH₄J₂SO₄, 1 % NaCI, 10 mM Tris-HCI, pH 7.0) on ice with periodical shaking until the beads were dissolved (about 30 min). The bead slurry containing suspended cells was diluted 10000 times in LB and 50 μl of the suspension were plated out on an LB-Agar plate (50 μg/ml ampicillin). After incubation over 18 hours at 37⁰C 104 colonies were counted on plates. This indicates that each of the beads contained 10⁶ cells on the average.

Example 2: Production of 40000 samples in 400 μm beads for shotgun sequencing of a microbial genome.

Step 1 : Library creation

Genomic DNA of a microbial species with a genome size of approximately 4.2 Mbp is isolated, purified, sheared and standardized by isolation of DNA fragments of an average length of 2 to 3.5 kb. Said DNA fragments are then ligated with a high copy number vector. The ligated plasmids with the desired inserts are separated from self-Iigated plasmids and plasmids containing di- or oligomers by gel-electrophoresis. The purified plasmid is then transformed into competent E. coli cells. Cells are cured by incubating them for 30 minutes in LB at 30⁰C.

Step 2: Embedment of cells

The cured cells are separated from LB by centrifugation, resuspended in saline to a concentration of 2 cells per μl and then mixed in a ratio of 1 to 1 with a sterile filtered 4 % alginate solution containing 5 % of magnetic nanoparticles. Approximately 150 ml of the alginate solution is then prilled into an aqueous hardening solution of 50 mM BaCI₂ (Encapsulator from company Nisco, Zurich, Switzerland, flow rate of 6.5 ml / min, excitation frequency of 2500 Hz). The beads are then matured (incubation in the hardening solution for 2 hours at 4°C).

Step 3: In-bead cell amplification

Beads are recovered from the hardening solution by sieving, washed with an excess of demineralized water and incubated in 150 ml of 50 % SOB medium containing 1 mM BaCb (30⁰C, continuously stirred tank reactor with bead retention). After 20 hours of incubation beads are recovered from the reactor by filtration. The bead quality is checked by light microscopy: 2 % of the beads contain a globular colony of a diameter of roughly 100 μm (~10⁶ cells), roughly 2 % of the beads are disrupted due to colony outgrowth, and roughly 0.02 % of all beads contain more than one colony.

Step 4: Separation of empty and filled beads The beads are washed with an excess of 1 mM BaCI₂ and then incubated in 10 mM BaCI₂ (4°C). After 1 h the beads are recovered by sieving, briefly washed in 1 mM BaCI₂, and filled into a measuring glass filled with 1 mM BaCI₂ solution and Percol (adjusted density of 1.0255 g / ml). Upon incubation in Percol colonized beads sediment within 30 minutes while empty beads are found in the top phase. Non colonized beads are discarded by decanting and roughly 100000 colonized beads are recovered and washed in 1 mM BaCI₂.

Step 5: In-bead cell lysis

Colonized beads are incubated in 0.1 M NaOH and 0.5 % dodecylmaltosid for 10 minutes at 75°C. Beads are recovered by sieving, incubated in an excess of water for 2 hours (4°C) to allow low molecular weight cell lysis products to leave the beads by diffusion, recovered by sieving and incubated in 1 mM BaCI₂ for 1 hour (4°C).

Step 6: Sequencing Beads containing lysed colonies are incubated in a reagent mixture for terminator cycle sequencing containing 20 % BigDye V3.1 reaction ready mix (Applied Biosystems, Foster City, CA₁ US), 20 % 5X reaction buffer (Applied Biosystems, Foster City, CA, US), 1.6 pmol oligonucletide primer and 10 % v/v beads containing cell lysates. The sample is incubated in a thermocycler with an initial step of 96°C for 2 minutes followed by at least 30 repetitions of 96°C for 10 seconds, 50°C for 5 seconds and 60⁰C for 4 minutes.

Step 7: Removal of labelled ddNTPs

Beads are recovered from the sequencing mixture by removal of the reaction mix from the vessel, briefly washed with water and incubated in an excess of water for 20 minutes (4°C) in order to remove non-reacted ddNTPs from the beads by diffusion.

Step 8: Sorting and bead dispensing

The non-reacted ddNTPs are removed and the beads are suspended in Tris-buffer (1 I) with 1 mM BaCI₂ and 10 % v/v glycerol. Beads are then sorted with a COPAS Plus (Union Biometrica, Geel, Belgium) (effective sorting rate of 1.5 Hz) according to the fluorescence intensity. Poorly or non fluorescent beads are sorted out (about 50 %). Those that contain sufficient amounts of labelled fragments (> 400 pg) for analysis by an ABI 373OxI capillary electrophoresis unit (Applied Biosystems, Foster City, CA, US) are dispensed (one beads per well) into 384-well microtiter plates.

Step 9: Recovery of labelled sequencing products from the beads To each microtiter (MT) plate well 5 μl of demineralized water are added. Plates are then stored at 4°C allowing labelled DNA fragments (200 pg; 50 % isolated yield) to leave the beads by diffusion. After 24 hours beads are recovered from the wells by means of a magnet and plates are directly analyzed by capillary electrophoresis

Example 3: Production of 5000000 samples in 150 μm beads for shotgun sequencing of a microbial genome.

Step 1 : Library creation This step is analogous to step 1 of example 2 except for the sequencing target which in this case is a eukaryotic species with a genome size of approximately 700 Mbp.

Step 2: Embedment of cells This step is analogous to step 2 of example 2 except for the alignate solution which does not contain magnetic particles, the concentration of cells in the cell suspension (200 cells per nl), the volume of the alginate / cell mixture (800 ml) and for the prilling parameters (flow rates 1.2 ml / min; production frequency 8000 Hz).

Step 3: In-bead cell propagation

Beads are recovered from the hardening solution by sieving, washed with an excess of demineralized water, and incubated in 8 L of 50 % SOB medium containing 1 mM BaCI₂ and 1 mM of hydrogen peroxide (30⁰C₁ magnetically stirred 10 L carboy (Schott, Mainz, Germany), forced supply of pressurized air-oxygen by a dispenser). After 4 hours of incubation (in bead colony size roughly 10 to 100 cells) beads are recovered from the reactor by filtration and briefly washed with water.

Step 4: Separation of empty and colonized beads

The beads are washed with an excess of 1 mM BaCb and then incubated (4°C) in a 5 I measuring glass filled with 10 mM BaCI₂ containing 2 % hydrogen peroxide. After 1 hour beads that approached the surface (~5 million colonized beads) are recovered by sieving and briefly washed in 1 mM BaCI₂. The non-colonized beads at the bottom phase are discarded.

Step 5: In-bead cell lysis

This step is analogous to step 5 of example 2.

Step 6: PCR

Beads containing lysed colonies are incubated in a reagent mixture for PCR amplification of DNA regions of interest. The reagent mixture consists of 1x reaction buffer, 200 μM of each dNTP, 25 nM of each oligonucleotide primer and 250 units/ml of Taq polymerase. 0.1 mM BaCI₂ is added to the PCR solution in order to stabilize the beads. The samples are incubated in a thermocycler with an initial step of 94°C for 2 minutes followed by 30 cycles of [94°C for 30 seconds, 50⁰C for 30 seconds and 72°C for 1 minute]. Beads are recovered from the solution of PCR reagents and washed with 0.1 mM BaCI₂ for 30 minutes at 4°C in order to remove non-reacted dNTPs and oligonucleotide primers from the beads by diffusion.

Step 7: Sequencing This step is analogous to step 6 of example 2

Step 8: Removal of labelled ddNTPs Step 8 is analogous to step 7 of example 2

Step 9: Bead storage

50 aliquots of roughly 100000 beads (-0.2 ml) are filled into ice-cold 2 ml Eppendorf- cups. 1 ml of 5 mM BaCI₂ with 20 % glycerol (ice cold) is added, cups are shock-frozen in liquid nitrogen and stored at -80⁰C.

Step 10: Sorting and bead dispensing

This step is analogous to step 9 of example 2 except that at the beginning bead aliquots are allowed to thaw in a water bath (3O⁰C, 5 min) and that effective sorting frequencies are adjusted to 2 Hz.

Step 11 : Recovery of labelled sequencing products from the beads This step is analogous to step 10 of example 2.

Example 4: Production of 10000000 samples in 50 μm beads for shotgun sequencing of a eukaryotic genome.

Step 1 : Library creation

This step is analogous to step 1 of example 2 except for the sequencing target which in this case is a eukaryotic species with a genome size of approximately 2000 Mbp and for the plasmid used for ligation because this plasmids constitutively expresses a catechol- 2,3-dioxygenase.

Step 2: Embedment of cells

This step is analogous to step 2 of example 3 except for the cell concentration in the suspension which is 2 cells per nl, for the volume of the alginate / cell mixture which is 100 ml and for the prilling parameters (flow rates 0.3 ml / min; production frequency 50 kHz). Step 3: Labelling of colonized beads

Beads are recovered from the hardening solution by sieving and washed with an excess of 1 mM BaCI₂. The beads are then incubated in 1 L 10 mM Tris-buffer (pH 7) with 5 % glycerol, 1 mM BaCI₂ and 10 mg/l catechol in a carboy flask (2 L Schott-flask, magnetically stirred). Upon incubation of the beads for 5 hours colonized beads acquire a yellow tint whereas non-colonized beads remain transparent.

Step 4: Separation of empty and colonized beads The beads are recovered, washed with 5 % glycerol, suspended in 5 % glycerol and then subjected to sieving for removal of beads with a diameter of > 50 μm. The < 50 μm bead fraction is then subjected to FACS for isolation of fluorescent beads (20000000; roughly 2 % of all beads).

Step 5: In-bead cell lysis

This step is analogous to step 5 of example 2.

Step 6: PCR

Beads containing lysed colonies are incubated in a reagent mixture for PCR amplification of DNA regions of interest. The reagent mixture consists of 1x reaction buffer, 200 μM of each dNTP, 25 nM of each oligonucleotide primer and 250 units/ml of Taq polymerase.

0.1 mM BaCI₂ is added to the PCR solution in order to stabilize the beads. The samples are incubated in a thermocycler with an initial step of 94°C for 2 minutes followed by

30 cycles of [94°C for 30 seconds, 50⁰C for 30 seconds and 72°C for 1 minute]. The beads are recovered from the solution of PCR reagents and washed with 0.1 mM BaCi₂ for 30 minutes at 4°C in order to remove non-reacted dNTPs and oligonucleotide primers from the beads by diffusion.

Step 7: Sequencing This step is analogous to step 6 of example 2

Step 8: Removal of labelled ddNTPs

This step 8 is analogous to step 7 of example 2.

Step 9: Bead storage 50 aliquots of roughly 2000000 beads (-0.2 ml) are filled into ice-cold 2 ml Eppendorf- cups. 1 ml of 5 mM BaCI₂ with 20 % glycerol (ice cold) is added, cups are shock-frozen in liquid nitrogen and stored at -8O⁰C.

Step 10: Sorting and bead dispensing

This step is analogous to step 9 of example 2 except that at the beginning bead aliquots are allowed to thaw in a water bath (30⁰C, 5 min) and that sorting/dispensing is done with a FACS instead of a COPAS at a frequency of ~3 Hz.

Claims

1. A method for sequencing nucleic acids, comprising:

(a) providing one or more capsules, each comprising one or more identical transformed cell(s) with at least one copy of a transformed nucleic acid therein,

(b) lysis of the cell(s) in the capsule, and

(c) sequencing the transformed nucleic acid(s) after cell lysis in the capsule.

2. The method of claim 1 , wherein said transformed cell comprises at least two copies of a nucleic acid.

3. The method of claim 1 or 2 comprising the additional step of multiplying the transformed nucleic acid(s) prior and/or after the lysis of the cell(s) in the capsule.

4. The method of claim 3, wherein the multiplication of the transformed nucleic acid(s) is effected by propagation of the transformed cell before cell lysis.

5. The method of claim 3, wherein the multiplication of the transformed nucleic acid(s) is effected by amplification of the transformed nucleic acid(s) after cell lysis.

6. The method of claim 5, wherein the amplification is effected by PCR (polymerase chain reaction) or RCA (rolling cycle amplification).

7. The method of any one of claims 3 to 6, wherein the multiplication is effected by propagation of the transformed cell before lysis and subsequent amplification of the transformed nucleic acid(s) after cell lysis.

8. The method of any one of claims 1 to 7, wherein the nucleic acids produced in the sequencing reaction are labelled, preferably labelled with a light absorbing label, more preferably with a fluorescent label.

9. The method of any one of claims 1 to 8, wherein the multiplied and/or sequenced nucleic acids are purified while present in the capsule.

10. The method of claim 9, wherein the purification is effected by extraction of the capsule.

11. The method of claim 9 or 10, wherein the capsule is separated from a liquid or solid medium by centrifugation, sieving, flotation, sedimentation, filtration, or magnetic binding.

12. The method of any one of claims 1 to 11 , wherein the capsule is provided with reactant(s) during and/or after encapsulation of the cell.

13. The method of claim 12, wherein the reactant(s) is (are) suitable for (i) the multiplication of the cell(s), (ii) the multiplication of nucleic acid(s), and/or (iii) the sequencing and/or labelling of nucleic acids.

14. The method of claim 12 or 13, wherein the capsule is provided with reactants or catalysts that are produced by the cell, preferably macromolecular compounds, more preferably nucleic acids or proteins, and even more preferably enzymes that catalyse reactions of nucleic acids.

15. The method of any one of claims 12 to 14, wherein the reactant(s) is(are) selected from the group consisting of growth factors, nutrients, antibiotics, buffers, enzymes, primers, labels, nucleotides, proteins, cell preservative agents, particulates for separation by gravimetric methods, or magnetic particles.

16. The method of any one of claims 1 to 15, wherein the transformed cell is a living or a dead cell.

17. The method of claim 15, wherein the transformed cell is selected from procaryotes or eucaryotes.

18. The method of claim 17, wherein the transformed cell is a procaryotic cell.

19. The method of claim 18, wherein the transformed cell is a bacterial cell.

20. The method of claim 17 or 18, wherein the procaryotic cell is selected from eubacteria, preferably from the group consisting of Salmonella typhimurium, Serratia marcescens, Streptomyces lividans, Pseudomonas sp, Bacillus sp, and Escherichia sp, and more preferably Escherichia coli and Bacillus subtilis.

21. The method of claim 17, wherein the transformed cell is a eucaryotic cell.

22. The method of claim 21 , wherein the transformed cell is selected from the group consisting of mammalian cells, preferably immortalized mammalian cells, CHO cells, 293 cells, hybridoma cells, HELA cells, BHK cells, NIH3T3 cells and COS cells, insect cells, preferably Drosophila Schneider cells, Drosophila Kc, and Sf9 cells, fungal cells, preferably Candida albicans, Candida methylica, Candida utilis,

Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe, Saccharomyces cerevisia, Pichia pastoris, and Aspergillus niger, more preferably Saccharomyces cerevisia, Pichia pastoris, and Aspergillus niger.

23. The method of any one of claims 1 to 22, wherein the capsules provided are produced by a method for the production of single droplets.

24. The method of claim 23, wherein the capsules provided have an average diameter in the range of 10 to 1000 μm, more preferably in the range of 20 to 700 μm, and most preferably in the range of 50 to 400 μm.

25. The method of claim 24, wherein the standard deviation of the capsule diameter of at least 1000 beads produced in one batch is ± 50 %, preferably ± 25 %, and more preferably ± 5 %.

26. The method of any one of claims 1 to 22, wherein the capsules provided are produced by suspension polymerisation.

27. The method of claim 26, wherein the capsules have a size-distribution in the range of 0.1 to 1000 μm, preferably in the range of 1 to 400 μm, and more preferably in the range of 2 to 200 μm.

28. The method of any one of claims 1 to 27, wherein the capsules have a pore size that is permeable for all or part of the reactants employed.

29. The method of any one of claims 1 to 28, wherein the material of the capsule comprises one or more material(s) selected from the group consisting of hydrogel- forming polymers, preferably poly(diallyldimethylammonium chloride), poly(ethyleneimine), polylysine, cellulose derivatives, preferably carboxymethylcellulose and cellulose esters, agaroses, alginates, carrageenans, polyacrylamides, acrylic acids, and polysaccharides, more preferably alginates, pectinates, chitosans, and carrageenans.

30. The method of claim 29, wherein the material is alginate, preferably calcium- barium- and/or strontium alginate, more preferably barium or strontium alginate.

31. The method of any one of claims 1 to 30, wherein the number of the copies of the transformed nucleic acids after cell lysis lies in the range of 2 to 2 x 10⁸.

32. The method of claim 31 , wherein the number of the copies of the transformed nucleic acid after cell lysis is selected from the group consisting of at least 20, 100, 1000, 10⁴, 10⁵, 10⁶, and 10⁷.

33. The method of any one of claims 1 to 32, wherein the lysis is effected by a method selected from the group consisting of freezing and thawing, enzymatic lysis, ultrasonification, pH-dependent lysis, lysis by hydrogen peroxide and detergent based lysis.

34. The method of any one of claims 1 to 33, wherein the transformed nucleic acid is a DNA or RNA, preferably a DNA, more preferably DNA that is derived from a shotgun library, an EST-library, an environmental library or a cDNA bank.

35. The method of any one of claims 1 to 36, wherein the transformed nucleic acid(s) comprise(s) one or more primer-specific regions for primer selective amplification and/or sequencing, preferably it (they) comprise(s) flanking primer-specific regions.

36. Use of a method of any one of claims 1 to 35 for the sequencing of transformed nucleic acids.