WO2020141144A1 - Procédé d'enrichissement pour séquençage - Google Patents

Procédé d'enrichissement pour séquençage Download PDF

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Publication number
WO2020141144A1
WO2020141144A1 PCT/EP2019/087061 EP2019087061W WO2020141144A1 WO 2020141144 A1 WO2020141144 A1 WO 2020141144A1 EP 2019087061 W EP2019087061 W EP 2019087061W WO 2020141144 A1 WO2020141144 A1 WO 2020141144A1
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particles
type
stranded
stranded oligonucleotide
oligonucleotide
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PCT/EP2019/087061
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English (en)
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Matthias Wahl
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Qiagen Gmbh
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Publication of WO2020141144A1 publication Critical patent/WO2020141144A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention belongs to the field of sample purification, in particular for enriching particles comprising desired nucleic acids such as surface-bound double-stranded amplicons.
  • the described technology is useful for a variety of applications and assays, in particular sequencing applications, e.g. for enriching particles comprising surface-bound clonally amplified template molecules.
  • Small particles also referred to as microspheres, are a commonly used tool for nucleic-acid based applications in the fields of basic biological research, biomedical research, applied testing, and molecular diagnostics. Applications include, but are not limited to, clonal amplification of specific DNA fragments on the surface of microspheres by polymerase chain reaction or other amplification methods, and specific isolation of nucleic acids/nucleic acids with oligo-conjugated microspheres by hybridization-based methods. Such particles are widely used in sequencing applications.
  • NGS Next generation sequencing
  • NGS technologies such as pyrosequencing, sequencing by synthesis or sequencing by ligation.
  • NGS technologies, NGS platforms and common applications/fields for NGS technologies are e.g. reviewed in Voelkerding et al (Clinical Chemistry 55:4 641- 658, 2009), Metzker (Nature Reviews/ Genetics Volume 11 , January 2010, pages 31-46), Goodwin et al., (Nature Reviews, June 2016, Vol. 17: pp.
  • next-generation sequencing approaches share common sample preparation steps to provide the nucleic acid template that is then sequenced and usually include steps such as nucleic acid extraction, optionally target enrichment, sequencing library preparation (wherein the comprised template molecules are usually provided with adapter sequences at one or both ends) and clonal amplification.
  • the particles used in emulsion PCR comprise a single-stranded (capture) oligonucleotide that can hybridize to the template molecules, usually to the adapter sequence that is incorporated into the template molecule during library preparation, and serves as a primer during amplification.
  • emulsion PCR small reaction compartments are formed in which (ideal case) one single template molecule and one single bead is present; thereby, this single molecule can be bound to the particle and amplified by PCR.
  • a single-strand of the template molecule becomes associated with the particle, followed by PCR-based amplification of the template molecule.
  • the droplet essentially provides a closed reaction compartment for the PCR and contains after amplification one particle associated with amplified nucleic acids (amplicons) that derived from the same template molecule. Both particles and template are statistically distributed in the droplet. As a consequence, situations could arise in which (a) multiple templates or (b) multiple particles are present in a droplet.
  • compartments which comprise an amplicon- containing particle also referred to in the art as“live bead”
  • compartments that comprise a particle that lacks an amplicon also referred to in the art as“null bead”.
  • the oil phase is removed or the emulsion is“broken” in order to release the particles comprising the amplified nucleic acids (see e.g. WO2014/186152 and EP 3 170 903).
  • This breaking step releases live beads as well as null beads.
  • the null beads must be depleted by enriching the live beads. Removal of the null beads is challenging.
  • One method to enrich the live beads is to selectively bind the live beads to another type of particle, also referred to as“enrichment bead”, whereby a larger particle complex is formed.
  • Null beads do not specifically bind to the enrichment beads and are therefore depleted in a subsequent size selective purification step, which enriches the complexes comprising the enrichment beads and the live beads due to their larger size (see e.g. WO2014/186152). However, despite performing such purification/enrichment step, null beads are still present at a relatively high concentration in the enriched live bead sample (e.g. up to 50%). In a downstream sequencing process the non-depleted null beads create significant problems. Each null bead without usable sequence will be deposited onto the available spaces on the flow cell (thereby taking up available spaces in the flow cell), thereby competing with (decreasing the number of) live beads that will provide usable sequence.
  • WO 2015/170187 teaches to add enzymes such as single-stranded nucleases, to degrade the capture oligonucleotides on the null beads. Yet, there is still a need for further methods that improve the purification of the live beads.
  • the invention aims at avoiding drawbacks of the prior art methods.
  • it is an object to increase the efficiency of the enrichment of amplicon-covered particles from non- amplicon covered particles for next generation sequencing (NGS) applications.
  • NGS next generation sequencing
  • the present invention is based on the finding that enrichment of a first type of particles comprising double-stranded nucleic acid molecules (e.g. live beads) from a composition additionally containing a second type of particles comprising a single-stranded oligonucleotide (e.g. null beads) can be significantly improved by adding a hybridizing agent capable of forming a hybrid with the single-stranded oligonucleotide prior to and/or during separation of the first type of particles. Addition of the hybridizing agent advantageously reduces the carry-over of the second type of particles during the enrichment process.
  • the hybridizing agent blocks the single-stranded oligonucleotide and thereby hinders it from interacting with the first type of particles and/or the third type of particles which may be used during the enrichment process.
  • the hybridizing agent thereby improves the depletion of the second type of particles during the enrichment process of the first type of particles.
  • the technology is particularly suitable for enriching live beads from null beads after clonal amplification using e.g. emulsion PCR. It provides particles of the first type with higher purity allowing for a more efficient downstream sequencing.
  • the technology of the invention is effective, reliable and can be easily integrated into existing enrichment and sequencing workflows. Additional time-consuming processing steps are not required because the hybridizing agent blocking the single-stranded oligonucleotide present on the second type of particles (null beads) can be easily integrated into existing workflows and may even be included in commonly used processing reagents, such as a wash reagent and/or enrichment reagent.
  • the hybridizing agent can be easily combined with automated sequencing platforms without requiring an additional processing step and/or space in the platform. It thereby makes an important contribution to the art.
  • the present disclosure provides a method for enriching a first type of particles comprising double-stranded nucleic acid molecules from a composition additionally containing a second type of particles comprising a single-stranded oligonucleotide, wherein the method comprises
  • the present disclosure provides a method of amplifying a template and enriching the provided amplicon, wherein the method comprises:
  • a particle comprising a single-stranded oligonucleotide, wherein said single-stranded oligonucleotide is capable of acting as a primer and wherein the particle comprises multiple copies of said single stranded oligonucleotide on the particle surface, and
  • compartments comprise one or both of (i) and (ii) but do not comprise (iii) a template molecule;
  • the present disclosure provides a kit for enriching a first type of particles comprising double-stranded nucleic acid molecules from a composition additionally containing a second type of particles comprising a single-stranded oligonucleotide, wherein the kit comprises:
  • hybridizing agent capable of forming a hybrid with the single-stranded oligonucleotide of the second type of particles.
  • the present disclosure is directed to the use of a hybridizing agent in a method for enriching a first type of particles comprising double-stranded nucleic acid molecules from a composition which additionally contains a second type of particles comprising a single-stranded oligonucleotide for blocking the single-stranded oligonucleotide of the second type of particles by forming a hybrid.
  • the double-stranded nucleic acid molecules are preferably double-stranded amplicons as are provided after clonal amplification.
  • Fig. 1 Illustrates amplification by particle-based emulsion PCR.
  • Aqueous compartments inside oil are produced that serve as small PCR reaction units as is known in the art.
  • Fig. 1a illustrates a single compartment (e.g. aqueous droplet in oil) which comprises a single template molecule carrying e.g. sequencing adapters at each end and a base particle which comprises a single-stranded oligonucleotide on its surface. Illustrated is a single strand of the template molecule.
  • a double-stranded template molecule can be rendered single-stranded by denaturation before preparing the emulsion so that a single-stranded template molecule is introduced into the compartment or a double-stranded template molecule can be introduced into the compartment which is then denatured e.g. by heating within the compartment to provide two separate strands so that one strand of the template molecule can anneal via a complementary sequence to the single-stranded oligonucleotide present on the surface of the base particle.
  • Suitable embodiments are well-known in the art and can be used in the context of the present invention.
  • the base particle is preferably provided as a microsphere, also referred to herein as bead.
  • the base particle comprises multiple copies of the single- stranded oligonucleotide, also referred to as single-stranded capture oligonucleotide, as it is capable of capturing the template molecule when single-stranded by hybridizing to a complementary sequence, e.g. to an adapter sequence, of the template molecule.
  • a preferred embodiment of the single-stranded (capture) oligonucleotide is illustrated in the enlarged box.
  • the single-stranded (capture) oligonucleotide may comprise a spacer region adjacent to the particle surface (the spacer region may be provided by a stretch of the same nucleotide, e.g.
  • the compartment furthermore comprises agents necessary to perform the PCR, such as a polymerase, dNTPs, a buffer and preferably a second primer which may comprise an affinity partner of an interaction pair, such as preferably biotin. Also a mixture of accordingly modified (e.g. biotin) and unmodified primers may be used.
  • agents necessary to perform the PCR such as a polymerase, dNTPs, a buffer and preferably a second primer which may comprise an affinity partner of an interaction pair, such as preferably biotin.
  • a mixture of accordingly modified (e.g. biotin) and unmodified primers may be used.
  • the single-stranded template molecule hybridizes via the adapter sequence to the hybridizing region of the single-stranded (capture) oligonucleotide, whereby the template is captured to the base particle.
  • the 3' end of the single- stranded oligonucleotide serves as a primer and is elongated during polymerization using the sequence of the template molecule as template, thereby providing a complementary strand.
  • a double-stranded amplicon comprising the original strand of the template molecule and the complementary strand resulting from the elongation of the single- stranded (capture) oligonucleotide which served as first primer.
  • the double-stranded amplicon is then denatured, whereby the original strand of the template molecule is released while the elongated complementary strand remains via the elongated single-stranded (capture) oligonucleotide tightly anchored to the particle surface.
  • the original strand of the template molecule can hybridize again to a“free” single-stranded (capture) oligonucleotide, where it serves again as template for the single-stranded (capture) oligonucleotide, thereby generating again a double-stranded amplicon.
  • a second primer in solution may hybridize to the 3’ end of the elongated complementary strand that is anchored to the particle surface, and is extended towards the particle, thereby also providing a double- stranded amplicon.
  • template nucleic acid molecules are provided in the emulsion at a concentration that is below one template molecule per compartment to avoid that multiple template molecules are present per compartment. Therefore, after performing the emulsion PCR, there are compartments comprising a particle of the first type, which comprises multiple copies of the same double-stranded amplicon (“live bead”), as well as compartments comprising a particle of the second type which comprise a single-stranded oligonucleotide but no double-stranded amplicon as there is no template for amplification (“null bead”).
  • the particles of the second type correspond to the base particles. The different types of particles are illustrated in Fig. 1f.
  • the first type of particles must be enriched from the second type of particles, as second type of particles would take up valuable space in the subsequent sequencing process.
  • the present invention advantageously improves the enrichment of the first type of particles comprising the double-stranded amplicon by adding a hybridizing agent that blocks the single-stranded oligonucleotide on the null beads thereby avoiding unspecific binding that can lead to unwanted carry-over of the null beads.
  • Fig. 2 Illustrates the workflow for enriching the first type of particles (live beads) from the second type of particles (null beads).
  • Fig. 2a illustrates the emulsion provided after performing the emulsion PCR. It comprises compartments comprising a particle of a first type (live bead) and compartments comprising a particle of the second type (null beads, corresponding to the base particle).
  • the particles of the first type may comprise a first affinity partner of an interaction pair, in the shown embodiment biotin.
  • the first affinity partner preferably biotin
  • the first affinity partner can be introduced by using a correspondingly modified reverse (second) primer during amplification, which incorporates the affinity partner into the double-stranded amplicon, whereby the particle of the first type becomes covered with said affinity partner (preferably biotin).
  • the particles of the second type do not comprise a double- stranded amplicon resulting from amplification (as there was no template) and therefore, do not comprise said affinity partner.
  • the particles of the second type correspond to the base particles and accordingly, only comprise the single-stranded (capture) oligonucleotide. As is illustrated in Fig. 2b, the particles are released from the compartments, e.g. by breaking the emulsion.
  • the released particles may be further purified, e.g. by performing one or more washing steps to remove the oil.
  • a composition which comprises particles of the first type as well as particles of the second type.
  • An embodiment for enriching the particles of the first type from said composition while depleting particles of the second type is illustrated in Fig. 2c.
  • the composition comprising the particles of the first and second type is contacted with a third type of particles (also referred to herein as enrichment beads).
  • the particles of the third type may be spherical.
  • the third type of particles is preferably larger than the first type of particles and the second type of particles (e.g. 10-15pm versus 0.5-3pm, e.g. 15pm versus 1 pm).
  • the enrichment particles specifically bind the first type of particles, whereby an association of particles is formed, also referred to herein as complex or aggregate. Due to its larger size, this complex comprising the third type of particles with the bound first type of particles can be separated from the second type of particles e.g. using a porous device, such as a nylon mesh, as is illustrated in Fig. 2.
  • the third type of particles may comprise a second affinity partner of an interaction pair, e.g. a biotin binding polypeptide (such as streptavidin or avidin) in case the particles of the first type comprise biotin as is shown in the illustrated embodiment.
  • a biotin binding polypeptide such as streptavidin or avidin
  • the particles of the second type cannot effectively bind to the third type of particles and are thus removed in the size-selective separation step.
  • a considerable amount of particles of the second type are nevertheless found in the enriched fraction of the first type of particles.
  • the second type of particles non-specifically bind or interact with the first type of particles and/or the third type of particles via the single- stranded (capture) oligonucleotide that is present on the surface of the second type of particles. Due to this unspecific binding/interaction, second type particles become associated with the complexes and are carried over into the enriched fraction of the first type of particles.
  • the inventors identified the single-stranded (capture) oligonucleotide present on the surface of the null beads as root cause for non-efficient removal of the null beads.
  • the single- stranded oligonucleotide which is usually present in a high density on the particles may cause unspecific binding of the second type particles, so that they are carried over into the fraction of enriched first type particles. These non-specific interactions negatively affect the separation and thus enrichment efficiency.
  • the present invention advantageously reduces the amount of carry-over of null beads by blocking the single-stranded (capture) oligonucleotide that is present in a large amount on the surface of the second type particles.
  • the present invention thereby renders the enrichment process significantly more efficient, as the hybridizing agent diminishes the unspecific interaction of second type particles.
  • a hybridizing agent as taught herein preferably provided by one or more oligonucleotides reverse complimentary to the single- stranded oligonucleotide present on the second type of particles, second type particles (null beads) are more efficiently depleted in the size-selective separation step of the enrichment process.
  • the hybridizing agent may be added during one or more of the steps that are illustrated in Fig. 2. E.g. the hybridizing agent may be added when breaking the emulsion and/or during washing of the particles that were separated from the broken emulsion (see Fig. 2b). Additionally or alternatively, the hybridizing agent may be added when adding or processing the third type of particles in order to enrich the first type of particles (see Fig. 2c).
  • the hybridizing agent may be simply included into one or more of the processing reagents, e.g. a washing reagent or the enrichment reagent. This is advantageous, because the technology of the present invention can be easily implemented into existing processing workflows. There is not requirement for additional, time consuming processing steps. All steps of the enrichment process can be performed at room temperature as the hybridizing agent can hybridize to the single-stranded oligonucleotide at room temperature.
  • Fig. 3 Shows different embodiments of the hybridizing agent and the resulting hybrid that can be formed with the single-stranded (capture) oligonucleotide of the second type of particles.
  • the hybridizing agent may comprise and may thus be provided by one or more oligonucleotides capable of hybridizing to the single-stranded (capture) oligonucleotide.
  • the hybridizing agent may be provided by a single-stranded oligonucleotide that hybridizes over the entire length with the single-stranded (capture) oligonucleotide (i.e. the hybridizing region and, if present, the spacer region - see also Fig.
  • the oligonucleotide of the hybridizing agent may also hybridize to the outer end of the single-stranded (capture) oligonucleotide, thereby providing an outer blunt end wherein a portion of the single-stranded (capture) oligonucleotide is not involved in hybrid formation.
  • the spacer region may not be involved in the formation of the hybrid or may only be partially involved in hybrid formation.
  • the oligonucleotide of the hybridizing agent may hybridize over the entire hybridizing region of the single-stranded (capture) oligonucleotide, as is illustrated in Fig. 3b, or at least a portion thereof as long as an outer blunt end is formed.
  • Fig. 3c and Fig. 3d illustrate embodiments that are related to the embodiment illustrated in Fig. 3a, where the oligonucleotide of the hybridizing agent is either longer or shorter than the single-stranded (capture) oligonucleotide, thereby providing a hybrid with a sticky end.
  • the oligonucleotide may hybridize over the entire length of the single-stranded (capture) oligonucleotide (as shown in Figs. 3c and 3d) or may hybridize to a portion thereof whereby a portion of the single-stranded (capture) oligonucleotide is not involved in the formation of the hybrid (as illustrated in Figs. 3e and 3f).
  • the oligonucleotide of the hybridizing agent is longer than the single-stranded (capture) oligonucleotide.
  • the hybridizing agent is complementary to the hybridizing region and optionally, at least a portion of the spacer region.
  • a sticky end, if present in the formed hybrid, is short, preferably £ 5nt, such as £ 4nt, £ 3nt, preferably £2nt or 1 nt, and therefore does not contribute to unspecific binding of the second type of particles (null beads) to the first type of particles (live beads) and/or the third type of particles (enrichment beads).
  • the hybridizing agent may comprise and thus may be provided by two or more different oligonucleotides, wherein each oligonucleotide is capable of hybridizing to a different region of the single-stranded (capture) oligonucleotide (embodiments are illustrated in Figs. 3g to 3j).
  • the two or more different oligonucleotides of the hybridizing agent are located adjacent to each other in the formed double-stranded hybrid.
  • a double-stranded hybrid is generated that may span at least a portion of or the entire hybridizing region (and optionally a portion of or the entire spacer region, if present) of the single-stranded (capture) oligonucleotide. Gaps may be present between the hybridized oligonucleotides of the hybridizing agent. If nucleotide gaps are present between the individual adjacent oligonucleotides, they are smaller than the length of the oligonucleotides.
  • Adjacent oligonucleotides are preferably spaced apart not more than 5nt, 4nt, 3nt, 2nt or 1 nt from each other in the formed hybrid.
  • all or at least two oligonucleotides of the hybridizing agent are contiguous to each other in the formed double-stranded hybrid and thus, the first oligonucleotide directly follows and is next to the terminal nucleotide of the previous oligonucleotide.
  • no nucleotide gaps are present between the adjacent probe molecules in such a contiguous setting.
  • Such contiguous short oligonucleotides closely resemble a longer oligonucleotide molecule, wherein however, no phosphodiester bond is present between the contiguous nucleotides of adjacent oligonucleotides, also referred to as nick.
  • Portions of the single- stranded (capture) oligonucleotide may remain single-stranded, e.g. at the outer or inner end.
  • the spacer region of the single-stranded (capture) oligonucleotide or a portion thereof (if present) may remain single-stranded.
  • a sticky end may be formed at the outer end, as has been explained above.
  • a hybridizing agent comprising at least two oligonucleotides
  • Figs. 3g to 3i Several embodiments of a hybridizing agent comprising at least two oligonucleotides are illustrated in Figs. 3g to 3i
  • Fig. 3i illustrates an embodiment with two contiguous oligonucleotides spanning at least the hybridizing region of single-stranded (capture) oligonucleotide and forming an outer blunt end.
  • Fig. 3j illustrates an embodiment, wherein three antisense oligonucleotides are used as hybridizing agent.
  • the hybridizing agent may make use of a combination of the above described embodiments.
  • Fig. 4 illustrates the workflow used in the example.
  • an antisense oligonucleotide hybridizing to the single-stranded capture oligonucleotide was added to the enrichment buffer to reduce unspecific binding of the null beads during the enrichment process (see large arrow).
  • the example demonstrates that more live beads were recovered by the workflow employing the present invention, leading to an increase in output of the whole sequencing system. The present invention therefore significantly improves existing sequencing workflows.
  • the present disclosure provides a method for enriching a first type of particles comprising double-stranded nucleic acid molecules from a composition additionally containing a second type of particles comprising a single-stranded oligonucleotide, wherein the method comprises
  • the method according to the first aspect improves the enrichment of first type of particles comprising double-stranded nucleic acid molecules from a composition comprising the first type of particles and a second type of particles comprising a single-stranded oligonucleotide by improving the depletion of the second type of particles.
  • This is achieved by the addition of the hybridizing agent which forms a hybrid with the single-stranded oligonucleotide on the second type of particles, whereby said oligonucleotide is blocked. This reduces the carry over of particles of the second type during the enrichment process as is demonstrated in the examples.
  • the method is particularly suitable for enriching and purifying clonally amplified templates that are attached to a particle (live beads) by removing particles without an amplicon (null beads).
  • particle compositions comprising first type and second type of particles as defined herein are e.g. formed in the preparation of samples for next generation sequencing.
  • Such particles are e.g. generated during clonal amplification, using e.g. particle-based emulsion PCR, which is widely applied in next generation sequencing platforms. Therefore, the double-stranded nucleic acid molecule present on the surface of a particle of the first type is preferably a double-stranded amplicon.
  • An“amplicon” is a product of an amplification reaction.
  • An amplicon is typically double-stranded, but can be rendered single-stranded if desired.
  • a single particle usually comprises multiple copies of the same double-stranded amplicon on its surface.
  • template molecules e.g. library
  • a mixture of different particles of the first type is provided, wherein each particle of the first type comprises multiple copies of the same double-stranded amplicon but wherein different particles of the first type comprise different double-stranded amplicons, as they originate from different template molecules.
  • the particles of the second type comprise multiple copies of the same single-stranded oligonucleotides, but no double- stranded amplicon. All embodiments disclosed herein particularly apply and refer to this preferred embodiment.
  • the method allows and improves enriching particles of a first type comprising double- stranded nucleic acid molecules from a composition additionally comprising a second type of particles comprising a single-stranded oligonucleotide.
  • the method preferably comprises providing a composition including particles comprising a double-stranded amplicon (first type) and particles comprising a single-stranded oligonucleotide but no double-stranded amplicon (second type).
  • Such composition is e.g. provided/obtained after performing a particle-based emulsion PCR.
  • the particles of the first type comprising the double-stranded amplicon are obtained when the template is clonally amplified on the particle surface (also referred to as“live beads”), while the second type of particle comprises a single-stranded oligonucleotide but does not comprise a clonally amplified template (also referred to as “null beads”).
  • An according composition is provided by the emulsion as such or is provided after breaking the emulsion and separating the first and second type of particles from the broken emulsion.
  • the hybridizing agent may be added to this composition, e.g. during a washing step, to form a hybrid with the single-stranded oligonucleotide that is present on the surface of the second type of particles.
  • the first type of particles and the second type of particles may originate from the same type of base particles which comprises the single-stranded oligonucleotide. It is preferred that the base particle comprises only one unique single-stranded oligonucleotide.
  • the surface of the base particles comprises multiple copies of the single-stranded oligonucleotide.
  • the base particle comprises at least 500 or at least 1000 copies of the single-stranded oligonucleotide, e.g. at least 5000, at least 10,000, at least 15,000, at least 20,000, at least 50,000 or at least 100,000 copies.
  • a high copy number of the single-stranded oligonucleotide on the base particles is preferred.
  • the number may lie e.g. in a range of 1000 to 15,000,000 copies of the single-stranded oligonucleotide per base particle.
  • the density of the surface bound single-stranded oligonucleotides is selected from 500 to 500,000 oligonucleotides/pm 2 , 750-200,000 oligonucleotides/pm 2 , or 1000-100,000 oligonucleotides/pm 2 . Suitable embodiments are well-known in the art and can be used in the context of the present invention.
  • the surface of the base particles may be coated with the single-stranded oligonucleotide as is known in the art.
  • the first type of particles may differ from the second type of particles in that said particles comprise a double-stranded amplicon, while the particles of the second type correspond to the base particles that were e.g. used for clonal amplification (see also Fig. 1).
  • the base particle (and accordingly the second type of particles) may comprise a large number of the same single-stranded oligonucleotide that is complementary to a region of the template molecule and that can serve as a primer for amplification. Extension of the single-stranded oligonucleotide by a polymerase using the hybridized strand of the template molecule as template provides a double-stranded amplicon that is attached to the particle.
  • the single- stranded oligonucleotide that serves as a primer is in the first type of particles partially or fully incorporated into the double-stranded amplicon.
  • the presence of the double-stranded amplicon is the only difference between the first and second type of particles.
  • a single particle of the first type usually comprises multiple copies of the same double-stranded amplicon covalently attached to the particle surface and different particles of the first type may comprise different double- stranded amplicons (originating from different template molecules).
  • the particles may be of any convenient size and material. They may be made of an inorganic material, a natural polymer or synthetic polymer.
  • Examples include but are not limited to cellulose, cellulose derivatives, acrylic resins, silica materials, glass, silica gel, polystyrene, gelatine, polyvinyl pyrrolidone, copolymers of vinyl and acrylamide, polystyrene, polystyrene cross-linked with divinylbenzene or the like, polyacrylamides, latex gels, dextran, rubber, plastics, nitrocellulose, metals, cross-linked dextrans (e.g., Sephadex) agarose gel (Sepharose), and other solid support materials known to those of skill in the art.
  • the particles may have an average size within a range of 0.1 pm to 50 pm.
  • the average size is within the range of 0.3pm to 20 pm, 0.5pm to 10pm and preferably 0.5pm to 5 pm.
  • a particle size in the range of 0.5pm to 3pm, such as 0.7pm to 3pm such as e.g. 1 pm is particularly suitable for sequencing applications.
  • the particles may be magnetic and therefore respond in a magnetic field.
  • the use of magnetic particles simplifies the processing of the particles by using a magnet.
  • the particles may be superparamagnetic, paramagnetic, ferromagnetic or ferrimagnetic.
  • the first type of particles comprises double-stranded nucleic acid molecules on the particle surface.
  • a particle of the first type comprises a double-stranded amplicon on its surface.
  • this includes a reference to multiple copies of the same amplicon.
  • a single particle of the first type of particles preferably comprises multiple copies of the same double-stranded amplicon.
  • a particle comprises only a single type of amplicon, as is e.g. provided as result of clonal amplification in a particle-based emulsion PCR (see Fig. 1).
  • the number of double-stranded amplicons present on the surface of a particle of a first type correlates with the number of single- stranded oligonucleotides present on the surface of a base particle. It is referred to the above disclosure which also applies to the number of amplicons on the surface of a particle of a first type. Typically, hundreds or thousands to millions of double-stranded amplicons are present on the surface of a particle of a first type after successful amplification, which is preferred for sequencing applications.
  • the first type of particles may originate from the same type of base particles as the second type of particles.
  • the originally single- stranded oligonucleotide of the base particle is in the first type of particles extended during the amplification process using a strand of the template molecule as template and becomes at least partially incorporated in a double-stranded hybrid.
  • a spacer region if present, may or may not remain single-stranded when the amplicon is generated.
  • the first type of particles may additionally comprise a single-stranded oligonucleotide, if not all single-stranded oligonucleotides were incorporated in a double-stranded amplicon. If present, such remaining residual single-stranded oligonucleotides are preferably present in a low amount.
  • the particles of the first type may be magnetically responsive.
  • the second type of particles may have the same characteristics as the base particles disclosed herein.
  • a particle of the second type comprises multiple copies of the same single- stranded oligonucleotide.
  • the particles of the second type may be coated with the same single-stranded oligonucleotide.
  • the second type of particles (and base particles) may also comprise more than one type of single-stranded oligonucleotide on the same particle surface.
  • the single-stranded oligonucleotides may comprise the same hybridizing region but may differ in their spacer region.
  • all particles of the second type comprise multiple copies of the same single-stranded oligonucleotide(s).
  • Methods for attaching single- stranded oligonucleotides to a given particle surface are known in the art.
  • the single-stranded oligonucleotide is covalently attached.
  • a single-stranded oligonucleotide may have an overall length within the range of 10nt to 200nt. It may have a length within a range of 10nt to 150nt or 15nt to 100nt. Preferred ranges include 15nt to 75nt and 20nt to 50nt. Particularly preferred is a length within the range of 20nt to 35nt or 25nt to 30nt. These ranges are particularly suitable for use as single-stranded (capture) oligonucleotide for clonal amplification.
  • the single-stranded oligonucleotide is exposed on the particle surface and is accessible for hybridization.
  • the single-stranded oligonucleotide may serve as a single-stranded capture oligonucleotide for capturing a single-stranded template molecule during clonal amplification in an emulsion PCR.
  • a particle of the first type is provided from the base particle comprising the single-stranded (capture) oligonucleotides.
  • no template is present, no amplicons are provided and the base particle corresponds to a particle of the second type.
  • the particles of the second type do not comprise a double-stranded amplicon.
  • the second type of particles only comprise the single-stranded oligonucleotide(s).
  • a single-stranded oligonucleotide provided on the surface of the second type of particles (and base particles) may comprise different functional regions, such as a spacer region and a hybridizing region.
  • the single-stranded oligonucleotide comprises a spacer region nearby the particle surface and a hybridizing region located 3’ thereto (see Fig. 1a, enlargement). Being located at the proximity of the particle surface the spacer region may compensate irregularities on the particle surface and thereby ensures that the hybridizing region of the oligonucleotide is well exposed on the particle surface and is sterically accessible for hybridization to e.g. a template molecule.
  • Adequate exposure of the single-stranded oligonucleotide can also be achieved by appropriate attachment strategies which ensure that the single-stranded oligonucleotide is exposed and well-accessible for hybridization.
  • the spacer region of the single-stranded oligonucleotide may have a length within the range of 3nt to 50nt, preferably 5nt to 20nt, more preferably 7nt to 15nt. Typically, it will have a length around 10nt.
  • any sequence may be chosen that suits the purpose of spacing the particle surface and the hybridizing region of the single-stranded oligonucleotide.
  • the spacer region comprises of consists of a nucleotide stretch of the same nucleotide, e.g. A, T/U, G, or C, preferably T.
  • the single-stranded oligonucleotide may comprise a hybridizing region.
  • the hybridizing region can be used to capture a template molecule for clonal amplification. It may be complementary to a portion of the template molecule, said portion preferably corresponding to an adapter sequence of a sequencing library provided at the end of the template molecule (see e.g. Fig. 1).
  • the hybridizing region is designed to enable base pairing with the target molecule, preferably a comprised adapter sequence.
  • the hybridizing region may be of any suitable length and may lie within a range of 10nt to 200nt or 10nt to 100nt.
  • the range is selected from 10nt to 50nt, 12nt to 30nt and 12nt to 25nt, more preferably 15nt to 25nt and 15nt to 20nt.
  • the hybridizing region hybridizes without mismatches to the complementary portion of the template molecule, such as e.g. an adapter sequence provided at the end of the template molecule.
  • the single-stranded oligonucleotide that is exposed on the particle surface may lead to unspecific binding during the enrichment process, resulting in a carry over of the second type of particles into the enriched fraction comprising the first type of particles.
  • This unspecific binding is in the present technology reduced by adding the hybridizing agent which forms a hybrid with the single-stranded oligonucleotide. Due to this hybrid formation, unspecific binding of the second type of particles is reduced.
  • the hybridizing agent may be added at one or more steps of the enrichment method prior to and/or during separation of the first type of particles from the second type of particles (see also Fig. 2). Before discussing further details of the hybridizing agent and the formed hybrid, the separation step is described in further detail which results in an enrichment of the first type of particles.
  • particles of the first type which preferably comprise a double-stranded amplicon (e.g. live beads) are separated from second type of particles (e.g. null beads).
  • the separation provides an enriched fraction of first type of particles.
  • Suitable embodiments of the separation step were illustrated in Fig. 2 and are also known in the art (see e.g. WO 2014/186152).
  • the separation comprises binding the first type of particles to a third type of particles and separating the third type of particles with the bound first type of particles from second type of particles.
  • at least a portion of said third type of particles bind two or more particles of said first type of particles (see Fig. 2).
  • the third type of particles with the bound first type of particles provides an association of particles, also referred to herein as complex of aggregate.
  • the formed complex/association of third type of particles and bound first type of particles is larger than the second type of particles. This facilitates separation by a size-selective separation step (e.g. filtration or density gradient centrifugation).
  • the third type of particles is preferably larger than the first type of particles and the second type of particles (which may originate from the same base particles).
  • the third type of particles is at least 5 times larger. It may be at least 7 times or at least 10 times larger. E.g. the third type of particles may be between approx. 10 to 20 times larger.
  • the particles of the third type may have an average size of at least 5pm or at least 7pm.
  • the particles of the third type have a size of at least 10 pm or at least 12pm, such as about 15pm.
  • the first and second type of particles have a size of approximately 0.7pm to 3pm in diameter and the third type of particles have a size of at least 10pm (e.g. in the range of 10pm to 20pm, such as 15pm).
  • the third type of particles may be magnetic and accordingly may be magnetically- responsive.
  • the particles may be superparamagnetic, paramagnetic, ferrimagnetic or ferromagnetic.
  • the use of magnetic particles allows separation of the third type of particles with the bound first type of particles using a magnet.
  • Binding of the first type of particles to the third type of particles may be reversible, thereby allowing to recover the first type of particles from the third type of particles.
  • Embodiments are known in the art and are also described herein.
  • the first type of particles specifically binds to the third type of particles.
  • the second type of particles is not capable of specifically binding to the third type of particles.
  • the third type of particles may selectively interact with the first type particles, thereby increasing the overall size in comparison to the first and/or second type particles.
  • unspecific binding of the second type of particles to the third type of particles and/or the first type of particles nevertheless occurs in prior art methods, leading to an inefficient separation and hence, inefficient enrichment of first type particles. This unspecific interaction is reduced according to the teachings of the invention by adding a hybridizing agent at one or more steps of the process.
  • the hybridizing agent is added to the composition when adding the third type of particles and/or the reagent (e.g. enrichment solution/buffer) that supports the binding of the first type particles to the third type particles.
  • the hybridizing agent which may be provided by at least one oligonucleotide reverse complementary to the single-stranded oligonucleotide of the second type of particles, may be included in the enrichment reagent that is used in order to provide suitable conditions for binding the first type of particles to the third type of particles.
  • specific binding of the first type of particles to the third type of particles is mediated by an interaction pair.
  • the interaction pair may be an affinity interaction pair, wherein one partner specifically binds the other partner of the interaction pair.
  • the interaction pair may be selected from (i) biotin and a biotin binding polypeptide, (ii) antibody and antigen and (iii) ligand and receptor. Other options are readily available to the skilled person.
  • the interaction pair is preferably provided by biotin and a biotin binding polypeptide such as streptavidin or avidin.
  • the first partner of the interaction pair is associated with the double-stranded nucleic acid (preferably amplicon) of the first type of particles and the second partner of the interaction pair is associated with the third type of particles.
  • the partners of the interaction pair interact with each other, the first type of particles is specifically bound to the third type of particles.
  • the particles of the second type do not comprise the first partner of the interaction pair, they cannot bind specifically to the third type of particles.
  • the first partner of the interaction pair e.g. biotin
  • amplification may involve the use of a primer in solution that comprises the first partner of the interaction pair (e.g. biotin).
  • one strand of the double-stranded amplicon comprises the first partner.
  • the primer is incorporated into the double-stranded amplicon during the amplification process, the resulting double-stranded amplicon provided on a particle of the first type comprises the first partner (e.g. biotin), usually at its outer end (see Fig. 2).
  • the first partner e.g. biotin
  • a mixture of primers comprising the first partner and unmodified primers may be used as second primer.
  • the particles of the first type which comprise a double-stranded amplicon become covered with the first partner of the interaction pair, while the second type of particles, which do not comprise a corresponding amplicon, lack the first partner and therefore, cannot specifically bind to the third type of particles during enrichment.
  • the third type particles which comprise the second partner of the interaction pair e.g. streptavidin
  • the first partner of the interaction pair is biotin which is associated with the double-stranded amplicon of the first type of particles and the second partner is a biotin binding polypeptide, preferably streptavidin or avidin, associated with the third type of particles (see e.g. Fig. 2).
  • biotin binding polypeptide preferably streptavidin or avidin
  • Separation and thus enrichment of first type of particles from second type of particles preferably comprises size selection. Separation may comprise separating the third type of particles with the bound first type of particles based on their larger size compared to second type of particles.
  • the third type of particles with the bound first type of particles are separated by passing the composition through a porous device, wherein the third type of particles with the bound first type of particles are retained by the porous device, while particles of the second type pass through.
  • the porous device can be a filter.
  • the filter may be a mesh, preferably a single layer mesh. It may be a nylon mesh.
  • the suitable pore size depends on the size of the second type of particles (as they must pass through the pores) and the third type of particles, because at least the formed association of the third type of particles with bound first type of particles must be retained by the porous device (so that it does not pass through the pores).
  • the third type of particles cannot pass the pores.
  • Suitable pore sizes can be chosen by the skilled person in view of the chosen size of the first, second and third type of particles.
  • the pore size may be e.g. within a range of 1 pm to 50pm, e.g. 5pm to 25pm, 7pm to 20pm or preferably 8pm to 15 pm, such as 10-11 pm.
  • the porous device may be comprised in a column, e.g. spin column.
  • the spin column may be centrifuged so as to facilitate separation.
  • vacuum could be applied to the column to suck the composition with the particles of the second type through the porous device, while the third particles with the bound first type of particles are retained.
  • separation of the third type of particles with the bound first type of particles comprises density centrifugation.
  • the composition may be contacted with a relatively dense medium (e.g. glycerol) and subjected to centrifugation, whereby the third type of particles with the bound first type of particles are separated from the second type of particles.
  • separation of the third type of particles with the bound first type of particles is performed using a magnet. This is e.g. feasible, if the third type of particles are magnetic and the first and second type of particles are not magnetic. As the first type of particles specifically bind to the third type of particles, they are separated together with the magnetic third type of particles while the unbound second type of particles are not recovered by the magnetic separation.
  • the separated third particles with the bound particles of the first type may be further processed. E.g. they may be washed after separation by performing at least one washing step. The washing step may further deplete second type of particles.
  • the method may further comprise releasing the particles of the first type from the third type of particles.
  • the release step comprises reversing the specific binding of the first type of particles from the second type of particles. Suitable methods are known to the skilled person.
  • particles of the first type labelled with biotin may be released from the third type of particles comprising a biotin binding protein such as streptavidin by denaturation, e.g. via treatment with a base such as NaOH.
  • the particles of the third type may then be separated from the first type of particles.
  • the first type of particles that are eluted from the third type of particles may pass through the pores, while the third type of particles are retained, thereby achieving separation.
  • Density centrifugation and/or magnetic separation may also be used.
  • Suitable embodiments for enrichment are also described in WO 2014/186152 to which it is referred.
  • the third type of particles may be used for enriching the first type particles after clonal amplification, preferably after breaking the emulsion and after recovering and purifying the particles, whereby a purified composition comprising first and second type particles is provided.
  • a crucial step in the method according to the first aspect therefore is the addition of a hybridizing agent capable of hybridizing to the single-stranded oligonucleotide that is present on the second type of particles, whereby a hybrid is formed.
  • Blocking the single-stranded oligonucleotide by forming a hybrid advantageously reduces the carry-over of second type of particles that results from unspecific binding/interactions mediated by the single-stranded oligonucleotide thereby rendering the depletion of said particles more efficient as is demonstrated by the examples.
  • Hybrid formation blocks and thereby hinders the single-stranded oligonucleotide from non-specifically interacting with e.g. the first and/or the third type of particles. It thereby reduces the amount of second type of particles in the separated fraction of the first type of particles.
  • the hybridizing agent may hybridize to a portion or the full length of the single-stranded oligonucleotide present on the second type particles.
  • the hybridizing agent shall not be limited to a particular molecule structure. Any hybridizing agent capable of forming a hybrid with the single-stranded oligonucleotide of second type particles may be applied according to this invention.
  • Hybridization may be achieved by hydrogen bonding between nucleobases. Such base pairing may be based on Watson-Crick base pairing or other modes of pairing, such as Hoogsteen base pairing. Therefore, double helices or triple helices may be formed.
  • the hybridizing agent is provided by at least one single-stranded oligonucleotide.
  • the oligonucleotide may be an antisense oligonucleotide that is reverse complementary to the single-stranded oligonucleotide or a portion thereof (see Fig. 3), resulting in formation of a hybrid structure.
  • the at least one oligonucleotide may be selected from various types, such as DNA, RNA, PNA, LNA or HNA, wherein preferably the oligonucleotide is selected from the group consisting of DNA or RNA, more preferably DNA.
  • the hybridizing agent may comprise nucleobases selected from A, G, T/U, and C, as well as from any chemical moiety capable of hybridizing to one or more of A, G, T/U and C.
  • Chemical moieties that are capable of hybridizing to one or more of A, G, T/U and C may be selected from artificial nucleic acids, such as peptide nucleic acids, morpholino nucleic acids, locked nucleic acids, glycol nucleic acids and threose nucleic acids, or from derivatives of natural nucleic acids, such as iso-configurations, daminopyrimidine, and xanthine.
  • Suitable embodiments of the hybridizing agent comprising one or more single-stranded oligonucleotides are illustrated in Fig. 3 and were explained above and it is referred to this disclosure.
  • the at least one oligonucleotide is at least partially complementary to the single- stranded oligonucleotide of the second type of particles.
  • a single-stranded oligonucleotide of the hybridizing agent may have a length within a range of 5nt to 200nt, 5nt to 150nt, 10nt to 100nt and 10nt to 50nt.
  • the length is within a range of 10nt to 35nt, e.g. 15nt to 30nt.
  • the length depends on the length of the single-stranded oligonucleotide of the second type particles and/or whether two or more oligonucleotides provide the hybridizing agent and are involved in hybrid formation with the single-stranded oligonucleotide. If two or more oligonucleotides are used, the individual oligonucleotides may be shorter compared to using a single oligonucleotide as the combination of oligonucleotides when hybridized to the single- stranded oligonucleotide provide the hybrid and block the single-stranded oligonucleotide.
  • the hybridizing agent is provided by a single oligonucleotide capable of forming a hybrid with the single-stranded oligonucleotide of the second type particles.
  • the length and thus the number of nucleotides (nt) of the oligonucleotide may be adjusted to the number of nucleotides of the single-stranded oligonucleotide of the second type of particles.
  • the single-stranded oligonucleotide of the hybridizing agent and the single-stranded oligonucleotide of the second type of particles are identical in length so that the hybrid is formed over the entire length of the single-stranded oligonucleotide of the second type of particles.
  • the oligonucleotide has a length that differs from the length of the single-stranded oligonucleotide of the second type of particles wherein the difference in the number of nucleotides is £ 15nt, £ 10nt, £ 7nt or £5nt.
  • a hybrid is formed with a portion of the single-stranded oligonucleotide of the second type of particles.
  • the length of the oligonucleotide is within a range of 10nt to 35nt, e.g. 15nt to 30nt and the single-stranded oligonucleotide present on the surface of the second type of particles has a length within the same range.
  • the hybridizing agent comprises two or more different single-stranded oligonucleotides capable of hybridizing at least to a portion of the single- stranded oligonucleotide of the second type of particles.
  • the two or more different single- stranded oligonucleotides may differ in at least one nucleotide from each other.
  • the at least two single-stranded oligonucleotides may hybridize to different regions of the single-stranded oligonucleotide of the second type of particles. Preferably, they hybridize adjacent to each other to the single stranded oligonucleotide. Suitable embodiments were also described in conjunction with Fig. 3 to which it is referred.
  • the hybridizing agent forms a hybrid over at least 60%, at least 70%, preferably at least 80%, at least 90% or over the entire length of the single- stranded oligonucleotide of the second type particles.
  • the hybridizing agent forms a hybrid over at least 60%, at least 70%, preferably at least 80%, at least 90% or over the entire length of the single- stranded oligonucleotide of the second type particles.
  • it can be sufficient to block only a portion thereof by hybrid formation.
  • at least the outer end of the single-stranded oligonucleotide may form part of the resulting hybrid and preferably provides a hybrid with an outer blunt end.
  • a double-stranded hybrid is formed over 3 60%, 3 70%, 3 80%, 3 90% or 3 95% of the single-stranded oligonucleotide.
  • the hybrid may be formed over the entire length (100%) of the single-stranded oligonucleotide of the second type particles.
  • the formed hybrid leaves a portion of the single-stranded oligonucleotide single-stranded.
  • the remaining single-stranded region may be £ 40%, £ 30%, £0% or £%.
  • the single-stranded portion may have a length of £ 10nt, £ 8nt or £ 5nt.
  • the single-stranded portion may be provided at the 5’ end of the single-stranded oligonucleotide, wherein the 5’end corresponds to the inner end directed towards the particle surface.
  • a single-stranded portion may also be located at the outer (3’) end of the single-stranded oligonucleotide, wherein the outer end correspond to the end of the oligonucleotide directed away from the particle surface.
  • a sticky end is formed.
  • the sticky end is too short to result in unspecific binding.
  • a partially single-stranded region is flanked by double-stranded hybrid regions. Suitable embodiments are described above in conjunction with Fig. 3.
  • the formed hybrid comprises a blunt end, wherein the blunt end is formed at the outer end of the single-stranded oligonucleotide, wherein the outer end corresponds to the end of the oligonucleotide that is directed away from the particle surface. It corresponds to the 3’ end of the single-stranded oligonucleotide that can be elongated during amplification.
  • the formed hybrid comprises at least one mismatch. In a different embodiment, the formed hybrid does not comprise a mismatch in the formed double- stranded region.
  • the hybridizing agent may be added e.g. prior to and/or during separation of the first type of particles. It may be added in two or more steps of the enrichment process to improve the depletion result. According to one embodiment, the hybridizing agent is contacted two or more times with the particles prior to separation and/or is used in excess during at least one step. It is thereby ensured that the single-stranded oligonucleotides present on the second type of particles are efficiently blocked by the hybridizing agent prior to separation. Suitable embodiments are described in conjunction with Fig. 2 and also elsewhere herein.
  • the hybridizing agent is used within the method in an amount so that at least a portion of the single-stranded oligonucleotides present on the second type of particles form a hybrid with the hybridizing agent.
  • a hybrid is preferably formed with the majority of the single-stranded oligonucleotides present on the surface of the second type particles.
  • Suitable concentration ranges for the hybridizing agent can be determined by the skilled person and can be adjusted by the skilled person to the assumed number of second type particles in the composition.
  • the % of blocked single-stranded oligonucleotides present during separation lies in the range of 50-100%, 60-100%, 70-100% , 80-100% and preferably 90-100%.
  • the hybridizing agent is added in an oversaturated regime, which means that the amount of hybridizing agent is usually higher than the amount of single-stranded oligonucleotide molecules expected to be present after amplification.
  • the molar concentration ratio of hybridizing agent to single-stranded oligonucleotide may be greater than 1.
  • the hybridizing agent may also hybridize to any remaining single-stranded oligonucleotides if present on the first type particles (e.g. residual amounts present after clonal amplification).
  • the amount of second type particles in the separated fraction of first type particles is £ 40%, £ 35% or £ 30%.
  • the amount of second type particles in the separated fraction of first type particles may be £ 25% or £ 20%.
  • the method according to the first aspect is preferably carried out subsequent to an emulsion amplification reaction.
  • the principle of the particle-based emulsion PCR is illustrated in Fig. 1.
  • the hybridizing agent preferably is provided by one or more oligonucleotides reverse complementary to the single-stranded oligonucleotide. It may be added after amplification to the composition comprising first and second type particles.
  • the hybridizing agent is comprised in a reagent that is added for processing to the composition.
  • concentration of the hybridizing agent in a reagent is in a range selected from 1 nM and 100 mM, 10 nM and 50 mM, 100 nM and 1 mM.
  • a reagent comprises the hybridizing reagent, such as e.g.
  • a single antisense oligonucleotide or a combination of two or more oligonucleotides in a final concentration selected from 2mM to 50mM, 5mM to 35mM and 7mM to 25mM, such as e.g. 10 mM.
  • the suitable concentration for the hybridizing agent may be adjusted depending on the number of applied reagents that comprise the hybridizing agent. For instance, if the hybridizing agent is applied in a single solution, the concentration of the oligonucleotide may be adjusted to a higher concentration compared to an embodiment wherein the hybridizing agent is comprised in more than one applied reagent.
  • the hybridizing agent may be added at least once, or two or more times.
  • the hybridizing agent may be supplied multiple times by using multiple solutions comprising the hybridizing agent, which can be used in sequential or non-sequential steps.
  • the hybridizing agent is present in a wash reagent that is used for washing the particles after breaking the emulsion and/or in an enrichment reagent (e.g. buffer) used before and/or during the size-selective separation of the third type of particles to which the first type of particles are bound.
  • an enrichment reagent e.g. buffer
  • Conditions are used that allow hybridization of the hybridizing agent to the single-stranded oligonucleotides.
  • the hybridizing agent hybridizes to the single-stranded oligonucleotides which are usually present at a high density on the second type particles that do not comprise double-stranded amplified template nucleic acids, thereby reducing unspecific binding and unwanted carry over of the second type particles during the enrichment step.
  • the present method thereby overcomes prior art drawbacks. It can be easily integrated into existing sequencing workflows because the hybridizing agent, which is preferably provided by at least one reverse complementary antisense oligonucleotide, can be added as additive to reagents that are already used in the workflow. It can be used at room temperature. The hybridizing agent is effective and easy to store. Therefore, the enrichment step of prior art methods (e.g.
  • WO 2014/186152 is improved, also in comparison to alternative methods such as an enzyme- based removal of the capture oligonucleotide (e.g. WO 2015/170187).
  • the hybridizing agent is inexpensive, in particular in comparison to enzyme-based strategies. It is moreover very effective.
  • the small oligonucleotides of the hybridizing agent can easily and quickly reach the single-stranded particles on the cell surface.
  • a separate processing step is not required with the present invention as the hybridizing agent may be added to one or more processing reagents that are anyhow used. This is an important advantage, in particular for automated workflows.
  • Another important advantage is that the hybridizing agent can be readily applied at various time points of existing processing and enrichment protocols, due to its high compatibility and robust functionality.
  • the method of the invention thus enables an efficient enrichment of first type particles through a cost-effective, simple and highly efficient reduction of the unspecific interaction of second type particles.
  • a hybridizing agent according to the invention By adding a hybridizing agent according to the invention, the single-stranded oligonucleotide of second type particles is blocked, disabling unspecific interaction via arbitrary base pairing.
  • the present disclosure provides a method of amplifying a template and enriching the provided amplicon, wherein the method comprises:
  • a particle comprising a single stranded oligonucleotide, wherein said single-stranded oligonucleotide is capable of acting as a primer and wherein the particle comprises multiple copies of said single-stranded oligonucleotide on the particle surface, and
  • compartments comprise one or both of (i) and (ii) but do not comprise (iii) a template molecule;
  • the method according to the first aspect is particularly suitable in order to enrich particles comprising a clonally amplified template (“live beads”) from particles not comprising a clonally amplified template (“null beads”).
  • Effective depletion of second type particles not comprising an amplified template improves the subsequent sequencing.
  • the space in a flow cell of a NGS sequencing device is less occupied by second type particles, which are irrelevant for nucleic acid sequencing, but are more occupied by first type particles comprising the amplified target nucleic acid.
  • more clonally amplified nucleic acid molecules can be sequenced per sequencing run, enhancing the overall throughput and cost-efficiency.
  • Step (a) particle-based emulsion amplification is performed to amplify target nucleic acids for subsequent sequencing.
  • the steps of the present method are explained in the following: Step (a)
  • an emulsion comprising one or more aqueous compartments in oil.
  • the emulsion may be formed according to any suitable method known in the art. As these methods are well-known and systems are also commercially available, they do not need any detailed description (see e.g. W02005/073410 and WO2014/186152, herein incorporated by reference).
  • the emulsion may be a heat stable water-in-oil emulsion.
  • the emulsion is heat stable to allow thermal processing/cycling, e.g., to at least 94°C, at least 95°C, or at least 96°C.
  • the compartments may be provided by microdroplets having a size between 1 and 100pm.
  • At least a portion of said compartments comprise in a single compartment (i) amplification agents, (ii) a particle comprising a single-stranded oligonucleotide, wherein said single- stranded oligonucleotide is capable of acting as a primer and wherein the particle comprises multiple copies of said single stranded oligonucleotide on the particle surface, and (iii) a template molecule.
  • These compartments provide upon amplification a particle of the first type, comprising multiple copies of the double-stranded amplicon on its surface.
  • Other compartments comprised in the emulsion comprise (i) and (ii) but do not comprise (iii) a template molecule. As these compartments lack a template molecule, no double-stranded amplicon can be provided on the particle upon amplification.
  • the particles comprising the single-stranded (capture) oligonucleotide are suspended as base particles in a heat stable water-in-oil emulsion. Suitable and preferred embodiments of the base particles (see description of the second type of particles) and the single-stranded oligonucleotide were described above in conjunction with the method according to the first aspect and it is referred to the above disclosure.
  • the template nucleic acids and the base particles are configured to associate with each other to reversibly bind.
  • a template nucleic acid molecule binds to the base particle via hybridization.
  • the template nucleic acids preferably comprise adapter sequences 5’ and/or 3’ of the target nucleic acid, i.e.
  • template nucleic acids comprise the same adapter sequences 5’ and/or 3’, preferably at both ends, as are usually provided in a sequencing library.
  • At least one of the adapter sequences of the template molecule is capable of hybridizing to the single-stranded (capture) oligonucleotide.
  • the base particles comprise multiple copies of the same single-stranded oligonucleotide thereby ensuring that after clonal amplification, the double-stranded amplicons are present on the particle surface in high density. Suitable embodiments are also described above.
  • the single-stranded (capture) oligonucleotide may hybridize to various different template nucleic acids of a sequencing library, as the hybridization is mediated through the adapter sequence of the target nucleic acid sequence, which can be configured be the same for the provided template nucleic acids.
  • the surface of the base particles (and second type of particles) may be provided with two or more different single-stranded oligonucleotides. If different types of single-stranded oligonucleotides are used, they may differ e.g. in the sequence and/or length of the spacer region, if present.
  • the hybridizing region of the different single-stranded oligonucleotides is preferably identical to allow efficient hybridization of the template molecule during amplification, e.g. to the adapter sequence of the template molecule, and to provide the generated amplicons in a high density at the particle surface.
  • each compartment comprises on average less than one template molecule.
  • the emulsion can be configured to statistically comprise one base particle per compartment and/or less than one template nucleic acid molecule per compartment.
  • Such a dilution ratio maximizes the frequency of amplifying only one template molecule per compartment in order to generate a clonally amplified template on a single particle that is homogeneous.
  • a particle of a first type is generated that comprises multiple copies of the same double stranded amplicon derived from the same template molecule.
  • the majority of the compartments comprised in the emulsion may include only one template molecule and one base particle.
  • the compartment furthermore comprises amplification agents and thus the agents that are required for amplifying the template nucleic acid within the compartment.
  • Amplification reactions include thermal amplification reactions, preferably polymerase chain reaction (PCR) or isothermal reactions.
  • PCR polymerase chain reaction
  • a preferred example would be amplification agents for performing a PCR.
  • the agents preferably comprise a polymerase (preferably DNA polymerase), dNTPs, salts and buffers. Embodiments are known to the skilled person and do not need any detailed description.
  • a reverse transcriptase is additionally required for a RNA template.
  • the amplification agents may comprise a primer, also referred to herein as second primer. It may function as reverse primer in solution (see also Fig. 1).
  • This second primer is capable of hybridizing to a portion of the template nucleic acid molecule.
  • the template molecule comprises sequencing adapters at the 5’ and 3’ end.
  • the single-stranded (capture) oligonucleotide of the base particle preferably hybridizes to the adapter sequence at the 3’ end of the single-stranded template molecule where it functions as a first primer. Details were described above and it is referred to this disclosure.
  • the second primer hybridizes to the 3’ end of a newly synthesized strand, wherein this newly synthesized strand comprises the single-stranded oligonucleotide bound to the particle.
  • a polymerization reaction may be induced.
  • the second primer in solution may thus hybridize to the 3’ end of the reverse strand of the template molecule that is obtained by elongation of the single-stranded capture oligonucleotide using the single-stranded template molecule as template (see Fig. 1). This portion may correspond to the adapter sequence in the reverse strand.
  • multiple copies of the double-stranded amplicon may be formed quickly.
  • the second primer may comprise a first partner of an interaction pair, such as e.g. biotin. It is referred to the above detailed disclosure which also applies here.
  • the second primer is biotinylated such that one strand of the amplified template comprises, preferably terminates with a biotin.
  • an amplicon is provided on the particle that comprises the first partner of an interaction pair. Labelling the generated amplicon with a first partner of an interaction pair may also be achieved by using e.g. labelled nucleotides for amplification.
  • the amplicon may also be provided with the first partner of the interaction pair after amplification.
  • providing the generated amplicon with a first partner of an interaction pair facilitates the subsequent enrichment of the successfully amplified first type of particles (live beads) which can specifically bind to third type of particles comprising the second partner of the interaction pair (e.g. a biotin binding polypeptide such as streptavidin or avidin).
  • a biotin binding polypeptide such as streptavidin or avidin
  • Step (b) comprises exposing said emulsion to amplification conditions.
  • nucleic acid amplification is performed by PCR.
  • the emulsion may be exposed to any suitable thermocycling conditions and/or processes known in the art.
  • the amplification of the template nucleic acid by PCR in a particle-based emulsion PCR may be performed as is known in the art (see also Fig. 1).
  • first type particles comprising a high density of double-stranded amplicons where a compartment comprises a template molecule. This is achieved by a high density of the single-stranded oligonucleotides on the surface of the base particles and a sufficiently high number of amplification cycles to achieve that the majority or all provided single-stranded oligonucleotides are after successful amplification provided with a double-stranded amplicon.
  • the template DNA is amplified until hundreds to million copies, i.e.
  • double- stranded amplicons are provided on the surface of the particle.
  • Embodiments were disclosed above and it is referred to the respective disclosure.
  • a portion of the single- stranded oligonucleotides bound to the particle may remain single-stranded without an associated double-stranded amplicon after amplification.
  • the amount, if at all present, is usually minor considering that many copies of the template molecule are provided during amplification which will then hybridize to the single-stranded oligonucleotides present on the particle surface.
  • substantially all single-stranded oligonucleotides are after amplification provided with a double-stranded amplicon.
  • the first type of particles comprises a double-stranded amplicon (“live beads”) and the second type of particles comprises the single-stranded oligonucleotide but no double-stranded amplicon (“null beads”).
  • the second type of particles essentially corresponds to the base particles that were added when forming the emulsion.
  • Optional step (c) comprises recovering the comprised particles from the emulsion prior to performing enrichment step (d).
  • Step (c) is preferably performed and may comprise breaking the emulsion and recovering the comprised particles from the broken emulsion. After separating the particles from the broken emulsion, step (c) may comprise one or more washing steps to remove residual traces of the oil phase from the nucleic acid molecules. Hence, the recovered particles may optionally be further purified in step (c), e.g. washed, prior to performing step (d).
  • the emulsion is “broken”. This step is well-known in the art and is also referred to as“de-emulsification”. The breaking or de-emulsification leads to the complete or partial separation of the water-in-oil emulsion into oil and water layers.
  • Breaking the emulsion may involve the use of an inorganic or organic de-emulsifier, and processes that treat emulsions mechanically. Breaking the emulsion may involve the use of additional oil to cause the emulsion to separate into two phases. The oil phase is then removed, and a suitable organic solvent is added. After mixing, the oil/organic solvent phase is removed. This step may be repeated several times. Finally, the aqueous layers above the particles are removed. The particles may be washed, e.g. with an organic solvent and buffer and then washed again in buffer.
  • Suitable organic solvents include alcohols such as methanol, ethanol, isopropanol and the like.
  • the emulsion is broken by the addition of organic phase that solubilizes both aqueous phase and the oil/detergent and the homogenous solution removed after centrifugation or magnetic separation. The workup is usually then followed by washes with aqueous buffers, such as PBS with additional detergent (e.g. Tween-20). Such method is e.g. described in WO 2014/186152.
  • a further method for breaking an emulsion after performing a particle-based emulsion PCR is described in EP 3 170 903 and may be used.
  • an anionic surfactant may be added which may be selected from the group consisting of: alkyl sulfates and alkyl carboxylates.
  • the anionic surfactant is provided to a water-in-oil emulsion in a final concentration of between approx. 1 - 50 wt.-%, preferably approx. 2 - 40 wt.-%, more preferably approx. 3 - 30 wt.-%, more preferably approx. 4 - 20 wt.-%, more preferably approx. 5 - 15 wt.-%, most preferably approx. 10 wt.-%.
  • the anionic surfactant is added to the water-in-oil emulsion in a concentration which ensures an effective breaking and removal of the oil phase. It is to be understood that the surfactant may be added to the emulsion in several steps where, possibly, different amounts or concentrations may be added.
  • the particles may be recovered from the broken emulsion by one or more of sedimentation, preferably centrifugation, filtration or application of a magnetic field (if the particles are magnetic). Separation may be achieved by sedimenting the particles, preferably assisted by centrifugation, and removal of the supernatant.
  • step (c) may encompass centrifugation to separate the comprised particles from the broken emulsion.
  • the particles comprise magnetic properties, as described above, thereby allowing magnetic processing for separation and optionally washing.
  • the particles may be collected by a magnet and the supernatant removed. Examples for magnetic processing of the particles for recovery from the broken emulsion are described in EP 3 170 903. This measure has the advantage that a convenient and simple separation of the captured nucleic acids can be achieved by applying a magnetic field to the broken solution.
  • Step (c) may comprise multiple breaking and washing cycles.
  • the hybridizing agent is added in step (c). In embodiments, it is added during breaking the emulsion.
  • the particles are washed one or more times in step (c) and the hybridizing agent is added during washing the particles. It may be added during one or more of the washing steps performed in step (c). Suitable washing steps and wash reagents for washing the particles are described in WO2014/186152 and EP 3 170 903.
  • the particles may be washed e.g. with a mixture of an organic solvent (e.g. methanol, ethanol, isopropanol) and annealing buffer (e.g. 100mM Tris-HCL, 1 M NaCI, 0.2% non-ionic detergent (e.g. Tween 20), pH 8), followed by washing with annealing buffer.
  • an organic solvent e.g. methanol, ethanol, isopropanol
  • annealing buffer e.g. 100mM Tris-HCL, 1 M NaCI, 0.2% non-ionic detergent (e.g. Tween 20), pH 8
  • washing with aqueous buffers such as PBS with additional detergent (e.g. a non-ionic detergent such as Tween 20).
  • additional detergent e.g. a non-ionic detergent such as Tween 20
  • washing with an aqueous buffer such as TE buffer, comprising an anionic surfactant such as ammonium lauryl sulfate followed by washing with TE buffer.
  • washing of the particles after recovery from the emulsion with the enrichment buffer that is subsequently used for enriching the first type of particles from the second type of particles using a third type of particles.
  • the hybridizing agent may advantageously be included into at least one wash reagent that is used for washing the particles in step c).
  • the hybridizing agent is added to a wash reagent that establishes conditions that allow hybridization of the hybridizing agent to the single-stranded oligonucleotides of the second particles.
  • Suitable wash reagents and conditions can also be determined by the skilled person following the disclosure of the present application.
  • the hybridizing agent may be comprised in a wash reagent in a final concentration of at least 1 mM, at least 5mM or at least 10mM.
  • the hybridizing agent is preferably included in a high concentration in the wash reagent to achieve good saturation of the single-stranded oligonucleotides by forming a hybrid.
  • Step (b) provides with the emulsion a composition comprising first type of particles and second type of particles.
  • step (c) provides a further purified composition comprising first and second type of particles.
  • template molecules e.g. a library
  • a mixture of different particles of the first type is provided, wherein each particle of the first type comprises multiple copies of the same double-stranded amplicon but wherein different particles of the first type comprise different double-stranded amplicons, as they originate from different template molecules.
  • the second type of particles essentially corresponds to the base particles.
  • Step (d) comprises enriching particles of the first type using the method according to the first aspect.
  • This method, the hybridizing agent and the formed hybrid were described in detail above and it is referred to the respective disclosure which also applies here.
  • Step (d) depletes second type particles.
  • enriching preferably comprises specifically binding the first type of particles to a third type of particles.
  • the hybridizing agent is added when adding the third type of particles and/or when separating said third type of particles with the bound first type of particles from the remaining particles.
  • the hybridizing agent is comprised in an enrichment reagent that is added to the composition when adding the third type of particles.
  • the enrichment reagent supports binding of the first type of particles to the third type of particles.
  • the enrichment reagent establishes conditions supporting the specific binding of the first type of particles to the third type of particles.
  • binding is preferably mediated by an interaction pair such as preferably biotin and a biotin binding polypeptide such as streptavidin.
  • Including the hybridizing agent in the enrichment reagent is advantageous, because no additional steps need to be performed and the hybridizing agent can be integrated into existing work-flows.
  • Suitable enrichment buffers are disclosed e.g. in WO 2014/186152 and are also commercially available.
  • the hybridizing agent may be comprised in the enrichment reagent such as e.g. an enrichment buffer in a final concentration of at least 1 mM, at least 5mM or preferably at least 10mM.
  • the recovered particles of the first type comprising the double-stranded amplicons can be subsequently used for sequencing.
  • the sequencing steps are preferably performed on each individual bead.
  • the complement strand of the amplicon may dissociate, thereby rendering particle-bound single-stranded DNA templates for sequencing.
  • a kit for enriching a first type of particles comprising double-stranded nucleic acid molecules from a composition additionally containing a second type of particles comprising a single-stranded oligonucleotide, wherein the kit comprises:
  • hybridizing agent capable of forming a hybrid with the single-stranded oligonucleotide of the second type of particles.
  • the kit can be advantageously used in the methods according to the first and second aspect.
  • Additional compounds may be one or more buffers for washing and/or a buffer suitable for allowing the interaction of the interaction pair, e.g. an enrichment buffer. As disclosed above, one or more of these buffers may comprise the hybridizing agent described above. A further compound may be a buffer suitable for allowing releasing associated particles.
  • the kit may comprise components to conduct the size-selective separation step, such as an enrichment column.
  • the third type of particles may comprise a biotin binding polypeptide, such as streptavidin or avidin. Suitable embodiments are known in the art and are also commercially available.
  • the kit may furthermore include a reagent suitable for allowing binding between biotin and the biotin binding protein.
  • the reagent may be an enrichment buffer as disclosed above.
  • the reagent may comprise the hybridizing agent, or the hybridizing agent may be added thereto before the reagent is used.
  • Embodiments and suitable concentrations were disclosed above and it is referred to the respective disclosure.
  • the hybridizing agent may be comprised in the enrichment reagent in a final concentration of at least 1 mM, at least 5mM or preferably at least 10mM. Suitable concentration ranges include 2mM to 50mM, 5mM to 35mM and 7mM to 25mM, such as e.g. 10 mM.
  • the kit may comprise a reagent for breaking an emulsion. Details of such reagent for breaking an emulsion obtained in a particle-based emulsion PCR were described above.
  • the reagent may comprise the hybridizing agent, or the hybridizing agent may be added thereto before the reagent is used.
  • the kit may furthermore comprise at least one wash reagent.
  • the wash reagent may comprise the hybridizing agent, or the hybridizing agent may be added thereto before the reagent is used.
  • the hybridizing agent may be comprised in the wash reagent in a final concentration of at least 1 mM, at least 5mM or at least 10mM. Suitable concentration ranges include 2mM to 50mM, 5mM to 35mM and 7mM to 25mM, such as e.g. 10 mM.
  • the wash reagent may be for washing the first type of particles and second type of particles after breaking the emulsion. Suitable embodiments were disclosed above and it is referred to the respective disclosure.
  • the kit may comprise a reagent suitable for releasing bound particles of the first type from the particles of the third type. Details were described above and it is referred to the respective disclosure.
  • the kit may furthermore comprise base particles comprising a single-stranded oligonucleotide.
  • the hybridizing agent comprised in the kit is capable of forming a hybrid with the single-stranded oligonucleotide of the base particles. Details were described above and it is referred to the respective disclosure which also applies here.
  • a base particle comprises multiple copies of the single-stranded oligonucleotide and preferably is coated therewith.
  • the base particles may correspond to the second type particles described in detail above and it is referred to the respective disclosure.
  • the base particles may be used in a particle-based emulsion PCR as described above.
  • the kit may furthermore comprise amplification agents, such as in particular PCR agents. Details were described above.
  • the amplification agents may comprise a second primer, which preferably comprises a first partner of an interaction pair, such as e.g. biotin.
  • the present disclosure is directed to the use of a hybridizing agent in a method for enriching a first type of particles comprising double-stranded nucleic acid molecules from a composition which additionally contains a second type of particles comprising a single-stranded oligonucleotide for blocking the single-stranded oligonucleotide of the second type of particles by forming a hybrid.
  • the double-stranded nucleic acid molecules are preferably double-stranded amplicons as are provided after clonal amplification.
  • a hybridizing agent in a method for enriching a first type of particles comprising a double-stranded amplicon from a composition which additionally contains a second type of particles comprising a single-stranded oligonucleotide but no double-stranded amplicon for blocking the single-stranded oligonucleotide of the second type of particles by forming a hybrid.
  • nucleic acid templates for sequencing as well as their preparation are well known in the art and therefore, do not need to be described herein in detail.
  • Common steps for preparing the nucleic acid template for sequencing usually include steps such as nucleic acid isolation from a biological sample and library preparation, optional target enrichment, and clonal amplification (described in detail above).
  • the technology of the present invention may be integrated into a sequencing workflow comprising the steps of (1) nucleic acid extraction, (2) optionally but preferably target enrichment, (3) library preparation, (4) clonal amplification, (5) next generation sequencing and (6) data analysis.
  • Nucleic acids may first be isolated from a biological sample or an artificial nucleic acid sample e.g. generated by PCR may be provided.
  • Biological samples from which nucleic acids can be isolated include but are not limited to cell-containing samples and cell-free biological samples.
  • Exemplary biological samples include, but are not limited to, whole blood; blood derived samples such as plasma, serum, blood cells, buffy coat; swabs; urine; sputum; saliva; semen; lymphatic fluid; amniotic fluid; cerebrospinal fluid; peritoneal effusions; pleural effusions; biopsy samples; fluid from cysts; synovial fluid; vitreous humor; aqueous humor; bursa fluid; eye washes; eye aspirates; pulmonary lavage; lung aspirates; animal, including human or plant tissues, including but not limited to, liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, cell cultures, as well as lysates, extracts, or materials and
  • nucleic acids Materials obtained from clinical or forensic settings that contain nucleic acids are also within the intended meaning of the term “sample”.
  • the extracted nucleic acids may be DNA and/or RNA.
  • the isolated nucleic acid may be extracellular nucleic acids, such as extracellular DNA, that can be isolated from cell-free or cell-depleted body fluid sample. RNA is usually transcribed into cDNA.
  • the step of nucleic acid isolation may be followed by target enrichment, as is well-known in the art.
  • the target region corresponds to the region which is supposed to be sequenced and accordingly, which is supposed to be covered by the enriched target sequences in order to obtain the sequence information for the target region of interest.
  • the regions of interest may be amplified using PCR. If the sequencing library is made of genomic DNA, a target region of interest usually consists of one or more genomic regions.
  • the present invention can be applied not only to coding exons in a genome but to any arbitrarily defined sequence portion of a genome or even metagenome.
  • the present invention can also be applied to the transcriptome and to cDNAs derived from the transcriptome. Accordingly, the target region of interest may also correspond to one or more transcripts, miRNAs, tumor derived nucleic acids or any other nucleic acid sequences of interest that are supposed to be sequenced.
  • the nucleic acid template is a DNA template.
  • the DNA template may have been prepared from RNA by reverse transcription.
  • the DNA template may have a length in a range selected from 50 to 1000, 50 to 800 and 50 to 500 nucleotides (the length refers to bp (base pairs) in case of a double-stranded molecule). Suitable sizes can be obtained by fragmentation. Suitable techniques are well-known in the art.
  • the method may comprise preparing a sequencing library.
  • a sequencing library comprises a plurality of double-stranded nucleic acid molecules and is suitable for massive parallel sequencing and accordingly, is suitable for next generation sequencing.
  • the plurality of double-stranded nucleic acid molecules present in the sequencing library may be linear.
  • a sequencing library which is suitable for next generation sequencing can be prepared using methods known in the prior art. Usually, methods for preparing a sequencing library suitable for next generation sequencing includes fragmenting the DNA followed by DNA repair and end polishing and, finally, often NGS platform-specific adaptor ligation.
  • the DNA such a genomic DNA, cell-free DNA or cDNA
  • the length of the fragments can be chosen based on the sequencing capacity of the next generation sequencing platform that is subsequently used for sequencing.
  • the obtained fragments have a length of 1500bp or less, 1000bp or less, 750bp or less, 600bp or less and preferably 500bp or less as this corresponds to the sequencing capacity of most current next generation sequencing platforms.
  • the obtained fragments may have a length that lies in a range of 20 to 550bp, 50 to 500bp, preferably 100 to 400bp, more preferred 150 to 350bp.
  • Respective fragment sizes are particularly suitable for genomic DNA, also considering that the size of an exon is approx. 150bp to 200bp in length and respective short fragments can be efficiently sequenced using common next generation sequencing platforms.
  • longer fragments can be used, e.g. if using next generation sequencing methods which allow longer sequence reads.
  • smaller fragment sizes e.g. starting from 15bp
  • the fragmented DNA can be repaired afterwards and end polished using methods known in the prior art, thereby providing for example blunt ends or A overhangs.
  • adapters are ligated at the 5' and/or 3' ends of the DNA fragments, preferably at both ends of the obtained fragments.
  • the specific design of the adapters depends on the next generation sequencing platform to be used and for the purposes of the present invention, basically any adaptors used for preparing sequencing libraries for next generation sequencing can be used.
  • at least one adapter sequence is provided which allows hybridization to at least a portion of the single-stranded oligonucleotide of the base particles.
  • the adapter sequences provide a known sequence composition allowing e.g. subsequent library amplification and/or sequencing primer annealing.
  • adaptors double-stranded or partially double-stranded nucleic acids of known sequence can be used.
  • the adapters may have blunt ends, cohesive ends with 3’ or 5’overhangs, may be provided by Y shaped adapters or by stem-loop shaped adapters.
  • the adaptors have a length of at least 7, preferably at least 10, preferably at least 15 bases.
  • the adapter length preferably lies in a range of 10 to 100 bases, preferably 15 to 75 bases, more preferred 20 to 60 bases. Either the same or different adaptors can be used at the 3’ and 5’ end of the fragments.
  • at least one of the adapters is at the sense or antisense strand complementary to the single-stranded oligonucleotide of the base particles.
  • the adapters may also provide an individual index thereby allowing the subsequent pooling of two or more target enriched sequencing libraries prior to sequencing.
  • the nucleic acid template to be amplified by emulsion PCR may thus be a population of DNA such as e.g. a DNA library. It is preferred that each member of the DNA population has a common nucleic acid sequence at the first end and a common nucleic acid sequence at the second end. This can be achieved by providing an adapter DNA sequence at each end.
  • the nucleic acid template may be provided by a PCR product comprising adapter sequences at each end.
  • the present method can be embedded within a sequencing workflow.
  • an enriched fraction of first type of particles is provided, wherein the particles comprise double-stranded amplicons.
  • the double-stranded amplicons result from clonal amplification so that the amplicons on a single particle are the same or are essentially the same (in view of potential amplification errors) as they were obtained from the same original template molecule.
  • particles are generated, wherein each particle comprises only one type of amplicon with a high density.
  • Particles without any amplified target nucleic acids are depleted in the enrichment step.
  • the methods may further comprise using the enriched fraction of first type of particles as a sample for sequencing.
  • the method may accordingly encompass sequencing the amplicons attached to the particle.
  • Sequencing may be performed using any known sequencing method and sequencing device. For instance, a GeneReader System (Qiagen) may be used. Importantly, this invention is not limited to a particular applied sequencing device capable of sequencing amplicons attached to a particle as substrate. In one embodiment, sequencing is performed by a sequencing by synthesis method.
  • next generation sequencing of the amplicons bound to the first type particles is performed.
  • the amplified nucleic acids comprised in the first type nucleic acids may be sequenced.
  • the enriched particles of the first type may be subjected to conditions that induce denaturation of the double-stranded amplicons to generate single-stranded template nucleic acids.
  • primers may be present when denaturation occurs.
  • An exemplary sequencing process comprises the hybridization of a primer, and the preparation of a flow cell. Afterward the sample is loaded into the device and sequencing is started.
  • the GeneReader sequencing method is applied, which may comprise FASTQ-file generation.
  • the sequencing may be followed by a data analysis step.
  • the various methods and processes described above are automated.
  • the enriching method may be performed using an automated sample processing system.
  • the system may have regions for particular tasks, e.g. centrifugation, to which and from which materials, e.g. tubes containing particles, are moved by a robotic arm or the like.
  • the regions may have platforms, drawers, or decks.
  • the commercially available QIAcube from QIAGEN is equipped with an automated centrifuge and pipetting system which can be programmed to do all or a portion of the method steps with limited human intervention. Automated systems suitable for processing magnetic particles are also known in the art.
  • the methods of the present invention may also be applied for particles used for other nucleic-acid based applications in the fields of basic biological research, biomedical research, applied testing, and molecular diagnostics.
  • Applications include, but are not limited to, clonal amplification of specific DNA fragments on the surface of microspheres by polymerase chain reaction or other amplification methods, and specific isolation of nucleic acids/ nucleic acid with oligonucleotide-conjugated particles by hybridization-based methods.
  • the method could also be used for decreasing unspecific binding observed for other hybridization based products of nucleic acids (e.g Oligotex mRNA kits).
  • Further potential applications include enrichment of emulsion PCR live beads (i.e. first type particles), enrichment of particles with desired DNA/RNA sequences, and/or capture of specific DNA and RNA targets (the method is applicable to non-particle based methods for capturing nucleic acids, e.g. via the surface of a tube).
  • the term“particle” refers to discrete, small objects that may be in various shapes, such as a sphere, capsule, polyhedron, and the like. It preferably is a sphere. In case of a sphere, it is not contemplated to have a perfectly round sphere but rather an approximate round or ellipsoidal shape, which may have a rough and/or structured surface. Moreover, the particles may be polydisperse having different properties within one or multiple populations of applied particles. Moreover, the term“bead” may be interchangeably used with the term“particle”.
  • first type particle or“particles of a first type” may be interchangeably used with the term“first type of particles”
  • the term“second type particle” or“particles of a second type” may be interchangeably used with the term“second type of particles”
  • the term“third type particle” or“particles of a third type” may be interchangeably used with the term“third type of particles”.
  • a hybridizing agent consisting of an antisense oligonucleotide is added to the enrichment buffer at 10 mM final concentration.
  • the enrichment buffer is part of Clonal Amplification kit, Cat. No. 185001.
  • the antisense oligonucleotide is reverse complementary to the single-stranded (capture) oligonucleotide of the base particles used in the particle based emulsion PCR.
  • the antisense oligonucleotide of the hybridizing agent has the following sequence: 5’ ACT GGC CGT CGT TTT ACA AAA AAA AAA 3’
  • the antisense oligonucleotide is added to the particles (composition comprising live beads and null beads) generated using the QIAGEN GeneReader workflow after breaking the emulsion and before separating the live beads from the null beads.
  • Method according to the invention same configuration, however with the modification that the antisense oligonucleotide was added as hybridizing agent to the enrichment buffer prior to starting the enrichment (process step is highlighted in Fig. 4 by a box and arrow).
  • Table 1 Results of evaluation of hybridizing agent in the GeneReader workflow. All values are normalized to the control condition. *Each flow cell is corresponding to an independent enrichment experiment.

Abstract

La présente invention concerne des procédés d'enrichissement avantageux qui sont appropriés dans le domaine du séquençage de nouvelle génération, des particules comprenant un amplicon à séquencer étant séparées et ainsi enrichies à partir de particules qui ne comprennent pas un amplicon. Le procédé bloque les oligonucléotides monocaténaires présents sur les particules ne comprenant pas un amplicon par addition d'un agent d'hybridation qui forme un hybride avec l'oligonucléotide simple brin, ce qui permet de réduire les interactions non spécifiques conduisant à un report indésirable des particules ne comprenant pas d'amplicon dans la fraction enrichie de particules comprenant un amplicon.
PCT/EP2019/087061 2018-12-31 2019-12-27 Procédé d'enrichissement pour séquençage WO2020141144A1 (fr)

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