[go: up one dir, main page]

WO2025146377A1 - Procédé de codage à barres d'acide nucléique et optique - Google Patents

Procédé de codage à barres d'acide nucléique et optique Download PDF

Info

Publication number
WO2025146377A1
WO2025146377A1 PCT/EP2024/087838 EP2024087838W WO2025146377A1 WO 2025146377 A1 WO2025146377 A1 WO 2025146377A1 EP 2024087838 W EP2024087838 W EP 2024087838W WO 2025146377 A1 WO2025146377 A1 WO 2025146377A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
barcode
optical
particles
sequencing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/087838
Other languages
English (en)
Inventor
Tuomas Pertti Jonathan KNOWLES
Georg MEISL
Catherine Kejia XU
Jochen Guck
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften
Cambridge Enterprise Ltd
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften
Cambridge Enterprise Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Max Planck Gesellschaft zur Foerderung der Wissenschaften, Cambridge Enterprise Ltd filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften
Publication of WO2025146377A1 publication Critical patent/WO2025146377A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/149—Particles, e.g. beads
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/179—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a nucleic acid

Definitions

  • the present disclosure relates to methods of, and apparatus for, optical and nucleic acid barcoding of material, such as biological material, methods of associating information about material obtained prior to preparation of the material for nucleic acid sequencing with information about the material obtained by means of said nucleic acid sequencing.
  • non-sequence derived information about biological material such as cells
  • sequence derived information about biological material at the level of a single set of biological material, such as a single cell. This is in part because information about the identity of the set of biological material, such as a single cell, is irrecoverably lost during the sequencing process.
  • a method of optical and nucleic acid barcoding of material comprising: providing a first nucleic acid barcode identifiable by sequencing means; providing an optical barcode identifiable by optical means and comprising a second nucleic acid barcode identifiable by said sequencing means; providing material from which nucleic acid molecules are derivable; partitioning the first nucleic acid barcode, the optical barcode and the material within a partition; wherein the material within the partition can be uniquely identified by said optical means using the optical barcode; wherein the first nucleic acid barcode is configured to separately combine with nucleic acid molecules derived from the material and with the second nucleic acid barcode, such that both the nucleic acid molecules derived from the material and the optical barcode encapsulated with the material within the partition can be uniquely identified by said sequencing means using the first nucleic acid barcode, such that the nucleic acid molecules derived from the material identified by said sequencing means can be associated with the material identified by said optical means.
  • the optical barcode is formed from one or more different optical barcode particles configured to be uniquely identifiable by said optical means.
  • the second nucleic acid barcode is formed from one or more different second nucleic acid barcode molecules.
  • the one or more different second nucleic acid barcode molecules are provided on respective different one or more second carrier particles.
  • the one or more second different carrier particles are optical barcode particles configured to be uniquely identifiable by said optical means and together form the optical barcode.
  • the optical barcode particles are differentiated from each other by one or more of size, shape, optical density, patterning, texture, refractive index, mass density, colour, and/or fluorescence.
  • the optical barcode particles comprise one or more fluorescent dyes.
  • different optical barcode particles comprise different proportions of the one or more fluorescent dyes.
  • the optical barcode particles are substantially spherical or substantially polyhedral.
  • the optical barcode particles comprise a plurality of optically identifiable particles within the optical barcode particles providing texture to the optical barcode particles.
  • the optical barcode is formed from a selection of one or more different particles from a plurality of different possible particles.
  • the selection of the one or more different particles forming the optical barcode is random.
  • the selection is non-predetermined.
  • the particles forming the optical barcode are formed from polyacrylamide.
  • the first nucleic acid barcode is provided on a first carrier particle.
  • a method of optical and nucleic acid barcoding of material comprising: providing a first carrier particle comprising molecules of a first nucleic acid barcode identifiable by sequencing means; providing one or more second carrier particles comprising molecules of different respective one or more second nucleic acid barcodes identifiable by said sequencing means; providing material from which nucleic acid molecules are derivable; encapsulating the first carrier particle, the one or more second carrier particles and the material within a droplet; wherein the one or more second carrier particles are configured to be uniquely identifiable by optical means, such that material within the droplet can be uniquely identified by said optical means using the one or more second carrier particles; wherein the molecules of the first nucleic acid barcode are configured to separately combine with nucleic acid molecules derived from the material and with molecules of the one or more second nucleic acid barcodes, such that the nucleic acid molecules derived from the material and the one or more second carrier particles encapsulated with the material within the partition can be uniquely identified by said sequencing means using the first nu
  • a method of associating information about material obtained prior to preparation of the material for nucleic acid sequencing with information about the material obtained by means of said nucleic acid sequencing comprising: the method of any preceding aspect; obtaining first information about the material within the partition and uniquely identifying the material by said optical means using the one or more second carrier particles; obtaining second information about the material within the partition, from nucleic acid molecules derived from the material, by sequencing means and uniquely identifying the nucleic acid molecules derived from the material and the one or more second carrier particles encapsulated with the material within the partition by said sequencing means using the first nucleic acid barcode; and associating the nucleic acid molecules derived from the material identified by said sequencing means with the material identified by said optical means.
  • an apparatus for optical and nucleic acid barcoding of material comprising: a microfluidics system comprising: a first inlet channel for providing particles forming first nucleic acid barcodes identifiable by sequencing means, within a first liquid phase; a second inlet channel for providing particles forming an optical barcode identifiable by optical means and comprising a second nucleic acid barcode identifiable by said sequencing means, within the first liquid phase; a third inlet channel for providing particles of material from which nucleic acid molecules are derivable, within the first liquid phase; a fourth inlet channel for providing a second liquid phase immiscible with the first liquid phase; a partition forming portion configured to encapsulate a single first nucleic acid barcode, an optical barcode and a single particle of material within a droplet suspended within the second liquid phase; wherein that material within the droplet can be uniquely identified by said optical means using the optical barcode; wherein the first nucleic acid barcode is configured to separately combine with nu
  • the apparatus further comprises control means configured to control the apparatus to form the droplet comprising a single first nucleic acid barcode, an optical barcode and a single particle of material.
  • control means is configured to control the flow rate of the first to fourth inlet channels.
  • the apparatus further comprises said optical means.
  • the apparatus further comprises the particles forming first nucleic acid barcodes, and/or the particles forming an optical barcode.
  • Fig. 1 shows a schematic illustration of a method according to the disclosure
  • Fig. 2 illustrates the combinatorial approach to optical barcoding of the disclosure
  • Fig. 3 illustrates an example chemical structure of the optical barcode particles
  • Fig. 4 illustrates an example apparatus according to the disclosure
  • Fig. 5 illustrates an example apparatus according to the disclosure
  • Fig. 6 shows example images of cells under shear stresses
  • Fig. 7 illustrates identification of an optical barcode
  • Fig. 8 illustrates examples of optical barcode beads with different textures.
  • Methods according to the disclosure may allow information derived from nucleic acid sequencing about material to be paired with other information about the material obtained prior to preparation of the material for sequencing, e.g. prior to disruption of the material to extract nucleic acid. Accordingly, data can more easily be obtained relating to a single set of material.
  • the material may be, or may comprise, biological material.
  • Biological material may refer to material derived from biological organisms. Examples of biological material may include a single cell, or cluster of cells, DNA, RNA, proteins or biopolymers.
  • the material may also be, or may comprise, synthetic material, including synthetic versions of the above example biological materials.
  • the material may comprise both biological and synthetic material. In general, the material may be biologically relevant material. Examples are described below which generally relate to biological material, in particular, cells.
  • Fig. 1 shows a schematic illustration of a method according to the disclosure.
  • a first nucleic acid barcode 11 an optical barcode 2 comprising a second nucleic acid barcode 21 and biological material 3 are provided.
  • these are encapsulated within a droplet 4.
  • the biological material 3 is associated with a nucleic acid barcode (namely, the first nucleic acid barcode) and an optical barcode.
  • the first nucleic acid barcode 11 is configured to separately combine with nucleic acid molecules 31 derived from the biological material 3 and with the second nucleic acid barcode 21. As shown in the bottom right of Fig. 1, this produces two sets of nucleic acid sequences, namely a first set of nucleic acid sequences comprising a sequence of a nucleic acid molecule derived from the biological material and the sequence the first nucleic acid barcode, and a second set of nucleic acid sequences comprising a sequence of the second nucleic acid barcode and the sequence the first nucleic acid barcode.
  • sequencing is simultaneously performed for a relatively large amount of biological material, e.g. as opposed to a single set of biological material, e.g. a cell. Accordingly, the sequencing process may produce the above described first and second sets of sequences together with additional sequences relating to different biological material.
  • the first and second sets of sequences for a particular set of biological material are identifiable and differentiable by the first nucleic acid barcode. Accordingly, the first and second sets of sequences can be identified and paired. Accordingly, information about the biological material 3 based on the nucleic acid molecules 31 derived from the biological material can be paired with the optical barcode corresponding to the same biological material. Accordingly, information about the biological material that is obtained prior to sequencing and which is associated with the biological material based on the optical barcode can associated with the information about the same biological material based on sequencing of the nucleic acid molecules 31 derived from the biological material.
  • the information about the biological material that is obtained prior to sequencing is not limited to any particular type of information, provided it can be associated with the biological material based on the optical barcode. This association may occur before at the same time as, or after the information is obtained.
  • the droplet comprising the biological material and the optical barcode may be tracked in a microfluidics device such that obtaining the information and reading the optical barcode can be performed separately and associated based on the tracking. If the obtaining of information results in the breaking up of the droplet and/or disruption of the optical barcode, then the reading of the optical barcode may be performed first, for example.
  • information about the biological material may be obtained before the biological material is encapsulated in a droplet with the optical barcode. For example, in some cases it may be possible to determine which droplet the biological material is later encapsulated in, e.g. based on a transit time of the biological material through a droplet forming apparatus.
  • the first nucleic acid barcode 11 is identifiable by sequencing means and may be provided by any suitable means known in the art. As shown in Fig. 1, the first nucleic acid barcode 11 may be provided on a first carrier particle 1.
  • the first carrier particle 1 may be formed from a polymer, such as a hydrogel.
  • the first carrier particle 1 may be formed from polyacrylamide.
  • the first carrier particle 1 may be formed from a microsphere bead, such as that described in Wang, Y. et al. Dissolvable polyacrylamide beads for high-throughput droplet DNA barcoding. Advanced Science 7, 1903463 (2020).
  • the first nucleic acid barcode may be formed from DNA or RNA, for example.
  • the surface of the first carrier particle 1 may be configured, e.g. functionalised, to allow attachment of the first nucleic acid barcode 11 thereto.
  • the attachment of the first nucleic acid barcode 11 to the carrier particle 1 may be provided by one or more linker molecules, for example.
  • the process of attachment may be facilitated by one or more enzymes, for example, such as DNA ligase or polymerase.
  • the optical barcode 2 may be identifiable by optical means.
  • the optical means may comprise one or more of bright-field microscopy, or fluorescence microscopy, for example. However, other suitable means may be used alternatively or additionally.
  • the optical barcode 2 may be provided by one or more different particles 2A-2C (also referred to as ID beads). These may be configured to be uniquely identifiable by said optical means. Accordingly, the biological material 3 within the droplet 4 may be identified based on the combination of different particles 2A-2C within the droplet 4.
  • the second nucleic acid barcode 21 may be formed from one or more different nucleic acid barcode molecules, namely those associated with the one or more different particles 2A-2C forming the optical barcode.
  • the different second nucleic acid barcode molecules each allow their respective particle to be identified by sequencing. Accordingly, the different particles 2A-2C identified allow identification of the optical barcode 2 they form.
  • Each different particle 2A-2C has a different nucleic acid barcode molecule.
  • a large number of different optical barcodes can be provided by a relatively small number of different possible particles 2A-2C, by combining the different possible particles in different ways. This is illustrated by Fig. 2.
  • the left-hand side of Fig. 2a shows individual barcoding, wherein an optical barcode is provided by a single optical barcode particle.
  • the number of different possible optical barcode particles corresponds linearly to the number of possible optical barcodes (also three).
  • the right-hand side of Fig. 2a shows combinatorial barcoding as disclosed herein, wherein the number of different possible optical barcode particles (e.g. three, as shown) can be combined in many different ways to increase to the number of possible optical barcodes (e.g. seven, as shown).
  • the number of possible optical barcodes is determined by the number of possible combinations of possible optical barcode particles. How the number of possible optical barcodes increases with the number of possible optical barcode particles is shown in Fig. 2b, on a log scale.
  • the different optical barcode particles 2A-2C may be optically identifiable and distinguishable from each other in any suitable way.
  • the different optical barcode particles 2A-2C may be differentiable from each other based on one or more of size, shape, optical density, patterning, texture, refractive index, mass density, colour (light absorption), and/or fluorescence. As described above, these features may be determined by one or more of bright-field microscopy and fluorescence microscopy, for example. Texture may refer to whether, or to what extend the optical barcode particles comprise a plurality of (optically identifiable) smaller particles.
  • optical barcode particles with different, distinguishable, textures are shown in Fig. 8 (scale bar: 20 pm).
  • the optical barcode particles are beads made from polyacrylamide, with polystyrene beads of varying sizes (0.5, 1, and 2 pm diameter from left to right) and concentrations trapped inside.
  • Fig. 2c illustrates how optical biomarker particles may be differentiated - different optical biomarker particles are shown in different columns.
  • the top row illustrates that the shape and size of the particles may be determined by bright-filed microscopy.
  • the second to fourth rows illustrate that fluorescence characteristics at different wavelengths may be determined by fluorescence microscopy. As illustrated, the shape, size and fluorescence alone may not differentiate all particles. However, these characteristics may be combined, resulting in fully distinguishable particles.
  • the optical barcode particles may be differentiable based on the ratio of fluorescence at different wavelengths, for example.
  • the optical barcode particles may comprise one or more different fluorescent dyes, for example.
  • the presence and ratios of the different fluorescent dyes may determine ratio of fluorescence at different wavelengths.
  • Figs. 2d and 2f show clusters of optical barcode particles based on the ratio of fluorescence detected at 525 nm and 593 nm, and 593 nm and 700 nm, respectively.
  • the ratio of two different dyes were varied. As shown, this provided seven distinctly identifiable particles based on fluorescence alone. This multiplicatively combines with variations in other features such as shape, size etc.
  • Fig. 2e the ratio of three different dyes were varied.
  • the particles were exposed to excitation laser light at 488 nm.
  • the dyes may be one or more of Atto-488, SCy3, SCy5, DY-51 IXL, and DY-521XL, for example.
  • optical barcodes, and/or the optical barcode particles forming the optical barcodes may be identified from images (e.g. using any combination of the imaging methods described above) by image processing software.
  • the image processing software may comprise a machine learning (or artificial intelligence) model trained to identify optical barcodes, and/or the optical barcode particles forming the optical barcodes.
  • Fig. 7 illustrates artificial intelligence-based detection and identification of optical barcode beads (four different sizes/shapes: light blue, dark blue, pale pink, and dark pink), nucleic acid barcode beads (white), and cells (orange) within microfluidic droplets (scale bar: 60 pm).
  • the optical barcode particles 2A-2C may each comprise a second carrier particle on which the second optical barcode 21 is provided.
  • the second carrier particle may be formed from a polymer, such as a hydrogel.
  • the second carrier particle may be formed from polyacrylamide.
  • the second carrier particle may be formed from a microsphere bead, such as that described in Wang, Y. et al. Dissolvable polyacrylamide beads for high- throughput droplet dna barcoding. Advanced Science 7, 1903463 (2020).
  • the second nucleic acid barcode may be formed from DNA or RNA, for example.
  • the surface of the second carrier particle may be configured, e.g. functionalised, to allow attachment of the second nucleic acid barcode 21 thereto.
  • the attachment of the second nucleic acid barcode 21 to the second carrier particle may be provided by one or more linker molecules, for example.
  • the process of attachment may be facilitated by one or more enzymes, for example, such as DNA ligase or polymerase.
  • the second nucleic acid barcode molecules need to be able to bind to the first nucleic acid barcode molecules, which enables downstream analysis. This binding can occur while second nucleic acid barcode is still attached to the second carrier particle, or the second nucleic acid barcode molecules may be cleaved from the second carrier particles in a controlled way.
  • the particle-barcode linkage needs to be stable, in that it does not break unless a specific trigger to do so is applied.
  • the trigger could be light to induce photo-cleavage, or a reducing agent to break disulphide bonds.
  • the trigger could be the introduction of a competitive binding molecule. It is to be noted that such a trigger needs to exist only if the nucleic acid barcode needs to be released from the carrier particle.
  • Each of the second carrier particles may be provided in one of a plurality of different possible shapes.
  • Each of the different shapes should be relatively distinct.
  • the shapes may be spherical or polyhedral.
  • the different shaped carrier particles may be formed from the same base materials.
  • the different shapes may be determined by the conditions under which the particles are formed.
  • polymerisation may be performed under static conditions to form spherical particles or under centrifugal force to form polyhedral particles (e.g. as described in Weisgerber, D. W., Hatori, M., Li, X. & Abate, A. R. Polyhedral particles with controlled concavity by indentation templating.
  • Each of the second carrier particles may be provided in one of a plurality of different possible sizes. Each of the different sizes should be relatively distinct. For example, the shapes may be small, medium and large, relatively.
  • the different sized carrier particles may be formed from the same base materials. The different sizes may be determined by the conditions under which the particles are formed.
  • the second carrier particles may be formed by droplet microfluidics and their size may be determined by the dimensions of the microfluidics device in which they are formed.
  • Each of the second carrier particles may comprise one or more different fluorescent dyes.
  • the different fluorescent dyes may be molecules configured to emit fluorescence at different predetermined wavelengths when excited by excitation light.
  • the fluorescent dyes may be attached to a surface of the second carrier particles.
  • the surface of the second carrier particle may be configured, e.g. functionalised, to allow attachment of the one or more different fluorescent dyes thereto.
  • the attachment of the one or more different fluorescent dyes to the second carrier particle may be provided by one or more functionalised groups attached to the dye, for example.
  • Fig. 3 illustrates an example chemical structure of the optical barcode particles.
  • the second carrier particles are synthesised from polyacrylamide. They are formed by droplet microfluidics to allow precise control of bead size and homogeneity.
  • the aqueous phase may contain acrylamide, N,N'-bis(acryloyl)cystamine as a cross linking agent, N-(3 -aminopropyl )methacrylamide hydrochloride, DNA with an acrydite modification at the 5’ end, and a free radical polymerisation initiator (e.g. ammonium persulphate (APS)).
  • the oil phase may contain surfactant and a polymerisation catalyst (e.g.
  • the second carrier particles may be incubated with N-hydroxysuccinimide-functionalised dyes configured to attach to the surface of the second carrier particle.
  • the DNA barcode may be released from the particle by Dithiothreitol (DTT) as a reducing agent used for the cleavage of disulfide bonds.
  • DTT Dithiothreitol
  • Fig. 4 illustrates an example apparatus 100 for generating droplets comprising the first nucleic acid barcode, the optical barcode and the biological material.
  • the apparatus comprises respective inlet channels for each of the biological material (referred to as cells), the first nucleic acid barcode (referred to as barcoded hydrogels) and the optical barcode (referred to as ID beads).
  • Each of these components may be provided within a first liquid phase, which may be an aqueous phase.
  • an inlet e.g. two on opposing sides, as shown
  • oil a second liquid phase
  • An inlet channel e.g. two on opposing sides, as shown
  • the components of the droplet may be encapsulated within the droplet at a droplet forming portion 101 of the apparatus.
  • the droplet forming portion 101 may be a junction between the first and second liquid phases.
  • the first and second liquid phases may be mixed at the droplet forming portion 101 in such a way that a droplet is formed, as shown in Fig. 4.
  • the components of the droplet may be mixed in the first liquid phase in a mixing porting 102 preceding the droplet forming portion 101, as shown.
  • the flow rate through each of the inlet channels may be controlled to form a droplet comprising a single first nucleic acid barcode, an optical barcode and a single particle of biological material.
  • the biological material may be provided as a particle of biological material, e.g. a cell. As shown in Fig. 4, the particles of biological material may provide one particle at a time to the mixing portion. A predetermined time gap may be provided between successive particles of biological material.
  • the first nucleic acid barcode may be provided as a particle and these particles may provide one particle at a time to the mixing portion. A predetermined time period may be provided between successive particles. The time period between the particles of biological material and the particles providing the nucleic acid barcode is preferably be the same.
  • the optical barcode may be formed from one or more optical barcode particles of different types.
  • the different types of optical barcode particles may be mixed within the stream prior to entering the mixing potion 102 of the apparatus, such that the optical barcode particles in each droplet are selected randomly and in a non-predetermined manner.
  • the flow rate of the optical barcode inlet may be configured such that a plurality of optical barcode particles enters the mixing portion 102 to be encapsulated in a droplet.
  • the apparatus 100 may comprise an imaging portion 103 at which the droplets may be imaged to read the optical barcode.
  • the imaging portion 103 may comprise a brightfield imaging region and a fluorescence detection region.
  • the microfluidic channel at the imaging region 103 may be constricted, e.g. narrower than the surrounding microfluidic channel. This constriction may prevent multiple optical barcode particles passing through the imaging region at the same time. This may prevent multiple optical barcode particles being excited by the excitation light source simultaneously, which may result in mixed fluorescent signals and inaccurate optical barcode reading.
  • Fig. 5 shows a microfluidic chip design of an example apparatus 200.
  • inlets 210, 220, 230, 240, 250 and corresponding inlet channels 211, 221, 231, 241, 251 may be provided respectively for each of the biological material, optical barcode particles, nucleic acid barcode particles, buffer and continuous liquid phase.
  • Components forming the droplets are provided within a dispersed phase.
  • Droplets of the dispersed phase are formed in the continuous phase at a droplet forming portion 260, which may comprise a junction between the continuous phase and the dispersed phase.
  • the junction may comprise a central channel configured to flow the dispersed phase and two opposing side channels configured to inject the continuous phase into the central channel.
  • Droplets may be incubated in an incubation channel 271 between the droplet forming portion 260 and an outlet 270. Droplets may be collected and removed from the apparatus via the outlet 270, e.g. for performing sequencing.
  • An imaging portion 280 may be provided at a portion of the output channel 271.
  • methods according to the disclosure may allow information about biological material, such as a cell, derived from nucleic acid sequencing to be paired with other information about the biological material obtained prior to preparation for sequencing, e.g. disruption of the biological material to extract nucleic acid.
  • the other information about the biological material is not particularly limited provided that it can be paired with the optical barcode.
  • An example use of the above described methods may be to pair mechanical properties of the biological material to biochemical information obtained by sequencing.
  • the mechanical properties may include transit time of the biological material (e.g. a cell) through a constriction and/or changes in shape of the biological material in response to mechanical stress.
  • the mechanical stress may be shear stress, e.g. provided by a viscous medium flowing through a constricted microfluidic channel.
  • Fig. 6 shows example images of cells under shear stresses to determine their mechanical properties. Cells with a high stiffness are not much affected by the shear stresses and remain roughly spherical, whereas softer cells deform into a bullet-like shape.
  • the apparatuses shown in Figs. 4 and 5 are configured to obtain this information.
  • the inlet channel 211 for the cells is constricted.
  • the buffer is mixed with the cell prior to entering this constriction to provide the viscous medium imparting shear stresses.
  • the constriction is also provided within an imaging portion, e.g. for bright filed imaging. The transit time of a cell from the constriction determines which droplet the cell is encapsulated in. This allows the cell to later be associated with an optical barcode, as described above.
  • the sequencing process may be performed.
  • the first nucleic acid barcode preferably combines with the second nucleic acid barcode and nucleic acid molecules derived from the biological material while the droplet is intact. This ensures that the correct first nucleic acid barcode combines with the correct optical barcode and nucleic acid molecules derived from the correct biological material.
  • Reagents may be provided that are configured to enable this combination.
  • the reagents may be configured to facilitate release of nucleic acid barcode molecules form respective carrier particles, extract the nucleic acids derived from the biological material (e.g. by cell lysis) and the combining of the first nucleic acid barcode molecules with the second nucleic acid barcode molecules and nucleic acid molecules derived from the biological material. Additionally, the reagents may be configured to enable sequencing of the combined nucleic acid molecules. The specific reagents may be determined by the sequencing method used.
  • the reagents may be provided within the first liquid phase, for example with the biological material, first nucleic acid barcode, and/or optical barcode.
  • the preparation of sequencing-ready DNA libraries from droplets is known in the art.
  • the viscous buffer contains DTT to release DNA from both the nucleic acid barcode particles and the optical barcode particles upon droplet formation.
  • the buffer containing both the nucleic acid and optical barcode particles also contains the reagents for cell lysis to release mRNA, alongside reagents for reverse transcription (e.g. dNTPs).
  • the nucleic acid barcode molecules can then anneal to each other, and their sequences are combined into a single nucleic acid molecule by reverse transcription with template switching.
  • the droplet emulsion can then be broken, since the droplet encapsulation information is now written into the nucleic acid sequences.
  • the resulting cDNA is then processed to yield a sequencing-ready DNA library.
  • the detailed steps involved are amplification by PCR followed by size-based separation of optical barcode-derived and biological material-derived sequences.
  • Adaptors for next-generation sequencing are added to optical barcode-derived DNA by PCR.
  • Biological material- derived sequences are fragmented, followed by dA-tailing and adaptor ligation, and PCR amplification. However, this sequence of steps is not fixed, and variations are possible.
  • the first nucleic acid barcode, the optical barcode and the biological material are encapsulated within a droplet.
  • encapsulation into a droplet represents only one example of partitioning.
  • Alternative methods in which the first nucleic acid barcode, the optical barcode and the biological material are otherwise partitioned within a partition are also possible within the scope of the disclosure.
  • partitions may be formed in well-plates or microfluidic compartments rather than in droplets.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de codage à barres d'acide nucléique et optique de matériau, le procédé comprenant : la fourniture d'un premier code à barres d'acide nucléique identifiable par des moyens de séquençage ; la fourniture d'un code-barres optique identifiable par des moyens optiques et comprenant un deuxième code-barres d'acide nucléique identifiable par lesdits moyens de séquençage ; la fourniture d'un matériau à partir duquel des molécules d'acide nucléique sont dérivables ; le partitionnement du premier code à barres d'acide nucléique, du code à barres optique et du matériau à l'intérieur d'une partition ; le matériau à l'intérieur de la cloison pouvant être identifié de manière unique par ledit moyen optique à l'aide du code à barres optique ; le premier code à barres d'acide nucléique étant conçu pour se combiner séparément avec des molécules d'acide nucléique dérivées du matériau et avec le second code à barres d'acide nucléique, de telle sorte que les deux molécules d'acide nucléique dérivées du matériau et le code à barres optique encapsulé avec le matériau, à l'intérieur de la partition, peuvent être identifiés de manière unique par ledit moyen de séquençage à l'aide du premier code à barres d'acide nucléique, de telle sorte que les molécules d'acide nucléique dérivées du matériau identifié par ledit moyen de séquençage peuvent être associées au matériau identifié par ledit moyen optique.
PCT/EP2024/087838 2024-01-04 2024-12-20 Procédé de codage à barres d'acide nucléique et optique Pending WO2025146377A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB2400115.8A GB202400115D0 (en) 2024-01-04 2024-01-04 Method
GB2400115.8 2024-01-04

Publications (1)

Publication Number Publication Date
WO2025146377A1 true WO2025146377A1 (fr) 2025-07-10

Family

ID=89901429

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/087838 Pending WO2025146377A1 (fr) 2024-01-04 2024-12-20 Procédé de codage à barres d'acide nucléique et optique

Country Status (2)

Country Link
GB (1) GB202400115D0 (fr)
WO (1) WO2025146377A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019099908A1 (fr) * 2017-11-17 2019-05-23 10X Genomics, Inc. Procédés et systèmes pour associer des propriétés physiques et génétiques de particules biologiques
WO2020086510A1 (fr) * 2018-10-22 2020-04-30 The General Hospital Corporation Analyse monocellulaire multiplexée utilisant des particules de capture d'arn optiquement codées
US20230390770A1 (en) * 2020-11-09 2023-12-07 The General Hospital Corporation High Throughput Microfluidics for Analysis of Immune Cell Activation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019099908A1 (fr) * 2017-11-17 2019-05-23 10X Genomics, Inc. Procédés et systèmes pour associer des propriétés physiques et génétiques de particules biologiques
WO2020086510A1 (fr) * 2018-10-22 2020-04-30 The General Hospital Corporation Analyse monocellulaire multiplexée utilisant des particules de capture d'arn optiquement codées
US20230390770A1 (en) * 2020-11-09 2023-12-07 The General Hospital Corporation High Throughput Microfluidics for Analysis of Immune Cell Activation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WANG, Y. ET AL.: "Dissolvable polyacrylamide beads for high-throughput droplet DNA barcoding", ADVANCED SCIENCE, vol. 7, 2020, pages 1903463
WEISGERBER, D. W.HATORI, M.LI, X.ABATE, A. R.: "Polyhedral particles with controlled concavity by indentation templating", ANALYTICAL CHEMISTRY, vol. 94, 2022, pages 7475 - 7482

Also Published As

Publication number Publication date
GB202400115D0 (en) 2024-02-21

Similar Documents

Publication Publication Date Title
Moragues et al. Droplet-based microfluidics
Ding et al. Recent advances in droplet microfluidics
ES2998086T3 (en) Method of generating monodisperse emulsions
Salomon et al. Droplet-based single cell RNAseq tools: a practical guide
US10890581B2 (en) Substrate-mediated reactors for bioassays
EP3271713B1 (fr) Coalescence sur puce massivement parallèle de microémulsions
EP3835431B1 (fr) Procédés pour codage à barres combinatoire
CN107407691B (zh) 用于单细胞中核酸靶标的分子条码化的装置和系统
US20220041967A1 (en) Real-time monitoring of single cell or events
US11814619B2 (en) Compositions, methods, and systems for single cell barcoding and sequencing
Zhang et al. Microwell array chip-based single-cell analysis
Sklar Flow cytometry for biotechnology
WO2022256612A1 (fr) Compositions, procédés et systèmes pour le codage et le séquençage unicellulaires
CN110678558B (zh) 整体性检测单个细胞的非破坏性测量信息和基因组相关信息的方法
Duncombe et al. Droplet barcoding: tracking mobile micro-reactors for high-throughput biology
US20240344989A1 (en) Improvements in or relating to a method or an apparatus for detecting an interaction between a biological entity and a molecule
WO2025146377A1 (fr) Procédé de codage à barres d'acide nucléique et optique
US20220233413A1 (en) Monodisperse emulsions templated by three-dimensional structured microparticles and methods of making the same
US20230018158A1 (en) Pre-templated instant partitioning of dna-encoded libraries
WO2024091930A1 (fr) Systèmes et procédés miniaturisés de criblage d'échantillons
Dhar et al. Research highlights: microfluidic-enabled single-cell epigenetics
WO2018067789A1 (fr) Dispositif d'affichage en hydrogel
WO2024091949A1 (fr) Procédés et systèmes de criblage d'effecteur codé
JP2017143783A (ja) 並列反応用懸濁液、並列反応方法、スクリーニング方法および検査方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24828837

Country of ref document: EP

Kind code of ref document: A1