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WO2019060685A1 - Système et procédé d'analyses de phénotype et de séquençage polynucléotidique de particules biologiques par l'intermédiaire d'un code-barres déterministe - Google Patents

Système et procédé d'analyses de phénotype et de séquençage polynucléotidique de particules biologiques par l'intermédiaire d'un code-barres déterministe Download PDF

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Publication number
WO2019060685A1
WO2019060685A1 PCT/US2018/052172 US2018052172W WO2019060685A1 WO 2019060685 A1 WO2019060685 A1 WO 2019060685A1 US 2018052172 W US2018052172 W US 2018052172W WO 2019060685 A1 WO2019060685 A1 WO 2019060685A1
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biological particle
phenotype
polynucleotides
oligonucleotide
carrier
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Dmitry S. ANDREYEV
Boris L. ZYBAYLOV
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BioVentures LLC
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BioVentures LLC
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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
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • 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

Definitions

  • BP - biological particle with genetic content e.g., a cell, or an organelle like a mitochondrion.
  • Phenotype a set of information regarding functional properties, molecular composition, structure and morphology of an individual BP. For example, a phenotype may be assessable via on-flow optical analysis or imaging.
  • FACS Fluorescently Activated Cell Sorting or, broadly, any FC-based BP sorting.
  • Genome/transcriptome all polynucleotide (DNA/RNA) sequences in a given BP.
  • mSCS Massive (or high-throughput) Single Cell Sequencing, but for all BPs and not just cells.
  • mSCS reveals genome/transcriptome of each BP in large
  • Barcoding of polynucleotide molecules from each BP is performed by using oligonucleotide tags with unique sequences. Most commonly, barcoding systems employ a stream of beads with barcoding tags attached and each bead has random but unique barcoding sequence. Each analyzed BP is paired with a single bead in a microdroplet, where barcoding is performed. Then, microdroplets are combined and sequenced together. After sequencing of combined polynucleotides from multiple BPs, the barcodes allow identification of sequences from the same particle.
  • IDBC - Identifiable Deterministically Barcoding Carrier for deterministic barcoding which consists of an identifiable core and oligonucleotides of a pre-defined sequence attached to this core. "Identifiable” refers to the ability for each particular carrier to be identified (ID), such as by intrinsic unique combination of fluorophores or by RFID, or by its positioning on a chip or in a serial flow.
  • mSCS now provides unprecedented insight into cellular heterogeneity of gene expression, genetic makeup, extent of DNA damage, regulatory mutations, etc.
  • This genetic heterogeneity extends from the cell in general to cellular organelles like the mitochondrion.
  • genetic heterogeneity of BPs is translated to phenotypic heterogeneity.
  • the phenotype of cells can be measured in multiple ways, but FC provides for one of the most high- throughput methods for assessing cellular phenotypes (e.g., functional properties, morphology). Simultaneous knowledge of phenotype and genome/transcriptome of each BP in a large population would be transformative for biomedical research.
  • the present invention is beneficial for analysis of BPs, including but not limited to: host somatic cells, host transformed cells, pathogenic biota, symbiotic biota, mitochondria (especially in oocytes), spermatozoids, platelets (for
  • the present invention overcomes these limitations of current mSCS methodologies available to the biomedical research community.
  • Some broad ranging opportunities that this invention will advance include: analysis of cancer development such as polyclonal seeding in metastatic cancers, defining links between stem cells and cancer, understanding of therapy resistance development and manifestation of genetic causes of phenotypes, studies of individual virus/bacteria/pathogen-cell interactions, studies of complex microbial communities and their interactions with host, defining roles of mitochondrial heteroplasmy in compromising of apoptotic antiproliferation, etc.
  • analysis of cancer development such as polyclonal seeding in metastatic cancers, defining links between stem cells and cancer, understanding of therapy resistance development and manifestation of genetic causes of phenotypes, studies of individual virus/bacteria/pathogen-cell interactions, studies of complex microbial communities and their interactions with host, defining roles of mitochondrial heteroplasmy in compromising of apoptotic antiproliferation, etc.
  • Stem cell therapy is effective for treatment of certain pathologies, limited in part by potential carcinogenicity.
  • the stem cell population is necessarily heterogeneous due to DNA damage, mutation load, and environment-induced transcriptome variability. This heterogeneity manifests itself in phenotypic variations, sometimes carcinogenic.
  • in-depth analysis of genome and transcriptome manifestations in phenotype for large populations is essential.
  • Carcinogenesis is typically a stochastic process where a combination of genetic changes (often mediated via retroviruses, transposons, plasm ids, viral factors) and regulatory influence (changing
  • transcriptome leads to a malignant phenotype. It is critical to reveal genotype- transcriptome-phenotype links at the single-cell level in large cellular communities.
  • Cancer cells often have increased mutation rate and, as a result, increased adaptability.
  • the mutagenic nature of most cancer therapies increases the adaptability further.
  • Early detection of clones resistant to drugs and immunotherapies is essential, especially for dispersed cancer cells.
  • Detection of cells with an unusual metabolism, apoptotic signature, or senescent response combined with immediate knowledge of corresponding mutations in the genome/transcriptome of the suspected cells would help to identify particularly malignant clones, and subsequently modify therapy.
  • Bacterial community dynamics Bacterial communities, including the human microbiome, are highly heterogeneous. The reaction of microbiomes to changing environments such as abundance of proteins, fat, host digestion-resistant and digestible carbohydrates, presence of toxins, therapeutic agents, or introduction of new possibly pathogenic species is extremely complex. Bacteria can adapt their phenotype via individual or collective response, dedicating particular organisms for suicidal release of biocides, or exhibiting regulatory mutations. There is currently no technology available to deeply investigate these complex and cell-specific changes without directly measuring functional properties, morphology, biomolecule content, transcriptome, and genome of every bacterium in a very large community.
  • Mitochondria heteroplasmy studies can define links between mtDNA mutations and damage, transcription, functionality, and biomolecule content. Investigation of how a cell tags individual mitochondria for mitophagy, replication, or initiation of apoptotic processes is essential for
  • the present invention is directed to a system and method for integration of polynucleotide sequencing and phenotype analyses of biological particles via deterministic barcoding.
  • the system and method preserves links between non- destructively assessable (e.g. optically) phenotype of the biological particle and the sequencing data of the polynucleotide content of the biological particle in a high- throughput analysis.
  • the present invention is directed to a system for analyzing the phenotype and the polynucleotide sequences of a biological particle, comprising a phenotype analyzer, wherein the phenotype analyzer is operable to determine a phenotype of the biological particle; a generator of identifiable, deterministically barcoding carriers, wherein a oligonucleotide barcode of pre-defined sequence is attached to the identifiable core of the barcoding carriers; a microreactor configured to receive the biological particle from the phenotype analyzer and to receive the barcoding carrier from the generator, to release the polynucleotides from the biological particle, to perform reverse transcription if needed for RNA analysis, to attach the oligonucleotide barcodes from the carrier to the polynucleotides from the biological particle, and to amplify the resulting barcoded polynucleotides; and a sequencer to sequence the barcoded polynucleotides.
  • the present invention is directed to a method for analyzing the phenotype and polynucleotide sequences of a biological particle, comprising the steps of non-destructively analyzing the biological particle to determine a phenotype of the biological particle; recording the phenotype of the biological particle;
  • a barcoding carrier wherein a oligonucleotide barcode with a pre-defined sequence is attached to the identifiable core of the barcoding carrier; passing the biological particle and the identifiable oligonucleotide barcode carrier to a
  • microreactor and pairing the barcoding carrier with the biological particle; lysing the biological particle to release the polynucleotides of the biological particle; reverse transcribing RNA from the biological particle (if needed), ligating the oligonucleotide barcode to the polynucleotides of the biological particle to form barcoded
  • polynucleotides sequencing the barcoded polynucleotides to determine sequences of the polynucleotides from the biological particle; and matching the phenotype of the biological particle to the sequences of the polynucleotides of the biological particle.
  • Fig. 1 A shows a schematic of a GIDBC.
  • the particular embodiment of the invention in Fig. 1A uses IDBC identification by position, reversible immobilization by magnetic field and photo activation for oligonucleotide growth.
  • Magnetic beads (circles) are shown reversibly trapped in chip surface indentations by external magnetic field (sparks). Trapped beads are individually illuminated for deprotection and nucleotides are subsequently attached.
  • Fig. 1 B is a top view of SU-8 master of a chip prototype with the microfluidic channel meandering over the photoprocessed area and the magnetic traps in the channel marked by arrows.
  • Figs. 2A-2C show schematics of an element of the cross-flow chip for oligonucleotide synthesis.
  • Fig. 2A shows after the reagent is introduced to the left reservoir and left region of the channels.
  • Fig. 2B shows after the reservoir is filled with insulating media to brake the electrical contact between the channels.
  • Fig. 2C shows after electric potential is applied to the upper channel's electrodes, which electroosmotically moves the reagent into the working region.
  • the upper horizontal channel in the chip may be used to add a nucleotide to all crossing in the upper row, then de-protect anchors on these crossings to subsequently add a nucleotide to some of them via the vertical channels.
  • Fig. 2D is a schematic illustrating tracked mixing synthesis.
  • An on-flow detector is used for tracking each IDBC through each round of nucleotide
  • a four-way valve directs one-quarter of the suspension of IDBCs to chambers for attachment of A, T, G, C nucleotides, correspondingly. After A, T, G and C nucleotide attachment, IDBCs are returned to the mixer from all four chambers and are ready for next step of barcode growth, As a result, a mixture of IDBCs with unique barcodes is complimented by a database where each IDBCs ID is linked to particular barcode sequence.
  • Fig. 3 is a schematic showing the components of the present invention in five blocks.
  • Block A is a cytometer or fluorescence-activated cell sorting device
  • Block B is a cell accumulator with a camera for verifying the position of reference beads (the darkest three circles)
  • Block C is a GIDBC with IDBC (e.g. beads) held in individual traps while pre-defined oligonucleotides are grown via selective photo activation using a DLP projector
  • Block D is a barcoding droplets generator
  • Block E is a synchronization detector.
  • Figs. 4A-4C show examples of integrated chip layouts.
  • Fig. 4A shows a complete instrument-on-chip with hydrodynamic traps.
  • Figs. 4B-4C show complete instrument layouts with magnetic traps.
  • Fig. 4B shows a layout with minimized outlets, while Fig. 4C shows a layout with a dual GIDBC and no loop morphology.
  • Fig. 5 is an example chip layout with all components integrated except the
  • the chip layout includes a separate on-flow IDBC detection area for synchronization (E1 ).
  • Figs. 6A-6E show layouts for two-layer PDMS chips.
  • Fig. 6A shows a mask for a 5 pm thick layer containing traps.
  • Fig. 6B shows a mask for 40 pm thick layer containing channels.
  • This layout is suitable for making both 105 mm (inner circle) and 130 mm (outer circle) 2-layer masters with extra chips at corners. It contains several kinds of chips, including chips for evaluating optimal geometry of channels and traps.
  • Fig. 6C shows a layout example containing five testing regions (3x3 mm squares filled with a meandering channel with traps to hold 900 and 1800 beads each), oval inlets with filters, rectangular inlets without filters, and on-flow detection region terminated by the outlet (center-left).
  • Fig. 6D shows an image of SU-8 master made using masks in Fig. 6A and Fig. 6B.
  • Fig. 6E is an image of a region of a completed PDMS/glass chip that is half- filled with running buffer. The traps are visible only in air-filled half of the channel due to higher difference in refractive index.
  • Fig. 7 A shows an example of a chip mask layout with hydrodynamic traps.
  • Fig. 7B is magnification of the traps from Fig. 7A.
  • Fig. 7C shows an element SU-8 master made with the layout of Fig. 7A and Fig. 7B.
  • the arrow marks a fluidic shortcut and a trap for a bead.
  • Figs. 8A-8D show magnetic traps of different morphologies.
  • Figs. 8A-8C show closer images of different channel-trap combinations for the chip layouts shown in Figs. 4C, 6C, and 6E.
  • Fig. 8D shows a region of a completed PDMS/glass chip made using the master shown on Fig. 6D and half-filled with running buffer. The traps are visible only in air-filled half due to a higher difference in refractive index.
  • Fig. 9 shows a schematic of GIDBC embodiment with independent EOF control of media flow in each horizontal channel.
  • the vertically-oriented side channels allow for parallel flushing during oligonucleotide synthesis.
  • Electrodes double triangles
  • the large circles represent electrolyte reservoirs. Pulse laser pumping could be used instead of EOF to selectively propel media.
  • the present invention is directed to a system and method of phenotype and polynucleotide sequencing analyses for
  • DNA/RNA-containing particles via deterministic barcoding The system and method is directed to the goal of preserving links between optically assessable individual BP's phenotypes and the BP's genome/transcriptome in a high-throughput analysis.
  • the deterministic barcoding approach of the present invention employs an IDBC for each BP and applies a unique and pre-defined barcode to each polynucleotide of the BP.
  • the IDBC is a key element of this invention.
  • the inventors considered combinations of identifiable cores and deterministic oligonucleotide growing procedures.
  • identifiable cores the inventors considered: (1 ) beads in serial flow where a particular IDBC is identified by a serial number based on its positioning in the consecutive outflow; (2) an array of on-chip micro wells or spots where the identity of a particular spot is defined by coordinates of the spot; (3) and beads with build-in unique identification tags that are identifiable on-flow.
  • the latter includes beads with: (a) multiple optical tags (e.g., fluorescent or surface-enhanced Raman tags); and (b) radio-frequency identifiable (RFID) beads.
  • multiple optical tags e.g., fluorescent or surface-enhanced Raman tags
  • RFID radio-frequency identifiable
  • Oligonucleotides with known, pre-defined sequences are then grown on the identifiable cores of IDBC discussed above to yield IDBC through one of the following growth procedures: (1 ) photo activation; (2) cross-flow synthesis; or (3) tracked mixing synthesis.
  • Photo activation is a well-established technology for generating pre-defined oligonucleotides on surface of a chip. Briefly stated, protective groups are removed in selected spots by light illumination and one nucleotide (A, T, G or C) is attached to growing oligonucleotides in the activated spots. This process is repeated for the other three nucleotides. Next, these four cycles are repeated N-times for N- nucleotide long oligonucleotides, generating up to 4 N unique spots.
  • Photo-activated synthesis can produce multitude spots with oligonucleotides with spot-specific, pre-defined sequences.
  • the number of the regions and thus different sequences is generally unlimited and depends only on size of the chip and resolution of the process.
  • DLP Digital Light Processing
  • This technology can be applied "as is” to oligonucleotide growth on chip spots (identifiable core option #2), and with changes to oligonucleotide growth on identifiable beads (identifiable core option #1 and #3). For the latter, reversible immobilization of beads in pre-defined spots of a chip is needed, as shown in Figs. 1A-1 B, 3, 6C, 6E, and 7A-9.
  • Beads can be reversibly immobilized (or trapped) in pre-defined spots of a microfluidic channel that are aligned over the photo processed chip area to place maximum number of these spots in the area.
  • dielectrophoretic, chemical, vacuum, and inertial-based immobilization approaches are all options.
  • magnetized (e.g. superparamagnetic) beads are held by external magnetic field in surface indentations or traps, as shown in Figs. 1 B, 3, 4B, 4C, 6C, and 6E.
  • hydrodynamic approach beads are held in smaller-than-beads microfluidic flow shortcuts, as shown in Figs. 4A and 7A-C.
  • dielectrophoretic approach beads are held in electrode-defined traps by alternating electromagnetic field with frequency defined by bead size.
  • beads are chemically immobilized using cleavable bonds, (e.g.
  • Fig. 1A magnetic immobilization as shown in Fig. 1A is the preferred option, closely followed by the hydrodynamic and dielectrophoretic approaches.
  • the inventors have identified the photo activation method to grow barcoding oligonucleotides on serial-flow IDBC with magnetic, hydrodynamic or dielectrophoretic trapping employed for positioning as the preferred embodiment of the present invention.
  • Cross-flow synthesis is an alternative for instances where photo activation is not suitable.
  • oligonucleotides are grown on the identifiable cores at intersections of individually-controlled microfluidic flows, as shown in Figs. 2A-C.
  • a chip with a grid of microfluidic channels with cores at channel intersections and electrodes placed at the end of each channel is suggested. Electrodes are used to generate EOF between each chosen pairs of electrodes and selectively drive media between them. Alternatively, laser pulsing can be used to drive this media.
  • Terminals of channels on each side of the chip are connected to reservoirs.
  • the reservoirs are step-wise filled with reagents for a particular protected nucleotide attachment to the growing tags and then with an insulated media.
  • the combination of alternating filling of intersecting channels and changing nucleotide type results in unique and known sequence on the IDBC at each intersection.
  • one set of channels may be filled by an anchor deprotecting reagent, while the other with the nucleotide-attaching reagent.
  • media can be driven by pulse laser.
  • Tracked mixing synthesis is a preferred method of growing oligonucleotides on the IDBC if cores with intrinsic ID (RFID or optical) are used.
  • Half of the IDBC sample can be modified by one
  • fluorophore mixed with an unmodified half, and this cycle repeated with other fluorophores.
  • Other means of identification e.g. RFID, magnetic, optical non- fluorescence, etc.
  • Combinations of different IDs e.g. fluorescent and RFID) further increase the number of unique core IDs.
  • IBCs generated through these cores and growth procedures enable several deterministic barcoding approaches which integrate non-disruptive phenotype analyses (e.g. flow cytometer or microscopic imaging) of biological particles with massive or high-throughput single cell sequencing analyses of the genome or transcriptome of the biological particles.
  • IDBC allows for individual labeling of each biological particle after nearly any phenotype analysis, and linking the phenotype of those biological particles to their individual sequencing data.
  • Population size of the analyzed biological particles is limited only by performance of sequencing, which is a fast growing field. This ensures that this invention will not only be useful in current biomedical research applications, but will be even more useful in the future ones. Even with current sequencing technologies, analyses could include millions of particles with smaller genomes/transcriptomes or small targeted genetic elements.
  • the deterministic (or known and pre-defined) barcoding of the present invention is capable of measuring any combination of optically-assessable phenotypic properties including but not limited to: (1 ) membrane potential, membrane fluidity, and membrane electropermeabilization; (2) intracellular pH, ions, and oxygen; (3) total DNA/RNA content, copy number variation, chromosome analysis, and sorting; (4) protein expression, protein modification, and localization; (5) presence of
  • the IDBCs are generated using beads in serial flow as the identifiable cores with photo activation as the procedure for
  • IDBCs in the system of the present invention can establish a one-to-one correspondence between high- throughput individual cell sequence information and flow cytometry data.
  • a FC collects and records single BP specific information and directs sorted BPs into the system via an optional cell accumulator.
  • a microfluidic chip reversibly immobilizes beads in a pre-defined array.
  • steps of photo-activated synthesis produce multitude of IDBCs containing oligonucleotides with a pre-defined, unique sequence for each identifiable core (i.e., bead ).
  • the outflow of IDBCs from this chip is combined with outflow of BPs from the BP accumulator in a chip in droplets (i.e. microreactors) that are being generated with IDBC-BP pairs for barcoding and sequencing.
  • Deterministic barcoding of BP's polynucleotides allows for matching sequencing data for these polynucleotides with cytometric and imaging data on the BP of polynucleotide origin.
  • the system is comprised of the following components:
  • Component A Flow cytometer with optional sorter ⁇
  • Component B BP accumulator with a BP and reference beads imager to confirm and track the relative positioning of the BPs and reference beads;
  • Component C GIDBC with another reference beads imager to confirm and track the relative positioning of the barcoding beads and the reference beads;
  • Component D Chip for water-in-oil droplet/microreactor generation, where release and barcoding of BP polynucleotides occurs;
  • Component E On-flow detector of reference beads for error correction if the relative positioning is incorrect.
  • the system allows for complete integration of all components on a single chip (as shown in Figs. 4A-C), combination of separate chip connected by
  • microfluidic lines and a combination of on-chip capillary based and macro-scale devices (as shown in Figs. 3 and 5).
  • PDMS chips including prototypes of separate GDBBs (Figs. 6C, 6E, and 7A-8D) complete instruments on- chips (Figs. 4A-C), and intermediate variants (e.g. a chip with all components except the GDBB (i.e. including microfluidics for FACS, BP- accumulator, droplet generator, and synchronization detector)) ( as shown in Fig. 5).
  • GDBB i.e. including microfluidics for FACS, BP- accumulator, droplet generator, and synchronization detector
  • a 2-layer soft lithography process with 5pm layer of traps and 40 m layer of channels, for core diameter from 5 to 10um was used.
  • the inventors manufactured a lithography mask for each layer (Figs. 6A-6B), SU-8 master (Fig. 6D) and PDMS casts.
  • the inventors cut PDMS casts to individual chip size and glued them to activated glass support or PDMS slabs to fabricate chips (Fig. 6E).
  • Resulting chips have a glass side for optical access and PDMS side for fluid lines connection and magnet influence (Fig. 1 B).
  • a flow cytometer is used for on-flow phenotype analysis and sorting BPs of interest. While most cytometers and other on-flow particle analyzers could be used, either a capillary-based scheme (Fig. 3A) or on-chip scheme (Figs. 4A-5) is preferable.
  • BPs can be driven through a microfluidic channel by pressure, EOF, laser pulsing or other means well-known to those skilled in the art.
  • the inventors suggest EOF and laser pulsing as primary driving means because they are fast responding and require no moving parts.
  • BPs can be detected in-channel or in a shear-flow cuvette by LIF/LS. In-channel detection is easier to implement and use, while detection in shear-flow cuvette could provide better signal to noise ratio and higher throughput.
  • Sorting can be activated by fluorescent, light scattering or any other signal from the detector and performed using flow redirection via EOF, laser pulsing driving or other means well-known to those skilled in the art.
  • EOF driving is easier to implement, while laser pulse driving is less dependent on channel wall surface chemistry and provide better throughput. As shown in Fig. 3, the portion of the sample that does not meet the sorting criteria is passed out of the system as waste.
  • An accumulator is optionally used to (i) timely decouple sorting and droplet generation to avoid cross-talk between flow drivers of different components, and (ii) imaging of reference beads for error correction.
  • BPs are loaded in a long microfluidic channel (Fig. 3) or more complex microfluidic systems (similar to shown in Figs. 7A-C and 9) and can be supplied in the same order to component D of the system at any convenient time. While loaded,
  • BPs with reference beads can be imaged on-chip to verify order and load via number of BPs between reference beads. Deep imaging can also provide additional information about BPs phenotype (e.g. BP shape), complementary to information acquired via on-flow detector.
  • BPs phenotype e.g. BP shape
  • BPs could be directly supplied to component D after sorting from block A.
  • this component with advanced imaging could be used not only in addition to, but also as a substitute to, block A.
  • phenotypic data is acquired exclusively via imaging, although this would also decrease capability of the system.
  • the core of the IDBCs are beads in serial flow where each particular bead is identified by a serial number (i.e. position in the outflow) to track its position relative to other carrier beads and reference beads (Figs. 3, 4A-C, 6C, 7A-C, and 8A-D).
  • a serial number i.e. position in the outflow
  • oligonucleotides are grown on the beads via photo activation (Fig. 1 ).
  • Fig. 1 reversible immobilization of beads in "traps" located in pre-defined spots on-chip is essential. These traps are positioned in a microfluidic channel that occupies as much of photo processed chip area as possible.
  • magnetic Figs. 1A-B, 2C, 4A-C, 6C, 6E, and 8A-9
  • hydrodynamic Fig. 7A-C
  • the geometry of the magnetic traps depends on the size of the beads and the hydrodynamic chip properties.
  • the inventors manufactured chips with channel widths from 20 to 80pm to find an optimal balance between load, seriality control and clogging resistance; 5-20pm size circular, triangular and trapezoidal trap shapes for optimal loading and release control; and 50 to 100pm inter-trap distance to evaluate possible optical cross-talk and to control seriality of elution with the inter-trap distance change (Figs. 6A-E).
  • the inventors also made chips with a set of hydrodynamic traps (Fig. 4A and Fig. 7A-C). Chips with dielectrophoretic traps were also considered, but were given lower priority.
  • GIDBC core trapping optimization For magnetic immobilization, traps can be not only isotropic (i.e. symmetrical relative media flow), but also anisotropic to facilitate immobilization and release, depending on media flow direction, and simplifying IDBC rotation management, if needed.
  • isotropic traps circular geometry is preferred, while triangular or trapezoidal traps are suggested for anisotropic traps (Figs. 8A-D).
  • a bead rotation in traps can be implemented. Rotation can be induced via
  • GIDBC bead release optimization Bead release will be induced by removing the trapping force while shear force of the media flow is present. However, additional means of release control and suppression of unwanted sorption can be implemented as needed. These include (1 ) inverted or alternating field s (2) reversing media flow for anisotropic magnetic traps as described and hydrodynamic traps, (3) ultrasonic driver and/or pulse laser illumination, (4) reversal of surface charge, (5) use of chaotropic agents, and (6) bead rotation.
  • GIDBC bead outflow seriality control For serial flow of the beads, it is important to ensure that the order of beads is retained in the outflow. For the monitoring of this order, the inventors suggest use of optically-labeled beads as a reference to mark positions of the beads on the chip. With these beads it is possible to determine whether the number of untagged beads between optically labeled ones remains the same in the outflow and to compare 2D-imaging of optically-tagged reference beads on traps in the bead processing regions of GIDBC with the data from on-flow LIF/LS detector (Figs. 3-5).
  • High-throughput bead processing For thousands of beads, the simplest, single-channel chips (Figs. 3, 4B, 4C, 6C, 6E, 8A-D) with pressure, laser pulse or EOF-driven elution is sufficient. However, for millions of beads and larger chips, flow resistance can be too high for driving via single channel. In this case, channels can be filled in parallel, and eluted subsequently (see example with hydrodynamic traps in Fig. 4A and Figs. 7A-C). A more universal scheme is presented in Fig. 9. In this design, channels can be filled and processed in parallel, and eluted sequentially. Inter-channel flow junctions can be blocked by solidifying media (e.g., photoresist) followed by pressure-driven elution.
  • solidifying media e.g., photoresist
  • EOF-driven chips would also allow for parallel loading and processing.
  • the excessive inter-channel electric junctions can be blocked by insulating media, allowing precisely-controllable, channel-by- channel EOF-driven elution.
  • Pulse-laser pumping could be employed as well.
  • Scaling up would also require higher resolution of optical processing where optical cross-talk could emerge.
  • Undesired light propagation suppression might be required via refractive index control, surface treatment for light extinction, and processing area size increases.
  • Each droplet should contain no more than one BP because all
  • polynucleotides in the droplet will have the same droplet-specific barcode and polynucleotides from multiple BPs in the same droplet will not be distinguished after sequencing.
  • a droplet with no BP causes no problems besides wasting efficiency.
  • the ratio of droplets to BPs is maintained high to statistically decrease the number of doublets and multiplets of BPs per droplet.
  • the sorter/accumulator can supply BPs to the droplet generator in a more controlled manner, greatly decreasing the amount of wasted droplets and barcoding beads.
  • Each droplet should also contain one barcoding bead. In a droplet without a bead, barcoding is impossible and BPs sequencing information will be lost. If a droplet contains multiple beads, different barcodes will be applied to polynucleotides from the same BP, causing problems with sequencing data processing in commonly employed chips. In contrast, in the preferred embodiment, synchronization and error correction (see below) can identify beads participating in such events and
  • Each droplet should also contain reagents for BP lysis, polynucleotide- barcode ligation, amplification, and sometimes reverse transcription. These reagents can be supplied with a stream(s) of water-based media, within barcoding beads or migrate into a droplet from oil environment.
  • RNA is reverse- transcribed (if necessary), and ligases are used to add the oligonucleotide from the IDBC to the polynucleotide of the BP.
  • the polynucleotides of the BP are now barcoded.
  • Amplification if necessary, can be performed in-droplet or after droplets merging and oil removal in preparation for sequencing.
  • a simplified secondary on-flow LIF/LS detector is implemented for droplet outflow analysis (Fig. 3, Fig. 4A-C, Fig. 5) for detecting and counting optically tagged reference beads. While using the same principles with LIF/LS detector of the cytometer/sorter, this detector needs a single scattering channel and two
  • fluorescence (or SERS)-tag detecting channels to detect reference beads, hence sensitivity can be low.
  • the data acquisition rate should correspond to droplet generation rate.
  • Basic imaging could also be beneficial for this detector.
  • low budget solutions such as a 3-color webcamera are sufficient for this component.
  • Errors may originate from the bead processing chip (i.e. GIDBC) because spots of oligonucleotide synthesis may be empty, occupied by multiple beads, or beads may sorb outside the spots. Errors may also originate from the in-drop pairing chip because the ratio of one BP to one bead per drop might be compromised. Errors may originate from the flow cytometer because doublets or multiplets of particles can be detected as a single event. In each of these cases, these errors will manifest themselves in mismatching of the indexes in the barcode database and cytometric data (i.e. phenotype data
  • particles e.g., reference beads, liposomes
  • BPs BPs
  • Any mismatch between expected bead sequence (based on FC data and BP-accumulator image) and the sequence of the tagged particle can be detected after sequencing. Then, an appropriate correction can be applied.
  • optically tagged reference beads can be added into the IDBC (one color) and to the BPs (another color), with the secondary on-flow detector (Figs. 3-5) controlling mismatches.
  • the beads it will measure the distance (number of un-marked beads) between the marked ones and compare this distance to the initial reference image of the GIDBC.
  • BPs it will measure whether the number of detected and sorted barcoding BPs (based on on-flow detector and BP-accumulator image) corresponds to the number of BPs between reference beads. Applied in parallel, these methods provide robust and reliable error correction.
  • identifiable cores are utilized.
  • this invention may be used with chips capable of confining and localizing reaction of biological particles with spots of barcoding media, including, but not limited to, micro wells on the chip surface that confine separate BPs or gel barriers introduced optically, electromagnetically or mechanically.
  • imaging is used to access phenotypes of BPs instead of on-flow methods.
  • IDBC with build-in unique identification tags that are identifiable on-flow e.g. RFID or optical tags, as described above
  • oligonucleotides can be grown using all mentioned methods, but tracked mixing synthesis is the most appropriate (as described above, see Fig. 2D).
  • Solution of these IDBC along with corresponding on-flow ID detector can be employed in the preferred embodiment instrument design instead of chip-based GIDBC, eliminating the need of multi-step oligonucleotide growing for each GIDBC bead load.
  • IBCs can instead be prepared in advance or even supplied as a disposable cartridge for analysis when using (i) loaded disposable GIDBC chip; (ii) chip with barcoding spots; or (iii) solution of IDBC with on-flow identifiable cores with appropriate database linking core ID (serial number, position or RFID/optical tags) of each IDBC element with the sequence of its oligonucleotide barcodes, as described above.
  • PINK1 acts as a gatekeeper. Biochem Biophys Res Commun, 2017.

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Abstract

La présente invention concerne un système et un procédé d'analyse d'un phénotype et de séquences polynucléotidiques d'une particule biologique. La particule biologique est optiquement analysée par un analyseur de phénotype, tel qu'un cytomètre d'écoulement, pour déterminer un phénotype de la particule biologique. Un support de code-barres oligonucléotidique est généré avec le code-barres oligonucléotidique fixé au support. Le code-barres oligonucléotidique présente une séquence prédéfinie. La particule biologique et le support de code-barres oligonucléotidique passent au niveau d'un microréacteur où la particule biologique est lysée pour libérer les polynucléotides de la particule biologique. Le code-barres oligonucléotidique est lié aux polynucléotides de la particule biologique pour former un complexe génétique. Le complexe génétique résultant est séquencé, et le phénotype de la particule biologique est adapté à la séquence des polynucléotides de la particule biologique.
PCT/US2018/052172 2017-09-22 2018-09-21 Système et procédé d'analyses de phénotype et de séquençage polynucléotidique de particules biologiques par l'intermédiaire d'un code-barres déterministe Ceased WO2019060685A1 (fr)

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