RU2756306C1 - System and method for phenotype analysis and polynucleotide sequencing of biological particles using deterministic barcoding - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: system and a method for analyzing a phenotype and a sequence of polynucleotides of a biological particle are proposed. The system contains an analyzer of a phenotype of a biological particle, a generator of an identifiable oligonucleotide coding carrier, a microreactor, a sequencer of a pre-determined sequence of an encoding oligonucleotide and a sequence of polynucleotides of a biological particle. The method includes optical analysis of a biological particle to determine the phenotype, recording the phenotype, generating a carrier of coding oligonucleotides, placing a biological particle and the carrier in the microreactor, lysis of a biological particle, ligation of coding oligonucleotides with polynucleotides of a biological particle, sequencing to determine the sequence of polynucleotides of a biological particle and comparing the phenotype of a biological particle with the sequence of polynucleotides of a biological particle.

EFFECT: inventions provide an accurate determination of genome/transcriptome and phenotype for each biological particle.

15 cl, 24 dwg

Description

The field of technology.

The present invention relates to a system and method for integrating polynucleotide sequencing and analyzing the phenotypes of biological particles by deterministic barcoding. In a high-throughput assay, the system and method maintain links between the phenotype of a biological particle, assessed by non-destructive methods (eg, optically), and the sequencing data of the polynucleotides contained in the biological particle.

This patent application grants priority to US Provisional Patent Application No. 62/562, 115, entitled "Inline Analysis with Deterministic Barcoding," filed September 22, 2017, and to US Provisional Application No. 62 / 577,905, entitled "Combining High Throughput Sequencing of Individual Particle Analysis with Other Methods for Analyzing Individual Particles by Deterministic Barcoding ”, filed October 27, 2017. A full description of these preliminary applications is available at the links provided in this application.

Definition of terms.

BP is a biological particle that contains genetic material (for example, a cell or organelle such as mitochondria).

Phenotype is the collection of information regarding the functional properties, molecular composition, structure, and morphology of an individual BP. For example, the phenotype can be studied using optical flow analysis or image analysis.

FC - flow cytometry.

FACS - Fluorescently activated cell sorting, or broadly any FC based BP sorting. Genome / Transcript - All polynucleotide (DNA / RNA) sequences in a given BP.

mSCS - mass (or high-throughput) sequencing of BP, individual cells or other BPs, not just cells. The mSCS is determined by the genome / transcript of each BP in large populations, most often by barcoding the polynucleotide content of each BP, followed by sequencing the cumulative encoded genetic material of the plurality of BPs. Barcoding of polynucleotide molecules from each BP is performed using oligonucleotide tags with unique sequences. Most often, barcoding systems use a stream of microparticles with attached coding tags (oligonucleotides) in a chip, and each microparticle carries a random but unique sequence for barcoding. Each analyzed BP is combined with one microparticle in a microdroplet, where barcoding is performed. The microdroplets are then combined and sequenced together. After sequencing the aligned polynucleotides from multiple BPs, the coding sequences allow identification of the original polynucleotide sequences derived from the same microparticle.

IDBC - An identifiable deterministically coding medium (coding oligonucleotides) for deterministic barcoding, which consists of an identifiable nucleus and oligonucleotides of a predetermined sequence attached to a given nucleus. "Identifiable" refers to the ability of each particular carrier to be identified (ID), for example, by a unique combination of fluorophores, or by RFID, or by its position on a chip or location in a stream sequence.

GIDBC is a generator of identifiable coding media (coding oligonucleotides) for deterministic barcoding.

LIF / LS - laser-induced fluorescence / light scattering for flow analysis.

FISH - fluorescent cytological in situ hybridization.

Prior art.

Influence of technology on the development of biomedical research. With the rapid development of bioanalysis and data processing methods, the study of aggregate-averaged properties of cells and organelles is becoming increasingly obsolete and replaced by high-throughput analysis of individual BPs. This large-scale shift in biomedical research is driven by an increase in biological data highlighting the importance of BP particle heterogeneity (eg, cell heterogeneity in a given population). Currently, mSCS provides an unprecedented understanding of cellular heterogeneity in gene expression, manifestation of genotype in phenotype, degree of DNA damage, regulatory mutations, etc. This genetic heterogeneity includes both cells as a whole and their organelles, for example, mitochondria. In response to environmental, biological, and cellular factors, the genetic heterogeneity of BP is transformed into phenotypic heterogeneity. Cell phenotype parameters can be measured in several ways, but FC is one of the most high-throughput methods for assessing parameters of cell phenotypes (eg, functional properties and morphology). The ability to simultaneously determine the phenotype and genome / transcript of each BP in a large population could radically change the paradigm of biomedical research.

The present invention is useful for BP analysis, including but not limited to BPs such as: somatic host cells, transformed host cells, pathogenic biota, symbiotic biota, mitochondria (especially in oocytes), spermatozoa, platelets (in mitochondrial content analysis), nuclei, nucleoli , ribosomes, viruses and exosomes.

In biomedical research, there is no and an urgent need for a tool or high-performance technological platform that can unambiguously correlate mSCS information for a specific cell with the phenotype parameters of the same cell. The randomness of the sequences of coding oligonucleotides in existing mSCS platforms does not provide identification of a particular BP containing polynucleotides, it only establishes whether the polynucleotides originate from the same BP or not. This does not allow correlating the measured FC phenotypic properties with the genome / transcriptome of individual BPs. Perhaps the closest technology that does map some phenotypic properties to individual BP transcriptomes is a recently published approach called CITE-SEQ, where tagging with an antibody-oligonucleotide complex allows limited phenotype identification using cell surface markers; however, this platform does not provide a comprehensive, high-throughput comparison of genome / transcriptome analysis and phenotype data of individual cells. The present invention overcomes these limitations of existing mSCS methodologies available to the biomedical research community.

New features and applicability. A precise definition of the genome / transcriptome and the corresponding phenotype for each BP in a heterogeneous population is necessary for a deeper understanding of many biomedical problems. Examples of opportunities that this invention will provide include: analysis of cancer development, in particular the formation of polyclonality in metastatic cancer, determination of the relationship between stem cells and carcinogenesis, understanding the mechanisms of development of resistance to therapy and manifestation of genotype in phenotype, studies of interactions of individual viruses / bacteria / pathogens with individual cells, investigating complex microbial communities and their interactions with the host, defining the role of mitochondrial heteroplasmidity in compromising apoptotic antiproliferation, etc. In the following paragraphs, we briefly highlight some of these biomedical applications to provide a sampling of the broad possibilities of this technology:

Determination of the relationship between stem cells and carcinogenesis. Stem cell therapy is effective in treating certain pathologies, but is partially limited by its potential carcinogenicity. The stem cell population is always heterogeneous due to DNA damage, mutational stress, and environmental-induced variability of the transcriptome. This heterogeneity manifests itself in phenotypic variations, sometimes carcinogenic. Consequently, a deep analysis of the manifestations of the genome and transcriptome in the phenotype is required for large populations.

Carcinogenesis studies. Carcinogenesis, as a rule, is a stochastic process, when a combination of genetic changes (often mediated by retroviruses, transposons, plasmids, viral factors) and regulatory influence (transcriptome change) leads to a malignant phenotype. It is very important to identify the genotype-transcriptome-phenotype relationships at the level of individual cells in large cell communities.

Research on polyclonal cancer. Cancer cells often have an increased mutation rate and, as a result, increased adaptability. The mutagenic nature of most cancer treatments further enhances adaptability. Early detection of drug and immunotherapy resistant clones is essential, especially for scattered cancer cells. Detection of cells with unusual metabolism, apoptotic signature or signs of aging, combined with direct detection of the corresponding mutations in the genome / transcriptome of the cells under study, will help to identify malignant clones and, therefore, modify therapy.

Studies of cellular-viral interactions. The interaction of cells with viral particles, in particular with oncoviruses, is a very heterogeneous process, in which only a small part of cells may be susceptible to infection, depending on the cellular status and transcriptome. Detection of cellular-viral interactions and subsequent phenotypic changes with direct determination of the corresponding transcriptome and the fate of the viral genetic material for each cell will be important for virulence studies.

Dynamics of bacterial communities. Bacterial communities, including the human microbiome, are highly heterogeneous. The response of microbiomes to a changing environment, such as the availability of proteins, fats resistant to digestion and assimilable carbohydrates, the presence of toxins, therapeutic agents, or the introduction of new, possibly pathogenic, biota species is extremely complex. Bacteria can adapt their phenotype through individual or collective responses, releasing certain organisms to self-destructively release biocides, or exhibiting regulatory mutations. Currently, there are no available technologies for in-depth study of these complex and specific changes in individual cells without targeted measurement of the functional properties, morphology, biomolecular content, transcyptome, and genome of each bacterium in the vast community.

Study of the role of mitochondrial heteroplasmy in the disturbance of the mechanisms of apoptosis / senescence / mitogenesis. Studies of mitochondrial heteroplasmy can determine the links between mtDNA mutations and their damage, transcription, functionality, and biomolecule content in mitochondria. The study of how the cell marks individual mitochondria for mitophagy, replication, or initiation of apoptotic processes is important for understanding the anticancer mechanisms and their progressive degradation during life, as well as for aging-related mitochondria-mediated nervous and muscle degeneration, infertility, and hereditary pathologies.

Thus, it follows from the prior art that a system and method is required that will allow unambiguous comparison of the mSCS information for a specific cell with the phenotype parameters of the same cell, correlating the measured FC phenotypic properties with the genome / transcriptome of individual BPs, with high productivity.

The present invention relates to a system and method for integrating polynucleotide sequencing and analyzing the phenotypes of biological particles by deterministic barcoding. In a high throughput assay, the system and method maintain links between the phenotype of a biological particle, assessed by non-destructive methods (eg, optically), and the sequencing data of the polynucleotides contained in the biological particle.

The technical result consists in providing the possibility of accurate non-destructive determination of the genome / transcriptome and the corresponding phenotype for each BP in a heterogeneous population. The system and method provide a comprehensive, high-throughput comparison of genome / transcriptome analysis and phenotype data of individual cells.

The technical result is achieved by the fact that the system for analyzing the phenotype and sequence of polynucleotides of a biological particle contains:

- a phenotype analyzer, where the specified phenotype analyzer is designed to determine the phenotype of the specified biological particle;

- generator of an identifiable oligonucleotide coding carrier, where the coding oligonucleotide with a predetermined sequence is attached to said carrier, an individual carrier is used for each biological particle, and a unique and predetermined barcode is used for each polynucleotide of each biological particle;

- a microreactor configured to receive said biological particle from said phenotype analyzer and receive said carrier of coding oligonucleotides from said generator;

- sequencer of said predetermined sequence of said coding oligonucleotide and said sequence of polynucleotides of said biological particle after said coding oligonucleotide is ligated with said polynucleotides of said biological particle

Unlike existing mSCS methods with particles carrying unique but random encoding oligonucleotides, the deterministic barcoding approach of the present invention uses IDBC for each BP and applies a unique and predefined barcode for each polynucleotide of each BP.

Said biological particle and said carrier of coding oligonucleotides are encapsulated in a drop in said microreactor.

The phenotype analyzer is contemplated to be a flow cytometer.

It is envisaged that the phenotype analyzer is a microscope.

It is envisaged that the carrier is a magnetic particle.

It is envisaged that the carrier is a microcell on the surface of the chip.

It is envisaged that the system contains an accumulator of biological particles between the specified phenotype analyzer and the specified microreactor.

The technical result is achieved in that the method for analyzing the phenotype and sequence of polynucleotides of a biological particle includes the following stages:

optical analysis of the specified biological particle to determine the phenotype of the specified biological particle;

recording the specified phenotype of the specified biological particle;

generating a carrier of coding oligonucleotides, where the coding oligonucleotide of a predetermined sequence is attached to said carrier; uses an individual carrier for each biological particle and applies a unique and predefined barcode for each polynucleotide of each biological particle;

placing the specified biological particle and the specified carrier of coding oligonucleotides in the microreactor;

lysis of said biological particle to release said polynucleotides of said biological particle;

ligating said coding oligonucleotides with said polynucleotides of said biological particle to form a genetic complex;

sequencing the specified genetic complex to determine the specified sequence of the specified polynucleotides of the specified biological particle;

comparing the specified phenotype of the specified biological particle with the specified sequence of the specified polynucleotides of the specified biological particle.

It is provided that said biological particle and said carrier of coding oligonucleotides are encapsulated in a drop in said microreactor.

Provided that the flow cytometer performs the step of optical analysis of the specified biological particle to determine the phenotype of the specified biological particle.

It is envisaged that the specified biological particle is supplied to the accumulator of biological particles before the specified biological particle is placed in the specified microreactor.

It is envisaged that the method comprises the step of analyzing the output from said microreactor for errors.

It is contemplated that said step of generating the carrier of the coding oligonucleotides comprises growing the oligonucleotide on the particle.

It is contemplated that the step of growing the oligonucleotide on the particle includes photoactivation.

Provided that includes the stage of immobilization of the specified particle in the trap on the chip.

It is contemplated that the step of generating the carrier of the coding oligonucleotides comprises growing the oligonucleotide on a chip.

The technical solution is illustrated by graphic materials, which show:

FIG. 1A shows a GIDBC scheme. A specific embodiment of the invention in FIG. 1A uses IDBC positional identification, reversible magnetic field immobilization, and photoactivation to grow oligonucleotides. Magnetic particles (circles) are shown reversibly trapped in the recesses of the chip surface by an external magnetic field (sparks). The captured particles are individually illuminated to remove the photo-removable protection and then nucleotides are attached to them. Designated: DLP-Projector - DLP Projector.

Fig. 1B shows a top view of an SU-8 master model of a prototype chip with a tortuous microfluidic channel in the photo-processing area and magnetic traps in the channel indicated by arrows.

Figures 2A-2C are schematic diagrams of a cross-flow chip for oligonucleotide synthesis. FIG. 2A shows how the reagent is introduced into the left reservoir and the left region / left ends of the channels. Figure 2B shows how the reservoir is filled with an insulating medium to break the electrical contact between the channels. FIG. 2C shows the electroosmotic movement of the reagent into the working area after an electrical potential is applied to the upper channel electrodes. The upper horizontal channel in the chip can be used to add nucleotide to all intersections in the upper row, and then to deprotect the anchors to grow oligonucleotides at these intersections, to subsequently add nucleotide to some of them via vertical channels.

Fig. 2D is a diagram illustrating the synthesis of a tracked mix. A flow detector is used to track each IDBC in each round of nucleotide attachment. The four-way valve directs one quarter of the IDBC suspension to the chambers for the attachment of nucleotides A, T, G, C, respectively. After nucleotides A, T, G and C are attached, the IDBCs are returned to the mixer from all four chambers and are ready for the next stage of growing the coding oligonucleotides. As a result, a mixture of IDBCs with unique barcodes is complemented by a database where each IDBC identifier is associated with a specific coding sequence.

3 is a diagram showing the components of the present invention in five blocks. Block A is a cytometer (FC) or fluorescence activated cell sorter (FACS), Block B is a cell accumulator with a chamber analyzing the positions of control microspheres (darkest three circles), Block C is a GIDBC with IDBC (e.g. microspheres), held in separate traps, while oligonucleotides with a predetermined sequence are grown by selective photoactivation using a DLP projector, the D block is the droplet generator in which the coding takes place, and the E block is the synchronization detector. Designated: LIF-detector - LIF-detector, EOF-sorter - EOF-sorter, Sample - Sample, Waste - Waste. Oil - Oil Cells - Cells, Beads - Microspheres.

FIG. 4A-4C show examples of the arrangement of integrated chips. FIG. 4A shows a chip layout containing all the components of a hydrodynamic trap tool. FIG. 4B-4C show the chip layouts containing all of the magnetic trap instrument components. FIG. 4B shows a layout with minimized connectors, and FIG. 4C shows a dual GIDBC layout with no loop in the morphology.

FIG. 5 shows an example of a chip diagram with all the integrated components except the GIDBC. The chip arrangement includes a dedicated IDBC flow detection area for synchronization (E1).

FIG. 6A-6E depict layouts for dual layer PDMS chips. 6A shows a mask for a 5 µm layer containing traps. 6B shows a mask for a 40 µm layer containing channels. This layout is suitable for making 105mm (inner circle) and 130mm (outer circle) double layer masters with additional chips at the corners. It contains several types of chips, including chips for evaluating the optimal geometry of channels and traps. 6C shows an example of an arrangement containing five test areas (3x3 mm squares filled with a tortuous channel with traps of 900 and 1800 microspheres each), oval inlets with filters, rectangular inlets without filters, and a flow detection area bounded by a connector (in the center left). Top right inset shows enlarged traps. FIG. 6D shows an SU-8 image of a master model made using the masks of FIGS. 6A and 6B. 6E shows an image of a finished PDMS / glass chip area that is half filled with a work buffer. Traps are only visible in the air-filled half of the channel due to the higher difference in refractive index.

7A shows an example of a mask arrangement for manufacturing a hydrodynamic trap chip. FIG. 7B is an enlarged view of the traps of FIG. 7A. FIG. 7C shows an SU-8 element of the master model made with the layout of FIG. 7A and FIG. 7B. The arrow denotes a microsphere trap based on a shortened trajectory microflow.

FIG. 8A-8D show magnetic traps of various morphologies. FIG. 8A-8C show enlarged views of various channel trap combinations for the chip arrangements shown in FIGS. 4C, 6C and 6E. FIG. 8D shows an area of a finished PDMS / glass chip made using the wizard shown in FIG. 6D and half filled with a work buffer. Traps are only visible in the air-filled half due to the higher difference in refractive index.

9 is a schematic diagram of an embodiment of GIDBC with independent EOF control of media flow in each horizontal channel. The vertically oriented side channels allow parallel washing during oligonucleotide synthesis. Electrodes (double triangles) provide independent EOF generation in individual horizontal channels after filling the vertical channels with an insulator. The large circles represent the electrolyte reservoirs. Pulsed laser pumping can be used instead of EOF to selectively pump the medium.

Implementation of the technical solution.

The present invention relates to a system and method for integrating polynucleotide sequencing and analyzing the phenotypes of biological particles by deterministic barcoding. In a high-throughput assay, the system and method maintain links between the phenotype of a biological particle, assessed by non-destructive methods (eg, optically), and the sequencing data of the polynucleotides contained in the biological particle.

More specifically, the present invention is directed to a system for analyzing the phenotype and polynucleotide sequences of a biological particle, including a phenotype analyzer, where the phenotype analyzer is for determining the phenotype of a biological particle; a generator of identifiable, deterministically coding media, where the coding oligonucleotide of a predetermined sequence is attached to an identifiable nucleus of the coding media; a microreactor configured to receive a biological particle from a phenotype analyzer and an identifiable coding carrier from a generator, to release polynucleotides from a biological particle, to perform reverse transcription if necessary for RNA analysis, to attach coding oligonucleotides from a carrier to polynucleotides from a biological particle, and for amplifying the resulting encoded polynucleotides; and a sequencer for obtaining sequences of encoded polynucleotides.

In addition, the present invention relates to a method for analyzing phenotypic and polynucleotide sequences of a biological particle, comprising the steps of non-destructive analysis of a biological particle to determine the phenotype of a biological particle; records of the phenotype of a biological particle; generating a coding medium in which a coding oligonucleotide with a predetermined sequence is attached to an identifiable nucleus of the coding medium; transferring the biological particle and an identifiable carrier of the coding oligonucleotide into the microreactor, and conjugation of the coding carrier with the biological particle; lysing the biological particle to release polynucleotides of the biological particle; reverse transcription of RNA from the biological particle (if necessary), ligating the coding oligonucleotide with the polynucleotides of the biological particle to form the encoded polynucleotides; sequencing the encoded polynucleotides to determine the sequences of the polynucleotides from the biological particle; and matching the phenotype of the biological particle to the polynucleotide sequences of the biological particle.

These and other features, objects and advantages of the present invention will become more apparent from a consideration of the following detailed description of the preferred embodiments and the appended claims, in conjunction with the drawings, as described below.

Private examples of implementation.

With reference to FIG. 1A-9, embodiments of the system and method of the present invention can be described. The present invention relates to a system and method for phenotype analysis and sequencing of polynucleotides for DNA / RNA-containing particles by deterministic barcoding. The system and method aims to preserve the links between optically scored individual BP phenotypes and the BP genome / transcriptome in a high-throughput assay. Unlike existing mSCS methods with particles carrying unique but random encoding oligonucleotides, the deterministic barcoding approach of the present invention uses IDBC for each BP and applies a unique and predefined barcode for each polynucleotide of each BP. Therefore, IDBC is a key element of this invention.

To develop IDBCs, the inventors considered combinations of identifiable nuclei and procedures for growing oligonucleotides with deterministic sequences. As identifiable nuclei, the inventors considered: (1) particles in a sequential stream, where a particular IDBC is identified by a sequence number based on its location in the sequential outflow; (2) an array of microwells or microzones on a chip, where the identity of a particular microzone is determined by the coordinates of the microzone; (3) and particles with embedded unique identification marks that can be identified in the stream. The latter case includes particles with: (a) multiple optical labels (eg, fluorescent labels or giant Raman labels); and (b) RFID particles.

Oligonucleotides with known, predetermined sequences are then grown on the identifiable IDBC cores described above to obtain IDBC by one of the following growth procedures: (1) photoactivation; (2) synthesis in crossed streams; or (3) mixing tracking synthesis.

Photoactivation is a well established technique for generating oligonucleotides with a predetermined sequence on the surface of a chip. Briefly, the protecting groups are removed in selected microzones under light illumination and one nucleotide (A, T, G, or C) is attached to growing oligonucleotides in the activated microzones. This process is repeated for the remaining three nucleotides. These four cycles are then repeated N times for N-nucleotide long oligonucleotides, generating up to 4N unique microzones.

Photoactivated synthesis can produce a variety of oligonucleotide regions with microzone-specific, predetermined sequences. The number of regions and, therefore, different sequences, as a rule, is not limited and depends only on the size of the chip and the resolution of the process. Currently, the most efficient approach to photoactivation uses digital light processing (DLP), which allows the formation of millions of independent microzones for unique sequences in a single processing.

This technology can be applied "as is" for the growth of oligonucleotides on microzones of the chip (identifiable nucleus variant No. 2), as well as, with modifications, for growing oligonucleotides on identifiable microparticles (identifiable nucleus variants No. 1 and No. 3). The latter requires reversible immobilization of microparticles in predetermined microzones of the chip, as shown in FIG. 1A-1B, 3, 6C, 6E and 7A-9.

Particles can be reversibly immobilized (or captured) in predetermined microzones of the microfluidic channel, which are organized in the photo-processing area on the chip so as to accommodate the maximum number of these microzones in this area. Possible magnetic, hydrodynamic, dielectrophoresis, chemical, vacuum and inertial approaches to immobilization. In the magnetic approach, magnetized (magnetically susceptible, eg, superparamagnetic) particles are held back by an external magnetic field in surface recesses or traps, as shown in FIG. 1B, 3, 4B, 4C, 6C and 6E. In the hydrodynamic approach, particles are held in microchannels of a shortened trajectory, with a cross section smaller than that of microparticles, as shown in FIG. 4A and 7A-C. In the dielectrophoresis approach, microparticles are held in traps defined by electrodes by means of an alternating electromagnetic field with a frequency determined by the particle size. In a chemical approach, particles are chemically immobilized using cleavable bonds (for example, covalent, ionic, hydrophobic, van der Waals, hydrogen, etc.), but this binding should not interfere with the synthesis of oligonucleotides and should be completely reversible. Gravity can be used for very low flows; and centrifugal force can be used on chips in a rotating holder. In the vacuum approach, microparticles are held in traps by external generation of reduced pressure in the traps.

Of these alternatives, magnetic immobilization, as shown in FIG. 1A, is the preferred option, followed by hydrodynamic and dielectrophoresis approaches. Thus, the inventors have identified a magnetic, hydrodynamic or dielectrophoresis retention photoactivation method used to position IDBC nuclei for growing coding oligonucleotides on IDBCs identified by position in a sequential stream as a preferred embodiment of the present invention.

Crossed flow synthesis is an alternative for cases where photoactivation is not suitable. In this alternative, oligonucleotides are grown on identifiable nuclei at the intersection of individually controlled microfluidic flows, as shown in FIG. 2A-C. It is proposed to use a chip with a grid of microfluidic channels, with the arrangement of nuclei at the intersections of the channels, and with electrodes placed at the end of each channel. Electrodes are used to generate EOF between each selected pair of electrodes and selectively control the medium between them. Alternatively, laser pulsation can be used to control such an environment. The ends of the channels on each side of the chip are connected to the reservoirs. Reservoirs are filled stepwise with reagents for attaching a specific protected nucleotide to growing labels (coding oligonucleotides), and then with isolated medium. The combination of alternating filling of intersecting channels and a change in the type of nucleotide to be attached leads to the growth of an oligonucleotide of a unique and predetermined sequence on IDBC at each intersection. For greater flexibility in the resulting sequences, one set of channels can be filled with an anchoring reagent and the other with a nucleotide attaching reagent. Alternatively, the medium can be controlled (pumped) by a pulsed laser.

Mix tracking synthesis is the preferred method for growing oligonucleotides on IDBCs when using cores with an internal identifier (RFID or optical). An example of an IDBC with an internal identifier is a nucleus with an identifier created by a combination of 16 fluorophores that are simultaneously found in many commercial cytometers and allow 216 = 65536 unique identifiers (for a binary tag). Half of the IDBC sample can be modified with one fluorophore, then mixed with the unmodified half, and this cycle is repeated with other fluorophores. Other means of identification are also possible (eg RFID, magnetic, optical non-fluorescent, etc.). Combinations of different identifiers (eg fluorescent and RFID) further increase the number of unique kernel identifiers.

In existing random barcode mSCS, a set of four reactors introduces one nucleotide at each step of the synthesis. After each step, all particles are mixed and divided into four fractions for the next step. But for IDBCs with core-embedded identifiers, the inventors propose to connect these reactors and a microfluidic mixer to a flow-through detector as shown in FIG. 2D. As a result, each IDBC transition between the mixing chamber and the reactors is recorded and a complete movement history is available for each IDBC, which determines the sequence of the coding oligonucleotides. Particles that received the same sequence (that is, followed the same path in all synthesis cycles) can be removed from the final mixture using a sorter activated by the same flow detector.

The IDBCs generated by these nuclei and oligonucleotide growth procedures provide several deterministic barcoding approaches that combine non-destructive phenotype analysis (e.g., flow cytometer or microscope image analysis) of biological particles with mSCS analysis of the genome or transcriptome of biological particles. IDBC allows each biological particle to be individually labeled after almost any phenotype analysis and to link the phenotype of these biological particles to their individual sequencing data. The population size of the analyzed biological particles is limited only by the sequencing performance, which is a rapidly growing area. This ensures that the present invention will not only be useful in current biomedical research applications, but will be even more useful in the future. Even with modern sequencing technologies, assays can include millions of particles with small genomes / transcriptomes or small target genetic elements. Deterministic (or known and predetermined) barcoding in the present invention is capable of measuring any combination of optically measurable phenotypic properties, including, but not limited to: (1) membrane potential, membrane fluidity, and membrane electropermeabilization; (2) intracellular pH, ion and oxygen content; (3) total DNA / RNA content, copy number variability, chromosome analysis and sorting; (4) protein expression, protein modification and protein localization; (5) the presence of intracellular, intranuclear and surface antigens; (6) enzymatic activity; (7) cell viability, volume and morphological complexity; (8) the presence of multidrug resistance; (9) adhesion (for example, a pathogen to a host cell); (10) parameters of apoptosis by measuring DNA degradation, mitochondrial membrane potential and changes in its permeability; and (11) the concentration of cellular pigments and transgenic products in vivo (eg, green fluorescent protein).

An embodiment of the invention.

In one preferred embodiment, IDBCs are generated using particles (eg, microspheres) in a sequential stream as identifiable photoactivated nuclei as a procedure for growing oligonucleotides on microspheres. Using these IDBCs in the system of the present invention can establish a one-to-one correspondence between polynucleotide sequence information of individual cells and flow cytometry data in a high throughput assay.

As shown in FIG. 3, FC collects and records information specific to an individual BP and routes the sorted BPs to the system via an additional cell accumulator. In parallel, the microfluidic chip reversibly immobilizes the microparticles in a predetermined array. Several photoactivated synthesis steps produce multiple IDBCs containing oligionucleotides with a predetermined unique sequence for each identified nucleus (ie, microspheres). The IDBC stream from this chip is combined with the BP stream from the BP accumulator in in-chip droplets (i.e. microreactors) that are generated with IDBC-BP pairs for barcoding and sequencing. Deterministic barcoding of BP polynucleotides allows sequencing data for these polynucleotides to be matched against cytometry and BP image analysis data from which the polynucleotides originated.

In this preferred embodiment, as shown in FIG. 3, where each component is labeled with a letter, the system consists of the following components:

Component A: Flow Cytometer with Optional Sorter;

Component B: BP battery with BP and reference microsphere imaging device to confirm and track the relative position of BP and reference microspheres;

Component C: GIDBC with another reference microparticle image analyzer to confirm and track the relative position of IDBC coding microparticles and reference microspheres;

Component D: Chip for generating water-in-oil droplets / microreactor where BP polynucleotides are released and barcode; and

Component E: In-line reference particle IDBC sensor to correct errors when relative positioning is incorrect.

The system allows full integration of all components on a single chip (as shown in Figures 4A-C), a combination of individual chips connected by microfluidic lines, and a combination of chip-based capillary and macroscopic devices (as shown in Figures 3 and 5).

The inventors have manufactured various PDMS chips (Fig. 6A, B, D), including prototype individual GDBBs (Fig. 6C, 6E and 7A-8D), integrated tools on a single chip (Fig. 4A-C), and intermediate options (for example, a chip with all components except GDBB (ie, including microfluidics for FACS, BP battery, droplet generator and timing detector) (as shown in FIG. 5).

The chips were fabricated using a two-layer soft lithography process with a 5 µm layer of traps and a 40 µm layer of channels for a core diameter of 5 to 10 µm. The inventors made a lithographic mask for each layer (FIGS. 6A-6B), a master model SU-8 (FIG. 6D) and PDMS impressions. The inventors cut PDMS castings to the size of a single chip and adhered them to an activated glass substrate or PDMS casting to make chips (Fig. 6E). The resulting chips have a glass side for optical access and a PDMS side for fluid lines and magnetism (FIG. 1B).

A detailed description of each component of the system and its alternatives is given below:

(A) Cytometer / sorter

A flow cytometer is used for flow phenotype analysis and sorting of BPs of interest. Although it is possible to use most cytometers and other in-line particle analyzers in the system, a capillary based circuit (FIG. 3A) or an on-chip circuit (FIGS. 4A-5) is preferred.

BP can be moved through the microfluidic channel using external pressure, EOF, laser pulsation, or other means well known to those skilled in the art. The inventors propose EOF and laser pulsation as the main motive means because they are quick to react and do not require moving parts. BP can be detected in a channel or in a hydrodynamic focusing cuvette using LIF / LS. Channel detection is easier to implement and use, while hydrodynamic focusing cuvette detection can provide better signal-to-noise ratio and higher throughput. Sorting can be activated with a fluorescent signal, a light scatter signal, or any other signal from a detector and is performed using flow redirection by EOF, laser pulsing, or other means well known to those of skill in the art. EOF control is easier to implement, while laser pulse control is less dependent on the chemical composition of the channel wall surface and provides better throughput. As shown in FIG. 3, the part of the sample that does not meet the sorting criteria is discharged from the system as waste.

(B) Cell accumulator

The cell accumulator is optionally used for (i) timely BP sorting and droplet generation to avoid mutual interference between flow drivers of different components, and (ii) visualization of BP and reference particles for error correction.

BPs are loaded into a long microfluidic channel (FIG. 3) or more complex microfluidic systems (similar to those shown in FIGS. 7A-C and 9) and can be fed in the same order to component D of the system at any convenient time. During loading BPs with reference particles can be displayed on the chip to check the sequence and volume of the loading by analyzing the amount of BP between the reference particles. In-depth image analysis can also provide additional information about BP phenotype (eg, BP shape) to complement the information obtained with the flow-through detector.

While this component is recommended for system enhancement, it is not absolutely necessary. If BPs are not used, then they can be directly directed to component D after sorting from block A. Conversely, this component with advanced image analysis can be used not only in addition, but also as a replacement for block A. In this case, phenotypic the data is collected exclusively through image analysis, although this will also diminish the capabilities of the system.

(C) GIDBC

In this preferred embodiment, the core of the IDBC is particles in a sequential stream, where each particular particle is identified by a serial number (i.e., position in the stream) to track its position relative to other carrier microparticles and reference particles (FIGS. 3, 4A-C, 6C, 7A -C and 8A-D). As described above, the coding oligonucleotides are grown on the particles by photoactivation (FIG. 1). In this embodiment, the reversible immobilization of particles in "traps" located in predetermined microzones on the chip is required. These traps are located in the microfluidic channel, which occupies as large a part of the photoprocessed area of the chip as possible. Of the possibilities described above, this preferred embodiment uses magnetic (FIGS. 1A-B, 2C, 4A-C, 6C, 6E and 8A-9) or hydrodynamic (FIGS. 7A-C) traps.

The geometry of the magnetic traps depends on the size of the microspheres and the hydrodynamic properties of the chip. For microspheres with a diameter of 8 µm, the inventors made chips with a channel width of 20 to 80 µm in order to find the optimal balance between loading, sequence control and clogging resistance; round, triangular and trapezoidal 5-20 micron traps for optimal control of loading and release; and a trap spacing of 50 to 100 µm to assess possible optical crosstalk and to monitor the elution sequence with varying trap spacing (FIGS. 6A-E). As an alternative embodiment, the inventors also made chips with a set of hydrodynamic traps (Fig. 4A and Fig. 7A-C). Dielectrophoresis trap chips have also been considered, but given a lower priority.

Optimizing the organization of kernel traps in GIDBC. For magnetic immobilization, traps can be not only isotropic (i.e., symmetric with respect to the flow of the medium), but also anisotropic to facilitate immobilization and release, depending on the direction of flow of the medium, and to simplify the rotation control of the IDBC, if necessary. For isotropic traps, circular geometry is preferred, and for anisotropic traps, triangular or trapezoidal traps are suggested (FIGS. 8A-D). To improve the coverage of microspheres with oligonucleotide labels, rotation of microspheres in traps can be implemented. Rotation can be caused by a superposition of the shear force of the flow of the medium and the force of capture (magnetic, dielectrophoresis, etc.). Alternatively, the optical deprotection of the anchors can asymmetrically expose the charged groups, creating an EOF based rotation pulse. In addition, the rotation of the electromagnetic field can cause an impulse if the microparticles are polarized appropriately.

Optimization of the release of microspheres in GIDBC. The release of the microspheres will be caused by the removal of the gripping force, in the presence of the shear force of the flow of the medium. However, if necessary, additional means of controlling the release and suppressing unwanted sorption can be implemented. These include (1) inverted or alternating field, (2) reversible fluid flow for anisotropic magnetic traps, as described, and hydrodynamic traps, (3) ultrasonic driver and / or pulsed laser illumination, (4) surface charge reversal, (5 ) the use of chaotropic agents; and (6) the rotation of the microspheres.

Controlling the sequence of the microspheres in the outgoing stream in the GIDBC: For a consistent stream of microspheres, it is important to ensure that the mutual order of the microspheres in the outgoing stream is maintained. To monitor this order, the inventors propose to use optically labeled microspheres as a reference to mark the positions of the microspheres on the chip. Using these reference microspheres, it is possible to determine whether the number of unlabeled microspheres between the optically labeled in the outgoing stream remains the same, and compare the 2D imaging of the optically labeled reference microspheres in traps in the general image processing area of the GIDBC microspheres with data from the LIF / LS flow detector (Fig. 3 -5).

Processing of microspheres with high productivity. For thousands of microspheres, the simplest single-channel chips (FIGS. 3, 4B, 4C, 6C, 6E, 8A-D) with elution under pressure, laser pulses or EOF are sufficient. However, for millions of microspheres and larger chips, the flow resistance may be too high to travel through a single channel. In this case, the channels can be filled in parallel and subsequently eluted (see the example with hydrodynamic traps in Fig. 4A and Fig. 7A-C). A more versatile circuit is shown in FIG. nine.

In this design, channels can be filled and processed in parallel and eluted sequentially. Inter-channel flow connections can be blocked with a solidifying medium (eg photoresist) followed by elution under pressure. EOF-driven chips also allow for parallel loading and processing. Excessive channel-to-channel electrical connections can be blocked with isolation media, allowing precise controlled EOF elution to be performed independently in each individual channel. Pulsed laser pumping can also be used. Scaling up would also require higher resolution optical processing where optical crosstalk can occur. Suppression of unwanted light propagation may be required through refractive index control, surface treatment to extinguish the light, and increase in the size of the treatment area.

(D) Generator of coding microreactors in droplets.

Chip circuits for water-in-oil droplet / microreactor generation (WIO) for barcoding BP polynucleotides are well developed in various commercial mSCS instruments. Briefly, an aqueous stream containing the coding microspheres analyzed by BP and all necessary reagents encounters two streams of oil (hydrophobic medium) supplied from the side at the junction, followed by a local narrowing of the channel (Figs. 4A-C and Fig. 5D), where water droplets in oil form.

Each drop should contain no more than one BP, because all polynucleotides in a drop will have the same barcode specific, and polynucleotides from multiple BPs in one drop will not be distinguished after sequencing. A drop without BP does not cause any problems other than loss of effectiveness. As a result, in commonly used chips, the ratio of drops to BP is kept high to statistically reduce the number of BP doublets and multiplets per drop. Conversely, in the preferred embodiment, using a sorter / accumulator, BP can be fed into the drip generator in a more controlled manner, significantly reducing lost drips and IDBC. This level of flow control is achievable because the excitation of the medium by laser pulses or EOF has a very low response time to signals from the LIF / LS flow detector, determined by the pulse width or the capacitance of the electrodes, respectively.

Each drop must also contain one IDBC. In a droplet without such a microsphere, barcoding is not possible and BP sequencing information will be lost. If a drop contains multiple IDBCs, different barcodes will be applied to polynucleotides from the same BP, which will cause problems with sequencing data processing when using traditional chips. In contrast, in a preferred embodiment, synchronization and error correction (see below) can identify the microparticles involved in such events and the processing of the sequencing data will not be affected.

Each drop should also contain reagents for BP lysis, ligation of the polynucleotide to the coding oligonucleotide, amplification, and sometimes reverse transcription. These reagents can be delivered by the stream (s) of an aqueous medium, within the IDBC, or migrate into droplets from an oily (hydrophobic) medium.

As with existing mSCS, after co-encapsulating BP, IDBC, and reagents in the drop, BP is lysed to release polynucleotides, RNA is reverse transcribed (if necessary), and ligases are used to add the coding oligonucleotide from IDBC to the BP polynucleotide. As a result, BP polynucleotides now carry a barcode. Amplification, if necessary, can be carried out in a drop or after droplet fusion and removal of the oily (hydrophobic) medium in preparation for sequencing.

(E) Auxiliary flow detector for synchronization and error correction.

A simplified secondary flow detector LIF / LS is designed for the analysis of droplets in the outflow stream (Fig. 3, Fig. 4A-C, Fig. 5), namely for the detection and counting of optically labeled reference microspheres. Using the same principles as the cytometer / sorter LIF / LS detector, this detector requires one light scatter detection channel and two fluorescent (or SERS) tag detection channels to detect reference microspheres, so the sensitivity may be low. The acquisition rate should match the rate at which the droplets are generated. The simplest image analysis can also be useful for this detector. As a result, low-budget solutions are enough for this component, such as a three-color webcam. The use of the data obtained in this block is described below in the section on synchronization and error correction.

Synchronization and error correction using optical and gene tags. Linking a database of coding sequences from GIDBC to cytometric data is error-prone and risky for analysis. Errors can occur due to the processing of microspheres (i.e., GIDBC) in the chip, because oligonucleotide synthesis microzones may not contain any microspheres, contain several microspheres, or microspheres can be sorbed outside the synthesis microzone. Errors can also be caused by the IDBC and BP interface chip in the drop, because the ratio of one BP to one IDBC per drop can be compromised. Errors can occur in a flow cytometer because doublets or multiplets of particles can be detected as one event. In each of these cases, these errors will manifest as a mismatch between the indexes in the database of coding sequences and cytometry data (that is, phenotype data associated with each BP and tracked by BP sequence within the system). These inconsistencies must be found and corrected. The inventors propose two independent correction mechanisms: pre-sequencing and post-sequencing.

For the method after sequencing, particles (eg, reference particles, liposomes) containing known polynucleotide labels and optical labels can be added to the analyzed BP. Any discrepancy between the expected sequence of reference particles in the BP stream (based on FC data and BP accumulator image) and the sequence of tagged particles (IDBC) can be detected after sequencing. Then you can apply the appropriate correction.

For the pre-sequencing method, optically labeled reference particles can be added to the IDBC (one color) and BP (another color), and analyzed with a secondary flow sensor (FIGS. 3-5) monitoring the inconsistencies. For IDBC, it will measure the distance (number of unlabeled IDBCs) between the marked (reference) ones, and compare this distance with the original GIDBC reference reference image. For BP, it will measure whether the number of detected and sorted for encoding coding BPs (based on the flow detector and BP battery image analysis) matches the number of BPs between the reference particles. Applied in parallel, these techniques provide reliable error correction.

Alternative options for implementation:

In alternative embodiments, other identifiable cores are used. For example, when an array of particles or microzones on a chip is used as an identifiable nucleus, where the identity of a particular microzone is determined by the coordinates of the microzone, the invention can be used with chips capable of limiting and localizing the reaction of biological BP particles with surface microzones containing coding oligonucleotides. Such microzones include, but are not limited to, microwells on the surface of the chip that delimit individual VRs, or gel barriers introduced optically, electromagnetically, or mechanically. In this approach, image analysis is used to access VR phenotypes instead of flow-through methods.

When IDBCs with embedded unique identification tags that can be identified in the stream (e.g. RFID or optical tags, as described above) are used as identifiable nuclei, oligonucleotides can be grown using all of the methods mentioned, but traceable mixing synthesis is most suitable (as described above, see Fig. 2D). A solution (Suspension) of these IDBCs, together with an appropriate flow-through identifier detector, can be used in a preferred instrument design instead of a chip-based GIDBC, eliminating the need for multistep oligonucleotide growth for each GIDBC particle load.

The present invention has been described with reference to some preferred and alternative embodiments, which are intended by way of example only and do not limit the full scope of the present invention. Although the elements of the proposed device are synergistic, some of them can be eliminated without completely losing functionality. For example, flow cytometry-based cell sorting can be eliminated with higher IDBC consumption and sequencer loading. Flow cytometry can be eliminated and replaced by image analysis of the biological particle accumulator (as described above). Even a flow cytometer, droplet generator, and particle-based IDBCs can be eliminated by combining the GIDBC and a biological particle accumulator and printing the IDBC array directly onto the surface of the chip.

In preferred or alternative embodiments, it is not necessary to grow the coding oligonucleotides on identifiable nuclei prior to each use of the system. Instead, IDBCs can be prepared in advance or even supplied as a disposable assay cartridge using (i) a preloaded IDBC disposable GIDBC chip; (ii) a chip with an array of surface microzones containing coding oligonucleotides; or (iii) suspensions of IDBCs with identifiable cores in the stream, with an appropriate database linking the core identifier (serial number, position, or RFID / optical tags) of each IDBC element with the sequence of its coding oligonucleotides, as described above.

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Claims (27)

1. A system for analyzing the phenotype and sequence of polynucleotides of a biological particle, which contains: - a phenotype analyzer, where the specified phenotype analyzer is designed to determine the phenotype of the specified biological particle; - generator of identifiable oligonucleotide coding carrier, where the coding oligonucleotide with a predetermined sequence is attached to the specified carrier, an individual carrier is used for each biological particle and a unique and predetermined barcode is applied for each polynucleotide of each biological particle; - a microreactor configured to receive said biological particle from said phenotype analyzer and receive said carrier of coding oligonucleotides from said generator; - a sequencer of said predetermined sequence of said coding oligonucleotide and said sequence of polynucleotides of said biological particle after said coding oligonucleotide is ligated with said polynucleotides of said biological particle. 2. The system of claim 1, wherein the phenotype analyzer is a flow cytometer. 3. The system of claim. 1, characterized in that the phenotype analyzer is a microscope. 4. The system of claim. 1, characterized in that the carrier is a magnetic particle. 5. The system of claim. 1, characterized in that the carrier is a microcell on the surface of the chip. 6. The system according to claim 1, characterized in that it contains an accumulator of biological particles between said phenotype analyzer and said microreactor. 7. A method for analyzing the phenotype and sequence of polynucleotides of a biological particle, including the steps: - optical analysis of the specified biological particle to determine the phenotype of the specified biological particle; - records of the specified phenotype of the specified biological particle; - generating a carrier of coding oligonucleotides, where the coding oligonucleotide of a predetermined sequence is attached to said carrier, an individual carrier is used for each biological particle, and a unique and predetermined barcode is applied for each polynucleotide of each biological particle; - placing the specified biological particle and the specified carrier of coding oligonucleotides in a microreactor; - lysis of said biological particle to release said polynucleotides of said biological particle; - ligating said coding oligonucleotides with said polynucleotides of said biological particle to form a genetic complex; - sequencing the specified genetic complex to determine the specified sequence of the specified polynucleotides of the specified biological particle; - comparing the specified phenotype of the specified biological particle with the specified sequence of the specified polynucleotides of the specified biological particle. 8. The method according to claim 7, characterized in that said biological particle and said carrier of coding oligonucleotides are encapsulated in a drop in said microreactor. 9. The method according to claim 7, wherein the flow cytometer performs the step of optical analysis of said biological particle to determine the phenotype of said biological particle. 10. The method according to claim 7, characterized in that said biological particle is supplied to a biological particle accumulator before said biological particle is placed in said microreactor. 11. The method according to claim 7, characterized in that it contains the step of analyzing the output from said microreactor for errors. 12. The method according to claim 7, wherein said step of generating the carrier of the coding oligonucleotides comprises growing the oligonucleotide on the particle. 13. The method of claim 12, wherein the step of growing the oligonucleotide on the particle comprises photoactivation. 14. A method according to claim 12, characterized in that it comprises the step of immobilizing said particle in a trap on a chip. 15. The method of claim 7, wherein the step of generating the carrier for the coding oligonucleotides comprises growing the oligonucleotide on a chip.

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