CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT/EP2018/056551, filed on Mar. 15, 2018, which claims the benefit of and priority to European Patent Application No. 17305297.8, filed on Mar. 17, 2017, the contents of all of which are incorporated by reference herein in their entirety. International Application No. PCT/EP2018/056551 was published under PCT Article 21(2) in English.

FIELD OF THE INVENTION

The present invention concerns processes for barcoding nucleic acids from single cells and processes for genotyping single cells having a phenotype of interest.

BACKGROUND OF THE INVENTION

Methods for analyzing multiple parameters of single cells in populations are of interest in a variety of contexts. In particular, the ability to analyze nucleic acids, optionally in combination with one or more other parameters, on a single-cell basis within a cell population is of broad interest to commercial and academic laboratories. Moreover, it is often necessary for this analysis to be performed in real-time in order to reveal the dynamic behavior of biological and biochemical processes.

Thus, technologies that can isolate, detect, and quantify individual components of a heterogeneous mixture in a highly parallel fashion are needed to meet these challenges. Conventional high-throughput platforms such as high-density microwell plates with robotic dispensing systems have been developed and widely used for high-throughput analysis, such as drug screening. However, they require expensive and bulky robotic machinery and suffer from sample evaporation and comparably large reaction volumes, which can waste precious biological samples or reagents.

Recently, microfabricated devices have emerged as a powerful experimental platform for performing a diverse range of biological and chemical assays in a high-throughput manner. These technologies often permit high-throughput analysis of a complex sample by partitioning a bulk solution into many isolated pico to nanoliter-sized compartments, or microreactors. However, post-analysis retrieval of individual samples is difficult to achieve. Furthermore, mixing of reagents in these devices either requires complex architecture or is often done in bulk before compartmentalization, which may prevent initial reaction products from co-localizing with their initiating target.

One approach is to compartmentalize reactions into discrete micron-sized droplets surrounded by an immiscible carrier fluid. Droplet-based microfluidics provides precise control over mixing of fluids, minimizes waste of precious reagents, and reduces evaporation and adsorption of molecules at the device walls.

White et al. (Proc Natl Acad Sci USA. 2011 Aug. 23; 108(34):13999-4004) describe a microfluidic device capable of performing RT-qPCR measurements of gene expression from hundreds of single cells per run, executing single-cell processing, including cell capture, cell lysis, reverse transcription, and quantitative PCR. However, incited to try smaller reaction volumes, White et al. (Proc Natl Acad Sci USA. 2011 Aug. 23; 108(34):13999-4004) performed 300 parallel RT-qPCR and demonstrated that RT is inhibited in volumes that are smaller than 5 nL in the reaction conditions tested. The researchers thus performed the RT reaction in 67 nL per cell and further claimed that the combination of RT and qPCR in a single reaction precludes large-scale transcriptome analysis and/or unbiased amplification.

Furthermore, a method using a simple axisymmetric flow-focusing device has been described for single cell mRNA capture by DeKosky et al. (Nat Med. 2015 January; 21(1):86-91). The RT and PCR reaction of the method take place in an emulsion. This process has proven to be efficient, yet requiring performing sequential 3 steps process.

Along the same line, Eastburn et al. (Anal Chem. 2013 Aug. 20; 85(16):8016-21) performed RT in small droplet volume, yet in a time consuming 3-4 steps process. They mentioned also RT inhibition in small volume from cell proteinase. To overcome this problem, they treat the cells with proteinase, dilute cell lysate, split drops, and pico-inject RT-PCR reagents.

Rotem et al. (PLoS One. 2015 May 22; 10(5):e0116328) encapsulate oligo in droplet population and fuse them to droplet containing cells while pico-injecting the RT enzymes and buffers. This 3 step process produces˜100 pL droplets allowing encapsulation of 100.000 cells in 3 h. Although this method relies on single cell cDNA labeling, the transcriptomic sequence data comes from an aggregate of multiple phenotypically and genotypically uncorrelated cells.

Macosko et al. (Cell. 2015 May 21; 161(5):1202-14) used droplet-based microfluidics to encapsulate cells together with lysis reagents and barcoded beads to capture mRNA in 1 nL drops. The beads are, however, used off chip to perform the conversion of the mRNA captured on beads into cDNA and they do not load into most of the formed droplet thus precluding analysis of rare cell populations. The third step of the method described by Macosko et al. is then devoted to library preparation and amplification.

The international patent application WO2015/164212 refers to a method for encapsulating and barcoding single cell nucleic acids. WO2015/164212 discloses that RT of mRNA is strongly inhibited in volumes that are smaller than 3 nL (Example 4 of WO2015/164212).

Labanieh et al. 2015 describes a microfluidic process using a microfluidic device to trap, analyse, and recover droplets using buoyancy. However, Labanieh et al. does not specify the analysis of single cell nucleic acids wherein the nucleic acids are reverse transcribed and therefore the genotype of single cells cannot be obtained using this method.

The patent application US2013/0323732 refers to methods and devices for assaying single cells and barcoding single cell cDNA. US2013/0323732 does not disclose a method wherein droplets are fused within a microfluidic chip.

Contrary to this, the inventors succeeded in developing an on chip microfluidic process for barcoding single cell nucleic acids, wherein single cell droplets are captured in individual compartments. The single cell droplets are further fused with droplets providing the reaction mixture for reverse transcription.

As mentioned above, the on chip process developed by the inventors allows accessing the phenotype of single cells. The inventors therefore further developed a process uniquely coupling phenotype information (protein expression level, cellular pathway activation/activity, ion channel/GPCR activities) with genotypic or epigenetic information, thus allowing determining the genotype of a single cell having a phenotype of interest.

The ability to image the array at each step in the process allows obtaining more quantitative data per single cell than with conventional methods and allows generating kinetic data during the phenotypic screen which can be then linked with the genotype of a particular single cell.

This process differs from the so far known prior art, such as Rotem et al., because in Rotem et al., for example, genomic information is obtained by sequencing an aggregate of multiple phenotypically and genotypically uncorrelated cells. Their method therefore does not disclose coupling phenotype information to the genotype of each single cell.

The international patent application WO2016145409 μl discloses a method wherein phenotypes and genotypes of a protein of interest may be correlated. In this method, different to the method of the inventors, a barcode is linked to, for example, a protein of interest or its binding partner, and at the same time to the nucleic acid encoding said protein of interest. The nucleic acids are then reverse transcribed to obtain barcoded cDNA, the barcoded cDNA is then sequenced. The protein of interest is in parallel phenotyped, by breaking the emulsion and performing a phenotypic assay, such as ELISA, or an essay using affinity columns. For the proteins that show the desired phenotype the barcode sequence may then be analyzed by sequencing. The phenotype is then linked to a genotype by matching the barcode of the protein of interest having a phenotype of interest with a cDNA having the same barcode.

However, as it will be understood by the skilled in the art, such a method does not allow to match a whole transcriptome of a single cell with the phenotype of a single cell or to access the genotypes of single cells having a phenotype which is not associated with the presence of one particular protein of interest.

Contrary to this the method of the inventors allows to phenotype, in one step, several single cells, and then to genotype, in parallel, those single cells by pooling their nucleic acids. Afterwards the genomic information is linked to the phenotype of a single cell having a phenotype of interest.

Furthermore, the process of the inventors allows determining any phenotype including a phenotype that is not necessarily associated with the presence of a single protein of interest but which might be associated with a change of activity of, for example certain pathways, and which results in any phenotype that might be determined using an assay, for example, antibody secretion rate, ion channel activity, GPCR activations, a change the Redox potential of a cell.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a process for genotyping single cells having a phenotype of interest comprising:providing a plurality of reservoirs, anda) for a plurality of reservoirs, providing, for each reservoir, at least one barcode sequence and at least one dye, wherein the at least one barcode sequence is associated with the color and the concentration of said at least one dye, and taking an image of the array thereby mapping the color and the intensity of the at least one dye for each reservoir; andb) for a plurality of reservoirs, providing, for each reservoir, at least one single cell and performing for each reservoir a phenotypic assay on said at least one single cell, and taking an image thereby mapping the phenotype of said at least one single cell for each reservoir;wherein step a) is performed before step b) or step b) is performed before step a); thenc) for a plurality of reservoirs, linking, for each reservoir, the phenotype of a single cell obtained in step b) to the color and the intensity of the at least one dye obtained in step a); andfor a plurality of reservoirs, reverse transcribing, for each reservoir, nucleic acids of said at least one single cell, to obtain single cell cDNA barcoded with said at least one barcode sequence which is associated with the color and concentration of the at least one dye; andlinking the genotype with the phenotype of said at least one single cell.

A second aspect of the present invention is directed to a microfluidic process for barcoding single cell nucleic acids, said method comprising:providing a microfluidic device comprising a chip comprising at least one microfluidic channel and a plurality of reservoirs,injecting into the inlet of the microfluidic channel a carrier fluid comprising a plurality of droplets of a first type dispersed in the carrier fluid, wherein the droplets of the first type are either single cell droplets or RT droplets, wherein at least some of the RT droplets comprise a reverse transcriptase and at least one oligonucleotide, and wherein at least some of the single cell droplets comprise one single cell, wherein said single cell comprises single cell nucleic acids,for a plurality of reservoirs, a first migration step, wherein at least one droplet of the first type among the plurality of droplets is moved into one reservoir of said plurality of reservoirs by buoyancy,injecting into the inlet of the microfluidic channel, a carrier fluid comprising a plurality of droplets of a second type dispersed in the carrier fluid, wherein the droplets of the second type are either single cell droplets or RT droplets, and wherein the droplets of the second type are RT droplets when the droplets of the first type are single cell droplets or the droplets of the second type are single cell droplets when the droplets of the first type are RT droplets,for a plurality of reservoirs, a second migration step, wherein, at least one part of at least one droplet of the second type enters into one reservoir of said plurality of reservoirs,for a plurality of reservoirs, fusing, in or at the edge of each reservoir, said at least one droplet of the first type with said at least one droplet of the second type, thereby resulting in a fused droplet,and further comprising the steps of:a) hybridizing, for each fused droplet, the at least some of the single cell nucleic acids with the at least one oligonucleotide in said fused droplet,b) reverse transcribing, in each fused droplet, at least some of the single cell nucleic acids present in said fused droplet, thereby resulting in single cell cDNA, andc) attaching at least one barcode sequence to the single cell cDNA obtained in step b), wherein said at least one barcode sequence encodes the identity of said single cell, orfurther comprising the steps of:a) hybridizing, for each fused droplet, at least some of single cell nucleic acids from at least one cell with the at least one oligonucleotide in said fused droplet, wherein said at least one oligonucleotide comprises at least one barcode sequence,b) reverse transcribing, in each fused droplet, at least some of the single cell nucleic acids present in said fused droplet, thereby resulting in barcoded single cell cDNA, wherein said at least one barcode sequence encodes the identity of said single cell.

