This disclosure demonstrates an approach that translates synthetic DNA codes to spatial codes registered in nanoliter microchambers for multiplexed measurement of nearly any type of molecular targets (e.g., miRNAs, mRNAs, intracellular and surface proteins) in single cells.

This disclosure demonstrates an approach that translates synthetic DNA codes to spatial codes registered in nanoliter microchambers for multiplexed measurement of nearly any type of molecular targets (e.g., miRNAs, mRNAs, intracellular and surface proteins) in single cells.

Implementations of a method for seeding sequence libraries on a surface of a sequencing flow cell that allow for spatial segregation of the libraries on the surface are provided. The spatial segregation can be used to index sequence reads from individual sequencing libraries to increase efficiency of subsequent data analysis. In some examples, hydrogel beads containing encapsulated sequencing libraries are captured on a sequencing flow cell and degraded in the presence of a liquid diffusion barrier to allow for the spatial segregation and seeding of the sequencing libraries on the surface of the flow cell. Additionally, examples of systems, methods and compositions are provided relating to flow cell devices configured for nucleic acid library preparation and single cell sequencing. Some examples include flow cell devices having a hydrogel with genetic material disposed therein, and which is retained within the hydrogel during nucleic acid processing.

Implementations of a method for seeding sequence libraries on a surface of a sequencing flow cell that allow for spatial segregation of the libraries on the surface are provided. The spatial segregation can be used to index sequence reads from individual sequencing libraries to increase efficiency of subsequent data analysis. In some examples, hydrogel beads containing encapsulated sequencing libraries are captured on a sequencing flow cell and degraded in the presence of a liquid diffusion barrier to allow for the spatial segregation and seeding of the sequencing libraries on the surface of the flow cell. Additionally, examples of systems, methods and compositions are provided relating to flow cell devices configured for nucleic acid library preparation and single cell sequencing. Some examples include flow cell devices having a hydrogel with genetic material disposed therein, and which is retained within the hydrogel during nucleic acid processing.

An apparatus includes a flow cell body, a plurality of electrodes, an integrated circuit, and an imaging assembly. The flow cell body defines one or more flow channels and a plurality of wells. Each flow channel is configured to receive a flow of fluid. Each well is fluidically coupled with the corresponding flow channel. Each well is configured to contain at least one polynucleotide. Each electrode is positioned in a corresponding well of the plurality of wells. The electrodes are operable to effect writing of polynucleotides in the corresponding wells. The integrated circuit is operable to drive selective deposition or activation of selected nucleotides to attach to polynucleotides in the wells to thereby generate polynucleotides representing machine-written data in the wells. The imaging assembly is operable to capture images indicative of one or more nucleotides in a polynucleotide.

An apparatus includes a flow cell body, a plurality of electrodes, an integrated circuit, and an imaging assembly. The flow cell body defines one or more flow channels and a plurality of wells. Each flow channel is configured to receive a flow of fluid. Each well is fluidically coupled with the corresponding flow channel. Each well is configured to contain at least one polynucleotide. Each electrode is positioned in a corresponding well of the plurality of wells. The electrodes are operable to effect writing of polynucleotides in the corresponding wells. The integrated circuit is operable to drive selective deposition or activation of selected nucleotides to attach to polynucleotides in the wells to thereby generate polynucleotides representing machine-written data in the wells. The imaging assembly is operable to capture images indicative of one or more nucleotides in a polynucleotide.

The invention is directed to a method to obtain the spatial location and sequence information of at least a part of a RNA or cDNA strand (006) in a sample comprising the steps a. hybridizing a first detection probe oligonucleotide (204) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the at least one RNA or cDNA strand, wherein the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide (205) comprising 5-100 nucleotides wherein a gap region (206) capable of binding oligonucleotides is created b. filling the gap region (206) in part with 1 to 16 barcode oligonucleotides comprising 4-20 nucleotides, wherein the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample c. partially hybridizing a second detection probe oligonucleotide (204′) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the same or cDNA strand and with the respective other end to the bridge oligonucleotide (205) to create a circular template d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers e. determining the sequence of nucleotides of the rolonies

The invention is directed to a method to obtain the spatial location and sequence information of at least a part of a RNA or cDNA strand (006) in a sample comprising the steps a. hybridizing a first detection probe oligonucleotide (204) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the at least one RNA or cDNA strand, wherein the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide (205) comprising 5-100 nucleotides wherein a gap region (206) capable of binding oligonucleotides is created b. filling the gap region (206) in part with 1 to 16 barcode oligonucleotides comprising 4-20 nucleotides, wherein the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample c. partially hybridizing a second detection probe oligonucleotide (204′) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the same or cDNA strand and with the respective other end to the bridge oligonucleotide (205) to create a circular template d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers e. determining the sequence of nucleotides of the rolonies

A method for generating region-specific amplification templates of a biological sample includes adding first oligonucleotide constructs and second oligonucleotide constructs to the biological sample. Each first or second oligonucleotide construct comprises a first or a second photoremovable cage molecule. The method further includes synthesising a complementary first strand from a template bound to target binding regions of each first oligonucleotide construct or each second oligonucleotide construct, scanning a first region of interest of the biological sample with a first focused light beam and a second region of interest of the biological sample with a second focused light beam to form uncaged first oligonucleotide constructs in the first region of interest and uncaged second oligonucleotide constructs in the second region of interest, synthesising a complementary second strand to form first amplification templates originating from the first region of interest and second amplification templates originating from the second region of interest.

A method for generating region-specific amplification templates of a biological sample includes adding first oligonucleotide constructs and second oligonucleotide constructs to the biological sample. Each first or second oligonucleotide construct comprises a first or a second photoremovable cage molecule. The method further includes synthesising a complementary first strand from a template bound to target binding regions of each first oligonucleotide construct or each second oligonucleotide construct, scanning a first region of interest of the biological sample with a first focused light beam and a second region of interest of the biological sample with a second focused light beam to form uncaged first oligonucleotide constructs in the first region of interest and uncaged second oligonucleotide constructs in the second region of interest, synthesising a complementary second strand to form first amplification templates originating from the first region of interest and second amplification templates originating from the second region of interest.