Addressable flow cell using patterned electrodes

Abstract

Disclosed are methods and systems concerning flow cells for sequencing a nucleic acid sample that may be characterized by the following components: a substrate having an inner surface facing a library sequencing region, and an outer surface; a plurality of a plurality of forward and reverse amplification primers immobilized over the inner surface and providing a nucleic acid library capture surface of the library sequencing region; a plurality of electrodes disposed along the inner surface directly under at least some of the forward and reverse amplification primers, and configured to provide, when charged, an electric field through the library capture surface and into the library sequencing region; electrical leads connected to the plurality of electrodes to permit the electrodes to be independently addressable; and fluidic couplings configured to deliver a plurality of nucleic acid libraries to the flow cell during different time periods.

What is claimed: 
     
         1 . A method of sequencing, the method comprising:
 introducing a first nucleic acid library to a library sequencing region of a flow cell comprising:
 a substrate having an inner surface facing the library sequencing region, 
 a plurality of forward and reverse amplification primers disposed over the inner surface and providing a nucleic acid library capture surface of the library sequencing region, and 
 a plurality of electrodes disposed along the substrate proximate the library capture surface; 
   applying a positive charge to a first set of one or more electrodes, from the plurality of electrodes, while the first nucleic acid library flows through the library sequencing region to thereby attract nucleic acids from the first nucleic acid library to the forward and reverse amplification primers disposed proximate the first set of one or more electrodes such that members of the first nucleic acid library hybridize to forward and reverse amplification primers proximate the first set of one or more electrodes, wherein members of the first nucleic acid library do not substantially hybridize to forward and reverse amplification primers located proximate electrodes that do not have positive charge applied;   introducing a second nucleic acid library to a library sequencing region flow cell; and   applying a positive charge to a second set of one or more electrodes, from the plurality of electrodes, while the second nucleic acid library flows through the library sequencing region to thereby attract nucleic acids from the second nucleic acid library to the forward and reverse amplification primers disposed proximate the second set of one or more electrodes such that members of the second nucleic acid library hybridize to forward and reverse amplification primers proximate the second set of one or more electrodes, wherein members of the second nucleic acid library do not substantially hybridize to forward and reverse amplification primers located proximate electrodes that do not have positive charge applied.   
     
     
         2 . The method of  claim 1 , further comprising one or both of:
 applying a negative charge to electrodes adjacent to the first set of electrodes while applying the positive charge to the first set of electrodes; and   applying a negative charge to electrodes adjacent to the second set of electrodes while applying the positive charge to the second set of electrodes.   
     
     
         3 . The method of  claim 1 , wherein introducing the first or second nucleic acid library comprises flowing the first or second nucleic acid library in a solution comprising one or more protective reagents that undergoes a redox reaction at an electric potential that is below the electric potential at which water electrolyzes, and wherein the redox reaction produces only products that are substantially benign to nucleic acids. 
     
     
         4 . The method of claim  43 , wherein said one or more protective reagents are selected from the group consisting of α-thioglycerol, dithiothreitol, hydroquinone, ferrocyanide, and β-mercaptoethanol. 
     
     
         5 . The method of  claim 1 , further comprising preparing the first nucleic acid library by:
 fragmenting a complex polynucleotide sample to generate a plurality of target polynucleotide fragments; and   ligating identical mismatched adapter polynucleotides to both ends of each of the different target polynucleotide fragments to form adapter-target constructs, wherein each mismatched adapter is formed from two annealed polynucleotide strands that form a bimolecular complex comprising at least one double-stranded region and a mismatched region comprising portions of both strands, wherein the ligating covalently attaches each strand of the at least one double-stranded region to each respective strand of each of the different target polynucleotide fragment to generate adapter-target constructs comprising covalently attached 5′ and 3′ adapter sequences.   
     
     
         6 . The method of  claim 1 , wherein each hybridized nucleic acid is amplified by:
 forming at least one nucleic acid template comprising the at least one nucleic acid to be amplified, wherein the at least one nucleic acid contains an oligonucleotide sequence Y at the 5′ end and an oligonucleotide sequence Z at the 3′ end, and the at least one nucleic acid carries a means for immobilizing the at least one nucleic acid to a solid support at the 5′ end;   mixing the at least one nucleic acid template, in the presence of the solid support, with one or more colony primers X, each of which can hybridize to the oligonucleotide sequence Z and carries a means for immobilizing the colony primer to the solid support at the 5′ end, whereby the 5′ ends of both the at least one nucleic acid template and the colony primers are immobilized to the solid support, wherein said 5′ ends of both the at least one nucleic acid template and the colony primers are immobilized to said solid support such that they cannot be removed by washing with water or aqueous buffer under DNA denaturing conditions; and   performing one or more nucleic acid amplification reactions on the immobilized nucleic acid template, so that nucleic acid colonies are generated.   
     
     
         7 . The method of  claim 6 , the method further comprising:
 moving one or more fluorescently labeled reagents through the flow cell into contact with the hybridized members of the first and second nucleic acid libraries, wherein the reagents comprise components to extend a second sequence complementary to the hybridized polynucleotides;   illuminating the hybridized polynucleotides with at least one excitation laser coupled through a fiberoptic device;   detecting, using at least one charge-coupled device (CCD) camera, fluorescence emissions of the fluorescently labeled reagents; and   determining, based on the fluorescence emissions, an identity of the second sequence.   
     
     
         8 . The method of  claim 1 , wherein the forward and reverse amplification primers are to hybridize specific gene sequences, wherein the specific gene sequences include a barcode region of the nucleic acid libraries, an adapter region of the nucleic acid libraries, or nucleic acid sequences of interest within the nucleic acid libraries. 
     
     
         9 . A system for sequencing, the system comprising:
 a solid support having a plurality of electrodes disposed thereon, the solid support including an attachment layer over the plurality of electrodes, the attachment layer having a library capture surface including a plurality of forward and reverse amplification primers immobilized thereon;   electrical leads connected to the plurality of electrodes to permit the electrodes to be independently addressable;   a fluid direction system for controllably delivering a plurality of polynucleotide libraries in a buffer with a reducing agent to the library capture surface during different time periods; and   a controller for controlling the fluid direction system and for delivering current and/or potential to the electrodes, wherein the controller is to:
 apply a positive charge to a first set of one or more of the plurality of electrodes while the fluid direction system delivers a first polynucleotide library along the solid support such that polynucleotides from the first polynucleotide library are attracted to forward and reverse amplification primers disposed proximate the first set of electrodes such that members of the first polynucleotide library hybridize to forward and reverse amplification primers proximate the first set of electrodes, wherein members of the first polynucleotide library do not substantially hybridize to forward and reverse amplification primers located proximate electrodes that do not have positive charge applied, and 
 apply a positive charge to a second set of one or more of the electrodes while the fluid direction system delivers a second polynucleotide library along the solid support such that polynucleotides from the second polynucleotide library are attracted to forward and reverse amplification primers disposed proximate the second electrode such that members of the second polynucleotide library hybridize to forward and reverse amplification primers proximate the second set of electrodes, wherein members of the second polynucleotide library do not substantially hybridize to forward and reverse amplification primers located proximate electrodes that do not have positive charge applied. 
   
     
     
         10 . The system of  claim 9 , wherein the controller is further to cause the fluid direction system to deliver the polynucleotide libraries to distinct lanes of the flow cell. 
     
     
         11 . The system of  claim 9 , wherein the controller is to apply the positive charge to the set of first or second electrodes that is at a voltage in the range of approximately 0.5-3V, produces a current in the range of approximately 250 nA-5 tA, or produces an electric field in the range of approximately 10-200 V/cm. 
     
     
         12 . The system of  claim 9 , where the plurality of electrodes comprises a conductor selected from the group consisting of gold, indium-doped tin oxide (ITO), silver, tin, titanium, copper, platinum, palladium, polysilicon, and carbon.