Time-based cluster imaging

Abstract

In an example method, a series of time-based clustering images is generated for a plurality of library fragments from a genome sample. Each time-based clustering image in the series is sequentially generated. To generate each time-based clustering image in the series: i) a respective sample is introduced to a flow cell, the respective sample including contiguity preserved library fragments of the plurality of library fragments, wherein the contiguity preserved library fragments are attached to a solid support or are attached to each other; ii) the contiguity preserved library fragments are released from the solid support or from each other; iii) the contiguity preserved library fragments are amplified to generate a plurality of respective template strands; iv) the respective template strands are stained; and v) the respective template strands are imaged.

1 .- 27 . (canceled) 
     
     
         28 . A method, comprising:
 generating a series of time-based clustering images for a plurality of contiguity-preserved library fragments from a genome sample, wherein each time-based clustering image in the series is sequentially generated by:
 introducing, to a flow cell, a respective sample including attached fragments, each attached fragment including some of the contiguity-preserved library fragments attached to each other via transposases; 
 removing the transposases, thereby releasing sub-fragments of the attached fragments, whereby the released sub-fragments seed onto a surface of the flow cell; 
 amplifying the seeded sub-fragments to generate a plurality of respective template strands; 
 staining the respective template strands; and 
 imaging the respective template strands. 
   
     
     
         29 . The method as defined in  claim 28 , wherein removing the transposases involves introducing sodium dodecyl sulfate into the flow cell. 
     
     
         30 . The method as defined in  claim 28 , wherein removing the transposases involves introducing proteinase K into the flow cell. 
     
     
         31 . The method as defined in  claim 28 , further comprising assigning each time-based clustering image in the series a temporal record of the introduction of the respective sample including the some of the contiguity-preserved library fragments, wherein the temporal record is a time stamp or a step number in a sequence. 
     
     
         32 . The method as defined in  claim 31 , further comprising using image subtraction to generate a resolved clustering image for each of the respective samples, wherein each resolved clustering image records a spatial location and orientation of the respective template strands associated with a different one of the samples. 
     
     
         33 . The method as defined in  claim 32 , further comprising:
 performing a sequencing operation on the flow cell including the respective template strands; and   
       grouping sequencing reads together in different groups based on the resolved clustering images. 
     
     
         34 . The method as defined in  claim 33 , further comprising linking the different groups to each of the different one of the samples based on the resolved clustering images. 
     
     
         35 . The method as defined in  claim 28 , wherein:
 prior to generating the series of time-based clustering images, the method further comprises:
 adding a liquid carrier to the attached fragments to form a mixture; and 
 diluting the mixture with the liquid carrier to generate a predetermined number of dilution samples to be introduced to the flow cell; and 
 the introduction of the respective sample involves fluidically directing one of the dilution samples to the flow cell. 
   
     
     
         36 . The method as defined in  claim 35 , wherein the mixture including the attached fragments is diluted to a predetermined volume based on i) a volume of the flow cell as a limiting dilution and ii) the predetermined number of dilution samples. 
     
     
         37 . The method as defined in  claim 28 , wherein:
 each of the sub-fragments includes a first sequence portion at a first end that hybridizes to a first primer sequence on the surface of the flow cell; and   prior to amplification, the method further comprises attaching a second sequence portion to each of the hybridized sub-fragments at a second end that is opposed to the first end, the second sequence portion being identical to a second primer sequence on the surface of the flow cell.   
     
     
         38 . A method, comprising:
 preparing a mixture including a plurality of contiguity-preserved library fragments of a genome sample, the plurality of contiguity-preserved library fragments being attached to solid supports or being attached to each other;   diluting the mixture to generate a predetermined number of dilution samples to be introduced to a flow cell; and   generating a time-based clustering image for at least one of the contiguity-preserved library fragments by:
 controlling diffusion of a first of the predetermined number of dilution samples into the flow cell; 
 initiating release of contiguity-preserved library fragments from a solid support or from each other in the first of the predetermined number of dilution samples; 
 amplifying the contiguity-preserved library fragments to generate a plurality of respective template strands; 
 staining the respective template strands; and 
 imaging the respective template strands. 
   
     
     
         39 . The method as defined in  claim 38 , further comprising:
 generating a second time-based clustering image for some other of the contiguity-preserved library fragments by:
 controlling diffusion of a second of the predetermined number of dilution samples into the flow cell and the first of the predetermined number of dilution samples out of the flow cell; 
 initiating release of the some other of the contiguity-preserved library fragments from the solid support or from each other; 
 amplifying the some other of the contiguity-preserved library fragments to generate a second plurality of template strands; 
 staining the second plurality of template strands; and 
 imaging the second plurality of template strands.