Time-based cluster imaging of amplified contiguity-preserved library fragments of genomic DNA

Patent metadata

Field	Value
Publication number	US-11714848-B2
Application number	US-202017600499-A
Country	US
Kind code	B2
Filing date	Dec 1, 2020
Priority date	Dec 2, 2019
Publication date	Aug 1, 2023
Grant date	Aug 1, 2023

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.

First claim

What is claimed is:

1. 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 some of the contiguity-preserved library fragments, wherein the some of the contiguity-preserved library fragments are attached to a solid support or are attached to each other;
initiating release of the some of the contiguity-preserved library fragments from the solid support or from each other;
amplifying the some of the contiguity-preserved library fragments to generate a plurality of respective template strands;
staining the respective template strands; and
imaging the respective template strands.

2. The method as defined in claim 1 , 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.

3. The method as defined in claim 2 , wherein the temporal record is a time stamp or a step number in a sequence.

4. The method as defined in claim 1 , 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.

5. The method as defined in claim 4 , further comprising storing the resolved clustering images.

6. The method as defined in claim 4 , further comprising:
performing a sequencing operation on the flow cell including the respective template strands for each of the plurality of library fragments; and
grouping sequencing reads together in different groups based on the resolved clustering images.

7. The method as defined in claim 6 , further comprising linking the different groups to each of the different one of the samples based on the resolved clustering images.

8. The method as defined in claim 1 , wherein:
prior to generating the series of time-based clustering images, the method further comprises:
adding a liquid carrier to the plurality of contiguity-preserved library 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.

9. The method as defined in claim 8 , wherein the mixture including the plurality of contiguity-preserved library 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.

10. The method as defined in claim 1 , wherein:
each contiguity-preserved library fragment is attached to the solid support; and
the release involves heating.

11. The method as defined in claim 1 , wherein:
each of the contiguity-preserved library fragments includes a first sequence portion at a first end that hybridizes to a first primer sequence on a surface of the flow cell; and
prior to amplification, the method further comprises attaching a second sequence portion to each of the hybridized contiguity-preserved library 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.

12. The method as defined in claim 1 , wherein:
each contiguity-preserved library fragment is attached to the solid support;
the solid support has a plurality of adapters attached thereto; and
the method further comprises preparing the contiguity-preserved library fragment attached to the solid support by:
tagmenting the genome sample in the presence of the solid support and a plurality of L-adapters, each L-adapter including a transferred strand and a non-transferred strand, thereby generating a plurality of sample fragments, whereby a respective transferred strand is incorporated to a 5′-end of each sample fragment and a respective non-transferred strand is hybridized to a portion of each adapter;
ligating the respective transferred strands to a respective one of the plurality of adapters;
digesting the non-transferred strand using a 5′-3′ exonuclease; and
attaching a partial Y-adapter to each of the transferred strands.

13. The method as defined in claim 12 , wherein ligating and digesting occur in a single, one pot protocol.

14. 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 clustering image for one of the contiguity-preserved library fragments by:
introducing a first of the dilution samples including some of the contiguity-preserved library fragments to the flow cell;
initiating release of the some of the contiguity-preserved library fragments from the solid support or from each other;
amplifying the some of the contiguity-preserved library fragments to generate a plurality of template strands;
staining the plurality of template strands; and
imaging the plurality of template strands.

15. The method as defined in claim 14 , wherein the mixture 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 to be introduced to the flow cell.

16. The method as defined in claim 14 , wherein:
the clustering image is a first time-based clustering image in a series of time-based clustering images for the plurality of contiguity-preserved library fragments;
each time-based clustering image in the series is sequentially generated; and
the method further comprises:
generating a second time-based clustering image for some other of the contiguity-preserved library fragments by:
introducing a second of the dilution samples including the some other of the contiguity-preserved library fragments to 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.

17. The method as defined in claim 16 , further comprising generating a predetermined number of time-based clustering images for a predetermined number of the contiguity-preserved library fragments by repeating the introducing, the initiating release, the amplifying, the staining, and the imaging with each other of the predetermined number of dilution samples.

18. The method as defined in claim 17 , further comprising assigning each of the predetermined number of time-based clustering images a temporal record of the introduction of the respective dilution sample.

19. The method as defined in claim 18 , wherein the temporal record is a time stamp or a step number in a sequence.

20. The method as defined in claim 17 , further comprising using image subtraction to generate a resolved cluster image for each of the dilution s

Assignees

• Illumina Cambridge Ltd

Inventors

• Morrell Natalie
• Slatter Andrew
• Thomson Vicki

Classifications

• B01L3/502761 Primary

  specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules · CPC title

• G06F16/58 Primary

  Retrieval characterised by using metadata, e.g. metadata not derived from the content or metadata generated manually · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q2304/12

  Ethidium · CPC title

• C12Q1/6855

  Ligating adaptors · CPC title