Methods and devices for sequencing nucleic acids in smaller batches

• US 9,145,589 B2
• Filed: 08/19/2014
• Issued: 09/29/2015
• Est. Priority Date: 02/05/2007
• Status: Active Grant

• Current Assignee: IsoPlexis Corporation (Bruker Corporation)
• Original Assignee: Intelligent Bio-Systems Incorporated. (Qiagen NV)
• Inventors: Gordon, Steven, Hagerott, Thomas, Golaski, Edmund, Olejnik, Jerzy
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/463,211
• Publication Number:
  US 20150038339A1
• Time in Patent Office: 406 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. A plurality of smaller flow cells is employed, each with a relatively small area to be imaged, in order to provide greater flexibility and efficiency.

Claims

1. A method for carrying out steps of a nucleic acid sequencing protocol, comprising:

a) providing;

i) a plurality of reservoirs, at least one of which comprising nucleic acid sequencing reagents, and at least one of which comprising wash buffer;

ii) first nucleic acids in a flow path of a first flow cell and second nucleic acids in a flow path of a second flow cell, said first and second nucleic acids comprising templates to be sequenced, each of said flow paths associated with an inlet port and outlet port, each of said inlet ports and outlet ports associated with tubing, said flow cells mounted on iii) a moveable support, said support configured to move said flow cells to iv) a plurality of stations, at least some of which are reagent delivery stations and at least one comprising a wash station, each station in fluidic communication with at least one of said reservoirs via tubing, each station comprising station ports capable of being aligned with said flow cell inlet and outlet ports, each station port comprising an elastomeric seal;

b) moving said moveable support under conditions such that said first flow cell is aligned with and connected to a reagent delivery station, such that said reagent delivery station ports are aligned with said flow cell inlet and outlet ports at interfaces sealed by said elastomeric seals, said seals defining an open area where fluids may flow, whereby said first flow cell is in fluid communication with at least a first reservoir of said plurality of reservoirs;

c) introducing reagent via one of said reservoirs into said first flow cell under conditions such that said reagent contacts said first nucleic acids in said flow path of said first flow cell;

d) disconnecting said first flow cell from said reagent delivery station associated with said first step;

e) moving said moveable support under conditions such that said first flow cell is aligned with and connected to a wash station, such that said wash station ports are aligned with said flow cell inlet and outlet ports at interfaces sealed by said elastomeric seals, said seals defining an open area where fluids may flow, whereby said first flow cell is in fluid communication with a wash reservoir comprising wash buffer, said open area defined by said seals of said wash station port being greater than said open area defined by said seals of said reagent delivery station, so as to allow any reagents trapped in the sealing area to be washed away, thereby preventing contamination of flow cells with reagents from a previous step; and

f) introducing wash buffer from said wash reservoir into said first flow cell.

2. The method of claim 1, wherein said disconnecting of step d) comprises raising said moveable support.

3. The method of claim 1, wherein said disconnecting of step d) comprising lowering the delivery reagent station.

4. The method of claim 1, wherein the fluid levels of said reservoirs are maintained below the level of the stations.

5. The method of claim 1, wherein said moveable support is a carousel.

6. The method of claim 5, wherein said moving of said carousel in step b) is in a clockwise direction.

7. The method of claim 6, wherein said carousel is subsequently moved in a counterclockwise direction.

8. The method of claim 1, further comprising aligning and connecting said second flow cell to said reagent delivery station, whereby said second flow cell is in fluid communication with said reagent reservoir of said plurality of reservoirs.

9. The method of claim 1, wherein said nucleic acid sequencing reagent comprise reversibly terminating nucleotide analogues, said analogues comprising removable chemical moieties capping the 3′

—

OH group.

10. The method of claim 9, wherein said reversibly terminating nucleotide analogues are contaminated by non-terminating nucleotides having free 3-OH groups.

11. The method of claim 10, further comprising, prior to introducing said analogues into said reservoir, purifying said reversibly terminating nucleotide analogues using chromatography to reduce the amount of non-terminating nucleotides present.