Alternative nucleotide flows in sequencing-by-synthesis methods

• US 9,416,413 B2
• Filed: 04/09/2013
• Issued: 08/16/2016
• Est. Priority Date: 06/11/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Schultz, Jonathan, Davidson, John
• Primary Examiner(s): Joike, Michele K
• Assistant Examiner(s): Hammell, Neil P
• Application Number:
  US13/859,673
• Publication Number:
  US 20130280702A1
• Time in Patent Office: 1,225 Days
• US Class Current: 1/1
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

A method for sequencing a polynucleotide strand by using sequencing-by-synthesis techniques. To address the problem of incomplete extension (IE) and/or carry forward (CF) errors that can occur in sequencing-by-synthesis reactions, an alternative flow ordering of dNTPs is used. In contrast to conventional flow orderings, the dNTPs are flowed in an ordering that is not a continuous repeat of an ordering of the four different dNTPs. This alternate flow ordering may reduce the loss of phasic synchrony in the population of template polynucleotide strands that result from IE and/or CF errors.

Claims

1. A method for improving homopolymer classification accuracy, comprising:

providing a template polynucleotide strand, a primer, and a polymerase in a microwell, the template polynucleotide strand being coupled to or associated with a bead;

successively exposing the template polynucleotide strand to a plurality of each of four kinds of dNTP reagent flows, each dNTP reagent flow having a single kind of dNTP, wherein the plurality of reagent flows are selected according to an ordering, the ordering being such that (a) a flow of one kind of dNTP reagent is always followed by a flow of a different kind of dNTP reagent, (b) at least one flow of each kind of dNTP reagent is followed by a flow of the same kind of dNTP reagent after a single intervening flow of a different kind of dNTP reagent, and (c) the number of flows of each of the four kinds of dNTP reagent flows in the ordering is the same, wherein the successively exposing comprises consecutively repeating the plurality of reagent flows according to the ordering one or more times; and

obtaining, using a sensor configured to provide information about reactions taking place in the microwell, an incorporation signal comprising values generally proportional to a number of dNTP incorporation(s) that result when a base in the template polynucleotide strand is complementary to one of the flowed dNTP reagents.

6. The method of claim 1, wherein the sensor is a pH-based sensor.

9. The method of claim 1, wherein the plurality of reagent flows in the ordering is chosen from 8, 12, 16, 20, 24, 28 or 32 dNTP flows.

2. A method for improving homopolymer classification accuracy, comprising:

(a) delivering a known nucleoside triphosphate precursor to a template-based primer extension reaction of a template polynucleotide strand, the known nucleoside triphosphate precursor being delivered according to a predetermined ordering of dNTP flows, the template polynucleotide strand being coupled to or associated with a bead loaded in a microwell;

(b) detecting incorporation of the known nucleoside triphosphate using a pH-based sensor configured to provide information about reactions taking place in the microwell and generate an incorporation signal comprising values generally proportional to a number of dNTP incorporation(s) occurring when the complement of the known nucleoside triphosphate is present in the template polynucleotide strand adjacent to the primer; and

(c) repeating steps (a) and (b) until each dNTP flow in the predetermined ordering of dNTP flows has been flowed;

wherein the predetermined ordering of dNTP flows is such that (i) a flow of one kind of dNTP is always followed by a flow of a different kind of dNTP, (ii) at least one flow of each kind of dNTP is followed by a flow of the same kind of dNTP after a single intervening flow of a different kind of dNTP, and (iii) the ordering includes four consecutive groups of three dNTP flows, the second dNTP flow in each of the four consecutive groups of three dNTP flows being different than the second dNTP flow in the other three consecutive groups of three dNTP flows, and the first and third dNTP flows in each of the four consecutive groups of three dNTP flows being both of the same kind of dNTP and being different than the second dNTP flow, wherein the delivering comprises consecutively repeating a delivery of the predetermined ordering of dNTP flows one or more times.

7. The method of claim 2, wherein each dNTP flow in the predetermined ordering of dNTP flows has a single kind of dNTP.

3. A method for improving homopolymer classification accuracy, comprising:

providing a polynucleotide strand, a primer, and a polymerase in a microwell, the polynucleotide strand being coupled to or associated with a bead;

successively exposing the polynucleotide strand coupled to or associated with the bead to flows of four different dNTPs, one kind of dNTP per flow, according to a first predetermined ordering, wherein the first predetermined ordering comprises sequential repetitions of the same sequence of four different dNTPs;

successively exposing the polynucleotide strand coupled to or associated with the bead to flows of a plurality of dNTPs, one kind of dNTP per flow, according to a second predetermined ordering, the second predetermined ordering being such that a flow of one kind of dNTP reagent is always followed by a flow of a different kind of dNTP reagent, and at least one flow of each kind of dNTP reagent is followed by a flow of the same kind of dNTP reagent after a single intervening flow of a different kind of dNTP reagent, wherein the second predetermined ordering occurs after the first predetermined ordering, and wherein the successive exposing of the polynucleotide strand according to the second predetermined ordering is consecutively repeated one or more times; and

obtaining, using a sensor configured to provide information about reactions taking place in the microwell, an incorporation signal comprising values generally proportional to a number of dNTP incorporation(s) that result when a base in the polynucleotide strand is complementary to one of the flowed dNTPs.

4. The method of claim 3, wherein the second predetermined ordering comprises an alternate ordering which is not a continuous repeat of the first predetermined ordering of the four different dNTPs.

5. The method of claim 3, further comprising successively exposing the polynucleotide strand coupled to or associated with the bead to flows of four different dNTPs, one kind of dNTP per flow, according to a third predetermined ordering, wherein the third predetermined ordering comprises an alternate ordering which is not a continuous repeat of the first predetermined ordering of the four different dNTPs or of the second predetermined ordering of the four different dNTPs and wherein the third predetermined ordering occurs after the first and second predetermined orderings.

8. The method of claim 3, wherein the sensor is a pH-based sensor.

10. The method of claim 3, wherein the plurality of dNTP flows in the second predetermined ordering is chosen from 8, 12, 16, 20, 24, 28 or 32 dNTP flows.

11. The method of claim 3, wherein the successive exposing of the template polynucleotide strand to a plurality of each of four kinds of dNTP reagent flows according to the second predetermined ordering is performed throughout a sequencing run.