Methods, systems, and computer readable media for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-10241075-B2
Application number	US-201113340490-A
Country	US
Kind code	B2
Filing date	Dec 29, 2011
Priority date	Dec 30, 2010
Publication date	Mar 26, 2019
Grant date	Mar 26, 2019

Abstract

A method for nucleic acid sequencing includes receiving a plurality of signals indicative of a parameter measured for a plurality of defined spaces, at least some of the defined spaces including one or more sample nucleic acids, the signals being responsive to a plurality of nucleotide flows introducing nucleotides to the defined spaces; determining, for at least some of the defined spaces, whether the defined space includes a sample nucleic acid; processing, for at least some of the defined spaces determined to include a sample nucleic acid, the received signals to improve a quality of the received signals; and predicting a plurality of nucleotide sequences corresponding to respective sample nucleic acids for the defined spaces based on the processed signals and the nucleotide flows.

First claim

The invention claimed is:

1. A method for nucleic acid sequencing, comprising:
for each of a plurality of defined spaces of a chip, a template, or a substrate, wherein each defined space is in communication with a respective sensor, and wherein at least some of the defined spaces comprise one or more sample nucleic acids, receiving at a processor, from the respective sensor of each defined space, a signal comprising a voltage measurement indicative of a hydrogen ion concentration, wherein the signal is in response to exposure of the corresponding defined space to a first hydrogen ion concentration associated with a first buffer or a first reagent, and then exposure of the corresponding defined space to a second hydrogen ion concentration associated with a second buffer or a second reagent;
first determining, for at least some of the defined spaces of the plurality of defined spaces, time delays in the signals, wherein the time delays are associated with diffusions resulting from the exposure of the defined spaces to the first hydrogen ion concentration and then the exposure to the second hydrogen ion concentration;
second determining whether the defined space comprises a sample nucleic acid or does not comprise a sample nucleic acid based on the determined time delays;
based on the second determining step, classifying into a first group the defined spaces that comprise a sample nucleic acid and a second group the defined spaces that do not comprise a sample nucleic acid;
receiving, at the processor, a second signal from the sensor of each defined space, wherein the second signal is responsive to a nucleotide flow introducing a nucleotide to the defined space for a plurality of nucleotide flows;
subtracting a noise component from the second signal for a respective defined space in the first group to form a noise-reduced signal, wherein the noise component is based on an average of one or more second signals corresponding to one or more defined spaces in the second group; and
predicting a plurality of nucleotide sequences corresponding to the sample nucleic acids for respective defined spaces in the first group, the predicting including estimating a number of nucleotide incorporations in response to the nucleotide flow based on the noise-reduced signal for each nucleotide flow in the plurality of nucleotide flows.

2. The method of claim 1 , wherein the second determining step, the defined space is determined to comprise a sample nucleic acid when the determined time delay is longer than the time delay determined from one or more neighboring defined spaces.

3. The method of claim 1 , wherein at least some of the defined spaces comprise a sample nucleic acid directly or indirectly coupled to a particle.

4. The method of claim 1 , wherein at least some of the defined spaces comprise a sample nucleic acid directly or indirectly coupled to a surface of the defined space.

5. The method of claim 1 , wherein the defined spaces comprise microwells in communication with the sensors in an integrated circuit chip.

6. The method of claim 1 , wherein the time delay of the signal spans a change in a hydrogen ion concentration in the defined space from the first hydrogen ion concentration to the second hydrogen ion concentration.

7. The method of claim 1 , wherein the second determining step comprises comparing a rate of change in a hydrogen ion concentration for the defined space with a modeled rate of change associated with an absence of a sample nucleic acid.

8. The method of claim 1 , further comprising, evaluating, for at least some of the defined spaces of the first group, whether the signals for the defined space are informative or non-informative, wherein a non-informative designation indicates that the defined space is not functional.

9. The method of claim 8 , further comprising evaluating, for at least some of the defined spaces of the first group evaluated to be informative, whether the defined space comprises a library fragment, a test fragment, or an ambiguous fragment.

10. The method of claim 1 , further comprising:
determining a flow-space vector derived from a series of initial signals in response to a set of initial nucleotide flows for at least one defined space, wherein the flow-space vector indicates an incorporation event or a non-incorporation for each nucleotide in the set of initial nucleotide flows; and
comparing the flow-space vector with at least two pre-defined vectors.

11. The method of claim 10 , wherein the at least two pre-defined vectors comprise:
a library fragment flow-space vector that would be expected to result if a predefined library fragment key sequence were subjected to the initial nucleotide flows; and
a test fragment flow-space vector that would be expected to result if a pre-defined test fragment key sequence were subjected to the initial nucleotide flows.

12. The method of claim 11 , wherein the library fragment flow-space vector and the test fragment flow-space vector are orthogonal under the set of initial nucleotide flows with respect to at least two nucleotides selected from nucleotides A, C, G, and T.

13. The method of claim 11 , wherein the library fragment flow-space vector and the test fragment flow-space vector are orthogonal under the set of initial nucleotide flows with respect to at least three nucleotides selected from nucleotides A, C, G, and T.

14. The method of claim 1 , wherein the predicting step comprises:
compiling the estimated numbers of nucleotide incorporations corresponding to each of the plurality of nucleotide flows into one or more sequences of consecutive base calls for the sample nucleic acid to form one or more reads.

15. The method of claim 14 , wherein the number of nucleotide incorporations is selected for each nucleotide from the group consisting of zero, one, two, and any other positive integer.

16. The method of claim 14 , wherein the predicting step comprises:
generating a quality metric for the base calls and for the reads; and
filtering out from the one or more reads at least one of:
any read that contains at least a threshold number of base calls failing to meet a minimal threshold of base call quality, and
any read that fails to meet a minimal threshold of read quality.

17. The method of claim 14 , wherein the predicting step comprises:
calculating at least a first quality predictor value and a second quality predictor value for a given base call, the first and second quality predictor values including a phase error and at least one other parameter selected from the group consisting of base position, local noise, read noise, multiple incorporations, and environment noise, wherein a phase error quality predictor indicates a number of incorporations of a same nucleotide in a previous nucleotide flow, wherein a base position quality predictor indicates a position of the given base in the read from the start of the nucleic acid sequence, wherein a local noise quality predictor indicates noise in a first vicinity of the given base in the nucleic acid sequence, wherein a read noise quality predictor is based on a mean and a standard deviation of 0-mers and 1-mers of the read, wherein a multiple incorporations quality predictor for multiple incorporations of the same nucleotide in a particular nucleotide flow, indicating a homopolymer region, assigns to a last base in the homopolymer region a value based on a total number of incorporations during the particular nucleotide flow and assigns a value of 1 to other bases in the homopolymer region, and wherein an environment noise quality predictor indicates no

Assignees

• Davey Melville
• Cawley Simon
• Williams Alan
• Life Technologies Corp

Inventors

• Davey Melville
• Cawley Simon
• Williams Alan

Classifications

• G01N27/4145 Primary

  specially adapted for biomolecules, e.g. gate electrode with immobilised receptors · CPC title

• G01N27/27

  Association of two or more measuring systems or cells, each measuring a different parameter, where the measurement results may be either used independently, the systems or cells being physically associated, or combined to produce a value for a further parameter · CPC title

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• G06F19/22

  Physics · mapped topic

• G16B30/20

  Sequence assembly · CPC title