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

• US 10,241,075 B2
• Filed: 12/29/2011
• Issued: 03/26/2019
• Est. Priority Date: 12/30/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Davey, Melville, Cawley, Simon, Williams, Alan
• Primary Examiner(s): Lin, Jerry
• Application Number:
  US13/340,490
• Publication Number:
  US 20120173159A1
• Time in Patent Office: 2,644 Days
• Field of Search: 702 20
• CPC Class Codes: G01N 27/27 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 Class Codes: G01N 27/4145 specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 30/20 Sequence assembly

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.

Claims

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 noise in a second vicinity of the given base in the nucleic acid sequence, where the first vicinity spans fewer bases than the second vicinity;

comparing the calculated first quality predictor value and the calculated second quality predictor value to corresponding pre-defined quality metrics of a look-up table, wherein the look-up table includes rows of pre-defined quality metrics and a quality score associated with each row;

selecting the row of the look-up table where the first and second quality predictor values are less than the corresponding pre-defined quality metrics in the selected row;

and

assigning the quality score associated with the selected row to the given base call.

18. The method of claim 17, wherein the predicting step comprises:

removing from the at least some of the base calls forming at least a portion of the one or more reads at least one of;

any base call identified as having a quality score below a quality score threshold, and

an adapter sequence; and

filtering out from the at least a portion of the one or more reads at least one of;

any read identified as a mixed template read, and

any read identified as having a median absolute residual value greater than a residual threshold value, thereby generating a filtered set of reads.

19. The method of claim 18, wherein the predicting step comprises aligning the filtered set of reads to predict a sample nucleic acid sequence assembled from the filtered set of reads for the sample nucleic acids.

20. The method of claim 14, wherein the predicting step comprises aligning the one or more reads to predict a sample nucleic acid sequence assembled from the one or more reads for the sample nucleic acids.

21. The method of claim 1, comprising transmitting, displaying, storing, printing or outputting to a user interface device, a computer readable storage medium, a local computer system or a remote computer system, information related to the predicted nucleotide sequences of the sample nucleic acids or other parameters, predictions, or data generated or used by the method.

22. A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, cause the processor to perform 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, 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.

23. The non-transitory machine-readable storage medium of claim 22, 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.

24. The non-transitory machine-readable storage medium of claim 23, 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.

25. The non-transitory machine-readable storage medium of claim 23, 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 noise in a second vicinity of the given base in the nucleic acid sequence, where the first vicinity spans fewer bases than the second vicinity;

comparing the calculated first quality predictor value and the calculated second quality predictor value to corresponding pre-defined quality metrics of a look-up table, wherein the look-up table includes rows of pre-defined quality metrics and a quality score associated with each row;

selecting the row of the look-up table where the first and second quality predictor values are less than the corresponding pre-defined quality metrics in the selected row; and

assigning the quality score associated with the selected row to the given base call.

26. The non-transitory machine-readable storage medium of claim 24, wherein the predicting step comprises:

removing from the at least some of the base calls forming at least a portion of the one or more reads at least one of;

any base call identified as having a quality score below a quality score threshold, and

an adapter sequence; and

filtering out from the at least a portion of the one or more reads at least one of;

any read identified as a mixed template read, and

any read identified as having a median absolute residual value greater than a residual threshold value, thereby generating a filtered set of reads.

27. The non-transitory machine-readable storage medium of claim 26, wherein the predicting step comprises aligning the filtered set of reads to predict a sample nucleic acid sequence assembled from the filtered set of reads for the sample nucleic acids.

28. A system, comprising:

a machine-readable memory; and

a processor configured to execute machine-readable instructions, which, when executed by the processor, cause the system to perform steps including;

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, 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.