# US 12,180,545 B2 · Methods and apparatus that increase sequencing-by-binding efficiency

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,180,545 B2
- Document ID: US12180545B2
- Status: Granted
- Kind: B2
- Application number: 17/876,893
- Filed: 2022-07-29
- Published: 2023-05-18
- Granted: 2024-12-31
- Earliest claimed: 2017-04-25
- Assignees: Pacific Biosciences of California, Inc.
- Applicants: Pacific Biosciences of California, Inc.
- Inventors: Sean Stromberg; John Vieceli; Kandaswamy Vijayan; Arnold Oliphant
- Web page: https://patentgrep.com/patent/US12180545B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6832: Enhancement of hybridisation reaction
- C12Q 1/6853: using modified primers or templates
- C12Q 1/6869: Methods for sequencing
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method of determining a nucleic acid sequence that includes steps of: (a) contacting a primed template nucleic acid with a series of mixtures for forming ternary complexes, wherein each of the mixtures includes a polymerase and nucleotide cognates for at least two different base types suspected of being present at the next template position of the template nucleic acid; (b) monitoring the next template position for ternary complexes formed by the series of mixtures, wherein a signal state indicates presence or absence of ternary complex formed at the next template position by each individual mixture, thereby determining a series of signal states that encodes a base call for the next template position; and (c) decoding the series of signal states to distinguish a correct base call for the next template position from an error in the base call.

## Claims

### Claim 1

A method of determining a nucleic acid sequence, comprising:
(a) contacting a primed template nucleic acid with a series of mixtures for forming ternary complexes, wherein the mixtures differ by the presence or absence of at least one type of labeled nucleotide cognate, and wherein each of the ternary complexes comprise the primed template, a polymerase, and a nucleotide cognate;
(b) examining the first and second mixtures separately to detect ternary complexes that produce the signals, wherein a signal state indicates presence or absence of ternary complex formed at the next template position by each individual mixture, thereby determining a series of signal states that encodes a base call for the next template position; and
(c) identifying the next correct nucleotide for the primed template nucleic acid molecule, wherein the next correct nucleotide is identified as a cognate of a first base type if ternary complex is detected in the first mixture but not the second mixture, and wherein the next correct nucleotide is identified as a cognate of a second base type if ternary complex is detected in the second mixture but not the first mixture.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first mixture comprises a polymerase.

### Claim 3 (depends on 2)

The method of claim 2, wherein the first and second mixtures comprise the same polymerase.

### Claim 4 (depends on 1)

The method of claim 1, wherein the series of signal states comprises an error correcting code.

### Claim 5 (depends on 4)

The method of claim 4, wherein the series of mixtures consists of three mixtures and the series of signal states is represented by three digits, each digit representing a signal state obtained from a mixture.

### Claim 6 (depends on 5)

The method of claim 5, wherein each of the signal states is represented by a binary digit, and wherein the error correcting code comprises a repetition code.

### Claim 7 (depends on 6)

The method of claim 6, further comprising correcting the erroneous base call by a majority vote between the three digits.

### Claim 8 (depends on 4)

The method of claim 4, wherein the series of mixtures consists of four mixtures and the series of signal states is represented by four digits, each digit representing a signal state obtained from a mixture.

### Claim 9 (depends on 8)

The method of claim 8, wherein each of the signal states is represented by a ternary digit, wherein the error correcting code comprises a Hamming code, and wherein the Hamming distance between valid base calls is three.

### Claim 10 (depends on 9)

The method of claim 9, further comprising correcting the erroneous base call to a valid base call having a code with the closest Hamming distance to the code for the erroneous base call.

### Claim 11 (depends on 4)

The method of claim 4, wherein the series of mixtures consists of five mixtures and the series of signal states is represented by five digits, each digit representing a signal state obtained from a mixture.

### Claim 12 (depends on 11)

The method of claim 11, wherein each of the signal states is represented by a binary digit, wherein the error correcting code comprises a Hamming code, and wherein each valid base call differs from other valid base calls by three digits.

### Claim 13 (depends on 1)

The method of claim 1, further comprising correcting the erroneous base call to a valid base call having a code with the closest Hamming distance to the code for the erroneous base call.

### Claim 14 (depends on 1)

The method of claim 1, wherein step (c) of identifying comprises disambiguation using an encoding scheme, and wherein the disambiguation comprises decoding of the series of signal states identifies the base call as being an erroneous base call.

### Claim 15 (depends on 14)

The method of claim 14, further comprising correcting the erroneous base call to make a valid base call for the next template position.

### Claim 16 (depends on 15)

The method of claim 15, wherein the correcting of the error comprises correlating a suspect signal state in the series of signal states with an aberration in step (a) or (b), and selecting the base call having the expected series of signal states with a change in the suspect signal state.

### Claim 17 (depends on 16)

The method of claim 16, wherein the aberration in step (b) is selected from the group consisting of a signal to noise ratio below a predetermined threshold, signal below a predetermined threshold, signal above a predetermined threshold, noise above a predetermined threshold, and detector malfunction.

### Claim 18 (depends on 16)

The method of claim 16, wherein the aberration in step (a) is selected from the group consisting of a fluidic delivery malfunction, temperature control malfunction, and reagent quality below a predetermined threshold.

### Claim 19 (depends on 1)

The method of claim 1, wherein the series of signal states comprises a repetition code, Hamming code, linear code or parity code.

### Claim 20 (depends on 1)

The method of claim 1, wherein each of the signal states is represented by a binary digit.

### Claim 21 (depends on 20)

The method of claim 20, wherein the binary digit comprises (i) symbols for presence and absence of a signal; (ii) symbols for signals emitted at two different wavelengths; (iii) symbols for signals having two different intensities; or (iv) symbols for signals resulting from excitation at two different wavelengths.

### Claim 22 (depends on 1)

The method of claim 1, wherein each of the signal states is represented by a ternary digit.

### Claim 23 (depends on 22)

The method of claim 22, wherein the ternary digit comprises (i) symbols for signals emitted at three different wavelengths; (ii) symbols for signals having three different intensities; or (iii) symbols for signals resulting from excitation at three different wavelengths.

### Claim 24 (depends on 1)

The method of claim 1, wherein each of the mixtures comprises nucleotide cognates for at least two and no more than three of four different base types suspected of being in the primed template nucleic acid.

### Claim 25 (depends on 1)

The method of claim 1, wherein each of the mixtures comprises nucleotide cognates for at least two and no more than two of four different base types suspected of being in the primed template nucleic acid.

### Claim 26 (depends on 1)

The method of claim 1, wherein the mixtures differ by the number or type of label attached to at least one type of nucleotide cognate.

### Claim 27 (depends on 1)

The method of claim 1, further comprising: (d) adding a next correct nucleotide to the primer of the primed template nucleic acid after step (b), thereby producing an extended primer.

### Claim 28 (depends on 27)

The method of claim 27, further comprising repeating steps (a) through (d) for the primed template nucleic acid that comprises the extended primer.

### Claim 29 (depends on 27)

The method of claim 27, wherein the next correct nucleotide that is added to the primer is a reversibly terminated nucleotide.

### Claim 30 (depends on 29)

The method of claim 29, further comprising repeating steps (a) through (d) for the primed template nucleic acid that comprises the extended, reversibly terminated primer.

### Claim 31 (depends on 30)

The method of claim 30, further comprising (e) removing the reversible terminator moiety from the extended, reversibly terminated primer after steps (a) through (d) are repeated.