# US 11,111,533 B2 · Generalized stochastic super-resolution sequencing

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- Number: US 11,111,533 B2
- Document ID: US11111533B2
- Status: Granted
- Kind: B2
- Application number: 16/294,696
- Filed: 2019-03-06
- Published: 2019-09-12
- Granted: 2021-09-07
- Earliest claimed: 2018-03-09
- Assignees: Illumina Cambridge Limited; Illumina, Inc.
- Applicants: Illumina Cambridge Limited; Illumina, Inc.
- Inventors: Gary Mark Skinner; Geraint Wyn Evans; Stanley S. Hong
- Web page: https://patentgrep.com/patent/US11111533B2

## CPC

- C12Q 1/6869: Methods for sequencing
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6408: {with measurement of decay time, time resolved fluorescence}
- G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates}
- G01N 2021/6432: {Quenching}
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- G01N 2021/6441: {with two or more labels}
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A method of sequencing a plurality of polynucleotides includes: attaching a single DNA template molecule to each of a plurality of attachment elements on a sample container, wherein the average distance between adjacent elements is less than Abbe's limit; applying a stochastic photo-switching chemistry to all of the molecules at the same time to cause the attached molecules to fluoresce in on and off events in up to four different colors by stochastic photo-switching; and imaging the on and off events in a color channel for each color in real-time as the on and off events are occurring for the attached molecules.

## Claims

### Claim 1

A method for sequencing polynucleotides comprising:
attaching a first DNA template molecule to a first attachment element of a plurality of attachment elements of a sample container and attaching a second DNA template molecule to a second attachment element of the plurality of attachment elements, wherein a distance between the first attachment element and the second attachment element is less than Abbe's limit;
providing a stochastic photo-switching chemistry to the sample container, the stochastic photo-switching chemistry comprising a set of nucleotides that have a 5′ diphosphate quencher molecule and 3′ phosphate block with a label moiety, a polymerase to incorporate a nucleotide of the set of nucleotides and cleave the 5′ diphosphate quencher molecule, and an enzyme to cleave the 3′ phosphate block with the label moiety;
imaging a series of on and off events for the first DNA template molecule in real-time as the on and off events are occurring, each on event occurring when the polymerase incorporates a nucleotide of the set of nucleotides and cleaves the 5′ diphosphate quencher molecule and each off event occurring when the enzyme cleaves the 3′ phosphate block with the label moiety;
imaging a series of on and off events for the second DNA template molecule in real-time as the on and off events are occurring, wherein the series of on and off events for the first DNA template molecule and the series of on and off events for the second DNA template molecule are stochastic and not synchronized; and
controlling a rate at which the on and off events occur;
wherein controlling the rate at which the on and off events occur comprises adjusting concentrations of nucleotides and enzymes used in the stochastic photo-switching chemistry so that a probability that an on event for a nucleotide base for the first DNA template molecule will occur at the same time as an on event for a nucleotide base for the second DNA template molecule is lower than a determined error rate in a sequencing application in which the method is applied.

### Claim 2 (depends on 1)

The method of claim 1, wherein each of the first attachment element and second attachment element of the plurality of attachment elements of the sample container is within a field of view of an imager used to image the on and off events such that imaging of the on and off events occurs at the same time for the first DNA template molecule and second DNA template molecule.

### Claim 3 (depends on 1)

The method of claim 1, further comprising, determining whether an illumination intensity of a detected on event in a color channel is greater than a predetermined threshold.

### Claim 4 (depends on 1)

The method of claim 1, further comprising, determining whether a spot size of a detected on event in a color channel is greater than a predetermined threshold.

### Claim 5 (depends on 1)

The method of claim 1, wherein the distance between the first attachment element and the second attachment element is less than about 20 nm.

### Claim 6 (depends on 1)

The method of claim 1, wherein the distance between the first attachment element and the second attachment element is within a range of about 2 nm to about 20 nm.