Generalized stochastic super-resolution sequencing

Patent metadata

Field	Value
Publication number	US-2021363578-A1
Application number	US-202117395294-A
Country	US
Kind code	A1
Filing date	Aug 5, 2021
Priority date	Mar 9, 2018
Publication date	Nov 25, 2021
Grant date	—

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.

First claim

We claim:

1 . An imaging system comprising:
a sample container comprising a plurality of attachment elements wherein a single DNA template molecule is attached to each of the attachment elements, and further wherein the average distance between adjacent attachment elements is less than Abbe's limit; and an imager positioned to image photo-switching occurring at the plurality of attachment elements by capturing on and off events in a plurality of color channels at the same time as the on and off events are occurring for the attached molecules when a stochastic photo-switching chemistry is applied to all of the attached molecules at the same time causing the attached molecules to fluoresce in the on and off events in up to four different colors.

2 . The imaging system of claim 1 , wherein the sample container comprises a flowcell that comprises the plurality of attachment elements at a plurality of sample locations.

3 . The imaging system of claim 1 , wherein each of the plurality of attachment elements on the sample container is within a field of view of the imager used to image the photo-switching such that the capturing of the on and off events occurs at the same time for the attached molecules at the plurality of attachment elements.

4 . The imaging system of claim 1 , the stochastic photo-switching chemistry applied to all of the attached molecules at the same time comprises a stochastic optical reconstruction microscopy, a DNA Points Accumulation for Imaging in Nanoscale Topography, or a direct stochastic optical reconstruction microscopy stochastic photoswitching chemistry.

5 . The imaging system of claim 1 , wherein concentrations of reagents for the stochastic photo switching are sufficient such that the probability that an on event for a given base for a given molecule will occur at the same time as an on event for the same base at a molecule adjacent to the given molecule is less than 0.5%.

6 . The imaging system of claim 1 , wherein a rate at which the on and off events occur yields a probability that an on event for a given base for a given molecule will occur at the same time as an on event for the same base at a molecule adjacent to the given molecule is lower than a determined error rate in a sequencing application in which the method is applied.

7 . The imaging system of claim 1 , further comprising, determining whether an illumination intensity or a spot size of a detected on event in a color channel is greater than a predetermined threshold.

8 . The imaging system of claim 1 , wherein the average distance between adjacent elements is less than about 20 nm.

Assignees

• Illumina Cambridge Ltd
• Illumina Inc

Inventors

• Skinner Gary Mark
• Evans Geraint Wyn
• Hong Stanley S

Classifications

• G01N2021/6441

  with two or more labels · CPC title

• G01N21/6458 Primary

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N2021/6432

  Quenching · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title