Sequencing by emergence

Abstract

Systems and methods for nucleic acid sequencing are provided. Nucleic acid is fixed in double-stranded linearized stretched form on a test substrate before being denatured into to single stranded form on the substrate to obtain adjacent fixed first and second strands of the nucleic acid. The strands are exposed to a respective pool of a respective oligonucleotide probe in a set of probes under conditions allowing for probes to form a heteroduplex with a corresponding complementary portion of the fixed first or second strand thereby giving rise to a respective instance of optical activity. An imager measures a location and duration on the substrate of this optical activity. The exposing and measuring is repeated for probes in the set of probes thereby obtaining a plurality of sets of positions. The nucleic acid sequence is determined from the plurality of sets of positions through compilation of the positions in the sets.

1 . A method of sequencing a nucleic acid, comprising:
 (a) fixing the nucleic acid in double stranded linearized stretched form on a test substrate thereby forming a fixed stretched double stranded nucleic acid;   (b) denaturing the fixed stretched double stranded nucleic acid to single stranded form on the test substrate thereby obtaining a fixed first strand and a fixed second strand of the nucleic acid, wherein respective bases of the fixed second strand lie adjacent to corresponding complementary bases of the fixed first strand;   (c) exposing the fixed first strand and the fixed second strand to a respective pool of a respective oligonucleotide probe in a set of oligonucleotide probes, wherein each oligonucleotide probe in the set of oligonucleotide probes is of a predetermined sequence and length, the exposing (c) occurring under conditions that allow for individual probes of the respective pool of the respective oligonucleotide probe to bind and form a respective heteroduplex with portions of the fixed first strand or the fixed second strand that is complementary to the respective oligonucleotide probe thereby giving rise to a respective instance of optical activity;   (d) measuring a location on the test substrate and a duration of each respective instance of optical activity occurring during the exposing (c) using a two-dimensional imager;   (e) repeating the exposing (c) and measuring (d) for respective oligonucleotide probes in the set of oligonucleotide probes, thereby obtaining a plurality of sets of positions on the test substrate, each respective set of positions on the test substrate corresponding to an oligonucleotide probe in the set of oligonucleotide probes; and   (f) determining the sequence of at least a portion of the nucleic acid from the plurality of sets of positions on the test substrate by compiling the positions on the test substrate represented by the plurality of sets of positions.   
     
     
         2 . The method of  claim 1 , wherein the exposing (c) occurs under conditions that allow for individual probes of the respective pool of the respective oligonucleotide probe to transiently and reversibly bind and form the respective heteroduplex with portions of the fixed first strand or the fixed second strand that is complementary to the individual probes thereby giving rise to an instance of optical activity. 
     
     
         3 . The method of  claim 1 , wherein each oligonucleotide probe in the set of oligonucleotide probes is bound with a label. 
     
     
         4 . The method of  claim 1 , wherein
 the exposing is in the presence of a first label in the form of a dye,   each oligonucleotide probe in the set of oligonucleotide probes is bound with a second label, and   the first label causes the second label to fluoresce when the first label and the second label are in close proximity to each other.   
     
     
         5 . The method of  claim 1 , wherein
 the exposing is in the presence of an intercalating dye, and   the respective instance of optical activity is from fluorescence of the intercalating dye intercalating into the respective heteroduplex between the oligonucleotide and the fixed first strand or the fixed second strand, wherein the respective instance of optical activity is greater than a fluorescence of the intercalating dye before it intercalates the respective heteroduplex.   
     
     
         6 . The method of  claim 1 , wherein
 more than one oligonucleotide probe in the set of oligonucleotide probes is exposed to the fixed first strand and the fixed second strand during a single instance of the exposing (c).   
     
     
         7 . The method of  claim 6 , wherein
 different oligonucleotide probes in the set of oligonucleotide probes that is exposed to the fixed first strand and the fixed second strand during the single instance of the exposing (c) are associated with different labels.   
     
     
         8 . The method of  claim 1 , wherein the exposing (c) is done for a first oligonucleotide probe in the set of oligonucleotide probes at a first temperature and the repeating (e) the exposing (c) and measuring (d) includes performing the exposing (c) and the measuring (d) for the first oligonucleotide at a second temperature. 
     
     
         9 . The method of  claim 1 , wherein
 the measuring the location on the test substrate comprises identifying and fitting the respective instance of optical activity with a fitting function to identify and fit a center of the respective instance of optical activity in a frame of data obtained by the two-dimensional imager, and   the center of the respective instance of optical activity is deemed to be the position of the respective instance of optical activity on the test substrate.   
     
     
         10 . The method of  claim 1 , wherein
 the respective instance of optical activity persists across a plurality of frames measured by the two-dimensional imager,   the measuring the location on the test substrate comprises identifying and fitting the respective instance of optical activity with a fitting function across the plurality of frames to identify a center of the respective instance of optical activity across the plurality of frames, and   the center of the respective instance of optical activity is deemed to be the position of the respective instance of optical activity on the test substrate across the plurality of frames.   
     
     
         11 . The method of  claim 1 , wherein
 the measuring the location on the test substrate comprises inputting a frame of data measured by the two-dimensional imager into a trained convolutional neural network,   the frame of data comprises the respective instance of optical activity among a plurality of instances of optical activity,   each instance of optical activity in the plurality of instances of optical activity corresponds to an individual probe binding to a portion of the fixed first strand or the fixed second strand, and   responsive to the inputting, the trained convolutional neural network identifies a position on the test substrate of each of one or more instances of optical activity in the plurality of instances of optical activity.   
     
     
         12 . The method of  claim 9 , wherein the measuring resolves the center of the respective instance of optical activity to a position on the test substrate with a localization precision of at least 20 nm. 
     
     
         13 . The method of  claim 1 , wherein the measuring (d) the location on the test substrate and the duration of the respective instance of optical activity measures more than 5000 photons at the location. 
     
     
         14 . The method of  claim 1 , wherein the respective instance of optical activity is more than a predetermined number of standard deviations over a background observed for the test substrate. 
     
     
         15 . The method of  claim 1 , wherein each respective oligonucleotide probe in the plurality of oligonucleotide probes comprises a unique N-mer sequence, wherein N is an integer in the set {1, 2, 3, 4, 5, 6, 7, 8, 9 and 10} and wherein all unique N-mer sequences of length N are represented by the plurality of oligonucleotide probes. 
     
     
         16 . The method of  claim 1 , wherein the nucleic acid is at least 140 bases in length and the determining (f) determines a coverage of the sequence of the nucleic acid sequence of greater than 70%. 
     
     
         17 . The method of  claim 1 , wherein the nucleic acid is at least 10,000 bases in length. 
     
     
         18 . The method of  claim 1 , wherein the test substrate is washed prior to repeating the exposing (c) and measuring (d), thereby removing a respective oligonucleotide probe from the test substrate prior to exposing the test substrate to another oligonucleotide probe in the set of oligonucleotide probes. 
     
     
         19 . The method of any  claim 1 , wherein the fixing (a) comprises applying the nucleic acid to the test substrate by molecular combing (receding meniscus), flow stretching nanoconfinement, or electro-stretching. 
     
     
         20 . The method of  claim 1 , wherein each respective instance of optical activity has an observation metric that satisfies a predetermined threshold. 
     
     
         21 - 30 . (canceled)