Method for sequencing single cells and reducing error rate

Abstract

The present disclosure relates in some aspects to real-time nucleic acid sequencing, including real-time sequencing, single molecule sequencing, long read sequencing, and / or single cell sequencing. Methods of reducing errors and methods of analyzing sequencing data obtained from sequencing methods are also described herein. In one aspect, provided herein is a nucleic acid sequencing method comprising: a) positioning a double-stranded nucleic acid to a position on a substrate; b) generating a first single-stranded nucleic acid and a second single-stranded nucleic acid from the positioned double-stranded nucleic acid; c) limiting diffusion of the first single-stranded nucleic acid and / or the second single-stranded nucleic acid; d) attaching the first single-stranded nucleic acid and the second single-stranded nucleic acid to a site near the location on the substrate; e) obtaining a sequencing read from the attached first single-stranded nucleic acid and a sequencing read from the attached second single-stranded nucleic acid. In some embodiments, the method further includes f) associating the sequence of the first single-stranded nucleic acid with the sequence of the second single-stranded nucleic acid, thereby determining the sequence of the double-stranded nucleic acid.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 353,590, filed on June 18, 2022, entitled “Methods for Low-Cost DNA Sequencing,” and U.S. Provisional Patent Application No. 63 / 353,591, filed on June 18, 2022, entitled “Sample Preparation Methods for Reduced Error Rates and Single-Cell Sequencing,” the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] The present disclosure generally relates to methods and compositions for determining the sequence of nucleic acid molecules, including methods and compositions for nucleic acid sequencing (such as single cell nucleic acid sequencing) and / or reducing nucleic acid sequencing error rates involving double-stranded nucleic acids. Background Art

[0004] Nucleic acid molecule analysis is an extremely complex task, which usually requires accurate and rapid characterization of a large number of nucleic acid molecules by high-throughput DNA sequencing. The determination of nucleic acid sequences remains a laborious and difficult task, especially compared to cheaper probe-based methods such as qPCR (also known as real-time PCR). Therefore, simplifying and reducing sequencing costs remains an important issue. The present disclosure addresses these and other needs. Summary of the invention

[0005] Known nucleic acid synthesis sequencing (SBS) methods are cyclic, requiring the signal of the labeled nucleotides incorporated in one cycle to be inactivated and the labeled nucleotides not incorporated in the cycle to be removed before introducing the labeled nucleotides for the next cycle. For example, in some existing methods, dye-labeled "A" nucleotides (e.g., dATP labeled with a first fluorophore) are introduced into a flow cell, incorporated and detected at specific spots in the flow cell (e.g., indicating bases "T" in the template molecule at these spots), then the dyes in the nucleotides incorporated at these specific spots are bleached (and the unincorporated dye-labeled nucleotides are removed from the flow cell), and then dye-labeled "T" nucleotides (e.g., dTTP labeled with a second fluorophore of a different "color" than the first fluorophore) are flowed into the flow cell to query the next base (e.g., base "A" at the 5' position of base "T" in the template molecule). In a particular cycle, a mixture of dye-labeled nucleotides can be introduced into the flow cell, for example, four fluorescent dyes, each of a different "color", can be used to label A, T, C, and G in the template molecule (as in a 4-channel SBS chemistry), or two different fluorescent dyes can be used (for example, in a two-channel SBS chemistry, "red" is used to represent C, "green" is used to represent T, "red" and "green" are displayed as "yellow" to represent A, and unlabeled represents G). In any case, these known SBS methods require inactivation of the fluorescent signal, for example by cleaving a fluorescently labeled reversible terminator on the incorporated nucleotide to allow incorporation of a nucleotide to query the next base. One or more washes are also performed between flow cell cycles, for example, to remove unincorporated nucleotides and / or cleaved fluorescent markers. These requirements and other requirements of the known SBS methods make their cost high (for example, compared to methods based on qPCR or antigen-antibody interactions) and limit their application, especially in sequencing-based diagnostics, for example, in response to pandemics such as COVID-19, where a large number of samples must be sequenced in a short period of time.

[0006] In one aspect, the present invention provides a method for nucleic acid sequencing, comprising: a) positioning a double-stranded nucleic acid to a location on a substrate; b) generating a first single-stranded nucleic acid and a second single-stranded nucleic acid from the positioned double-stranded nucleic acid; c) limiting the diffusion of the first single-stranded nucleic acid and / or the second single-stranded nucleic acid; d) attaching the first single-stranded nucleic acid and the second single-stranded nucleic acid to a site near the location on the substrate; e) obtaining a sequencing read from the attached first single-stranded nucleic acid and a sequencing read from the attached second single-stranded nucleic acid. In some embodiments, the method further comprises: f) correlating the sequence of the first single-stranded nucleic acid with the sequence of the second single-stranded nucleic acid to determine the sequence of the double-stranded nucleic acid.

[0007] In any of the foregoing embodiments, the method may include allowing or promoting diffusion of the first single stranded nucleic acid and / or the second single stranded nucleic acid before restricting diffusion of the first single stranded nucleic acid and / or the second single stranded nucleic acid.

[0008] In any of the foregoing embodiments, the positioning in a) may include non-covalently attaching the double-stranded nucleic acid to a position on the substrate. In any of the foregoing embodiments, the double-stranded nucleic acid may, but need not, be covalently attached to the substrate or covalently attached to a molecule fixed on the substrate. In any of the foregoing embodiments, only one strand of the double-stranded nucleic acid may be covalently attached to the substrate or covalently attached to a molecule fixed on the substrate.

[0009] In any of the foregoing embodiments, the positioning in a) may include using an electrode to attract and / or confine the double-stranded nucleic acid to the position on the substrate. In any of the foregoing embodiments, the electrode may be integrated into the substrate or the electrode may be provided separately from the substrate. In any of the foregoing embodiments, the electrode may be removed from the substrate.

[0010] In any of the foregoing embodiments, the positioning in a) may include hybridizing a region of the double-stranded nucleic acid with a nucleic acid probe that is directly or indirectly fixed to the substrate. In any of the foregoing embodiments, the double-stranded nucleic acid may include a single-stranded region. In any of the foregoing embodiments, the single-stranded region may be a loop, a bulge, or generated by partially melting a double-stranded nucleic acid.

[0011] In any of the foregoing embodiments, the double-stranded nucleic acid may be from a cell or tissue sample. In any of the foregoing embodiments, the double-stranded nucleic acid may be fragmented DNA. In any of the foregoing embodiments, the double-stranded nucleic acid may be free DNA or generated by fragmenting genomic DNA. In any of the foregoing embodiments, the double-stranded nucleic acid may be an amplification product of cellular DNA or RNA or free DNA. In any of the foregoing embodiments, the double-stranded nucleic acid may be from a single cell and spaced apart from double-stranded nucleic acids from other cells on the substrate.

[0012] In any of the foregoing embodiments, the location may be a random location on the substrate. In any of the foregoing embodiments, the location may be one of the locations in an ordered pattern on the substrate. In any of the foregoing embodiments, the location may be located in a protrusion or depression at the location on the substrate. In any of the foregoing embodiments, the location may be located on a bead at the location on the substrate.

[0013] In any of the foregoing embodiments, the first single-stranded nucleic acid and the second single-stranded nucleic acid may be generated in b) by melting the positioned double-stranded nucleic acid using heat, pH change, denaturing buffer, or any combination thereof.

[0014] In any of the foregoing embodiments, limiting the diffusion of the first single-stranded nucleic acid and the second single-stranded nucleic acid in c) may include capturing the first single-stranded nucleic acid and the second single-stranded nucleic acid by a nucleic acid probe directly or indirectly immobilized on the substrate. In any of the foregoing embodiments, the nucleic acid probe may be located at a site near the position on the substrate.

[0015] In any of the foregoing embodiments, limiting the diffusion of the first single-stranded nucleic acid and the second single-stranded nucleic acid in c) may include limiting the first single-stranded nucleic acid and the second single-stranded nucleic acid using an electrode. In any of the foregoing embodiments, the electrode may be integrated into the substrate or the electrode is provided separately from the substrate. In any of the foregoing embodiments, the electrode may be removed from the substrate.

[0016] In any of the foregoing embodiments, in d), the first single-stranded nucleic acid and the second single-stranded nucleic acid may be independently covalently and / or non-covalently attached to the site near the position on the substrate. In some embodiments, in a), only one of the first and second chains of the double-stranded nucleic acid is covalently fixed to the position on the substrate, and in b), the other chain is separated from the fixed chain and allowed to diffuse, and in c), its diffusion is restricted, and in d), it is attached to a site near the position. In some embodiments, in a), both chains of the double-stranded nucleic acid are non-covalently positioned at the position on the substrate, and in b), the two chains are separated from each other and allowed to diffuse, and in c), their diffusion is restricted, and in d), the two chains can be independently attached to the site near the position.

[0017] In any of the foregoing embodiments, in d), the site may be no more than about 8 μm, no more than about 6 μm, no more than about 4 μm, no more than about 2 μm, no more than about 1 μm, no more than about 0.5 μm, or no more than about 0.25 μm from the position. In any of the foregoing embodiments, one of the sites may be located at the position, and the other site may be no more than about 2 μm, no more than about 1 μm, no more than about 0.5 μm, or no more than about 0.25 μm from the position. In any of the foregoing embodiments, both sites may be no more than about 2 μm, no more than about 1 μm, no more than about 0.5 μm, or no more than about 0.25 μm from the position.

[0018] In any of the foregoing embodiments, the attached first single-stranded nucleic acid and the attached second single-stranded nucleic acid can be amplified on the substrate. In any of the foregoing embodiments, the attached first single-stranded nucleic acid and the attached second single-stranded nucleic acid can be clonally amplified to form amplicon clusters on the substrate and can be sequenced using the cluster-based sequencing method disclosed herein. In any of the foregoing embodiments, the attached first single-stranded nucleic acid and the attached second single-stranded nucleic acid can be amplified using a bridge amplification method.

[0019] In any of the foregoing embodiments, the attached first single-stranded nucleic acid and the attached second single-stranded nucleic acid may, but need not, be amplified on the substrate, or may be amplified only minimally on the substrate. In any of the foregoing embodiments, in e), the sequencing reads may be obtained using single molecule sequencing. In any of the foregoing embodiments, in e), the sequencing reads may be obtained using real-time sequencing, optionally, using single molecule real-time sequencing.

[0020] In any of the foregoing embodiments, in e), the sequencing reads may be obtained using synthetic sequencing, sequencing-by-binding, avidity sequencing, sequencing-by-ligation, and / or hybridization sequencing. In any of the foregoing embodiments, in e), the sequencing reads may be obtained by imaging the substrate and recording the optical signal in successive imaging cycles for each of the sites.

[0021] In any of the preceding embodiments, the optical signal of one of the sites may be optically distinguishable from the optical signal of another site.

[0022] In any of the foregoing embodiments, the method may include determining the sequence of the first single-stranded nucleic acid by comparing multiple sequencing reads from the attached first single-stranded nucleic acid, and optionally, the method includes aligning the multiple sequencing reads and / or generating a consensus sequence of the multiple sequencing reads.

[0023] In any of the foregoing embodiments, the method may include determining the sequence of the second single-stranded nucleic acid by comparing multiple sequencing reads from the attached second single-stranded nucleic acid, and optionally, the method includes aligning the multiple sequencing reads and / or generating a consensus sequence of the multiple sequencing reads.

[0024] In any of the foregoing embodiments, the sequence of the first single-stranded nucleic acid and the sequence of the second single-stranded nucleic acid can be determined independently of each other.

[0025] In any of the foregoing embodiments, the method may include comparing the sequence of the first single-stranded nucleic acid to the complement of the sequence of the second single-stranded nucleic acid, and / or comparing the sequence of the second single-stranded nucleic acid to the complement of the sequence of the first single-stranded nucleic acid.

[0026] In any of the foregoing embodiments, the method may include comparing the single-stranded consensus sequence of the first single-stranded nucleic acid to the single-stranded consensus sequence of the second single-stranded nucleic acid to generate a double-stranded consensus sequence, optionally wherein the comparison of the single-stranded consensus sequences is used to identify one or more errors in the sequence.

[0027] In any of the foregoing embodiments, the method may include identifying an overlapping sequence between the sequence of the first single-stranded nucleic acid and the complement of the sequence of the second single-stranded nucleic acid, and identifying a first non-overlapping sequence in the sequence of the first single-stranded nucleic acid and / or a second non-overlapping sequence in the complement of the sequence of the second single-stranded nucleic acid.

[0028] In any of the foregoing embodiments, the method may include identifying an overlapping sequence between the sequence of the second single-stranded nucleic acid and the complement of the sequence of the first single-stranded nucleic acid, and identifying a first non-overlapping sequence in the sequence of the second single-stranded nucleic acid and / or a second non-overlapping sequence in the complement of the sequence of the first single-stranded nucleic acid.

[0029] In any of the foregoing embodiments, the method may include assembling i) a sequence of the first single-stranded nucleic acid and a complementary sequence of the sequence of the second single-stranded nucleic acid, and / or ii) a sequence of the second single-stranded nucleic acid and a complementary sequence of the sequence of the first single-stranded nucleic acid into a sequence longer than a sequence of the first single-stranded nucleic acid and a sequence of the second single-stranded nucleic acid.

[0030] In any of the foregoing embodiments, the method may include correlating the sequence of the first single-stranded nucleic acid with the sequence of the second single-stranded nucleic acid during basecalling.

[0031] In any of the foregoing embodiments, the method can include correlating the sequence of the first single-stranded nucleic acid with the sequence of the second single-stranded nucleic acid after base calling.

[0032] In any of the foregoing embodiments, the method may include determining that the sequence of the first single-stranded nucleic acid and the sequence of the second single-stranded nucleic acid are derived from two strands of the same double-stranded nucleic acid localized to the substrate in a).

[0033] In one aspect, a nucleic acid sequencing method is provided herein, comprising: a) positioning a single cell or a cell nucleus to a position on a substrate; b) releasing nucleic acid from the positioned single cell or cell nucleus; c) limiting the diffusion of the nucleic acid; d) attaching the nucleic acid to a site at a position on the substrate or a site near the position; and e) obtaining sequencing reads from the attached nucleic acid to determine the sequence of the nucleic acid. In some embodiments, the nucleic acid from the positioned single cell or cell nucleus can be a double-stranded nucleic acid, or can be used to generate a double-stranded nucleic acid positioned on a substrate. The double-stranded nucleic acid can be analyzed using a nucleic acid sequencing method according to any of the foregoing embodiments.

[0034] In any embodiment herein, the nucleic acid molecule may include deoxynucleotides or derivatives or analogs thereof, and / or ribonucleotides or derivatives or analogs thereof. In any embodiment herein, the nucleic acid molecule may include DNA or RNA. In any embodiment herein, the methods disclosed herein may be used for direct RNA sequencing without first converting RNA to DNA (e.g., cDNA).

[0035] In any embodiment herein, polymerase can be DNA dependent polymerase and / or RNA dependent polymerase.In any embodiment herein, the same polymerase can be used for catalysis of multiple nucleotide incorporation events with the same nucleic acid molecule as template.In any embodiment herein, the same polymerase can be used for catalysis of multiple nucleotide incorporation events with different nucleic acid molecules as template, and different nucleic acid molecules can be provided on substrate for nucleic acid sequencing.In any embodiment herein, different polymerases can be used for catalysis of two or more nucleotide incorporation events with the same nucleic acid molecule as template.In any embodiment herein, different polymerases can be used for catalysis of two or more nucleotide incorporation events with different nucleic acid molecules as template, and different nucleic acid molecules can be provided on substrate for nucleic acid sequencing.In any embodiment herein, the nucleotide incorporation rate by one or more polymerases can be controlled.

[0036] In any embodiment herein, the one or more polymerases may include a DNA polymerase and / or an RNA polymerase. In any embodiment herein, the polymerase may have DNA-dependent DNA polymerase activity and / or RNA-dependent DNA polymerase activity. In any embodiment herein, the one or more polymerases may be selected from DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, Taq polymerase, KlenTaq polymerase, TopoTaq polymerase, Bst polymerase, rBST DNA polymerase, Bsu polymerase, T7 DNA polymerase, T7 RNA polymerase, T3 DNA polymerase, T3 RNA polymerase, T4 polymerase, T5 polymerase, polymerase, 9°N polymerase, KOD polymerase, Pfu DNA polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) polymerase, M2 polymerase, B103 polymerase, GA-1 polymerase, polymerase, N29 DNA polymerase, SP6 RNA polymerase, reverse transcriptase (optionally, III reverse transcriptase), and variants or derivatives thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings illustrate some embodiments of the features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way.

[0038] Figure 1 A schematic diagram showing double-stranded fragments of nucleic acid being attached, made single-stranded, and clustered for cluster-based sequencing. For example, dsDNA can be melted and one or both of the resulting ssDNA strands can be located, allowing reconstruction of the source dsDNA sequence by an algorithm.

[0039] Figure 2 An exemplary charge / electric field barrier configuration is shown, wherein reagents are located in regions labeled "A," "T," "G," and "C" (representing adenine nucleotides, guanine nucleotides, thymine nucleotides, and cytosine nucleotides, respectively). Rectangles located at the top of the regions with reagents and at the bottom of a main chamber (e.g., apyrase can be provided in the main chamber) represent electrodes, wherein the electrodes located at the top of the regions with nucleotides can be positively charged, for example, to attract "A," "T," "G," and "C" nucleotides, thereby confining negatively charged nucleotides in the respective regions. By converting the electrodes in the corresponding regions to be negatively charged, the nucleotides can be released and / or drawn into the main chamber, while the electrodes in the main chamber can be positively charged relative to this example.

[0040] Figure 3 An exemplary chamber is shown in which the reagent is confined in a vesicle ("reagent bubble"), which is represented as a circle in the figure. The reagent can be released by breaking the vesicle (e.g., using any one of the methods or combinations of methods disclosed herein, such as charge, heat, pH change, surfactant, etc.), and then enters the main chamber.

[0041] Figure 4It is shown that photobleaching and step counting can be used to determine the observed number of steps and homopolymer length. Step counting, including but not limited to exemplary algorithms from Shu et al. ("Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system," EMBO J. 2007 Jan 24; 26(2): 527–537, the entire contents of which are incorporated herein by reference for all purposes) and Mira et al. ("Counting the Number of Fluorophores Labeled in Biomolecules by Observing the Fluorescence-Intensity Transient of a Single Molecule," BCS J 78(9): 1612-18 (2005), the entire contents of which are incorporated herein by reference for all purposes), can be used to determine the observed number of steps, thereby determining the possible homopolymer length. DETAILED DESCRIPTION

[0042] All publications, including patent documents, scientific literature, and databases, mentioned in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was individually incorporated herein by reference. If the definitions listed herein contradict or are inconsistent with definitions in patents, applications, published applications, and other publications incorporated herein by reference, the definitions listed herein take precedence over the definitions incorporated herein by reference.

[0043] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0044] I. Overview

[0045] Provided herein are methods related to nucleic acid (e.g., DNA / RNA) sequencing that involve improving the error rate of reads generated from single-stranded or double-stranded nucleic acid fragments (e.g., dsDNA). Also provided herein are methods for isolating sequencing reads from single cells.

[0046] A. Reducing error rates and generating long reads

[0047] In some embodiments, the present invention provides a double-stranded nucleic acid sequencing method, comprising: a) weakly attaching a double-stranded nucleic acid from a sample to a surface; b) melting or denaturing the double-stranded nucleic acid into a single-stranded nucleic acid to generate a first single-stranded nucleic acid and a second single-stranded nucleic acid, which are complementary chains to each other and can no longer be attached to the surface; c) limiting the mutual diffusion of the first single-stranded nucleic acid and the second single-stranded nucleic acid and attaching them to the surface, wherein limiting the diffusion of the first single-stranded nucleic acid and the second single-stranded nucleic acid causes one or both of the chains to attach to the surface close to each other; d) obtaining spatially located reads of the first single-stranded nucleic acid and the second single-stranded nucleic acid, wherein, due to the limited diffusion of step c), the first single-stranded nucleic acid and the second single-stranded nucleic acid can be identified as belonging to the same double-stranded nucleic acid due to their proximity on the surface, and their reads are merged to improve accuracy.

[0048] In some embodiments, double-stranded nucleic acids (e.g., double-stranded nucleic acids derived from circulating free DNA) are weakly attached to the surface. In some embodiments, such surface attachment can use one of a variety of methods. For example, the surface can have a (weak) positive charge that attracts double-stranded nucleic acids. In some embodiments, the surface can have embedded electrodes that attract and / or attach double-stranded nucleic acids. In some embodiments, DNA can be partially melted and attached to probes (randomly or otherwise) located on the surface (see, e.g., Figure 1 ). In some instances, double-stranded fragments are attached to the surface and melted. Single strands can diffuse and attach to the surface over a very small distance, and diffusion can be limited by a variety of methods described herein. For cluster-based sequencing methods, clusters can be formed by one or two strands generated by double-stranded fragments, and clusters may or may not overlap. In a preferred embodiment, clusters are non-overlapping. In some embodiments, clusters are formed within a localized region.

[0049] In any embodiment herein, once attached to the surface, the double-stranded nucleic acid can be melted / denatured (e.g., using heat, or using changes in pH, buffer, or any combination thereof) to form single-stranded nucleic acids. In some embodiments, the surface with probes is prepared so that single-stranded nucleic acids are attached to these probes (e.g., as performed in cluster-based synthetic sequencing in US 10,370,652 B2, the entire contents of which are incorporated herein by reference for all purposes).

[0050] In any embodiment herein, a "probe-less" attachment method, such as charge-based attachment, can be used. In any embodiment herein, the surface can be prepared so that one or more single-stranded nucleic acids will not diffuse widely before being attached to a probe or surface. In any embodiment herein, the diffusion of single-stranded nucleic acids can be limited by using an electric field. In a specific embodiment, the electric field can attract one or more single-stranded nucleic acids to the surface. In some embodiments, the electric field can be formed using electrodes to form a barrier that prevents the single-stranded nucleic acids from diffusing widely. For example, negatively charged electrodes can be used to limit one or more single-stranded nucleic acids.

[0051] In any of the embodiments herein, once the nucleic acids are attached, clusters can be formed (eg, by bridge amplification) and then sequenced, such as by sequencing by synthesis or similar methods.

[0052] In some embodiments, double-stranded nucleic acid is randomly attached to the surface. In some embodiments, double-stranded nucleic acid is not randomly attached to the surface, and the surface can be patterned to allow the formation of an ordered array of double-stranded nucleic acids attached directly or indirectly to the surface. In some embodiments, the surface is a patterned flow cell surface. In some embodiments, the surface may include projections and / or depressions, such as posts and / or holes (wells).

[0053] In some embodiments, a bead-based cluster generation method can be used, including performing cluster amplification on beads and then loading the beads into an unstructured flow cell to form a 3D array of beads that can be imaged by confocal microscopy. Exemplary methods include those described in US2021 / 0040555, the entire contents of which are incorporated herein by reference for all purposes.

[0054] In some embodiments, a bead-based cluster generation method can be used, comprising loading beads onto a patterned flow cell comprising two or more wells, wherein each well is separated from any adjacent well by about 0.2 μm to about 2.0 μm, and each well comprises at least one particle comprising a plurality of oligonucleotide moieties covalently attached to the particle by a bioconjugation linker, wherein the bioconjugation linker is formed by a reaction between a particle polymer comprising a first bioconjugation reactive moiety and an oligonucleotide comprising a second bioconjugation reactive moiety, and wherein the average longest dimension of the particles is about 100 nm to 1000 nm, wherein the solid support comprises a polymer layer. Clusters are then grown on these beads and fluorescently imaged. Exemplary methods include those described in U.S. Pat. No. 11,629,380 B2, the entire contents of which are incorporated herein by reference for all purposes.

[0055] In any embodiment herein, in the image analysis sequence process, the signals at multiple positions on the surface are recorded, which can use super-resolution to obtain accurate positioning. In some embodiments, after base calling or as part of the base calling process, an algorithm is used to determine whether two adjacent templates (e.g., using Euclidean distance or other methods) are from the same source fragment. In some embodiments, the basis for determining whether two adjacent templates are from the same source fragment is their proximity. In some embodiments, as long as the read length is long enough to overlap between the sense strand and the antisense strand, it is possible to use to determine whether two adjacent templates are from the same source. In some embodiments, if it is determined that the two chains are from the same double-stranded nucleic acid source, the information of the two chains can be combined.

[0056] In some embodiments, the methods disclosed herein may include masking bases that are not paired when the strand sequences are switched to the same orientation (e.g., replacing them with "N"). In some embodiments, base quality scores (determined during the base calling process) may be used to select the most accurate base calls. In some embodiments, features extracted during the image analysis process may provide additional information to inform this process (e.g., circularity of clusters at positions where errors may occur, local background, or other errors).

[0057] In some embodiments, synthetic sequencing can be used, and errors in each strand may have different error characteristics. In some embodiments, this is due to the nature of phase lag errors in these methods. In some embodiments, early bases (e.g., bases toward the 3' end of the sequencing template) may be of higher quality and may be used in preference to later bases in the complementary strand. In some embodiments, determining whether two adjacent templates are from the same source fragment can be used to facilitate base calling, e.g., to achieve more accurate base calling.

[0058] In some embodiments, a number of algorithmic methods can be used to combine the above information during or after base calling. In some embodiments, determining whether two adjacent templates are from the same source fragment can increase the effective read length, where the chain is longer than the platform read length, but short enough to have overlap. In some embodiments, overlap can be used to locally assemble two adjacent reads into a longer read.

[0059] In some embodiments, determining whether two adjacent templates are from the same source fragment can be used for well-separated clusters. In some embodiments, information from clusters formed by sense strands and antisense strands (commonly referred to as mixed clusters or mixed template clusters) can be used. In some embodiments, HMM or other algorithms can be used to extract the most likely source template from the convolution signal of sense strands and antisense strands.

[0060] B. Single-cell sequencing and spatial sequencing

[0061] In some embodiments, isolating single cells and sequencing them is of interest for many applications. In any of the embodiments described herein, single cells are attached to a surface. In some embodiments, the attachment can be charge-based attachment, or binding attachment using monoclonal antibodies or polyclonal mixed antibodies.

[0062] In some embodiments, the attachment sites can be distributed randomly or in a pattern on the surface. In some embodiments, the patterned attachment sites can have a size such that only a single cell can attach to a single attachment site.

[0063] In some embodiments, once attached to the surface, the cells can be lysed (e.g., using SDS or other lysis buffers). In some embodiments, other methods (such as toxins, heat, pH, etc.) can also be used to disrupt the cell bilayer to lyse the cells. In some embodiments, the attachment site can include a reverse transcriptase attached to the surface (or otherwise). In some embodiments, the reverse transcriptase can be used to convert cellular RNA into DNA.

[0064] In some embodiments, the charge barrier around the cell can be used to limit the extracted material to the area near the cell location (e.g., attachment site) on the surface where the cell was previously attached. In some embodiments, the charge barrier can include negatively charged patterned electrodes that can repel negatively charged DNA / RNA and prevent it from diffusing from the cell location. In some embodiments, positive electrodes can also be used to pull DNA / RNA to the attachment site.

[0065] In some embodiments, the DNA / RNA can then be constrained to the surface, for example, by charge-based interactions or using probes (e.g., random probes, specific ligated adapters, polyT probes attached to introduced polyA tails, or targeted probes). The polyA tails can be those in mRNA molecules, or, alternatively, polyA sequences can be introduced for attachment to DNA molecules.

[0066] In some embodiments, an index sequence can be introduced into the restricted fragment. In some embodiments, the index can be introduced using known methods such as ligation or PCR-based methods. In some embodiments, the index can be patterned on a surface and released using a variety of methods including established methods (such as optical cleavage or release in other ways using heat / charge-based methods).

[0067] In some embodiments, any one or more of the above methods can be used in the context of spatial sequencing. In specific embodiments, cells maintain orientation in the source tissue, and nucleic acid molecules (e.g., cell DNA / RNA, probes of cell DNA / RNA, or connections and / or amplification products of probes or cell DNA / RNA) can migrate from cells or tissue samples and be captured at corresponding positions on an artificial substrate, for example, by a capture probe fixed on an artificial substrate.

[0068] The present disclosure relates to methods and systems for determining the nucleotide sequence of individual nucleic acid molecules using optical techniques in some aspects. In some embodiments, disclosed herein is a method for imaging the labeled nucleotides added to the nucleic acid molecules fixed on a substrate (e.g., solid surface), wherein sequencing by synthesis (SBS) is used to sequence the nucleic acid molecules. Any one or more of the labeled nucleotides can be labeled with only one marker (e.g., a fluorophore displayed as "red" or "green"), or can be labeled with one or more molecules of the same marker. In addition, any one or more of the labeled nucleotides can be labeled with two or more markers (e.g., a "red" first fluorophore and a "green" second fluorophore, so that the labeled nucleotides appear as "yellow"), and can be labeled with one or more molecules of each type of marker. For multi-labeled nucleotides, the ratio of different types of markers can be adjusted as needed, for example, so that the labeled nucleotides of unique markers with different ratios can be distinguished.

[0069] Before the 2000s, the most commonly used DNA sequencing method was dideoxy chain termination sequencing (Sanger et al., PNAS 74 (12): 5463-5467 (1977), the entire contents of which are incorporated herein by reference for all purposes). However, this method is time-consuming, labor-intensive, expensive, and has low throughput. To overcome some of these drawbacks, Solexa et al. developed massively parallel sequencing (MPS) methods (e.g., U.S. Pat. No. 7,115,400, the entire contents of which are incorporated herein by reference for all purposes). These methods sequence a large number of nucleic acids in parallel, greatly reducing the cost-per-base of sequencing.

[0070] However, key drawbacks remain. Specifically, while MPS has reduced the cost of sequencing per base, the cost per run is still high (>$400). Common MPS methods also work using amplified DNA clusters. Therefore, they are limited to DNA sequencing and cannot be directly used for RNA sequencing.

[0071] Single molecule sequencing (e.g., sequencing implemented by Pacific Biosciences, Helicos, etc.) solves some of these problems. However, these methods do not reduce running costs. In single molecule SBS, photobleaching is considered to be a method for inactivating labeled nucleotides to avoid signal accumulation (Braslavsky et al.). Counting discrete bleaching events is considered to be a method for solving multiple incorporations (e.g., U.S. Patent No. 6,221,592, the entire contents of which are incorporated herein by reference for all purposes). In these methods, the dye incorporated can be bleached to prevent signal accumulation, because the residual signal of the previous cycle can interfere with the detection of the subsequent cycle. Before adding labeled nucleotides to start a new cycle, photobleaching must be completed to remove all dye markers.

[0072] Existing SBS methods can only be used in a cycle sequencing setting, as photobleaching is done by exposing the nucleic acid strands to light, which may cause photodamage to the nucleic acid before sequencing is completed. Therefore, it is best to minimize the exposure of the strand being sequenced. Furthermore, it is unclear how to use photobleaching in a real-time sequencing setting, where nucleotides are introduced continuously rather than cyclically.

[0073] The present disclosure relates in some aspects to nucleic acid sequencing methods, in which dye inactivation (e.g., by photobleaching) limits signal accumulation, but generally does not complete before additional labeled nucleotides are incorporated into a given strand being sequenced. In some embodiments, as part of a nucleic acid sequencing method, a decrease in signal intensity (e.g., emission) caused by dye inactivation can be used to infer information about the strand (and complementary template strand) being synthesized. It should be noted that photobleaching and / or any other suitable dye inactivation method can be used. Exemplary photobleaching techniques are described in, for example, Chen et al., Mol Biol Cell, 25(22): 3630-42 (2014), the entire contents of which are incorporated herein by reference for all purposes.

[0074] In some embodiments, provided herein is a method for sequencing by synthesis in which a nucleic acid chain is attached to a solid surface and then extended by a polymerase (e.g., by a DNA polymerase or a reverse transcriptase) to incorporate a nucleic acid molecule (e.g., a nucleotide) containing a fluorescent (or other luminescent property) marker into the 3' end of a sequencing primer hybridized to the nucleic acid chain.

[0075] In some embodiments, the dye is imaged using an imaging platform that can distinguish a single dye at multiple locations on a substrate, and the "intensity" of the nucleic acid "spot" is determined. In some embodiments, the term "intensity" as used herein refers to a value calculated from the dye emission light of a single nucleic acid imaged as a "light spot". In some embodiments, the intensity may include emission light from one or more molecules of the same dye or different dyes, and corrections may be made, for example, to compensate for background signals such as background light (such as background fluorescence, such as autofluorescence). In some embodiments, the imaging system can be used to determine when a marker is incorporated (which results in an increase in intensity), and when a bleaching event occurs (which results in a decrease in intensity).

[0076] In some embodiments, the sequence of a single strand of nucleic acid can be determined probabilistically by algorithmically combining information about incorporation events (increases in intensity) and photobleaching events (decreases in intensity). This approach is simpler than current sequencing methods that require multiple reagent cycles and does not require nanofabricated surfaces.

[0077] In some embodiments, disclosed herein is a method of not completing photobleaching during a single incorporation / imaging cycle. In some embodiments, a gradual increase in signal intensity is used to record the incorporation of labeled nucleotides. In some embodiments, the photobleaching step can be used not only to provide information for determining the amount of incorporation, but also to provide information for determining the nucleotide sequence of the chain being synthesized. In some embodiments, a variety of markers can be used, wherein the labeled nucleotides can be distinguished from each other according to the type and / or quantity of one or more markers on the individual labeled nucleotides. These markers can emit light at a specific wavelength, or produce a characteristic increase in signal intensity after filtering. In some embodiments, nucleotide incorporation events and signal inactivation events of incorporated nucleotides can be matched or paired. For example, a marker that produces a characteristic increase in signal intensity can cause a characteristic decrease in the corresponding signal intensity when the marker is bleached. In some embodiments, between two time points or by comparing the signal intensity between two detection time windows, the recorded intensity changes can reflect the type of labeled nucleotides incorporated and can be used to determine the complementary sequence of the chain being sequenced.

[0078] In some embodiments, the labeled nucleotides can be, but are not required to be, added in cycles. In some embodiments, the methods disclosed herein may include one or more cycles in which one or more labeled nucleotides are added, signals associated with nucleotide incorporation are detected, signals of incorporated nucleotides are inactivated, and the substrate is washed to remove the labeled nucleotides and optionally remove the cleaved labels, and then additional labeled nucleotides are added to sequence the next base. In the case of cyclic addition of one or more labeled nucleotides, a single label may be used to label the one or more labeled nucleotides in one cycle, for example, similar to dual-channel SBS chemistry, using "red" to represent C, "green" to represent T, "red" and "green" to represent A, and unlabeled G. In some embodiments, the methods disclosed herein may include using a single label and introducing labeled nucleotides in one or more cycles, wherein only labeled nucleotides containing one nucleotide type (such as A, T, C or G) and the single label are introduced into the sequencing reaction in each cycle or circulation, and nucleotide incorporation / non-incorporation is monitored in the one or more cycles. In these embodiments, a nucleotide introduced in one cycle is either signal-inactivated (if incorporated) or removed (if not incorporated) prior to the introduction of a nucleotide of the same or different type labeled with the same single label.

[0079] In some embodiments, the method disclosed herein is a sequencing method, such as a method for DNA or direct RNA sequencing. In some embodiments, the method can use a single detection channel, such as a single detection channel for detecting the signal intensity of multiple different markers. For example, a single channel is sufficient to detect and distinguish signals associated with two fluorophores ATTO532 and ATTO542, based on their characteristic intensity (such as the sum of relative fluorescence within a certain wavelength range). In some embodiments, the method is a single-channel sequencing method. In some embodiments, the method is non-terminated or non-cyclic. For example, the method does not require the use of a chain terminator (such as a reversible terminator that can reversibly terminate primer extension) or does not require a sequencing cycle including signal inactivation and / or marker removal. In some embodiments, the method uses labeled nucleotides, but does not require cleavage and / or removal of markers from incorporated nucleotides.

[0080] In some embodiments, labeled nucleotides can be added to the real-time sequencing method and imaged during the incorporation process. A labeled light spot can be created based on a point spread function (PSF) of a single emitter or a group of diffraction-limited emitters (e.g., multiple markers on a single strand of nucleic acid). The image can be recorded and segmented to determine the position of the light spot. Once the light spot is determined, the background signal (e.g., due to background fluorescence and / or autofluorescence) can be calculated and removed from the image of the light spot. Other signal artifacts (e.g., foreground illumination changes) can also be compensated. The characteristic signal of each light spot can be extracted. A variety of methods can be used to extract signals. For example, a characteristic signal can be obtained by extracting a peak value within the light spot, and / or by fitting a point spread function (e.g., a 2D Gaussian function) to the light spot profile and using a peak value or other features from the fit. In some embodiments, the characteristic value is referred to as "intensity" or "signal intensity", which can be used interchangeably in this article.

[0081] In some embodiments, the intensity of the light spot can be extracted in several frames to generate an intensity profile of the light spot (e.g., in the form of a time trajectory). In some embodiments, the intensity profile of the light spot (e.g., the time trajectory of the signal intensity) is generated by the labeled nucleotides incorporated into the chain being synthesized. The profile can be further corrected and processed to determine the nucleic acid sequence of the complementary template nucleic acid, which can be RNA or DNA.

[0082] In some embodiments, the labeled nucleotide is incorporated into the chain being synthesized (e.g., using a polymerase or reverse transcriptase). In some embodiments, the previously incorporated labeled nucleotide does not need to be photobleached before incorporating one or more subsequent labeled nucleotides. In some embodiments, a single strand of nucleic acid (5'-AATAG-3') is first attached to a surface, and then a polymerase or reverse transcriptase is used to incorporate the first labeled nucleotide ("A" in this example). The second labeled nucleotide ("T" in this example) can be present in a sequencing reaction before, during, and / or after the incorporation of the "A" nucleotide of the first label. Then, the "T" nucleotide of the second label can be incorporated before the "A" nucleotide of the first label is photobleached. Then, the third labeled nucleotide ("T" in this example) can be incorporated after the "A" nucleotide of the first label is photobleached and before the "T" nucleotide of the second label is photobleached. When the "T" nucleotide of the second label has not yet been photobleached, the "T" nucleotide of the third label can be bleached. Then, the "T" nucleotide of the second label is photobleached. A time trace of the signal detected at the light spot can be generated and used to determine the sequence of the nucleic acid strand, for example 5'-AAT-3', which is complementary to the synthetic 5'-ATT-3' sequence in the sequencing primer strand.

[0083] In some embodiments, labeled nucleotides may be incorporated and imaged under illumination (e.g., objective lens or prismatic TIRF illumination). In some embodiments, labeled nucleotides may be incorporated, and photobleaching of incorporated labeled nucleotides occurs randomly. In some embodiments, nucleotides comprising different bases may be labeled with the same marker. In some embodiments, markers having different excitation wavelengths and / or different emission wavelengths may be used to label nucleotides comprising different bases. In some embodiments, markers that produce different intensities at a given wavelength or within a given wavelength range may be used to label nucleotides comprising different bases.

[0084] In some embodiments, photobleaching and / or any suitable dye inactivation method can be used. For example, photodegradable fluorescent nucleotides can be used, as described in Meng et al., "Design and Synthesis of a Photocleavable Fluorescent Nucleotide 3'-O-Allyl-dGTP-PC-Bodipy-FL-510as a ReversibleTerminator for DNA Sequencing by Synthesis," J. Org. Chem. 71, 8, 3248-3252 (2006), the entire contents of which are incorporated herein by reference for all purposes. Other temperature or pH-based dye inactivation methods can also be used.

[0085] Photobleachable nucleotides may include 5-(3-aminoallyl)-2'-deoxyuridine-5'-triphosphate labeled with ATTO 532 triethylammonium salt (Jena Biosciences, Germany), or similar ATTO labeled nucleotides. The nucleotides may be introduced at a concentration compatible with the experimental conditions, such as 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM, or in a range between any of the above values. Nucleotides may be constructed in which photodamage is used to cause dye cleavage. Nucleotides may also be constructed to contain multiple emitters, providing different emitted light intensities. Such nucleotides may contain a cleavable element so that all emitters can be removed / inactivated simultaneously.

[0086] A suitable polymerase such as 9°N or related polymerases, or Klenow fragments, or III reverse transcriptase (Invitrogen) or another reverse transcriptase to incorporate nucleotides.

[0087] In some embodiments, nucleotides are labeled with markers that produce different intensities. In the process of nucleotide incorporation and imaging as described above using the markers of different intensities, tracks can be extracted from the acquired images. These markers will produce convolution signals of random photobleaching events. Both incorporation events (increase in intensity) and bleaching events (reduction in intensity) can provide information that helps determine the nucleotide sequence of the chain in synthesis and the complementary chain being sequenced. Nucleotide markers can be selected so that the markers display different emission light levels within the same wavelength range. For example, ATTO 532 and ATTO 542 can be used, and their relative emission light levels at 537nm are 0.443 and 0.104 respectively.

[0088] In some embodiments, the methods disclosed herein include controlling the photobleaching rate, for example, by using a free radical scavenger, such as β-mercaptoethanol (Yanagida et al., 1986, in Applications of Fluorescence in the Biomedical Sciences, Taylor et al. (eds) Adaln R. Liss Inc., New York, pp. 321) or glucose oxidase. For example, in some embodiments, the methods include adjusting the photobleaching rate so that the total emitted light remains below a threshold total value. In some embodiments, the methods disclosed herein include preventing the emitted light from saturating the image sensor well depth at a given exposure time.

[0089] For example, a hidden Markov model (HMM) capable of decoding a dinucleotide sequence in which nucleotides are labeled with different brightnesses can be used to analyze and deconvolute the time trace of signal intensity. "A" nucleotides are labeled with level 1 intensity, and "T" nucleotides are labeled with level 2 intensity (twice the intensity of "A"). This HMM using Viterbi or other decoders can be used to base call the intensity track. The transitions in this model represent the type of nucleotides incorporated. The state represents the intensity level obtained from the above intensity track. The state labeled P bThe transitions represent photobleaching events. The HMM can be used to model any combination of 3 nucleotide types irradiated at any one time. To simplify the example, only 2 nucleotide types ("A" and "T") are shown here, however the model can be extended to 4 nucleotides, where more than 3 nucleotide types are irradiated at any one time using known methods. Self-transitions are not shown, which will simulate stable states. Other states can be added to compensate for multiple bleaching events in a single sample. In some embodiments, states can be added to simulate dye self-quenching, blinking, photo-switching, and / or dye recovery. States can simulate the emission light intensity as a fixed value, a range, or as a Gaussian distribution. The incorporated transition probabilities can be fixed (determined experimentally) or fitted according to each experiment. Similarly, the photobleaching transition probability (P b ) can be fixed (determined experimentally) or fitted to each experimental dataset.

[0090] Although the HMM can be demonstrated using two transition types representing adenine (A), thymine (T), it can also be extended to use cytosine (C) and guanine (G) nucleotides. The HMM can also represent synthetic sequencing and photobleaching of RNA chains.

[0091] In some embodiments, the methods disclosed herein can be used to provide fast, inexpensive sequencing solutions, for example, in response to a pandemic such as COVID-19. This pandemic-scale sequencing method is comparable in cost to qPCR-based methods, with the cost per run being much lower than existing synthetic sequencing methods that rely on flow cell cycling. In some embodiments, the sequencing methods disclosed herein can be used to diagnose diseases or conditions, such as viral infections. In some embodiments, the sequencing methods disclosed herein overcome the limitations of qPCR-based methods and achieve improved detection accuracy.

[0092] In some embodiments, the low-cost sequencing methods provided herein (e.g., for pandemic response) can accurately detect multiple nucleic acid molecules, including viral RNA in biological samples. For example, biological samples can be processed to extract viral nucleic acids (e.g., RNA), while optionally depleting human nucleic acids (e.g., RNA). The extracted viral nucleic acids can be sequenced in a large-scale parallel, high-throughput manner using the methods disclosed herein. Therefore, using the methods including extracting RNA from patient samples and direct RNA sequencing according to some embodiments of the present disclosure, the presence / absence, amount, and sequence of viral nucleic acids can be quickly detected. In some embodiments, there is no need to reverse transcribe RNA into cDNA. In some embodiments, there is no need to perform multiple PCR on the extracted RNA or reverse transcribed cDNA. In some embodiments, before sequencing using the methods disclosed herein, there is no need to further process the extracted nucleic acids (e.g., RNA). For example, in some embodiments, it is not necessary to perform enzyme fragmentation (tagmented) and / or amplification on the extracted nucleic acids (e.g., RNA) before sequencing. In some embodiments, the methods provided herein can be used to sequence a nucleotide sequence of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides, or longer, with an error rate of less than about 10%, less than about 5%, or less than about 1% in about 100,000 to about 1 million sequenced reads.

[0093] II. Samples and Nucleic Acid Molecules

[0094] The nucleic acid molecules used in the methods described herein can be obtained from any suitable biological source, such as a tissue sample, a blood sample, a plasma sample, a saliva sample, a stool sample, or a urine sample. The polynucleotide can be a DNA or RNA molecule. In some embodiments, before the polynucleotide is hybridized with a sequencing primer, the RNA molecule is reverse transcribed into a DNA molecule. In some embodiments, the RNA molecule is not reverse transcribed, but hybridized with a sequencing primer and RNA sequencing is performed directly. In some embodiments, the nucleic acid molecule is free DNA (cfDNA), such as circulating tumor DNA (ctDNA) or fetal free DNA.

[0095] Examples of nucleic acid molecules include DNA molecules, such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, PCR products synthesized in situ, and RNA / DNA hybridization products. A DNA analyte can be a transcript of another nucleic acid molecule (such as DNA or RNA such as mRNA) present in a tissue sample.

[0096] Examples of nucleic acid molecules also include RNA molecules, such as various types of coding and non-coding RNA, including viral RNA. Examples of different types of RNA molecules include messenger RNA (mRNA), including nascent RNA, mRNA precursors, primary transcript RNA, and processed RNA, such as capped mRNA (e.g., with a 5'7-methylguanosine cap), polyadenylated mRNA (with a poly A tail at the 3' end), and spliced ​​mRNA (wherein one or more introns have been removed). Nucleic acid molecules disclosed herein also include non-capped mRNA, non-polyadenylated mRNA, and non-spliced ​​mRNA. The RNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as viral RNA).

[0097] In some embodiments, the nucleic acid molecule can be a denatured nucleic acid, wherein the denatured nucleic acid produced is single-stranded. Nucleic acid denaturation can be, for example, optionally carried out using formamide, heating, or using formamide and heating simultaneously. In some embodiments, the nucleic acid is not denatured when used in the methods disclosed herein.

[0098] In some embodiments, nucleic acid molecules can be extracted from cells, viruses, or tissue samples containing cells or viruses. Treatment conditions can be adjusted to extract or release nucleic acid molecules (such as RNA) from cells, viruses, or tissue samples.

[0099] III. Sequencing Methods

[0100] In some embodiments, a nucleic acid sequencing method is disclosed herein, the method comprising colony surface amplification (colony surface amplification) (e.g., using bridge amplification or isothermal amplification methods). Exemplary colony surface amplification methods include those disclosed in US 7,115,400, US 7,541,444, US 7,771,973, US 8,071,739, US 8,597,881, US 8,652,810, US 9,121,060, US 9,297,006, US 9,388,464, US10,370,652, US10,513,731 and US 2020 / 0399692, the entire contents of each patent are incorporated herein by reference for all purposes.

[0101] In some embodiments, amplification clusters of nucleic acid molecules (e.g., DNA) are formed on the surface. In some embodiments, the amplification cluster is clonal, and all nucleic acid chains in the cluster include at least one identical sequence to be determined, accepting polymerase errors (e.g., if nucleotide differences are introduced due to polymerase errors during clonal amplification, the sequences in the two chains can be considered as identical sequences). In some embodiments, the amplification cluster may include sequences from one or more concatemers, such as rolling circle amplification products of unit sequences comprising multiple copies or repetitions, and these copies or repetitions include at least one identical sequence to be determined, and can be cut out from the rolling circle amplification products.

[0102] In some embodiments, clusters and identical sequences shared between molecules (or shared by repeated sequences in the same molecule) can be sequenced, for example, using synthesis sequencing (SBS), binding sequencing (SBB), or sequencing using dye-labeled polymers with multiple identical nucleotides attached (e.g., affinity sequencing).

[0103] In some embodiments, in the methods disclosed herein, reversibly terminated nucleotides are incorporated into the synthesizing chain using a polymerase. In some embodiments, nucleotides are labeled with cleavable fluorophores so that each nucleotide type can be specifically detected. Once detected, the label can be removed, and blocking groups (such as terminators) can also be removed. In some embodiments, subsequent nucleotides can be incorporated and the complete sequence of the same sequence in the chain in the cluster can be determined.

[0104] In some embodiments, a cluster-based amplification method is provided herein. Cluster-based sequencing can generally provide more emission signals compared to traditional single-molecule methods. Cluster-based amplification can provide an improved signal-to-noise ratio (SNR) advantage and allow the use of cheaper and simpler cameras. This method also means that a certain degree of photodamage can be tolerated. If some molecules (chains) within the cluster are photodamaged, the remaining molecules can still provide enough signals to allow sequencing to continue and determine the sequence. However, a major limitation of cluster-based sequencing is phase lag (phasing).

[0105] In some synthetic sequencing methods, a first group of detectably labeled nucleotides (e.g., dNTPs) are introduced into a reaction chamber to contact template nucleotides hybridized with a sequencing primer in the reaction chamber, and a first detectably labeled nucleotide (e.g., A, T, C, or G nucleotide) is incorporated by a polymerase using a complementary nucleotide (a first nucleotide residue) in the template nucleotide as a template to extend the sequencing primer in the 5' to 3' direction. The signal from the first detectably labeled nucleotide can then be detected. The first group of nucleotides can be continuously introduced into the reaction chamber (e.g., a flow cell), but in order to allow the second group of detectably labeled nucleotides to be incorporated into the extended sequencing primer, the nucleotides in the first group of nucleotides that are not incorporated into the sequencing primer must generally be removed (e.g., by washing), and the second group of detectably labeled nucleotides must be introduced into the reaction chamber. Then, a second detectably labeled nucleotide (e.g., A, T, C, or G nucleotide) is incorporated by the same or different polymerase using a complementary nucleotide (a second nucleotide residue) in the template nucleotide as a template to extend the extended sequencing primer in the 5' to 3' direction. Therefore, in these methods, cycles of introduction and removal of detectably labeled nucleotides must be performed.

[0106] In contrast, in one embodiment of the method disclosed herein, the first detectable labeled nucleotide and the second detectable labeled nucleotide do not need to be introduced into the reaction chamber in a separate cycle. In some embodiments, when the first detectable labeled nucleotide is being mixed into the sequencing primer, the second detectable labeled nucleotide is already present in the reaction chamber. In some embodiments, when the second detectable labeled nucleotide is mixed into the extended sequencing primer, other molecules of the first detectable labeled nucleotide that have not yet been mixed into the template nucleotide / sequencing primer duplex fixed at a specific position are not removed. In fact, the second detectable labeled nucleotide can be a molecule of the first detectable labeled nucleotide that has not yet been mixed. For example, the first detectable labeled nucleotide can be an A nucleotide, and another A nucleotide can be a second detectable labeled nucleotide. Therefore, for the template nucleotide / sequencing primer duplex at a given position, it can be said that the detectably labeled nucleotide is continuously mixed in a non-circular manner.

[0107] In any of the embodiments herein, the template nucleotides used in the sequencing methods disclosed herein can be in a decimated cluster, wherein some of the template nucleotides in the cluster have been inactivated, such that the inactivated chain does not generate a signal associated with a nucleotide incorporation or non-incorporation event, and the inactivated chain remains "dark" throughout the process of single nucleotide real-time sequencing of non-inactivated chains within the cluster.

[0108] In some embodiments, a method for obtaining a sense strand and antisense strand sequence of a double-stranded nucleic acid such as dsDNA is provided herein. In some embodiments, double-stranded nucleic acids (such as dsDNA) are weakly restricted on a surface, such as on a solid substrate (such as a flow cell). In some embodiments, the method includes providing a single-stranded nucleic acid from a double-stranded nucleic acid, and the provided single-stranded nucleic acid (such as ssDNA) is attached to the surface. In some embodiments, one of the single-stranded nucleic acids or two single-stranded nucleic acids is at a distance (for example, no more than about 8 μm, no more than about 6 μm, no more than about 4 μm, no more than about 2 μm, no more than about 1 μm, no more than about 0.5 μm or no more than about 0.25 μm fixed distance) from the position of the original source double-stranded nucleic acid on the surface. In some embodiments, single-stranded nucleic acids are fixed or fixed at a certain position on the surface.

[0109] In some embodiments, a single-stranded nucleic acid (e.g., ssDNA fixed or fixed to a certain position on a surface) is further amplified, for example, using a bridge amplification process or rolling circle amplification, for example, amplified into clusters or concatemers (e.g., long continuous DNA molecules containing multiple copies of the same DNA sequence connected in series). In some embodiments, single-stranded nucleic acids and their amplicons are sequenced to obtain spatially positioned reads of the sense and antisense strands generated by the same double-stranded nucleic acid (e.g., dsDNA) (e.g., dsDNA fragments of a genome). In some embodiments, the sequences of the sense and antisense strands (e.g., sequencing reads and consensus sequences generated by sequencing reads can be compared and / or combined) to generate a single, higher-precision read. In some embodiments, the sequences of the sense and antisense strands are combined together to generate a single, longer read.

[0110] In some embodiments, the signals from the sense and antisense strands are combined using a model-based algorithm to produce a single higher accuracy read. In some embodiments, other features extracted from the image (e.g., circularity) are used in the model-based base calling process.

[0111] In some embodiments, an electric field is used to attract double-stranded nucleic acids (such as dsDNA) to a surface. In some embodiments, an electric field is used to attract single-stranded nucleic acids (such as ssDNA) to a surface.

[0112] In some embodiments, a charge / electric field barrier is used to confine a single-stranded nucleic acid (e.g., ssDNA). In some embodiments, a double-stranded nucleic acid (e.g., dsDNA) is attracted to a surface, a single-stranded nucleic acid (e.g., ssDNA) is generated from the double-stranded nucleic acid, and the single-stranded nucleic acid is confined to a certain position on the surface, for example, using a charge / electric field barrier.

[0113] In some embodiments, signals from sense and antisense single stranded nucleic acids (e.g., from the same double stranded nucleic acid) cannot be resolved, and thus a mixed signal is generated and detected at a location on the surface. In some embodiments, the mixed signal is base called using a model-based algorithm.

[0114] In some embodiments, provided herein is a method of attaching a cell (e.g., directly or indirectly, covalently or non-covalently) to a surface (e.g., using methods disclosed herein or any other suitable known method), wherein the cell is disrupted (e.g., the cell membrane and / or nuclear membrane is disrupted, e.g., by agents or conditions that disrupt the lipid bilayer), and an electric field is used to confine the cellular material to a certain location on the surface.

[0115] In some embodiments, a negative electric field around a cell attachment site is used to spatially confine cellular material on a surface. In some embodiments, cellular material is attracted to the surface. In some embodiments, a positive electric field is used to attract cellular material (e.g., nucleic acids such as negatively charged DNA and / or RNA) to a location on the surface and reduce diffusion of cellular material from that location on the surface. In some embodiments, once the cellular DNA / RNA is confined, an index is introduced.

[0116] A. Nucleotides and Nucleotide Analogs

[0117] In some embodiments, the methods disclosed herein include the use of one or more nucleotides or their analogs, including natural (native) nucleotides or nucleotide analogs or modified nucleotides (e.g., labeled with one or more detectable markers). In some embodiments, nucleotide analogs include nitrogenous bases, pentoses, and phosphate groups, wherein any component in the nucleotide can be modified and / or substituted. In some embodiments, the methods disclosed herein may include but are not required to use one or more non-incorporable nucleotides. Non-incorporable nucleotides can be modified to be incorporable nucleotides at any time point during the sequencing method.

[0118] Nucleotide analogs include, but are not limited to, α-phosphate modified nucleotides, α-β nucleotide analogs, β-phosphate modified nucleotides, β-γ nucleotide analogs, γ-phosphate modified nucleotides, caged nucleotides, or ddNTPs. Examples of nucleotide analogs are described in U.S. Pat. No. 8,071,755, the entire contents of which are incorporated herein by reference.

[0119] In some embodiments, the methods disclosed herein may include but are not required to use a terminator that reversibly prevents nucleotides from incorporating at the 3' end of a primer. One type of reversible terminator is a 3'-O-blocked reversible terminator. Here, the terminator portion is connected to the oxygen atom at the 3'-OH end of the 5-carbon sugar of the nucleotide. For example, U.S. Patents No. 7,544,794 and No. 8,034,923 (the disclosures of these patents are incorporated herein by reference) describe reversible terminator dNTPs, whose 3'-OH group is replaced by a 3'-ONH2 group. Another type of reversible terminator is a 3'-unblocked reversible terminator, in which the terminator portion is connected to the nitrogenous base of the nucleotide. For example, U.S. Patent No. 8,808,989 (the disclosure of which is incorporated herein by reference) discloses a specific example of a base-modified reversible terminator nucleotide, which can be used in combination with the methods described herein. Other similar reversible terminators that can be used in conjunction with the methods described herein include those described in US Pat. Nos. 7,956,171, 8,071,755, and 9,399,798, all of which are incorporated herein by reference.

[0120] In some embodiments, the methods disclosed herein may include, but do not require, the use of nucleotide analogs with terminator moieties that irreversibly prevent nucleotide incorporation into the 3' end of the primer. Irreversible nucleotide analogs include 2', 3'-dideoxynucleotides, ddNTPs (ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3'-OH group of dNTPs necessary for polymerase-mediated synthesis.

[0121] In some embodiments, the methods disclosed herein may include, but do not require, the use of non-incorporable nucleotides comprising a blocking moiety that inhibits or prevents the nucleotide from forming a covalent bond with another nucleotide (the 3'-OH of the primer) during the incorporation step of a nucleic acid polymerization reaction. The blocking moiety can be removed from the nucleotide to allow the nucleotide to be incorporated.

[0122] In some embodiments, the methods disclosed herein may include but are not required to use 1, 2, 3, 4 or more nucleotide analogs present in the SBS reaction. In some embodiments, the nucleotide analogs are replaced, diluted or isolated in the incorporation step. In some embodiments, the nucleotide analogs are replaced by natural nucleotides. In some embodiments, the nucleotide analogs are modified in the incorporation step. The modified nucleotide analogs can be similar or identical to natural nucleotides.

[0123] In some embodiments, the methods disclosed herein may include but are not required to use nucleotide analogs having a different binding affinity to polymerase than natural nucleotides. In some embodiments, the nucleotide analogs have different interactions with the next base than natural nucleotides. Nucleotide analogs and / or non-incorporable nucleotides can base pair with the complementary base of the template nucleic acid.

[0124] In some embodiments, one or more nucleotides can be marked with distinguishable and / or detectable tags or markers. The tag can be distinguished by the difference of its fluorescence, Raman spectrum, charge, quality, refractive index, emitted light, length or any other measurable characteristics. The tag can be attached to one or more different positions on the nucleotide, as long as the fidelity combined with the polymerase-nucleic acid complex is enough to correctly identify the complementary base on the template nucleic acid. In some embodiments, the tag is attached to the core base of the nucleotide. Or, the tag is attached to the gamma phosphate position of the nucleotide.

[0125] Detectable markers can be applicable to small-scale detection and / or to high-throughput screening. Therefore, suitable detectable markers include but are not limited to radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds and dyes. Detectable markers can be qualitative detection (such as optical detection or spectral detection), or quantitative detection. Qualitative detection generally includes a detection method that confirms the presence or existence of detectable markers, while quantitative detection generally includes a detection method with a value such as intensity, duration, polarization and / or other characteristics that can be quantified (for example, numerable report). In some embodiments, detectable markers are combined with another part such as nucleotides or nucleotide analogs, and can include fluorescence, colorimetric or chemiluminescent markers.

[0126] In some embodiments, the detectable label can be attached to another moiety such as a nucleotide or nucleotide analog. In some embodiments, the detectable label is a fluorophore. For example, the fluorophore can be from the group consisting of: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA), 350, 430, 488, 532, 546, 555, 568, 594, 633, 647, 660, 680, 700, 750, Allophycocyanin (APC), AMCA / AMCA-X, 7-aminoactinomycin D (7-AAD), 7-amino-4-methylcoumarin, 6-aminoquinoline, Aniline Blue, ANS, APC-Cy7, ATTO-TAG TM CBQCA, ATTO-TAG TM FQ, Auramine O-Feulgen, BCECF (high pH), BFP (blue fluorescent protein), BFP / GFP FRET, BOBO TM -1 / BO-PRO TM -1.BOBO TM -3 / BO-PRO TM -3. TMR, TR-X, 530 / 550, 558 / 568, 564 / 570, 581 / 591, 630 / 650-X, 650-665-X, BTC, calcein, calcein blue, calcein red TM , Calcium Green-1 TM , Calcium Orange TM , White, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthylfluorescein, 6-carboxyrhodamine 6G, 5-carboxytetramethylrhodamine (5-TAMRA), carboxy-X-rhodamine (5-ROX), Cascade Cascade Yellow TM 、CCF2(GeneBLAzer TM ), CFP (cyan fluorescent protein), CFP / YFP FRET, Chromomycin A3, Cl-NERF (low pH), CPM, 6-CR6G, CTC formazan, Cychrome (PE-Cy5), Dansylamine, Dansyl cadaverine, Dansyl chloride, DAPI, Dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18 (5)), DIDS, Dil (DilC18 (3)), DiO (DiOC18 (3)), DiR (DilC18 (7)), Di-4 ANEPPS, Di-8 ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (red fluorescent protein), EBFP, ECFP, EGFP, -97 alcohol, eosin, erythrosine, ethidium bromide, ethidium homodimer-1 (EthD-1), europium (III) chloride, 5-FAM (5-carboxyfluorescein), Fast Blue, fluorescein-dT phosphoramidite, FITC, Fluo-3, Fluo-4, Fluor-Gold TM (High pH), Fluor-Gold TM (low pH), Fluor-Jade, 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red TM (High calcium), Fura Red TM / Fluo-3、GeneBLAzer TM (CCF2), GFP red shift (rsGFP), GFP wild type, GFP / BFP FRET, GFP / DsRed FRET, Hoechst 33342 & 33258, 7-hydroxy-4-methylcoumarin (pH 9), 1,5IAEDANS, Indo-1 (high calcium), Indo-1 (low calcium), Indodicarbocyanine, Indotricarbocyanine, JC-1, 6-JOE, JOJO TM -1 / JO-PRO TM -1, LDS 751(+DNA), LDS 751(+RNA), LOLO TM -1 / LO-PRO TM -1. Fluorescent yellow, LysoSensor TM Blue (pH5), LysoSensor TM Green (pH 5), LysoSensor TM Yellow / blue (pH 4.2), green, red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green TM (MagnesiumGreen TM )、Marina 4-Methylumbelliferone, Mithramycin, green, orange, Red, NBD (amine), Nile Red, Oregon 488. Oregon 500. Oregon 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (chlorophyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (propidium iodide), PKH26, PKH67, POPO TM -1 / PO-PRO TM -1. POPO TM -3 / PO-PRO TM -3, propidium iodide (PI), PyMPO, pyrene, Pyronin Y, Quantam Red (PE-Cy5), Quinacrine Mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), Red Fluorescent Protein (DsRed), Resorufin, RH 414, Rhod-2, Rhodamine B, Rhodamine Green TM , Rhodamine Red TM , rhodamine phospholipid protein, rhodamine 110, rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, -1 (high pH), -2. -1 (high pH), -1 (low pH), sodium green TM , #1, #2, 11. 13. 17. 45. blue, green, Orange, 5-TAMRA (5-carboxytetramethylrhodamine), tetramethylrhodamine (TRITC), / -X, -X (NHS ester), Thiadicarbocyanine, Thiazole Orange, -1 / TO- -1. -3 / TO- -3.TO- -5, tricolor (PE-Cy5), TRITC (tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (yellow fluorescent protein), -1 / YO- -1. -3 / YO- -3, 6-FAM (fluorescein), 6-FAM (NHS ester), 6-FAM (azide), HEX, TAMRA (NHS ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 542, ATTO550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5' 700, 5' 800, 5' 800CW (NHS ester), WellRED D4 dye, WellRED D3 dye, WellRED D2 dye, 640 (NHS ester) and Dy750 (NHS ester).

[0127] Detectable markers can be directly detectable (such as radioisotope labels or fluorescent markers) by themselves, or in the case of enzyme labels, can be indirectly detectable, such as by catalyzing the chemical changes of substrate compounds or compositions, and substrate compounds or compositions are directly detectable. The marker can emit a signal or change the signal passed to the marker, so that the presence or absence of the marker can be detected. In some cases, coupling can be achieved by a joint, and the joint can be cleavable, such as photocleavable (such as cleavable under ultraviolet light), chemically cleavable (such as by a reducing agent, such as dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP)) or enzymatically cleavable (such as by esterase, lipase, peptidase or protease).

[0128] B. polymerase

[0129] Polymerases that can be used to implement the disclosed technology include naturally occurring polymerases and any modified variants thereof, including but not limited to mutants, recombinants, fusions, genetic modifications, chemical modifications, synthetics and the like. Naturally occurring polymerases and modified variants thereof are not limited to polymerases that retain the ability to catalyze polymerization reactions. In some embodiments, naturally occurring polymerases and / or their modified variants retain the ability to catalyze polymerization reactions. In some embodiments, naturally occurring polymerases and / or their modified variants have special properties that enhance their ability to DNA sequencing, including enhanced binding affinity to nucleic acids, reduced binding affinity to nucleic acids, enhanced catalytic rate, reduced catalytic rate, etc. Mutant polymerases include polymerases in which one or more amino acids are replaced by other amino acids (natural or non-natural amino acids) and polymerases in which one or more amino acids are inserted or deleted.

[0130] In some embodiments, the methods disclosed herein may include but do not require the use of a modified polymerase containing an external label (e.g., an exogenous detectable marker) that can be used to monitor the presence and interaction of the polymerase. In some embodiments, the intrinsic signal of the polymerase can be used to monitor their presence and interaction. Therefore, the provided method can include monitoring the interaction of the polymerase, nucleotides, and template nucleic acid by detecting the intrinsic signal of the polymerase. In some embodiments, the intrinsic signal is a light scattering signal. For example, the intrinsic signal includes the natural fluorescence of certain amino acids (such as tryptophan).

[0131] In some embodiments, the methods disclosed herein may include the use of an unlabeled polymerase and monitoring in the absence of an exogenous detectable label associated with the polymerase. Under certain reaction conditions, some modified polymerases or naturally occurring polymerases may only incorporate a single nucleotide and maintain binding to the primer-template after incorporation of a single nucleotide.

[0132] In some embodiments, method disclosed herein may include using a polymerase that is not labeled by an exogenous detectable marker (such as a fluorescent marker). After using protein separation technology to purify polymerase at least in part, the marker can be chemically connected to the structure of the polymerase by a covalent bond. For example, an exogenous detectable marker can be chemically connected to the polymerase by the free sulfhydryl or free amine moiety of the polymerase. This may relate to being chemically connected to the polymerase by the side chain of a cysteine ​​residue or by the free amino group of the N-terminus. In some preferred embodiments, the fluorescent marker attached to the polymerase helps the positioning of the polymerase, which is very important for determining whether the polymerase is positioned to the point position on the array corresponding to the fixed primer template nucleic acid. Fluorescent signal does not need to change absorption or emission characteristics because of combining any nucleotide, and in some embodiments, absorption or emission characteristics will not be changed because of combining any nucleotide. In some embodiments, the signal emitted by the marker polymerase remains consistent in the presence and absence of any nucleotide studied as the next possible correct nucleotide.

[0133] As used herein, the term polymerase and variants thereof also refers to a fusion protein comprising at least two interconnected portions, for example, one portion comprising a peptide that catalyzes the polymerization of nucleotides into a nucleic acid chain, which is connected to another portion, the other portion comprising a second moiety, such as a reporter enzyme or a processivity-modifying domain. For example, T7 DNA polymerase comprises a nucleic acid polymerization domain and a thioredoxin binding domain, wherein thioredoxin binding increases the processivity of the polymerase. Without the binding of thioredoxin, T7 DNA polymerase is a distributive polymerase with a processivity of only one to a few bases. Although DNA polymerases differ in detail, their overall shape resembles a hand, with specific regions called fingers, palms, and thumbs; during the process of synthesizing nucleic acids, there are similar overall structural transitions, including the movement of the thumb and / or finger domains.

[0134] DNA polymerases include, but are not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases, and bacteriophage DNA polymerases. Bacterial DNA polymerases include E. coli DNA polymerase I, E. coli DNA polymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, and E. coli DNA polymerase V, the Klenow fragment of E. coli DNA polymerase, Clostridium stercorarium (Cst) DNA polymerase, Clostridium thermocellum (Cth) DNA polymerase, and Sulfolobus solfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases include DNA polymerase α, DNA polymerase β, DNA polymerase γ, DNA polymerase δ, DNA polymerase ε, DNA polymerase η, DNA polymerase ζ, DNA polymerase λ, polymerase σ, DNA polymerase μ and DNA polymerase κ, as well as Revl polymerase (terminal deoxycytidine transferase) and terminal deoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerase, PZA DNA polymerase, phi-15 DNA polymerase, Cpl DNA polymerase, Cp7 DNA polymerase, T7 DNA polymerase and T4 polymerase. Other DNA polymerases include thermostable and / or thermophilic DNA polymerases, such as DNA polymerases isolated from Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus sp.) GB-D polymerase, Thermotoga maritima (Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase, Sulfolobus acidocaldarius DNA polymerase, Thermococcus go N-7 DNA polymerase, Pyrodictium polymerase, Methanococcus wollastonii (Methanococcus voltae DNA polymerase, Methanococcusthermoautotrophicum DNA polymerase, Methanococcus jannaschii DNA polymerase, Desulfurococcus strain TOK DNA polymerase (D.TokPol), Pyrococcus abyssi DNA polymerase, Pyrococcus horikoshii DNA polymerase, Pyrococcusislandicum DNA polymerase, Thermococcus fumicolans DNA polymerase, Aeropyrumpernix DNA polymerase, and heterologous DNA polymerases DP1 / DP2. Engineered and modified polymerases are also suitable for use with the disclosed technology. For example, the extremely thermophilic marine archaeon Thermococcus 9°N (e.g., Therminator from New England Biolabs, Inc.) can be used. DNA polymerase; Ipswich, MA). Other useful DNA polymerases, including 3PDX polymerase, are disclosed in U.S. Pat. No. 8,703,461, the entire disclosure of which is incorporated by reference.

[0135] RNA polymerases include, but are not limited to, viral RNA polymerases, such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and K11 polymerase; eukaryotic RNA polymerases, such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V; and archaeal RNA polymerases.

[0136] Reverse transcriptases include, but are not limited to, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2 reverse transcriptase from human immunodeficiency virus type 2, M-MLV reverse transcriptase from Moloney murine leukemia virus, AMV reverse transcriptase from avian myeloblastosis virus, and telomerase reverse transcriptase that maintains eukaryotic chromosome telomeres.

[0137] C. Sequencing reaction

[0138] In some embodiments of the synthetic sequencing (SBS) method disclosed herein, the first labeled nucleotide that has been incorporated will not be inactivated (e.g., by removal and / or photobleaching of the label) before the introduction and / or incorporation of the next second labeled nucleotide. The first labeled nucleotide and the second labeled nucleotide may contain the same base or different bases. The first labeled nucleotide and the second labeled nucleotide may be introduced into the sequencing reaction mixture in any order at the same time or at different time points. In addition, the first labeled nucleotide and the second labeled nucleotide may be introduced separately (e.g., in a suitable solvent such as water), or may be mixed and introduced with another sequencing reagent (e.g., one or more other labeled nucleotides and / or one or more unlabeled nucleotides). The first labeled nucleotide and the second labeled nucleotide may also contain the same base or different bases. In some embodiments, nucleotides that have not yet been incorporated at a residue corresponding to a base in the template nucleic acid (e.g., because the first labeled nucleotide has been incorporated into the residue) are not removed from the sequencing reaction mixture before the introduction and / or incorporation of the second labeled nucleotide. In some embodiments, the first labeled nucleotide and the second labeled nucleotide (and optionally labeled nucleotides for subsequent bases in the query template) are provided in the same sequencing reaction mixture, and the first labeled nucleotide, the second labeled nucleotide, and optionally any subsequent labeled nucleotides are incorporated sequentially in a continuous manner.

[0139] Thus, unlike existing SBS methods, some embodiments of the methods disclosed herein use continuous introduction and / or incorporation of nucleotides (e.g., fluorescently labeled A, T, C, and / or G nucleotides) without requiring label inactivation and / or washing steps between successive incorporation events for a given template nucleic acid molecule to be sequenced. Instead, in some embodiments, label inactivation (e.g., by cleavage and / or photobleaching of the label) of the first incorporated nucleotide can occur randomly throughout the continuous nucleotide incorporation process, e.g., before, during, or after incorporation of the second, third, fourth, or subsequent labeled nucleotides.

[0140] Nucleic acid sequencing reaction mixtures, or simply "reaction mixtures", typically include reagents commonly present in polymerase-based nucleic acid synthesis reactions. The reaction mixture may include other molecules, including but not limited to enzymes. In some embodiments, the reaction mixture contains any reagents or biomolecules commonly present in nucleic acid polymerization reactions. Reaction components may include but are not limited to salts, buffers, small molecules, detergents, crowding agents, metals, and ions. In some embodiments, the properties of the reaction mixture can be manipulated by electrical, magnetic, and / or vibrational means.

[0141] The methods provided herein may further include, but do not require, one or more washing steps, temperature changes, mechanical vibrations, pH changes, or optical stimulations other than dye illumination or photobleaching. In some embodiments, the washing steps include contacting the substrate and nucleic acid molecules, primers and / or polymerases with one or more buffers, detergents, protein denaturants, proteases, oxidants, reducing agents, or other agents capable of crosslinking or releasing crosslinks (such as crosslinks within the polymerase or crosslinks between the polymerase and the nucleic acid). Methods and compositions for nucleic acid sequencing are known, such as described in U.S. Pat. Nos. 10,246,744 and 10,844,428, the entire contents of which are incorporated herein by reference for all purposes.

[0142] Reaction mixture reagents may include, but are not limited to, enzymes (such as polymerases), dNTPs, template nucleic acids, primer nucleic acids, salts, buffers, small molecules, cofactors, metals, and ions. The ions may be catalytic ions, divalent catalytic ions, non-catalytic ions, non-covalent metal ions, or combinations thereof. The reaction mixture may include salts that ionize in aqueous solution to generate monovalent cations, such as NaCl, KCl, potassium acetate, ammonium acetate, potassium glutamate, or NH4Cl. The reaction mixture may include an ion source, such as Mg 2+ , Mn 2+ 、Co 2+ 、Cd 2+ and / or Ba 2+ ions. The reaction mixture may include tin, Ca 2+ 、Zn 2+ , Cu 2+ 、Co 2+ , Fe 2+ and / or Ni 2+ Or other divalent non-catalytic metal cations. In some embodiments, the reaction mixture may include a metal cation that can inhibit the formation of a phosphodiester bond between the primer template nucleic acid molecule and the homologous nucleotide. In some embodiments, the metal cation can be used to (e.g., at a suitable concentration) slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0143] In some embodiments, the sequencing reaction conditions include contacting the nucleic acid molecule and the primer with a buffer for regulating osmotic pressure. In some embodiments, the reaction mixture includes a buffer for regulating osmotic pressure. In some embodiments, the buffer is a high salt buffer, including a monovalent ion of about 50mM to about 1,500mM, such as a monovalent metal ion (such as potassium ion or sodium ion) at a concentration of about 100mM to about 1,500mM. A buffer with a salt concentration in the range of about 200mM to 1,000mM can also be used. In some embodiments, the buffer also includes a glutamate ion source (such as potassium glutamate). In some embodiments, the buffer includes a stabilizer. In some embodiments, the stabilizer is a non-catalytic metal ion (such as a divalent non-catalytic metal ion). Non-catalytic metal ions useful in this case include but are not limited to calcium, strontium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium and / or terbium. In some embodiments, the non-catalytic metal ion is strontium, tin or nickel. In some embodiments, the sequencing reaction mixture comprises strontium chloride or nickel chloride.In some embodiments, a stabilizer can be used (eg, at an appropriate concentration) to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0144] The buffer may include Tris, trimethylglycine (Tricine), HEPES, MOPS, ACES, MES, phosphate-based buffers and acetate-based buffers. The reaction mixture may include a chelating agent such as EDTA, EGTA, etc. In some embodiments, the reaction mixture includes a cross-linking agent.

[0145] In some embodiments, the interaction between the polymerase and the template nucleic acid can be manipulated by adjusting sequencing reaction parameters such as ionic strength, pH, temperature, or any combination thereof, or by adding a destabilizing agent to the reaction. In some embodiments, a destabilizing agent can be used (e.g., at an appropriate concentration) to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0146] In some embodiments, high salt (e.g., 50mM to 1,500mM) and / or pH changes are used to destroy the stability of the complex between the polymerase and the template nucleic acid. In some embodiments, the reaction conditions are conducive to the stability of the complex between the polymerase, the template nucleic acid, and the labeled nucleotide. For example, the pH of the reaction mixture can be adjusted from 4.0 to 10.0 to facilitate the stability of the complex between the polymerase, the template nucleic acid, and the labeled nucleotide. In some embodiments, the pH of the reaction mixture is 4.0 to 6.0. In some embodiments, the pH of the reaction mixture is 6.0 to 10.0. In some embodiments, suitable salt concentrations and / or suitable pH can be selected to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0147] In some embodiments, the reaction mixture comprises a competitive inhibitor, wherein the competitive inhibitor can reduce the occurrence of multiple incorporation events in a detection window. In one embodiment, the competitive inhibitor is a non-incorporable nucleotide. In one embodiment, the competitive inhibitor is an aminoglycoside. The competitive inhibitor can replace the nucleotide or catalytic metal ion in the active site, so that the competitive inhibitor occupies the active site, preventing or slowing down the incorporation of the nucleotide. In some embodiments, both incorporable nucleotides and competitive inhibitors are introduced, so that the ratio of incorporable nucleotides and inhibitors can be adjusted to regulate the incorporation rate of a single nucleotide at the 3' end of the primer. In some embodiments, competitive inhibitors can be used to (e.g., at low concentrations) slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0148] In some embodiments, the reaction mixture comprises at least one nucleotide molecule that cannot be incorporated into nucleotides. In some embodiments, the reaction mixture comprises one or more nucleotide molecules that cannot be incorporated into the primer of the primer template nucleic acid molecule. Such nucleotides that cannot be incorporated include, for example, monophosphate nucleotides. For example, nucleotides may contain a triphosphate group modification that makes nucleotides unincorporable. Examples of nucleotides that cannot be incorporated can be found in U.S. Patent No. 7,482,120, the entire contents of which are incorporated herein by reference. In some embodiments, primers may not contain free hydroxyls at their 3' ends, so that primers cannot be incorporated into any nucleotides, and thus any nucleotides cannot be incorporated. In some embodiments, primers may be treated so that they contain free hydroxyls at their 3' ends, to allow nucleotides to be incorporated. In some embodiments, nucleotides that cannot be incorporated can be used to (for example, at low concentrations) slow down but not completely inhibit or prevent nucleotides from being incorporated, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0149] In some embodiments, the reaction mixture comprises at least one nucleotide molecule that can be incorporated into a nucleotide but has a slower incorporation rate than the corresponding naturally occurring nucleoside triphosphate (such as NTP or dNTP). For example, such a nucleotide that can be incorporated at a slower rate can include a diphosphate nucleotide. For example, a nucleotide can contain a modification to a triphosphate group so that the incorporation rate of the nucleotide is slower. In some embodiments, a nucleotide that can be incorporated at a slower rate can be used to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0150] In some embodiments, the reaction mixture comprises a polymerase inhibitor. In some embodiments, the polymerase inhibitor is a pyrophosphate analog. In some embodiments, the polymerase inhibitor is an allosteric inhibitor. In some embodiments, the polymerase inhibitor is a DNA aptamer or an RNA aptamer. In some embodiments, the polymerase inhibitor competes with the catalytic ion binding site in the polymerase. In some embodiments, the polymerase inhibitor is a reverse transcriptase inhibitor. The polymerase inhibitor can be an HIV-1 reverse transcriptase inhibitor or an HIV-2 reverse transcriptase inhibitor. HIV-1 reverse transcriptase inhibitor can be (benzo [d] thiazol-2-yl substituted by 4 / 6-halogen / MeO / EtO-) thiazolidine-4-one. In some embodiments, the polymerase inhibitor can be used for (e.g., at low concentrations) to slow down but not completely inhibit or prevent nucleotide incorporation, thereby reducing multiple nucleotide incorporation events in a single detection window.

[0151] In some embodiments, the contacting step is facilitated by using a chamber such as a flow cell. The methods and apparatus described herein can use next generation sequencing technology (NGS), which allows large-scale parallel sequencing. In some embodiments, a single DNA molecule is sequenced in a large-scale parallel manner in a reaction chamber. A flow cell can be used, but it is not necessary. Liquid reagents flow through the flow cell, which contains an internal solid support surface (such as a plane) for convenient reagent exchange. Fixed on the inner surface of the flow cell are one or more primer template nucleic acids, which will be sequenced or detected using the procedures described herein. A typical flow cell includes a microfluidic valve that allows liquid reagents (such as components of the "reaction mixture" discussed herein) to be delivered to the inlet. Liquid reagents can be discharged from the flow cell through an exit port.

[0152] In some embodiments, the reaction chamber disclosed herein may include a reagent wall, an imaging zone, and an optional outlet, the outlet being configured to remove one or more molecules of a polymerase, a first detectably labeled nucleotide, a second detectably labeled nucleotide, and / or one or more other reagents from the imaging zone. In some embodiments, the device may include one or more vents, but there is no outlet or outlet for the reaction mixture. In some embodiments, the method disclosed herein does not include a step of removing a liquid reagent, for example, from a reaction chamber such as a flow cell, by an outlet or an outlet.

[0153] The method disclosed herein can be but need not be used in combination with any NGS sequencing method. NGS sequencing techniques include, but are not limited to, pyrophosphate sequencing, synthetic sequencing using reversible dye terminators, oligonucleotide probe ligation sequencing, and ion semiconductor sequencing. Nucleic acids (such as DNA or RNA) from a single sample can be sequenced individually (singleplex sequencing), and nucleic acids (such as DNA or RNA) from multiple samples can be pooled together and sequenced (multiplex sequencing) as indexed genomic molecules in a single sequencing run to generate up to hundreds of millions of sequence reads. Examples of sequencing techniques that can be used to obtain sequence information according to the present method are further described herein.

[0154] Some sequencing technologies are commercially available, such as the hybridization sequencing platform from Affymetrix Inc. (Sunnyvale, Calif.), the synthetic sequencing platforms from 454 Life Sciences (Bradford, Conn.), Illumina / Solexa (Hayward, Calif.), and Helicos Biosciences (Cambridge, Mass.), and the ligation sequencing platform from Applied Biosystems (Foster City, Calif.). In addition to single-molecule sequencing using synthetic sequencing from Helicos Biosciences, other single-molecule sequencing technologies include, but are not limited to, SMRT from Pacific Biosciences. TM Technology, ION TORRENT TM Technology and nanopore sequencing technology developed by Oxford Nanopore Technologies and others.

[0155] Although the automated Sanger method is considered a "first generation" technique, Sanger sequencing, including automated Sanger sequencing, may also be employed in the methods described herein. Other suitable sequencing methods include, but are not limited to, nucleic acid imaging techniques, such as atomic force microscopy (AFM) or transmission electron microscopy (TEM).

[0156] In some embodiments, the disclosed methods can be used in conjunction with massively parallel sequencing of nucleic acid molecules using Illumina synthesis sequencing and reversible terminator-based sequencing chemistry. In some embodiments, the methods disclosed herein can use a flow cell having a slide with lanes.

[0157] After sequencing the nucleic acid molecule, the sequence reads of a predetermined length (e.g., at least about 15 bp) are located by mapping (aligning) with a known reference sequence or genome (e.g., a viral sequence or genome). Many computer algorithms can be used for sequence alignment, including but not limited to BLAST, BLITZ, FASTA, BOWTIE, or ELAND (Illumina, Inc., San Diego, Calif., USA).

[0158] In one illustrative but non-limiting embodiment, the methods described herein may include obtaining sequence information of nucleic acids in a test sample, for example, using a single molecule sequencing technique similar to Helicos True Single Molecular Sequencing (tSMS) technology. In tSMS technology, a DNA sample is cut into chains of approximately 100 to 200 nucleotides, and a polyA sequence is added to the 3' end of each DNA chain. Each chain is labeled by adding fluorescently labeled adenine nucleotides. The DNA chains are then hybridized to a flow cell containing millions of oligo-T capture sites fixed to the surface of the flow cell. In some embodiments, the density of the templates can be approximately 100 million templates / cm 2 The flow cell is then loaded into an instrument such as the HeliScope TM In a sequencer, a laser illuminates the surface of a flow cell, revealing the location of each template. A CCD camera maps the location of the templates on the surface of the flow cell. Then, the template fluorescent marker is cleaved and washed away. The sequencing reaction is initiated by introducing a DNA polymerase and fluorescently labeled nucleotides. An oligo-T nucleic acid is used as a primer. The polymerase incorporates the labeled nucleotides into the primer in a template-directed manner. The polymerase and unincorporated nucleotides are removed. Templates with directed incorporation of fluorescently labeled nucleotides can be identified by imaging the surface of the flow cell. After imaging, a cleavage step removes the fluorescent marker, and the process is repeated with additional fluorescently labeled nucleotides until the desired read length is achieved. Sequence information is collected with each nucleotide addition step. Whole genome sequencing by single-molecule sequencing technology excludes or generally avoids PCR-based amplification when preparing sequencing libraries, and these methods allow for direct measurement of samples rather than measurements of copies of the sample.

[0159] In another illustrative but non-limiting embodiment, the methods described herein may include obtaining sequence information of nucleic acids in a test sample, similar to the single molecule real-time (SMRT) method of Pacific Biosciences. TM ) sequencing technology. In SMRT sequencing, the continuous incorporation of dye-labeled nucleotides is imaged during DNA synthesis. A single DNA polymerase molecule is attached to the bottom surface of a separate zero-mode wavelength detector (ZMW detector), which can obtain sequence information when phosphate-linked nucleotides are incorporated into a growing primer chain. The ZMW detector includes a closed structure that can observe the incorporation of a single nucleotide by a DNA polymerase against a background in which fluorescent nucleotides rapidly diffuse outside the ZMW detector (e.g., within microseconds). It usually takes a few milliseconds to incorporate nucleotides into a growing chain. During this period, the fluorescent marker is excited and produces a fluorescent signal, and then the fluorescent marker is cleaved. The measurement of the corresponding dye fluorescence indicates which base is incorporated. This process is repeated to provide a sequence.

[0160] In some embodiments, the sequencing methods disclosed herein can regulate the interaction of polymerase with nucleotides and template nucleic acids (and the rate of nucleotide incorporation) to reveal the identity of the next base while controlling the chemical addition of nucleotides. In some embodiments, the SBS reaction conditions include multiple primer template nucleic acids, polymerases, nucleotides, or any combination thereof. In some embodiments, multiple nucleotides include 1, 2, 3, 4 or more types of different nucleotides, such as dATP, dTTP (or dUTP), dGTP and dCTP. In some embodiments, multiple template nucleic acids are single molecules fixed on a substrate for single molecule sequencing.

[0161] In some embodiments, the method may further include contacting the nucleic acid molecule with a substrate to immobilize the nucleic acid molecule. In some embodiments, the immobilization density of the nucleic acid molecule may be at least about 250 nm. 2 One molecule, at least about 200nm 2 One molecule, at least about 150nm 2 One molecule, at least about 100nm 2 One molecule, each at least about 90nm 2 One molecule, each at least about 80nm 2 One molecule, at least 70nm 2 One molecule, at least about 60nm 2 One molecule, at least 50nm 2 One molecule, at least 40nm 2 One molecule, at least about 30nm 2 One molecule, at least about 20nm 2One molecule, every at least about 10nm 2 One molecule, each at least about 5nm 2 One molecule or one molecule per area between any two of the above values. Methods and compositions for arranging biomolecules on a substrate, for example, as described in US2005 / 0042649 (the entire contents of which are incorporated herein by reference for all purposes), can be used in the methods disclosed herein.

[0162] In some embodiments, nucleic acid molecules, polymerase molecules and / or sequencing primers may be provided on a substrate for super-resolution signal detection. For example, two nucleic acid molecules to be sequenced may be located at two points close to each other. If only one point emits light at any time, a positioning-based technique may be used to resolve the point position to a sub-diffraction limit resolution, thereby assigning the detected signal (such as light emission) to different molecules / chains in the synthesis. In this case, the nucleic acid molecules to be sequenced may be separated by about 20 nm per 100 nm. 2 One molecule, every 15nm 2 One molecule, every 10nm 2 One molecule, each at least about 5nm 2 Densities of one molecule or higher are packed onto the substrate.

[0163] In some embodiments, the detectable marker may include one or more scintillation markers, which can be used to achieve super-resolution positioning of the sequenced nucleic acid chain during single-molecule level sequencing. In some embodiments, markers with different scintillation properties can be used to label one or more nucleotides. In some embodiments, the detectable marker may include one or more markers that exhibit random scintillation (also known as photoluminescence intermittent), such as quantum dots. The scintillation phenomenon may be caused by local electric fields, non-radiative Auger recombination and / or surface trap induced recombination due to high excitation power. The scintillation can be light-induced or spontaneous, such as Stefani et al., "Quantification of photoinduced and spontaneous quantum-dot luminescence blinking," Physical Review B 72, 125304 (2005), the entire contents of which are incorporated herein by reference for all purposes. It is generally believed that inherent quantum dot blinking interferes with fluorescence quenching analysis, and there are some techniques that can be used to limit intermittent fluorescence. In some embodiments herein, scintillation markers (such as quantum dots) can be used, for example, when the density of nucleic acid molecules on the substrate is high. In instances where two nucleotides with scintillation markers or one nucleotide with a scintillation marker and another nucleotide with a non-scintillation marker are incorporated at two adjacent sites, the signals detected at one or more time points when only one of the two markers emits light can be used to distinguish the positions of the two adjacent sites.

[0164] In some embodiments, a subset of nucleic acid molecules on a substrate (e.g., nucleic acid chains to be sequenced) may be active at one or more time points. In some embodiments, at any one time point, a first subset of nucleic acid molecules on a substrate is active (e.g., allowing nucleotides to be incorporated into sequencing primers using single-stranded sequences as templates), while a second subset of nucleic acid molecules on a substrate is not active (e.g., not allowing nucleotides to be incorporated into sequencing primers using single-stranded sequences as templates). In some embodiments, at one or more time points, a first subset of nucleic acid molecules on a substrate is activated (e.g., by a first set of polymerases and / or primer molecules) for nucleotide incorporation, while a second subset of nucleic acid molecules on a substrate is not activated (e.g., by a first set of polymerases and / or primer molecules), so that only signals associated with the first subset of nucleic acid molecules are detected. At one or more other time points, the second subset of nucleic acid molecules on the substrate is activated (e.g., by the second set of polymerases and / or primer molecules) for nucleotide incorporation, while the first subset of nucleic acid molecules on the substrate is not activated (e.g., by the second set of polymerases and / or primer molecules), so that only signals associated with the second subset of nucleic acid molecules are detected. In some embodiments, the first set and the second set of polymerases and / or primer molecules can be introduced at different time points, such as in consecutive cycles, with optional washing steps between cycles (e.g., to remove one set of polymerases and / or primer molecules for SBS of the first subset of chains before introducing the next set of polymerases and / or primer molecules for SBS of the second subset of chains). In some embodiments, nucleotide incorporation templated for a particular chain can be performed in a non-cyclic manner as described herein, regardless of whether the particular chain being sequenced belongs to the first subset or the second subset.

[0165] In some embodiments, the substrate may include beads, planar substrates, solid surfaces, flow cells, semiconductor chips, wells, columns, chambers, channels, through holes, nanopores, or any combination thereof. In some embodiments, the substrate may include micropores, microcolumns, microchambers, microchannels, or any combination thereof.

[0166] D. Signal inactivation

[0167] In some embodiments, one or more incorporated nucleotides may be randomly inactivated in a non-cyclic manner (e.g., by photobleaching and / or cleavage of the label). In some embodiments, for a given labeled nucleotide, once the label is cleaved or inactivated, the signal intensity associated with the nucleotide (if any) will no longer change, for example, no longer respond to the light that bleaches the labels on other nucleotides. For example, in one embodiment, after the fluorescent dye of a specific dye-labeled nucleotide is photobleached (so the fluorescence intensity associated with the dye-labeled nucleotide drops from a first intensity to a lower second intensity), the photobleached dye-labeled nucleotide will not recover to the first fluorescence intensity. In some embodiments, the fluorescence intensity of the photobleached dye-labeled nucleotide remains at a second intensity, which may be zero; that is, the photobleached dye may become "non-luminescent", for example, its signal is below a certain threshold or is undetectable, and will not recover. In some embodiments, in the methods disclosed herein, the increase in signal intensity due to a nucleotide incorporation event will not be detected as an increase in signal intensity due to the recovery of the photobleached dye from the bleached state. In some embodiments, the dye photobleached herein is prevented from recovering from the bleached state, so that the increase in signal intensity is attributed to the recovery of nucleotide incorporation rather than photobleaching. In some embodiments, for each marker that has been inactivated (such as photobleached), the inactivation is complete because the inactivated marker will not recover. In some embodiments, markers located at multiple positions (some of which may contain the same marker and others may contain different markers) will not be inactivated (such as photobleached) at the same time or in the same time window (such as the same cycle). On the contrary, in the method disclosed herein, markers located at different positions can be randomly inactivated, so that at a given time point or within a given time window, markers at all positions of the substrate will not be completely inactivated, and for each marker, the signal inactivation is complete or will be complete (for example, no signal recovers from the inactivated state).

[0168] In some embodiments where the dye can be recovered from an inactive state (e.g., after photobleaching), the probability of recovery can be modeled and used during base calling. In some embodiments, the probability of recovery is modeled using a reference-based correction. Methods for dye recovery from photobleaching have been described in, for example, Braslavsky et al., "Sequence information can be obtained from single DNA molecules," PNAS 100(7):3960-64 (2003), the entire contents of which are incorporated herein by reference for all purposes.

[0169] In some embodiments, the gradual change of the luminescence of the fluorophore at a specific point over time (e.g., the gradual increase and / or decrease of the signal intensity) can be detected and / or monitored. In a specific point and a given time window or time point (e.g., an imaging window in frames / exposure meters), the increase in signal intensity (e.g., due to the incorporation of nucleotides) and / or the reduction in signal (e.g., due to photobleaching events) can partially or completely offset each other. In some embodiments, the incorporation of labeled nucleotides can cause an increase in the characteristic signal intensity of the marker and / or base into which the labeled nucleotide has been incorporated. For example, a nucleotide can be labeled with a marker having a characteristic signal intensity of the nucleotide base, and the signal intensity of the marker can be distinguished from the signal intensity of the marker on another nucleotide with a different base. In some embodiments, the signal inactivation of the labeled nucleotide (e.g., by cleavage and / or photobleaching of the marker) can cause a reduction in the characteristic signal intensity of the marker and / or base of the labeled nucleotide with the signal inactivation.

[0170] E. Deconvolution and Base Calling

[0171] In some embodiments, each type of nucleotide (e.g., nucleotides containing A, T / U, C, or G) can be labeled with a different fluorophore so that the emission of a particular fluorophore will pass through one filter while being rejected by all other filters. An exemplary high-throughput sequencing platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence is described in Chen et al., PNAS 111 (2) 664-669 (2014), the entire contents of which are incorporated herein by reference for all purposes.

[0172] In some embodiments, the method provided herein includes the use of a marker with different intensities (e.g., brightness) within a certain wavelength range. When used in combination with an appropriate optical filter, different dyes can be recorded as different intensities using a single fixed optical filter and camera. The advantage of doing so is that the optical system can be made simpler and cheaper. This labeling scheme can be used for a real-time environment (such as no circulation, no terminator) where each nucleotide is randomly incorporated and bleached. For example, the dye on the incorporated nucleotide may not be completely bleached (or otherwise randomly removed) before the subsequent nucleotide is incorporated. In some aspects, the composition of the base (e.g., a continuous nucleic acid sequence) can be determined by a real-time sequencing method, in which nucleotides are randomly incorporated and markers are randomly bleached. In some embodiments, imaging is continuous so as to observe all incorporation events. In some cases, the average incorporation rate can be adjusted (e.g., by nucleotide concentration and / or polymerase activity) so that multiple incorporations are unlikely to occur in a single frame. Similarly, the photobleaching rate can also be adjusted (e.g., by laser intensity or oxygen scavenging additives).

[0173] Although photobleaching may occur in either order, incorporation and photobleaching events are matched. At the single-molecule level, photobleaching can occur with a fixed probability at each time point. By tracking incorporation events and photobleaching events, the nucleic acid sequence of the synthesizing strand and the complementary template strand can be determined. Hidden Markov models (HMMs) can be used to deconvolute the detected signal intensity over time to detect incorporation and photobleaching events.

[0174] In some embodiments, the net change in signal intensity at a specific point and a given time window or time point can be associated with an event at the specific point, for example, the incorporation of a new labeled nucleotide and the photobleaching of one or more incorporated labeled nucleotides. One or more incorporated labeled nucleotides can maintain an arbitrary distance from the newly incorporated labeled nucleotide, for example, 0, 1, 2, 3, 4, 5 or more nucleotide residues. In some embodiments, the net change in signal intensity can be deconvoluted into one or more increases and / or one or more decreases in the characteristic signal intensity of nucleotide incorporation events (such as incorporation of nucleotides labeled with a specific fluorophore) and signal inactivation events (such as photobleaching of the same or another specific fluorophore), respectively.

[0175] In some embodiments, the inactivation step and / or the detection step can be carried out when the detectably labeled nucleotides are continuously provided to contact nucleic acid molecules and / or primers. In some embodiments, the detection step is carried out in real time when nucleotide incorporation and signal inactivation (e.g., photobleaching) events occur. In some embodiments, the detection step is carried out without using multiple switchable optical filters (each optical filter is used to detect different detectable labels). In some embodiments, the detection step can use a dichroic filter to divide the light signal into multiple channels, thereby detecting different detectable labels in each channel. In some embodiments, the detection step can use a total internal reflection fluorescence (TIRF) microscope to carry out. In some embodiments, the signal in the detection step can compensate for the background signal.

[0176] In some embodiments, using time track to carry out nucleotide identification can include probabilistically identifying the nucleotides of the first, second, third and / or fourth detectable labels. In some embodiments, the probabilistic identification step can include assigning a signal intensity state to each detectable label and decoding the time track. In some embodiments, the signal intensity state corresponds to a fixed value of signal intensity (for example, the relative fluorescence sum in an excitation wavelength range). In some embodiments, the signal intensity state corresponds to the range of signal intensity. In some embodiments, the signal intensity state corresponds to the Gaussian distribution of signal intensity. In some embodiments, decoding time track can include pairing the detectable labeled inactivation event of the nucleotides mixed in the incorporation event with the incorporation event. In some embodiments, decoding time track can include using the conversion probability between two states of signal intensity, and the conversion can include the incorporation event, inactivation event (for example, photobleaching) of the same marker or different markers at the substrate position, or the incorporation event and the inactivation event. In some embodiments, the conversion probability between the two states of signal intensity is fixed. In some embodiments, the conversion probability between the two states of fitting signal intensity.

[0177] In some embodiments, a hidden Markov model (HMM) can be used to analyze one or more incorporation events and / or one or more inactivation events at one or more substrate positions by observing the transition between signal intensity states and states. In some embodiments, using HMM includes providing the transition probability between the states of signal intensity caused by nucleotide incorporation and label bleaching, wherein individual label bleaching is not expected to recover. For example, HMM can model the first state with two current unbleached label luminescences, one on the nucleotide of the first detectable label incorporated, and the other on the nucleotide of the second detectable label incorporated. In this example, the first state can be converted to the second state in which the label on the nucleotide of the first detectable label incorporated is bleached, or converted to the third state in which the label on the nucleotide of the second detectable label incorporated is bleached. The first state may also be converted to the fourth state due to the incorporation of the nucleotide of the third detectable label, and the label on the nucleotide of the first and second detectable labels incorporated is not bleached. In some embodiments, decoding of the time trajectory may include using the Viterbi algorithm to represent the HMM of incorporation and inactivation events.

[0178] In some embodiments, one or more sequence reads are about 10 bp, about 15 bp, about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, or about 500 bp. Mapping of sequence reads can be accomplished by comparing the read sequence to a reference sequence to determine the source of the sequenced nucleic acid molecule (e.g., from a virus such as a coronavirus, such as SARS-CoV-2). In some embodiments, sequence reads can be mapped to one or more reference sequences or genomes. For example, sequence reads generated using the methods disclosed herein for sequencing-based SARS-CoV-2 detection in samples can be preferentially mapped to a SARS-CoV-2 reference sequence or genome, rather than to a background of human sequences and other viral sequences. In some embodiments, a certain degree of mismatching (e.g., 0-2 mismatches per read, 2-5 mismatches per read, or 5 or more mismatches per read) can be allowed, and the degree of mismatching allowed can be selected and / or adjusted according to the application. In some embodiments, the degree of mismatching can be used to explain minor polymorphisms that may exist between a reference sequence or genome and a nucleic acid sequence in a mixed sample. In some embodiments, the degree of mismatching can be used to explain sequencing errors, such as technical errors, rather than actual differences in the sequence (e.g., sequence differences between two copies of a similar sequence in a sample). For example, errors may be introduced when nucleic acids are processed before or during a single-molecule sequencing reaction, and / or introduced due to the inherent error rate of the polymerase used in the reaction.

[0179] In some embodiments, the length of one or more sequence reads is no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, no more than 15, or no more than 10 nucleotides. In some embodiments, the determined nucleic acid molecule sequence length can be about 8, about 12, about 16, about 20, about 24, about 28, about 32, about 36, or about 40 nucleotides. In some embodiments, the determined nucleic acid molecule sequence length can be about 5 to about 50 nucleotides, such as about 10 to about 35 nucleotides, or about 15 to about 30 nucleotides.

[0180] In some embodiments, the method described herein also includes reporting the information determined using an analytical method and / or generating a report comprising the information determined using an analytical method. For example, in some embodiments, the method also includes reporting or generating a report comprising the identification of a variation in a polynucleotide derived from a subject (e.g., derived from a virus in an infected subject or a subject's genome) related to the subject. The information reported or the information in the report can be associated with the sequencing reads mapped to a reference sequence, the variation detected (e.g., the structural variation detected in the viral genome or the SNP or sequence variation detected), one or more assembly consensus sequences and / or the verification statistics of the one or more assembly consensus sequences. The report can be distributed to a recipient or the information can be reported to a recipient, such as a clinician, the subject or a researcher.

[0181] IV. Optical System

[0182] In some embodiments, provided herein is a total internal reflection fluorescence (TIRF) imaging system (e.g., a TIRF microscope system), and a method of using the TIRF imaging system to detect and process optical signals for nucleic acid (e.g., DNA or RNA) sequencing.

[0183] In some embodiments, an inexpensive and simple TIRF imaging system is provided herein that can be used in user-oriented analytical equipment, such as analytical equipment for nucleic acid sequencing. Existing TIRF platforms either use objective-based TIRF optical systems (expensive and usually require the use of immersion oil between the lens and the substrate (such as a coverslip)), or use prism-based TIRF optical systems (usually requiring low autofluorescence fused silica prisms, and immersion oil / optical oil between the substrate and the prism). In some embodiments, prism-based TIRF platforms are attractive because cheaper low numerical aperture (NA) objectives can be used. Numerical aperture (also known as objective side aperture) is the value of the microscope objective and condenser: NA = n × sin (μ) or n × sin (α), where n represents the refractive index of the medium between the front lens of the objective and the specimen, and μ or α is the half-angle aperture of the objective. The numerical aperture of a microscope objective is a measure of its ability to focus light and resolve fine specimen details at a fixed object distance.

[0184] However, in some embodiments, in user-facing devices, it is cumbersome and inconvenient to handle the oil on the prism. To address these issues, in some embodiments, the prism is embedded in the substrate. In some embodiments, the prism is used as a substrate, making this component disposable, but in the case of using a fused silica prism, this cost is too high. In some embodiments, the fused silica prism can be replaced with a low autofluorescence plastic (e.g., ZEONEX5000*). Plastics with minimal autofluorescence at a given excitation wavelength can be selected. In some embodiments, the prism comprises one or more optical quality plastic materials with low autofluorescence for detection by fluorescence and laser induced fluorescence techniques. For example, PDMS has relatively low autofluorescence compared to other common plastics and can be used as a prism in the TIRF imaging system disclosed herein. In some embodiments, the prism comprises one or more commercially available plastic chip materials, such as PMMA, COC, PC and / or PDMS. See, e.g., Piruska et al., “The autofluorescence of plastic materials and chips measured under laser irradiation,” Lab Chip, 2005, 5, 1348-1354, the entire contents of which are incorporated herein by reference for all purposes.

[0185] In some embodiments, the plastic prism may form part of a disposable flow cell or flow cell / kit. In some embodiments, the prism surface itself may be used as a substrate to attach the analyte to be imaged. In some embodiments, the prism may be bonded to a substrate.

[0186] In some embodiments, to reduce the effects of autofluorescence, an excitation filter may be used below and / or above the substrate. In some embodiments, the excitation filter is selected so that it passes the excitation wavelength and blocks autofluorescence. In some embodiments, the excitation filter may block at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the autofluorescence. Alternatively, or in addition to the excitation filter, an additive may be added to the prism plastic as a filter. In some embodiments, the system disclosed herein may be used in conjunction with an excitation filter.

[0187] In some embodiments, light-guided TIRF can be used.In some embodiments, a lightguide is integrated into the flow cell.

[0188] The above system can be used in diagnostic or life science equipment and other fields. In some embodiments, the optical system disclosed herein can form part of a DNA or RNA sequencing instrument. In some embodiments, the optical system disclosed herein can be integrated into a DNA or RNA sequencing system that wishes to use low-cost optical methods for single-molecule imaging. In some embodiments, the TIRF prism can be integrated into a disposable box or flow cell.

[0189] V. Low-cost nucleic acid sequencing methods

[0190] In some embodiments provided herein, methods and compositions for realizing low-cost nucleic acid (e.g., DNA and / or RNA) sequencing are provided. In some embodiments, improvements related to the fluidic system (fluidic system) in a simplified synthesis sequencing (SBS) sequencing platform are provided herein. In some embodiments, the sequencing platform can be unimolecular, or a collection method (e.g., sequencing involving clusters, polymerization, polymerase cloning (polony) or nanoballs, etc.) can be used. In some embodiments, the fluidic system can also be used for other applications where it is necessary to control the movement of reagents (e.g., controlling the movement of nucleotides from a first region to a second region in which nucleotides are incorporated by a polymerase).

[0191] A. Fluidics

[0192] In some embodiments, it is preferred to use flow cell integrated reagents or reagents tightly coupled to the flow cell. In some embodiments, the reagents are included in the flow cell or box system. In some embodiments, the reagents (e.g., nucleotides, such as modified nucleotides and / or unmodified nucleotides) are confined to the starting area (e.g., in the flow cell or box system) and transported to another area, such as in the flow cell or box system. In some embodiments, electrophoretic flow, diffusion, charge method or pressure can be used to move reagents (including nucleotides) from one area in, for example, a flow cell or box system to another area.

[0193] B. Charge barrier

[0194] In some embodiments, the reagents may be initially confined by a charge barrier (e.g., an electric field). For example, one or more negatively charged reagents (e.g., one or more nucleotides) may be confined in a chamber / region using a negatively charged electrode, or attracted to a chamber / region using a positively charged electrode. In some embodiments, charged electrodes, charged surfaces (e.g., statically charged surfaces), or barriers may be used in place of or in combination with electrodes.

[0195] In some embodiments, the charge can be used to release the reagent. For example, for negatively charged reagents (e.g., nucleotides), the positively charged electrode (attracting the negatively charged reagent) can be made more negatively charged (e.g., charge neutral or more negatively charged) to allow the reagent (e.g., nucleotide) to diffuse away from the electrode. For example, nucleotides can be allowed to diffuse freely from a first region (e.g., a reagent chamber where the electrode is located) into a second region (e.g., a region where nucleotides are incorporated by a polymerase, such as a main chamber).

[0196] In some embodiments, as an alternative, or in combination with any of the foregoing embodiments, a charged electrode can act as a barrier to prevent reagents (e.g., nucleotides) in a first region from entering a second region. For example, a negatively charged reagent (e.g., nucleotide) can be repelled by a negatively charged electrode (e.g., in a first chamber such as a reagent chamber) to prevent it from diffusing (e.g., entering a second chamber such as a main chamber). In some embodiments, the barrier charge can be removed (e.g., making it more positively charged), thereby allowing the reagent (e.g., nucleotide) to diffuse from the first region to the second region (e.g., a region where nucleotides are incorporated by a polymerase, such as a main chamber).

[0197] In some embodiments, in conjunction with any of the foregoing embodiments, additional electrodes can be used to assist the movement of reagents (e.g., nucleotides). In some embodiments, the additional electrodes can be located in the second region (e.g., the region where nucleotides are incorporated by a polymerase). In some embodiments, the additional electrodes can be charged to attract reagents (e.g., nucleotides) to move to the second region (e.g., the additional electrodes can be positively charged to attract one or more nucleotides).

[0198] In some embodiments, the electrode may comprise a conductive material, such as indium tin oxide (ITO). In some embodiments, the electrode may be formed using an indium tin oxide (ITO) coating or any suitable conductive material. In some embodiments, the electrode may be formed using a material deposited by vapor deposition or other methods. In some embodiments, other selectively statically or dynamically charged materials may be used to generate the electrode, and the material coating may be on glass, fused quartz, or any other surface. In some embodiments, the electrodes disclosed herein may comprise glass or fused quartz. For example, a glass electrode may be used to provide an ion-selective electrode made of a doped glass membrane that is sensitive to ions. In some embodiments, the electrode may be charged or discharged, and may also be mechanically inserted and / or removed (e.g., in an area such as a reagent chamber and / or in a main chamber).

[0199] In some embodiments, charge-based methods using electrodes can be used to confine reagents (e.g., nucleotides) until the reagent needs to be exposed, such as to a second region (e.g., an incorporation region). In some embodiments, electrodes are used to provide a reagent confinement region in which charged reagents are confined until the charge on the electrode changes to release and / or repel the reagent. In some embodiments, multiple reagent confinement regions can be used to expose multiple reagents to one or more other regions (e.g., an incorporation region).

[0200] For example, multiple reagent restriction regions can restrict different types of nucleotides. For example (see e.g. Figure 2 ), each of the four regions contains one of guanine (unmodified and / or modified guanine nucleotides), adenine (unmodified and / or modified adenine nucleotides), cytosine (unmodified and / or modified cytosine nucleotides), and thymine (unmodified and / or modified thymine nucleotides). In some embodiments, the reagent confinement region of each of these nucleotides can be exposed sequentially (or in combination) to a second region, such as an incorporation region in the main chamber.

[0201] In some embodiments, the beads comprising one or more reagents fixed thereon can move under the action of electric charge. In some embodiments, reagents can include beads, wherein one or more reagents are confined to the bead surface. The beads themselves and / or the reagents thereon can be charged so that the beads move under the action of electric charge. In some embodiments, the conditions in the second region can be such that the reagent attached to the bead surface can be released from the beads (e.g., from the bead surface) in the second region.

[0202] In some embodiments, the reagent delivery methods disclosed herein can be used to implement a fluidics system of a synthetic sequencing platform ( Figure 2 An exemplary configuration is shown. In some embodiments, labeled (or unlabeled) nucleotides can be sequentially exposed to one or more templates being synthesized. The incorporation of each nucleotide type can be detected sequentially.

[0203] In some embodiments, agents (e.g., nucleotides) can be confined in agent "bubbles". These bubbles can be formed by a confining layer around the agent. For example, the layer can be a lipid bilayer. The charge can be used to apply pressure to these agent bubbles, breaking the layer and releasing the confined agent. The charge can also be used to destabilize the layer (e.g., the stability of the lipid bilayer). For example, see Figure 3 .

[0204] In some embodiments, heat can be used (e.g., as an alternative or in combination with any of the foregoing embodiments) to break the reagent bubble and release the reagent. The released reagent (e.g., nucleotide) can then diffuse freely to the second region and / or be pulled under the action of the charge.

[0205] In some embodiments, a pH change can be used (e.g., as an alternative or in combination with any of the foregoing embodiments) to break the reagent bubble. In some embodiments, a surfactant can be used (e.g., as an alternative or in combination with any of the foregoing embodiments) to break the reagent bubble. In some embodiments, a lysis solution can be used (e.g., as an alternative or in combination with any of the foregoing embodiments) to break the reagent bubble. In some embodiments, SDS can be used (e.g., as an alternative or in combination with any of the foregoing embodiments) to break the reagent bubble. In some embodiments, an enzyme can be used (e.g., as an alternative or in combination with any of the foregoing embodiments) to break the reagent bubble. The bubble can be broken by an enzyme. For example, alpha hemolysin nanopores or other nanopores can introduce instability and break lipid bubbles, which can be used in the methods disclosed herein.

[0206] In some embodiments, the reagent bubble can be transported from one area to a second area under pressure. The surface charge can be introduced using the methods described in "Charge Barriers" herein to transport or restrict the bubble under the action of the charge. The reagent can be transported to a second area at a different temperature, a different pH, and / or containing other reagents (e.g., one or more enzymes) to break the reagent bubble.

[0207] In some embodiments, the layer (eg, lipid bilayer) may be doped with photosensitive components. Light (UV light, visible light, or other wavelengths of light) can be used to disrupt these photosensitive components and break open the bubble, releasing the enclosed agent.

[0208] In some embodiments, as described herein, a bubble-based confinement system can be used to sequentially expose the incorporation regions to nucleotides (or other agents) within a charge barrier.

[0209] C. Physical confinement in the external activation chamber

[0210] In some embodiments, reagents (e.g., nucleotides) can be confined in chambers. In some embodiments, these chambers can have externally activated valves or "doors."

[0211] In some embodiments, external pressure can be used to apply pressure to valves embedded in a flow cell or cartridge system, causing reagents to move under pressure, diffuse, or move to a second chamber (e.g., an incorporation chamber and / or an imaging chamber) under the action of other forces (e.g., charge). The incorporation chamber and the imaging chamber can be the same or different.

[0212] In some embodiments, heat may be applied to selectively open the reagent chamber. In some embodiments, thermal activation may be performed by expansion or contraction of the material forming the valve to selectively open the reagent chamber.

[0213] In some embodiments, any one or more chamber valves can be activated by light. In some embodiments, the valve material deforms when exposed to light (e.g., light of a specific wavelength).

[0214] D. Magnetic and optical tweezers reagent guidance

[0215] In some embodiments, optical tweezers can be used to guide reagents along a path (e.g., along a fixed or flexible path) from one area of ​​a flow cell or box to another area. In some embodiments, the path can be a fixed path created using a series of LEDs focused on the desired reagent path. In some embodiments, optical tweezers can be used to guide reagents, beads attached to reagents, and / or reagent bubbles.

[0216] In some embodiments, the reagents can be confined to magnetic beads, which can be manipulated under a magnetic field. The magnetic beads can be directed along a path (e.g., along a fixed or flexible path) from one area of ​​a flow cell or cartridge to another.

[0217] E. Determination of the length of a homopolymer run

[0218] In some embodiments, determining the length of homopolymer continuous fragments in synthetic sequencing is a problem worthy of attention, which can be solved using the methods disclosed herein. In some embodiments, determining the length is usually solved by using reversible terminators. In some embodiments, terminators prevent the incorporation of multiple nucleotides of the same base type.

[0219] In some embodiments, the methods provided herein provide a strategy for sequencing by synthesis (e.g., for single molecule sequencing such as sequencing by synthesis techniques based on real-time imaging of fluorescently labeled nucleotides as they are synthesized along a single nucleic acid template molecule). Exemplary sequencing by synthesis platforms include those described in U.S. Patent No. 7,056,661, PCT / US2022 / 034346 (published as WO2022 / 271701), US2010 / 0227327A1, and PCT / US2023 / 062148, all of which are incorporated herein by reference in their entirety for all purposes.

[0220] In some embodiments, multiple fluorescently labeled nucleotides are incorporated into the homopolymer being synthesized. In some embodiments, each incorporated nucleotide will cause the fluorescence intensity to increase stepwise. In some embodiments, the initial intensity provides an estimate of the number of homopolymers incorporated. In some embodiments, step counting (e.g., described in Mira et al., "Counting the Number of Fluorophores Labeled in Biomolecules by Observing the Fluorescence-Intensity Transient of a Single Molecule," BCSJ 78 (9): 1612-18 (2005) in the photobleaching step counting (step-counting-photo-bleaching), the entire contents of the document are incorporated herein by reference for all purposes) can be used to provide additional information on the number of nucleotide incorporations.

[0221] In some embodiments, the signal from the labeled nucleotide can be removed from the signal presented by the template (e.g., the strand being sequenced) by reducing or extinguishing the signal associated with the nucleotide (e.g., by bleaching the fluorescent label), for example, in a stepwise manner. In some embodiments, bleaching of the nucleotide label eliminates the requirement to cleave the label, simplifying the analysis. In one exemplary method, a graph showing the relationship between fluorescence intensity and time for one, two, three, or more steps in photobleaching can be generated (e.g., as shown in FIG. 1 ). Figure 4 ). The intensity can be plotted in arbitrary units, and an empirical histogram showing the distribution frequency of detected light spots can be grouped by the number of quantified photobleaching steps. A statistical model can be used to fit the empirical histogram of photobleaching steps and compare the empirical data with the model predictions.

[0222] In some embodiments, the gradual change of the fluorophore emission light at a specific point over time (e.g., a gradual increase and / or decrease in signal intensity) can be detected and / or monitored. At a specific point and in a given time window or time point (e.g., in an imaging window measured in frames / exposure), the increase in signal intensity (e.g., due to the incorporation of nucleotides) and / or the decrease in signal (e.g., due to photobleaching events) can partially or completely offset each other. In some embodiments, the incorporation of labeled nucleotides can cause the characteristic signal intensity of the marker and / or base into which the labeled nucleotide has been incorporated to increase. For example, a nucleotide can be labeled with a marker having a characteristic signal intensity of the nucleotide base, and the signal intensity of the marker can be distinguished from the signal intensity of the marker on another nucleotide with a different base. In some embodiments, the signal inactivation of the labeled nucleotide (e.g., by cleavage and / or photobleaching of the marker) can cause the characteristic signal intensity of the marker and / or base of the signal-inactivated labeled nucleotide to decrease.

[0223] In some embodiments, the net change in signal intensity at a specific point and a given time window or time point can be associated with an event at the specific point, for example, the incorporation of a new labeled nucleotide and the photobleaching of one or more incorporated labeled nucleotides. One or more incorporated labeled nucleotides can maintain an arbitrary distance from the newly incorporated labeled nucleotide, for example, 0, 1, 2, 3, 4, 5 or more nucleotide residues. In some embodiments, the net change in signal intensity can be deconvoluted into one or more increases and / or one or more decreases in the characteristic signal intensity of nucleotide incorporation events (such as incorporation of nucleotides labeled with a specific fluorophore) and signal inactivation events (such as photobleaching of the same or another specific fluorophore), respectively.

[0224] F. Surface Attachment

[0225] Provided herein are a variety of methods for attaching a chain being sequenced to a surface of a flow cell and / or sequencing region. In some embodiments, the chain can be non-covalently bound to the surface by charge-based interactions to synthesize on the surface, for example, as described in Belosludtsev et al., "DNA Microarrays Based on Noncovalent Oligonucleotide Attachment and Hybridization in Two Dimensions," Analytical Biochemistry 292 (2): 250-256 (2001), the entire contents of which are incorporated herein by reference for all purposes. In some embodiments, the chain after non-covalent binding can then be subjected to single molecule synthesis sequencing. In some embodiments, synthesis sequencing can use targeted primers and / or random primers.

[0226] In some embodiments, the template is captured from solution using a surface attachment step in which the single molecule probes are non-covalently attached to the surface using charge-based interactions. The probes can be targeted or random.

[0227] In some embodiments, a fluidic control / reagent dispensing system is provided herein that uses an electric charge to limit and guide one or more reagents. In some embodiments, the charge on the reagent is used to attract the reagent from the first area to the second area. In some embodiments, the charge on the reagent is used to limit the reagent in the first area and is removed to release the reagent to the second area. In some embodiments, the charge on the reagent is used to attract the reagent from the first area to the second area, and the charge on the reagent is also used to limit the reagent in the first area and is removed to release the reagent to the second area.

[0228] In some embodiments, the reagent is a nucleotide. In some embodiments, the charge is generated using one or more electrodes. In some embodiments, the charge is a static charge. In some embodiments, the charge is a combination of a static charge and a dynamic charge generated by one or more electrodes.

[0229] In some embodiments, the reagent is digested in the second region (e.g., the nucleotides are digested by apyrase, which catalyzes the hydrolysis of NTP / dNTP to generate NMP / dNMP and inorganic phosphate). In some embodiments, the reagent can be removed from the second region using any of the methods described herein.

[0230] In some embodiments, provided herein is a reagent dispensing system in which a reagent is confined using reagent vesicles (vesicles or vesicle-like structures) and in which the reagent confined in the vesicles can be selectively released.

[0231] In some embodiments, the bubble is a lipid bilayer. In some embodiments, the bubble is a lipid bilayer, and a lysis buffer is applied to release the reagent. In some embodiments, the bubble is a lipid bilayer, wherein heat is used to cause bilayer instability and release the reagent. In some embodiments, the bubble is a lipid bilayer, wherein charge or electric field is used to cause bilayer instability and release the reagent. In some embodiments, the bubble is a lipid bilayer, wherein pH changes are used to cause bilayer instability and release the reagent. In some embodiments, the bubble is a lipid bilayer, wherein a surfactant is used to cause bilayer instability and release the reagent. In some embodiments, the bubble is a lipid bilayer, wherein an enzyme is used to cause bilayer instability and release the reagent. In some embodiments, the bubble is a lipid bilayer, wherein a nanopore (e.g., α-hemolysin) is used to cause bilayer instability and release the reagent.

[0232] In some embodiments, the reagent bubble is directed under the influence of an electric charge / field.

[0233] In some embodiments, provided herein is a method of physically confining a reagent in a chamber having a reagent valve that can be opened (and optionally closed) to release the reagent.

[0234] In some embodiments, heat is used to deform and open the reagent chamber valve. In some embodiments, pH is used to deform and open the reagent chamber valve. In some embodiments, light is used to deform and open the reagent chamber valve. In some embodiments, pressure is applied from the outside to open the reagent chamber valve.

[0235] In some embodiments, provided herein is a method of using optical tweezers to guide a reagent from a confined area to a second area. Provided herein is a method of using a magnetic field to guide a reagent confined on a magnetic bead.

[0236] In some embodiments, the reagents are further confined in beads or vesicles (bubbles) and guided using optical tweezers.

[0237] In some embodiments, the agent is moved to a region where conditions allow release of the agent from the bead / vesicle. In some embodiments, the agent is moved to a region where conditions allow release of the agent from the bead.

[0238] In some embodiments, provided herein is a method in which the length of a continuous segment of a homopolymer is determined by the fluorescence intensity of multiple incorporations.

[0239] In some embodiments, the nucleotides undergo photobleaching. In some embodiments, the photobleaching step is used to help determine the number of nucleotides present. In some embodiments, sufficient time is allowed for complete bleaching. In some embodiments, the bleaching process is monitored to ensure that all labels are completely bleached.

[0240] In some embodiments, provided herein is a method of non-covalently attaching single-stranded DNA to a surface using charge interactions.

[0241] In some embodiments, the strand is a DNA fragment to be sequenced. In some embodiments, the strand is primer extended by a random primer to initiate synthesis sequencing. In some embodiments, the strand is primer extended using a targeted primer to initiate synthesis sequencing.

[0242] In some embodiments, the DNA probe is attached to a surface to capture a strand to be sequenced using a synthetic sequencing method. In some embodiments, the DNA probe attached to the surface is a random probe. In some embodiments, the DNA probe attached to the surface is a targeted probe.

[0243] VI. Compositions, Kits and Uses

[0244] Also provided herein are compositions and kits comprising one or more of the primers, nucleic acid molecules, substrates, nucleotides (including detectably labeled nucleotides), polymerases, and reagents (e.g., reagents required for one or more steps including hybridization, ligation, amplification, detection, sequencing, and / or sample preparation, as described herein, e.g., in Section III) for performing the methods provided herein.

[0245] The various components of the kit may be present in separate containers, or certain compatible components may be pre-combined into a single container. In some embodiments, the kit also includes instructions for using the components of the kit to perform the provided methods.

[0246] In some embodiments, the kit may contain reagents and / or consumables required for performing one or more steps of the provided method. In some embodiments, the kit contains reagents for sample processing, such as nucleic acid extraction, separation and / or purification, such as RNA extraction, separation and / or purification. In some embodiments, the kit contains reagents for enzymes and buffers for connection and / or amplification, such as ligases and / or polymerases. In some embodiments, the kit contains reagents for enzymes and buffers for primer extension and / or nucleic acid sequencing, such as polymerases and / or transcriptases. In some aspects, the kit may also include any reagent as described herein, for example, for regulating nucleotide incorporation rate and / or regulating the buffer component of signal inactivation (e.g., by photobleaching) rate. In some embodiments, the kit contains reagents for signal detection in sequencing, such as detectable markers and detectably labeled molecules. In some embodiments, the kit optionally also includes other components, such as nucleic acid primers, enzymes and reagents, buffers, nucleotides, modified nucleotides, and reagents for additional analysis.

[0247] In some respects, the embodiment provided can be applied to analyzing nucleic acid sequence, such as DNA and / or RNA sequencing, such as single molecule real-time DNA and / or RNA sequencing.In some respects, the embodiment can be applied to imaging or the detection method of multiple nucleic acid analysis.In some respects, the embodiment provided can be used for identifying or detecting the region of interest in target nucleic acid (for example, viral DNA or RNA).In some embodiments, the region of interest comprises one or more nucleotide residues, such as single nucleotide polymorphism (SNP), single nucleotide variation (SNV), substitutes such as single nucleotide substitution, mutation such as point mutation, inserts such as single nucleotide insertion, lacks such as single nucleotide lack, translocation, inversion, repeats and / or other sequences of interest.

[0248] In some respects, the embodiments may be applied to research and / or diagnostic applications such as characterizing or evaluating samples from subjects. The application of the provided method may include biomedical research and clinical diagnosis. For example, in biomedical research, applications include but are not limited to gene and genome analysis for biological research or drug screening. In clinical diagnosis, applications include but are not limited to detecting gene markers, such as diseases, immune responses, bacterial or viral DNA / RNA of patient samples, loss of gene heterozygosity, the presence of gene alleles indicating disease or health tendency, the possibility of responsiveness to treatment, or applications in personalized medicine or pedigree.

[0249] VII. Terminology

[0250] Specific terminology is used throughout this disclosure to explain various aspects of the described devices, systems, methods, and compositions.

[0251] Having described some illustrative embodiments of the present disclosure, it will be apparent to those skilled in the art that the foregoing is merely illustrative, not restrictive, and is presented by way of example only. Many modifications and other illustrative embodiments are within the scope of those of ordinary skill in the art, and these modifications and illustrative embodiment examples are considered to fall within the scope of the present disclosure. Specifically, although many of the examples presented herein relate to specific combinations of method behaviors or system elements, it should be understood that these behaviors and elements can be combined in other ways to achieve the same purpose.

[0252] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more".

[0253] As used herein, the term "about" refers to the usual error range of the corresponding numerical value known to those skilled in the art. Reference herein to "about" for a value or parameter includes (and describes) embodiments for that value or parameter itself.

[0254] Throughout this disclosure, aspects of the claimed subject matter are presented in a range format. It should be understood that the description in a range format is only for convenience and brevity, and should not be interpreted as an unchangeable limitation on the scope of the claimed subject matter. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and single values ​​within the range. For example, in the case of providing a numerical range, it can be understood that each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value in the specified range are included in the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in the claimed subject matter, subject to any explicitly excluded restrictions in the specified range. In the case where the specified range includes one or two limits, the range that does not include one or two of the limits is also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0255] The use of ordinal terms such as "first", "second", "third", etc. in the claims to modify claim elements does not by itself imply any priority, precedence or order of one claim element relative to another claim element, or the temporal order of performance of method acts, but is merely used as a marker to distinguish a claim element of a certain name from another element of the same name (but using ordinal terms) to distinguish these claim elements. Similarly, the use of a), b), etc. or i), ii), etc. in the claims does not by itself imply any priority, precedence or order of steps. Similarly, the use of these terms in the specification does not by itself imply any required priority, precedence or order.

[0256] The terms "nucleic acid" and "nucleotide" are intended to be consistent with their use in the art and include naturally occurring substances or functional analogs thereof. Particularly useful functional analogs of nucleic acids can hybridize with nucleic acids in a sequence-specific manner (e.g., can hybridize with two nucleic acids so that connection occurs between the two hybridized nucleic acids), or can be used as a template for replicating a specific nucleotide sequence. Naturally occurring nucleic acids typically have a backbone containing phosphodiester bonds. Analog structures can have alternative backbone connections including any of a variety of connection methods known in the art. Naturally occurring nucleic acids typically have deoxyribose (e.g., deoxyribose in deoxyribonucleic acid (DNA)) or ribose (e.g., ribose in ribonucleic acid (RNA)).

[0257] Nucleic acid can contain nucleotides of any of the multiple analogs of these sugar moieties known in the art. Nucleic acid can include natural nucleotides or non-natural nucleotides. In this regard, natural deoxyribonucleic acid can have one or more bases selected from adenine (A), thymine (T), cytosine (C) or guanine (G), and ribonucleic acid can have one or more bases selected from uracil (U), adenine (A), cytosine (C) or guanine (G). Useful non-natural bases that can be included in nucleic acids or nucleotides are known in the art.

[0258] When "probe" or "target" is used to refer to a nucleic acid or nucleic acid sequence, it is intended as a semantic identifier of the nucleic acid or sequence in the context of a method or composition, and does not limit the structure or function of the nucleic acid or sequence beyond that explicitly indicated.

[0259] The terms "oligonucleotide" and "polynucleotide" are used interchangeably and refer to single-stranded polymers of about 2 to 500 nucleotides in length. Oligonucleotides can be synthetic, or can be prepared enzymatically (e.g., by polymerization), or prepared using a "split-pool" method. Oligonucleotides can include ribonucleotide monomers (e.g., can be oligoribonucleotides) and / or deoxyribonucleotide monomers (e.g., oligodeoxyribonucleotides). In some instances, an oligonucleotide can include a combination of deoxyribonucleotide monomers and ribonucleotide monomers (e.g., a random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). The length of the oligonucleotide can be, for example, 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, or 400 to 500 nucleotides. The oligonucleotide can include one or more functional parts attached (e.g., covalently or non-covalently) to the polymer structure. For example, the oligonucleotide can include one or more detectable labels (e.g., radioisotopes or fluorophores).

[0260] The terms "detectable marker", "optical marker" and "label" are used interchangeably herein and refer to a directly or indirectly detectable part that is coupled to or can be coupled to another part (e.g., nucleotides or nucleotide analogs). Detectable markers can be directly detectable (e.g., radioisotope labels or fluorescent markers) themselves, or, in the case of enzyme labels, can be indirectly detectable, for example, by catalyzing the chemical changes of substrate compounds or compositions, which are directly detectable. Labels can emit a signal or change the signal delivered to the label, so that the presence or absence of the label can be detected. In some cases, coupling can be carried out by a joint, and the joint can be cleavable, such as photolysis (e.g., cleavable under ultraviolet light), chemical cleavage (e.g., by reducing agents such as dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP)) or enzyme cleavage (e.g., by esterase, lipase, peptidase or protease).

[0261] In some embodiments, the detectable label is a fluorophore, or includes a fluorophore. Exemplary fluorophores include, but are not limited to, fluorescent nanocrystals; quantum dots; d-rhodamine acceptor dyes, including dichloro [R110], dichloro [R6G], dichloro [TAMRA], dichloro [ROX], etc.; fluorescein donor dyes, including fluorescein, 6-FAM, etc.; cyanine dyes, such as Cy3B; Alexa dyes, SETA dyes, Atto dyes, such as Atto 647N, which form a FRET pair with Cy3B, etc. Fluorophores include, but are not limited to, MDCC (7-diethylamino-3-[([(2-maleimido)ethyl]amino)carbonyl]coumarin), TET, HEX, Cy3, TMR, ROX, Texas Red, Cy5, LC Red 705, and LC Red 640.

[0262] In some embodiments, the detectable label is a luminescent or chemiluminescent moiety, or includes a luminescent or chemiluminescent moiety. Common luminescent / chemiluminescent moieties include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. Under appropriate substrate (e.g., oxidizing agent plus chemiluminescent compound) conditions, these protein moieties can catalyze chemiluminescent reactions. It is known that many families of compounds can provide chemiluminescence under a variety of conditions. Non-limiting examples of chemiluminescent compound families include 2,3-dihydro-1,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- and dimethylamino[ca]benz analogs. These compounds emit light in the presence of alkaline hydrogen peroxide or calcium hypochlorite and a base. Other examples of chemiluminescent compound families include, for example, 2,4,5-triphenylimidazole, p-dimethylamino substituents and p-methoxy substituents, oxalates (such as oxalyl active esters), p-nitrophenyl, N-alkyl acridinium esters, luciferin, luciferase or acridinium esters. In some embodiments, the detectable label is a metal-based label or a mass-based label, or includes a metal-based label or a mass-based label.

[0263] The terms "hybridizing" and "anneal" and "annealing" are used interchangeably in this disclosure and refer to the pairing of substantially complementary or fully complementary nucleic acid sequences in two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence binds to a substantially complementary or fully complementary sequence through base pairing to form a hybrid complex. For the purposes of hybridization, two nucleic acid sequences are "substantially complementary" if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of their individual bases are complementary.

[0264] "Primer" is a single-stranded nucleic acid sequence whose 3' end can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed by RNA nucleotides for RNA synthesis, while DNA primers are formed by DNA nucleotides for DNA synthesis. Primers can also include both RNA nucleotides and DNA nucleotides (for example, in a random or designed pattern). Primers can also include other natural or synthetic nucleotides as described herein that can have additional functions. In some instances, a DNA primer can be used to initiate RNA synthesis, and vice versa (for example, an RNA primer can be used to initiate DNA synthesis). The length of the primer can be different. For example, a primer can be about 6 bases to about 120 bases. For example, a primer can include up to about 25 bases. In some cases, a primer can refer to a primer binding sequence.

[0265] "Nucleic acid extension" generally involves incorporating one or more nucleic acids (e.g., A, G, C, T, U, nucleotide analogs or derivatives thereof) into a molecule (e.g., but not limited to a nucleic acid sequence) in a template-dependent manner such that successive nucleic acids are incorporated by an enzyme (e.g., a polymerase or reverse transcriptase) to generate a newly synthesized nucleic acid molecule. Enzymatic extension can be performed by enzymes including, but not limited to, polymerases and / or reverse transcriptases. For example, a primer that hybridizes to a complementary nucleic acid sequence can be used to synthesize a new nucleic acid molecule by using the complementary nucleic acid sequence as a template for nucleic acid synthesis. Similarly, the 3' polyadenylated tail of an mRNA transcript that hybridizes to a poly (dT) sequence can be used as a template for the single-stranded synthesis of a corresponding cDNA molecule. In addition, a poly (dT) sequence can also be used as a sequencing primer for sequencing RNA molecules containing a poly (A) tail.

[0266] A "non-terminating nucleotide" or "incorporated nucleotide" may include a nucleic acid portion that can be attached to the 3' end of a polynucleotide using a polymerase or transcriptase and may have another non-terminating nucleic acid attached thereto using a polymerase or transcriptase without removing a protecting group or reversible terminator from the nucleotide. Naturally occurring nucleic acids are a type of non-terminating nucleic acid. Non-terminating nucleic acids may be labeled or unlabeled.

[0267] "PCR amplification" refers to the use of polymerase chain reaction (PCR) to generate copies of genetic material (including DNA and RNA sequences). Suitable reagents and conditions for implementing PCR are described in, for example, U.S. Patents Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the entire contents of each of which are incorporated herein by reference. In a typical PCR amplification, the reaction mixture includes genetic material to be amplified, an enzyme, one or more primers used in a primer extension reaction, and a reaction reagent. The oligonucleotide primer has a length sufficient to hybridize with complementary genetic material under annealing conditions. The length of the primer is usually dependent on the length of the amplification domain, but is usually at least 4 bases, at least 5 bases, at least 6 bases, at least 8 bases, at least 9 bases, at least 10 base pairs (bp), at least 11 bp, at least 12 bp, at least 13 bp, at least 14 bp, at least 15 bp, at least 16 bp, at least 17 bp, at least 18 bp, at least 19 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, and may be up to 40 bp or longer, wherein the length of the primer is usually 18 bp to 50 bp. The genetic material may be contacted with a single primer or a set of two primers (a forward primer and a reverse primer), depending on whether primer extension, linear amplification or exponential amplification of the genetic material is desired.

[0268] In some embodiments, the PCR amplification process uses a DNA polymerase. The DNA polymerase activity can be provided by one or more different DNA polymerases. In some embodiments, the DNA polymerase is from bacteria, for example, the DNA polymerase is a bacterial DNA polymerase. For example, the DNA polymerase can be from bacteria of the genus Escherichia, Bacillus, Thermophilus or Pyrococcus.

[0269] In some embodiments, PCR amplification may include reactions such as, but not limited to, strand displacement amplification, rolling circle amplification (e.g., multiple repeats may be cleaved from a rolling circle amplification product), ligase chain reaction, transcription-mediated amplification, isothermal amplification, and / or loop-mediated amplification.

[0270] In some embodiments, PCR amplification uses a single primer that is complementary to the 3' tag of the target DNA fragment. In some embodiments, PCR amplification uses a first primer and a second primer, wherein at least the 3' end portion of the first primer is complementary to at least a portion of the 3' tag of the target nucleic acid fragment, and at least the 3' end portion of the second primer displays the sequence of at least a portion of the 5' tag of the target nucleic acid fragment. In some embodiments, the 5' end portion of the first primer is not complementary to the 3' tag of the target nucleic acid fragment, and the 5' end portion of the second primer does not display the sequence of at least a portion of the 5' tag of the target nucleic acid fragment. In some embodiments, the first primer includes a first universal sequence and / or the second primer includes a second universal sequence.

[0271] The term "DNA polymerase" includes not only naturally occurring enzymes, but also all modified derivatives thereof, and also derivatives of naturally occurring DNA polymerases. For example, in some embodiments, the DNA polymerase can be modified to remove 5'-3' exonuclease activity. Sequence-modified derivatives or mutants of DNA polymerases that can be used include, but are not limited to, mutants that retain at least part of the function (e.g., DNA polymerase activity) of the wild-type sequence. Under different reaction conditions (e.g., temperature, template concentration, primer concentration, etc.), mutations can affect the activity characteristics of the enzyme, such as increasing or decreasing the polymerization rate. Mutations or sequence modifications can also affect the exonuclease activity and / or thermal stability of the enzyme.

[0272] Suitable examples of DNA polymerases that can be used include, but are not limited to, E. coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT TM DNA polymerase, DEEPVENT TM DNA polymerase, Taq DNA polymerase, Hot Start TaqDNA Polymerase, Crimson Taq DNA polymerase, Crimson Taq DNA polymerase, DNA polymerase, Quick- DNA polymerase, Hemo DNA polymerase, DNA polymerase, DNA polymerase, High-Fidelity DNA Polymerase, Platinum Pfx DNA Polymerase, AccuPrime Pfx DNA Polymerase, Phi29 DNA Polymerase, Klenow Fragment, Pwo DNA Polymerase, Pfu DNA Polymerase, T4 DNA Polymerase, and T7 DNA Polymerase.

[0273] In some embodiments, the genetic material is amplified by reverse transcription polymerase chain reaction (RT-PCR). The required reverse transcriptase activity can be provided by one or more different reverse transcriptases, suitable examples of which are not limited to: M-MLV, MuLV, AMV, HIV, ArrayScript TM , MultiScribe TM , ThermoScript TM and I, II, III and IV enzymes. "Reverse transcriptase" includes not only the naturally occurring enzyme but also all modified derivatives thereof, and also derivatives of the naturally occurring reverse transcriptase.

[0274] In addition, sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptases can be used for reverse transcription, including mutants that retain at least some functional activity (e.g., reverse transcriptase activity) of the wild-type sequence. The reverse transcriptase can be provided as part of a composition that includes other components, such as stability components that can enhance or improve the activity of the reverse transcriptase, such as one or more RNase inhibitors, DNA-dependent DNA synthesis inhibitors, such as actinomycin D. Many sequence-modified derivatives or mutants of reverse transcriptases, such as M-MLV, and compositions comprising unmodified and modified enzymes are commercially available, such as ArrayScript TM , MultiScribe TM , ThermoScript TM and I, II, III and IV enzymes.

[0275] Certain reverse transcriptases (e.g., avian myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase) can use both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as templates to synthesize complementary DNA strands. Therefore, in some embodiments, the reverse transcription reaction can use an enzyme (reverse transcriptase) that can use both RNA and ssDNA as templates for extension reactions, such as AMV or MMLV reverse transcriptase.

[0276] Example

[0277] The following examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0278] Example 1: Reducing the error rate of double-stranded nucleic acid sequencing

[0279] Double-stranded nucleic acid fragments (e.g., DNA fragments) are weakly attached to a surface. The double-stranded nucleic acid fragments become the first single-stranded nucleic acid and the second single-stranded nucleic acid after melting or denaturation. The first single-stranded nucleic acid and the second single-stranded nucleic acid are attached to the surface by using a probe or an electric field that binds nucleic acid. The diffusion of the first single-stranded nucleic acid and the second single-stranded nucleic acid on the surface is limited by the electric field. In a specific example, the first single-stranded nucleic acid and the second single-stranded nucleic acid are attached by using a positive charge on the surface, and the positive charge attracts negatively charged nucleic acid, while the diffusion is limited by using a negatively charged electrode to generate a negatively charged electric field between the double-stranded nucleic acid fragment junctions, thereby making the distance between the first single-stranded nucleic acid and the second single-stranded nucleic acid closer than the distance between other single-stranded nucleic acids generated by different double-stranded nucleic acids.

[0280] A cluster is formed by the first single-stranded nucleic acid and the second single-stranded nucleic acid. The cluster is used to perform spatial sequencing of the first single-stranded nucleic acid and the second single-stranded nucleic acid. For example, spatial sequencing can be completed by synthetic sequencing. During the image analysis part of sequencing, the position on the surface is recorded. After base calling or as a part of the base calling process, an algorithm is used to determine whether two adjacent templates are from the same nucleic acid, for example, using Euclidean distance or other methods. If it is determined that the two chains are from double-stranded nucleic acid fragments from the same source, the chain sequences are combined.

[0281] When the sequences from each strand are combined, mismatched bases are handled in a variety of ways. If the mismatch cannot be resolved, the base can be replaced with an "N". Alternatively, the base quality scores for each strand can be used to select the most accurate base call. Features extracted during image analysis can be used to influence base quality scores, such as the circularity of clusters at positions where errors may occur, local background, or other features that may cause errors. Due to the nature of phase-lag errors, errors on each strand may have different error characteristics, resulting in higher quality for early bases. When the sequences from each strand are combined, the early bases of each strand are prioritized over the later bases sequenced in the complementary strand, resulting in more accurate base calls. This approach allows for more accurate base calls while increasing the effective read length. When the strand is longer than the platform read length, but short enough to allow overlap between the reads from the first single-stranded nucleic acid and the second single-stranded nucleic acid, the overlap is used to assemble the two reads into a longer read. This process can be used for clusters formed by sense and antisense strands that are not well separated and are often referred to as mixed, where a hidden Markov model or other algorithm is used to extract the most likely source template from the convolved signals of the sense and antisense strands.

[0282] Example 2: Single-cell spatial sequencing

[0283] Single cells are isolated and attached to a surface. Cells are attached by charge-based attachment or using monoclonal or polyclonal antibodies or any other method for attaching cells to a surface. The attachment sites for single cells are distributed on the surface randomly or as a pattern. The size of the attachment sites is such that only a single cell can be attached to each attachment site. The attachment sites contain a reverse transcriptase for generating cDNA.

[0284] Once a single cell is attached, the single cell is lysed and nucleic acid is extracted from the single cell. Cell lysis can be performed by any method (e.g., SDS, lysis buffer, toxin, heat, pH, etc.). Once the cell is lysed, the reverse transcriptase bound to the surface of the attachment site converts the extracted RNA into cDNA.

[0285] The charge acts as a charge barrier around the cell to confine the extracted nucleic acids and cDNA to areas near the cell, such as the attachment site. The charge barrier includes a negatively charged patterned electrode, which repels negatively charged nucleic acids and prevents their diffusion, and a positive electrode, which pulls the negatively charged nucleic acids to confine them to the attachment site. Once the nucleic acids are confined on the surface, ligation can be used to introduce index sequences into the confined fragments, and then the nucleic acids are spatially sequenced.

[0286] The present disclosure is not intended to be limited to the scope of the disclosed specific embodiments, which are provided, for example, to illustrate aspects of the present disclosure. From the description and teachings herein, various modifications to the compositions and methods will become apparent. These changes can be implemented without departing from the true scope and spirit of the present disclosure, and are intended to fall within the scope of the present disclosure.

Claims

1. A nucleic acid sequencing method, comprising: a) positioning the double-stranded nucleic acid at a position on a substrate; b) generating a first single-stranded nucleic acid and a second single-stranded nucleic acid from the positioned double-stranded nucleic acid; c) limiting the diffusion of the first single-stranded nucleic acid and / or the second single-stranded nucleic acid; d) attaching the first single-stranded nucleic acid and the second single-stranded nucleic acid to sites near the position on the substrate; e) obtaining sequencing reads from the attached first single-stranded nucleic acid and sequencing reads from the attached second single-stranded nucleic acid; and f) correlating the sequence of the first single-stranded nucleic acid with the sequence of the second single-stranded nucleic acid to thereby determine the sequence of the double-stranded nucleic acid.

2. The method of claim 1, wherein the positioning in a) comprises non-covalently attaching the double-stranded nucleic acid to the location on the substrate, Optionally, wherein the double-stranded nucleic acid is not covalently attached to the substrate or is not covalently attached to a molecule immobilized on the substrate, or Optionally, only one strand of the double-stranded nucleic acid is covalently attached to the substrate or to a molecule immobilized on the substrate.

3. The method according to claim 1 or 2, wherein the positioning in a) comprises using an electrode to attract and / or confine the double-stranded nucleic acid to the position on the substrate, optionally wherein the electrode is integrated into the substrate or the electrode is provided separately from the substrate, optionally wherein the electrode is removable from the substrate.

4. The method according to any one of claims 1 to 3, wherein the positioning in a) comprises hybridizing a region of the double-stranded nucleic acid with a nucleic acid probe directly or indirectly fixed on the substrate, optionally wherein the double-stranded nucleic acid comprises a single-stranded region that is optionally a loop, a bulge, or generated by partially melting the double-stranded nucleic acid. The method according to any one of claims 1 to 4, wherein the double-stranded nucleic acid is derived from a cell or tissue sample.

6. The method according to any one of claims 1 to 5, wherein the double-stranded nucleic acid is fragmented DNA, optionally, wherein the double-stranded nucleic acid is free DNA or is generated by fragmenting genomic DNA.

7. The method according to any one of claims 1 to 6, wherein the double-stranded nucleic acid is an amplification product of cellular DNA or RNA or free DNA.

8. The method according to any one of claims 1 to 7, wherein the double-stranded nucleic acid is derived from a single cell and is separated from double-stranded nucleic acids derived from other cells on the substrate.

9. The method according to any one of claims 1 to 8, wherein the locations are random locations on the substrate.

10. The method according to any one of claims 1 to 8, wherein the location is one of a location of an ordered pattern on the substrate.

11. The method according to any one of claims 1 to 10, wherein the location is located in a protrusion or a depression at the location on the substrate.

12. The method of any one of claims 1 to 11, wherein the location is on a bead at the location on the substrate.

13. The method according to any one of claims 1 to 12, wherein the first single-stranded nucleic acid and the second single-stranded nucleic acid are generated in b) by melting the positioned double-stranded nucleic acid using heat, pH change, denaturing buffer or any combination thereof.

14. The method according to any one of claims 1 to 13, wherein limiting the diffusion of the first single-stranded nucleic acid and the second single-stranded nucleic acid in c) comprises capturing the first single-stranded nucleic acid and the second single-stranded nucleic acid by nucleic acid probes directly or indirectly immobilized on the substrate.

15. The method of claim 14, wherein the nucleic acid probe is located at a site near the location on the substrate.

16. The method according to any one of claims 1 to 15, wherein limiting the diffusion of the first and second single-stranded nucleic acids in c) comprises limiting the first and second single-stranded nucleic acids using electrodes, optionally wherein the electrodes are integrated into the substrate or the electrodes are provided separately from the substrate, optionally wherein the electrodes are removable from the substrate. 17 . The method according to any one of claims 1 to 16 , wherein in d), the first single-stranded nucleic acid and the second single-stranded nucleic acid are covalently and / or non-covalently attached at the site near the position on the substrate.

18. The method of any one of claims 1 to 17, wherein in d), the site is no more than about 8 μm, no more than about 6 μm, no more than about 4 μm, no more than about 2 μm, no more than about 1 μm, no more than about 0.5 μm, or no more than about 0.25 μm from the location.

19. The method of claim 18, wherein one of the sites is located at the location and the other site is no more than about 2 μm, no more than about 1 μm, no more than about 0.5 μm, or no more than about 0.25 μm from the location.

20. The method of claim 18, wherein both sites are no more than about 2 μm, no more than about 1 μm, no more than about 0.5 μm, or no more than about 0.25 μm from the location.

21. The method of any one of claims 1 to 20, wherein the attached first single-stranded nucleic acid and the attached second single-stranded nucleic acid are amplified on the substrate.

22. The method of any one of claims 1 to 21, wherein the attached first single-stranded nucleic acid and the attached second single-stranded nucleic acid are clonally amplified to form amplicon clusters on the substrate.

23. The method according to any one of claims 1 to 22, wherein the attached first single-stranded nucleic acid and the attached second single-stranded nucleic acid are amplified using a bridge amplification method.

24. The method of any one of claims 1 to 20, wherein the attached first single-stranded nucleic acid and the attached second single-stranded nucleic acid are not amplified or are only minimally amplified on the substrate.

25. The method of claim 24, wherein in e), the sequencing reads are obtained using single molecule sequencing.

26. The method of claim 24 or 25, wherein in e), the sequencing reads are obtained using real-time sequencing, optionally, single molecule real-time sequencing.

27. The method according to any one of claims 1 to 26, wherein in e), the sequencing reads are obtained using synthesis sequencing, binding sequencing, affinity sequencing, ligation sequencing and / or hybridization sequencing.

28. The method according to any one of claims 1 to 27, wherein in e), the sequencing reads are obtained by imaging the substrate and recording light signals in consecutive imaging cycles for each of the sites.

29. The method of any one of claims 1 to 28, wherein the optical signal of one of the sites is optically distinguishable from the optical signal of another site.

30. The method according to any one of claims 1 to 29, comprising determining the sequence of the first single-stranded nucleic acid by comparing multiple sequencing reads from the attached first single-stranded nucleic acid, optionally comprising aligning the multiple sequencing reads and / or generating a consensus sequence of the multiple sequencing reads.

31. The method according to any one of claims 1 to 30, comprising determining the sequence of the second single-stranded nucleic acid by comparing multiple sequencing reads from the attached second single-stranded nucleic acid, optionally comprising aligning the multiple sequencing reads and / or generating a common sequence of the multiple sequencing reads.

32. The method according to any one of claims 1 to 31, wherein the sequence of the first single-stranded nucleic acid and the sequence of the second single-stranded nucleic acid are determined independently of each other.

33. The method according to any one of claims 1 to 32, comprising comparing the sequence of the first single-stranded nucleic acid with the complement of the sequence of the second single-stranded nucleic acid, and / or comparing the sequence of the second single-stranded nucleic acid with the complement of the sequence of the first single-stranded nucleic acid.

34. The method of any one of claims 1 to 33, comprising comparing the single-stranded consensus sequence of the first single-stranded nucleic acid to the single-stranded consensus sequence of the second single-stranded nucleic acid to generate a double-stranded consensus sequence, optionally wherein comparison of the single-stranded consensus sequences is used to identify one or more errors in sequence.

35. The method according to any one of claims 1 to 34, comprising identifying: an overlapping sequence between the sequence of the first single-stranded nucleic acid and the complementary sequence of the sequence of the second single-stranded nucleic acid, and A first non-overlapping sequence in the sequence of the first single-stranded nucleic acid and / or a second non-overlapping sequence in the complementary sequence of the sequence of the second single-stranded nucleic acid.

36. The method according to any one of claims 1 to 35, comprising identifying: an overlapping sequence between the sequence of the second single-stranded nucleic acid and the complementary sequence of the sequence of the first single-stranded nucleic acid, and a first non-overlapping sequence in the sequence of the second single-stranded nucleic acid and / or a second non-overlapping sequence in the complementary sequence of the sequence of the first single-stranded nucleic acid.

37. The method according to claim 35 or 36 comprises assembling i) the sequence of the first single-stranded nucleic acid and the complementary sequence of the sequence of the second single-stranded nucleic acid, and / or ii) the sequence of the second single-stranded nucleic acid and the complementary sequence of the sequence of the first single-stranded nucleic acid into a sequence longer than the sequence of the first single-stranded nucleic acid and the sequence of the second single-stranded nucleic acid.

38. The method of any one of claims 1 to 37, wherein the sequence of the first single-stranded nucleic acid is correlated with the sequence of the second single-stranded nucleic acid during base calling.

39. The method of any one of claims 1 to 38, wherein the sequence of the first single-stranded nucleic acid is correlated with the sequence of the second single-stranded nucleic acid after base calling.

40. The method of any one of claims 1 to 39, comprising determining that the sequence of the first single-stranded nucleic acid and the sequence of the second single-stranded nucleic acid are derived from two strands of the same double-stranded nucleic acid localized to the substrate in a).

41. A nucleic acid sequencing method, comprising: a) localize single cells or nuclei to their position on the substrate; b) releasing nucleic acid from the localized single cell or cell nucleus; c) limiting the diffusion of said nucleic acid; d) attaching the nucleic acid to a site at or near the location on the substrate; as well as e) obtaining sequencing reads from the attached nucleic acid, thereby determining the sequence of the nucleic acid.

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