Chemical composition and method for using the same

Abstract

To provide a probe for sequencing, a reporter probe and a method in which nucleic acid sequencing that is quickly executed with no enzyme, no amplification and no library, and has a low error rate with a long read-out length.SOLUTION: A probe including a primary nucleic acid molecule which hybridizes with at least six secondary nucleic acid molecules, in which each of the plurality of secondary nucleic acid molecules hybridizes with at least five tertiary nucleic acid molecules, and each of the tertiary nucleic acid molecules includes at least one detectable label.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Application No. 62 / 424,887, filed November 21, 2016, U.S. Provisional Application No. 62 / 457,237, filed February 10, 2017, and U.S. Provisional Application No. 62 / 536,147, filed July 24, 2017, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Array List This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format, which is hereby incorporated by reference in its entirety. This ASCII copy was created on November 20, 2017, is named NATE033-001WO_SeqList_ST25.txt, and is 19,351 bytes in size. [Background technology]

[0003] Currently, there are various methods for sequencing nucleic acids, i.e., the process of determining the exact order of nucleotides in a nucleic acid molecule. Current methods require the nucleic acid to be amplified by enzymes (e.g., PCR) and / or by cloning. Further enzymatic polymerization is required to generate a signal that can be detected by optical detection means. Thus, there is a need in the art for a rapid, amplification-free, enzyme-free method of nucleic acid sequencing. The present disclosure addresses such a need. Summary of the Invention

[0004] The present disclosure provides sequencing probes, methods, kits, and devices that provide long read lengths, low error rates, rapid, enzyme-free, amplification-free, and library-free nucleic acid sequencing. The sequencing probes described herein include a barcode domain, where each position in the barcode domain corresponds to at least two nucleotides in the target binding domain. Additionally, the methods, kits, and devices have the ability to rapidly obtain results from samples. These features are particularly useful for sequencing in clinical settings. The present disclosure is an improvement over the disclosures disclosed in U.S. Patent Application Publication No. 2016 / 0194701, the entire contents of which are incorporated herein by reference.

[0005] The present disclosure provides a composition comprising: a) a target binding domain and a barcode domain, wherein the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, wherein at least six nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; at least two nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs; and a barcode domain comprising a synthetic backbone, wherein the barcode domain comprises at least three attachment positions, each attachment position being capable of identifying a complementary nucleic acid molecule. a first complementary primary nucleic acid molecule hybridizing to a first attachment position of the at least three attachment positions, the first complementary primary nucleic acid molecule comprising at least two domains and a linker modification, a first domain of which hybridizes to a first attachment position of the barcode domain and a second domain of which is capable of hybridizing to at least one second complementary secondary nucleic acid molecule, the linker modification being [ka] wherein the linker modification is located between the first domain and the second domain.

[0006] The present disclosure provides a method for the preparation of a composition comprising: a) a target binding domain and a barcode domain, wherein the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, wherein at least six nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; at least two nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs; and a barcode domain comprising a synthetic backbone, wherein the barcode domain comprises at least three attachment sites, each attachment site comprising at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can be bound, wherein the nucleic acid sequences of the at least three attachment sites are not bound by the target binding domain prior to binding. a first complementary primary nucleic acid molecule hybridizing to a first attachment position of the at least three attachment positions, the first complementary primary nucleic acid molecule comprising at least two domains and a linker modification, the first domain of which hybridizes to a first attachment position of the barcode domain and the second domain of which is capable of hybridizing to at least one second complementary secondary nucleic acid molecule, the linker modification being [ka] wherein the linker modification is located between the first domain and the second domain.

[0007] The present disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, at least six nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; at least two nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprises at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, the nucleic acid sequences of the at least three attachment sites determine the positions and attributes of the at least six nucleotides in the target nucleic acid to which the target binding domain binds, and each of the at least three attachment sites has a different nucleic acid sequence.

[0008] The present disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, at least six nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; at least two nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment positions, each attachment position comprises at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, each of the at least three attachment positions corresponds to two nucleotides of the at least six nucleotides in the target binding domain, and each of the at least three attachment positions has a different nucleic acid sequence, and the nucleic acid sequence of each of the at least three attachment positions determines the position and attribute of the corresponding two nucleotides of the at least six nucleotides in the target nucleic acid to which the target binding domain binds.

[0009] The present disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least 10 nucleotides and is capable of binding to a target nucleic acid, at least six nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least four nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprises at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, the nucleic acid sequences of the at least three attachment sites determine the positions and attributes of the at least six nucleotides in the target nucleic acid to which the target binding domain binds, and each of the at least three attachment sites has a different nucleic acid sequence.

[0010] The present disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least 10 nucleotides and is capable of binding to a target nucleic acid, at least six nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least four nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment positions, each attachment position comprises at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, each of the at least three attachment positions corresponds to two nucleotides of the at least six nucleotides in the target binding domain, and each of the at least three attachment positions has a different nucleic acid sequence, and the nucleic acid sequence of each of the at least three attachment positions determines the position and attribute of the corresponding two nucleotides of the at least six nucleotides in the target nucleic acid to which the target binding domain binds.

[0011] The synthetic backbone can comprise a polysaccharide, a polynucleotide, a peptide, a peptide nucleic acid, or a polypeptide. The synthetic backbone can comprise DNA. The synthetic backbone comprises single-stranded DNA. The sequencing probe comprises a single-stranded DNA synthetic backbone and a double-stranded DNA spacer between the target binding domain and the barcode domain. The double-stranded DNA spacer can be about 1 to about 100 nucleotides in length, or about 2 to about 50 nucleotides in length, or about 20 to 40 nucleotides in length. The double-stranded DNA spacer is about 36 nucleotides in length. The sequencing probe can comprise a single-stranded DNA synthetic backbone and a polymer-based spacer between the target binding domain and the barcode domain, the polymer-based spacer providing similar mechanical properties as the double-stranded DNA spacer.

[0012] The number of nucleotides in the target binding domain can be the same as, or less than, or more than the number of attachment positions in the barcode domain. Preferably, the number of nucleotides in the target binding domain is more than the number of attachment positions in the barcode domain. The number of nucleotides in the target binding domain can be at least 3 more than the number of attachment positions in the barcode domain, or at least 4 more than, or at least 5 more than, or at least 6 more than, or at least 7 more than, or at least 8 more than, or at least 9 more than, or at least 10 more than the number of attachment positions in the barcode domain. Preferably, the target binding domain comprises 8 nucleotides and the barcode domain comprises 3 attachment positions.

[0013] At least three, or at least four, or at least five, or at least six nucleotides in the target binding domain that can identify the corresponding nucleotides in the target nucleic acid molecule can be modified nucleotides or nucleic acid analogs. The modified nucleotides or nucleic acid analogs can be locked nucleic acids (LNA), bridged nucleic acids (BNA), propyne-modified nucleic acids, zip nucleic acids (ZNA®), isoguanines, isocytosines, or any combination thereof. The modified nucleotides or nucleic acid analogs are preferably bridged nucleic acids (LNA).

[0014] The at least two nucleotides without identifying a corresponding nucleotide in the target binding domain can be located before the at least six nucleotides in the target binding domain. The at least two nucleotides without identifying a corresponding nucleotide in the target binding domain can be located after the at least six nucleotides in the target binding domain. At least one nucleotide of the at least two nucleotides without identifying a corresponding nucleotide in the target binding domain is located before the at least six nucleotides in the target binding domain, and at least one nucleotide of the at least two nucleotides without identifying a corresponding nucleotide in the target binding domain is located after the at least six nucleotides in the target binding domain. That is, the at least two nucleotides without identifying a corresponding nucleotide in the target binding domain are adjacent to the at least six nucleotides in the target binding domain.

[0015] The at least two nucleotides having no corresponding nucleotides identified in the target binding domain can be universal bases, degenerate bases, or a combination thereof. At least two of the at least four nucleotides having no corresponding nucleotides identified in the target binding domain can be universal bases, degenerate bases, or a combination thereof.

[0016] Each attachment position in a barcode domain can comprise one attachment region. Each attachment position in a barcode domain can comprise two or more attachment regions.

[0017] Each attachment location in a barcode domain can contain the same number of attachment regions. Each attachment location in a barcode domain can contain a different number of attachment regions. At least one of the at least three attachment locations in a barcode domain can contain a different number of attachment regions than the other two. When an attachment location in a barcode domain contains two or more attachment regions, the attachment regions can be the same. When an attachment location in a barcode domain contains two or more attachment regions, the attachment regions can contain the same nucleic acid sequence. When an attachment location in a barcode domain contains two or more attachment regions, the attachment regions can be different. When an attachment location in a barcode domain contains two or more attachment regions, the attachment regions can contain different nucleic acid sequences.

[0018] Each nucleic acid sequence comprising each attachment region in the barcode domain is about 8 to about 20 nucleotides in length. Each nucleic acid sequence comprising each attachment region in the barcode domain is about 12 nucleotides in length. Each nucleic acid sequence comprising each attachment region in the barcode domain is about 14 nucleotides in length.

[0019] At least one, or at least two, or at least three of the at least three attachment positions in the barcode domain can be adjacent to at least one flanking single-stranded polynucleotide. Each of the at least three attachment positions in the barcode domain can be adjacent to at least one flanking single-stranded polynucleotide.

[0020] At least one, or at least two, or at least three of the attachment regions of the at least one attachment location can be integrated into a synthetic scaffold. Each attachment region of each of the at least three attachment regions can be integrated into a synthetic scaffold. At least one, or at least two, or at least three of the attachment regions of the at least one attachment location can branch off from the synthetic scaffold. Each attachment region of each of the at least three attachment regions can branch off from the synthetic scaffold.

[0021] The complementary nucleic acid molecule capable of directly or indirectly binding to at least one nucleic acid sequence in at least one attachment region in each attachment position can be either RNA, DNA or PNA. The complementary nucleic acid molecule is preferably DNA.

[0022] The complementary nucleic acid molecule can be a primary nucleic acid molecule that directly binds to at least one attachment region within at least one attachment position of the barcode domain.

[0023] The primary nucleic acid molecule can comprise at least two domains, a first domain capable of binding to at least one attachment region within at least one attachment location of the barcode domain, and a second domain capable of binding to at least one complementary secondary nucleic acid molecule. The primary nucleic acid molecule can comprise at least two domains, a first domain capable of binding to at least one attachment region within at least one attachment location of the barcode domain, and a second domain comprising a first detectable label and at least a second detectable label.

[0024] The primary nucleic acid molecule can hybridize to at least one attachment region in at least one attachment location of the barcode domain and to at least one, or two, or three, or four, or five, or more secondary nucleic acid molecules. Preferably, the primary nucleic acid molecule hybridizes to four secondary nucleic acid molecules. The primary nucleic acid molecule can hybridize to at least one attachment region in at least one attachment location of the barcode domain and to a first detectable label and at least a second detectable label. The first detectable label and the at least a second detectable label can have the same emission spectrum or different emission spectra.

[0025] The primary nucleic acid molecule may comprise a cleavable linker. The cleavable linker is preferably located between the first domain and the second domain. The cleavable linker may be a photocleavable linker (e.g., a UV light cleavable linker), a reducing agent cleavable linker, or an enzyme cleavable linker. The linker is preferably a photocleavable linker.

[0026] The secondary nucleic acid molecule can comprise at least two domains, a first domain capable of binding to a complementary sequence in at least one primary nucleic acid molecule and a second domain capable of binding to (a) a first detectable label and at least a second detectable label, or (b) at least one complementary tertiary nucleic acid molecule, or (c) a combination thereof.

[0027] The secondary nucleic acid molecule can hybridize to at least one primary nucleic acid molecule and at least one, or two, or three, or four, or five, or six, or seven, or more tertiary nucleic acid molecules. The secondary nucleic acid molecule is preferably hybridized to one tertiary nucleic acid molecule. The secondary nucleic acid molecule can hybridize to at least one primary nucleic acid molecule and can include a first detectable label and at least a second detectable label. The secondary nucleic acid molecule can hybridize to at least one primary nucleic acid molecule, at least one tertiary nucleic acid molecule, a first detectable label, and at least a second detectable label. The first detectable label and the at least a second detectable label can have the same emission spectrum or different emission spectra. When a secondary nucleic acid molecule hybridizes to at least one primary nucleic acid molecule, at least one tertiary nucleic acid molecule comprising a first detectable label and at least a second detectable label, the first detectable label and the at least second detectable label, the first detectable label and the at least second detectable label located on the surface of the secondary nucleic acid molecule can have the same emission spectrum, the first detectable label and the at least second detectable label located on the surface of the tertiary nucleic acid molecule can have the same emission spectrum, and the emission spectrum of the detectable label on the surface of the secondary nucleic acid molecule can be different from the emission spectrum of the detectable label on the surface of the tertiary nucleic acid molecule.

[0028] The secondary nucleic acid molecule may comprise a cleavable linker. The cleavable linker is preferably located between the first domain and the second domain. The cleavable linker may be a photocleavable linker (e.g., a UV light cleavable linker), a reducing agent cleavable linker, or an enzyme cleavable linker. The linker is preferably a photocleavable linker.

[0029] The tertiary nucleic acid molecule can comprise at least two domains, a first domain capable of binding to a complementary sequence in at least one secondary nucleic acid molecule and a second domain capable of binding to a first detectable label and at least a second detectable label.

[0030] The tertiary nucleic acid molecule can hybridize to at least one secondary nucleic acid molecule and can include a first detectable label and at least a second detectable label. The first detectable label and the at least second detectable label can have the same emission spectrum or can have different emission spectra.

[0031] The tertiary nucleic acid molecule may comprise a cleavable linker. The cleavable linker is preferably located between the first domain and the second domain. The cleavable linker may be a photocleavable linker (e.g., a UV light cleavable linker), a reducing agent cleavable linker, or an enzyme cleavable linker. The linker is preferably a photocleavable linker.

[0032] The present disclosure also provides a population of sequencing probes comprising a plurality of the sequencing probes disclosed herein, wherein each sequencing probe in the plurality of sequencing probes preferably comprises a different target binding domain and binds to a different region of a target nucleic acid.

[0033] The invention also provides a method of sequencing a nucleic acid, the method comprising: (1) hybridizing a first sequencing probe as described herein to a target nucleic acid, optionally immobilized at one or more locations on a substrate; (2) binding a first complementary nucleic acid molecule comprising a first detectable label and at least a second detectable label to a first of the at least three attachment locations of a barcode domain; (3) detecting the first detectable label and at least a second detectable label of the bound first complementary nucleic acid molecule; (4) identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid; (5) freeing the first complementary nucleic acid molecule comprising a detectable label by binding a first hybridizing nucleic acid molecule lacking a detectable label to the first attachment location, or contacting the first complementary nucleic acid molecule comprising a detectable label with a force sufficient to release the first detectable label and at least a second detectable label; and (6) contacting the first complementary nucleic acid molecule comprising a detectable label with a force sufficient to release the first detectable label and at least a second detectable label. (7) detecting the third detectable label and the at least fourth detectable label of the bound second complementary nucleic acid molecule; (8) optionally identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid; (9) identifying a linear order of at least six nucleotides for at least a first region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe by repeating steps (5)-(8) until a complementary nucleic acid molecule comprising two detectable labels is bound to each of the at least three attachment locations in the barcode domain and the two detectable labels of the bound complementary nucleic acid molecule are detected; and (10) optionally removing the sequencing probe from the immobilized target nucleic acid.

[0034] The method further includes (11) hybridizing a second sequencing probe to the target nucleic acid immobilized at one or more locations on the substrate, where the target binding domains of the first and second sequencing probes are different; (12) binding a first complementary nucleic acid molecule comprising a first detectable label and at least a second detectable label to a first of the at least three attachment locations of the barcode domain; (13) detecting the first detectable label and at least a second detectable label of the bound first complementary nucleic acid molecule; (14) optionally identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid; and (15) freeing the first complementary nucleic acid molecule or complex comprising the detectable label by binding the first hybridizing nucleic acid molecule lacking the detectable label to the first attachment location, or releasing the first detectable label and at least a second detectable label of the first complementary nucleic acid molecule or complex comprising the detectable label. (16) binding a second complementary nucleic acid molecule comprising a third detectable label and at least a fourth detectable label to a second of the at least three attachment locations of the barcode domain; (17) detecting the third detectable label and the at least a fourth detectable label of the bound second complementary nucleic acid molecule; (18) optionally identifying a location and identity of at least two nucleotides in the immobilized target nucleic acid; (19) identifying a linear order of at least six nucleotides for at least a second region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe by repeating steps (15)-(18) until a complementary nucleic acid molecule comprising two detectable labels is bound to each of the at least three attachment locations of the barcode domain and the two detectable labels of the bound complementary nucleic acid molecule are detected; and (20) optionally removing the second sequencing probe from the immobilized target nucleic acid.

[0035] The method can further include identifying a sequence of the immobilized target nucleic acid by assembling a linear order of the identified nucleotides in at least the first region and at least the second region of the immobilized target nucleic acid, respectively.

[0036] Steps (5) and (6) can be performed sequentially or simultaneously. The first detectable label and the at least second detectable label can have the same emission spectrum or different emission spectra. The third detectable label and the at least fourth detectable label can have the same emission spectrum or different emission spectra.

[0037] The first complementary nucleic acid molecule can include a cleavable linker. The second complementary nucleic acid molecule can include a cleavable linker. The first complementary nucleic acid molecule and the second complementary nucleic acid molecule can each include a cleavable linker. The cleavable linker is preferably a photocleavable linker. The releasing force can be light. UV light is preferred. The light can be provided by a light source selected from the group consisting of an arc lamp, a laser, a focused UV light source, and a light emitting diode.

[0038] The first complementary nucleic acid molecule and the first hybridizing nucleic acid molecule lacking a detectable label can comprise the same nucleic acid sequence. The first hybridizing nucleic acid molecule lacking a detectable label can comprise a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to the first attachment position in the barcode domain.

[0039] The second complementary nucleic acid molecule and the second hybridizing nucleic acid molecule lacking a detectable label can comprise the same nucleic acid sequence. The second hybridizing nucleic acid molecule lacking a detectable label can comprise a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to the second attachment position in the barcode domain.

[0040] In accordance with the present invention, a method for sequencing a nucleic acid includes: (1) hybridizing at least one first population of first sequencing probes, the population including a plurality of sequencing probes as described herein, to a target nucleic acid, optionally immobilized at one or more locations on a substrate; (2) binding a first complementary nucleic acid molecule, the first complementary nucleic acid molecule including a first detectable label and at least a second detectable label, to a first of at least three attachment locations of a barcode domain; (3) detecting the first detectable label and the at least a second detectable label of the bound first complementary nucleic acid molecule; (4) optionally identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid; and (5) freeing the first complementary nucleic acid molecule including the detectable label by binding a first hybridizing nucleic acid molecule lacking the detectable label to the first attachment location, or freeing the first complementary nucleic acid molecule including the detectable label by binding a first hybridizing nucleic acid molecule lacking the detectable label to the first attachment location. (6) binding a second complementary nucleic acid molecule comprising a third detectable label and at least a fourth detectable label to a second of the at least three attachment locations of the barcode domain; (7) detecting the third detectable label and the at least a fourth detectable label of the bound second complementary nucleic acid molecule; (8) identifying a location and identity of at least two nucleotides in the optionally immobilized target nucleic acid; (9) identifying a linear order of at least six nucleotides for at least a first region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe by repeating steps (5)-(8) until each of the at least three attachment locations in the barcode domain is bound by a complementary nucleic acid molecule comprising two detectable labels and the two detectable labels of the bound complementary nucleic acid molecule are detected; and (10) removing at least one first population of the first sequencing probe from the optionally immobilized target nucleic acid.

[0041] The method further includes (11) hybridizing at least one second population of a second sequencing probe comprising a plurality of sequencing probes described herein to a target nucleic acid optionally immobilized on a substrate at one or more locations, wherein the target binding domains of the first and second sequencing probes are different; and (12) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and at least a second detectable label to a first of the at least three attachment locations of the barcode domain. (13) detecting the first detectable label and at least a second detectable label of the bound first complementary nucleic acid molecule; (14) optionally identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid; and (15) freeing the first complementary nucleic acid molecule or complex containing the detectable label by binding the first hybridizing nucleic acid molecule lacking a detectable label to the first attachment site, or freeing the first complementary nucleic acid molecule or complex containing the detectable label by binding the first hybridizing nucleic acid molecule lacking a detectable label to the first attachment site, thereby freeing the first complementary nucleic acid molecule or complex containing the detectable label by binding the first detectable label and at least a second detectable label. (16) binding a second complementary nucleic acid molecule comprising a third detectable label and at least a fourth detectable label to a second of the at least three attachment locations of the barcode domain; (17) detecting the third detectable label and the at least a fourth detectable label of the bound second complementary nucleic acid molecule; (18) optionally identifying a location and identity of at least two nucleotides in the immobilized target nucleic acid; (19) identifying a linear order of at least six nucleotides for at least a second region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe by repeating steps (15)-(18) until a complementary nucleic acid molecule comprising two detectable labels is bound to each of the at least three attachment locations of the barcode domain and the two detectable labels of the bound complementary nucleic acid molecule are detected; and (20) optionally removing at least a second population of second sequencing probes from the immobilized target nucleic acid.

[0042] The method can further include identifying a sequence of the immobilized target nucleic acid by reconstructing the linear order of nucleotides identified in at least the first region and at least the second region, respectively, of the immobilized target nucleic acid.

[0043] Steps (5) and (6) can be performed sequentially or simultaneously. The first detectable label and the at least second detectable label can have the same emission spectrum or different emission spectra. The third detectable label and the at least fourth detectable label can have the same emission spectrum or different emission spectra.

[0044] The first complementary nucleic acid molecule can include a cleavable linker. The second complementary nucleic acid molecule can include a cleavable linker. The first complementary nucleic acid molecule and the second complementary nucleic acid molecule can each include a cleavable linker. The cleavable linker is preferably a photocleavable linker. The releasing force can be light. UV light is preferred. The light can be provided by a light source selected from the group consisting of an arc lamp, a laser, a focused UV light source, and a light emitting diode.

[0045] The first complementary nucleic acid molecule and the first hybridizing nucleic acid molecule lacking a detectable label can comprise the same nucleic acid sequence. The first hybridizing nucleic acid molecule lacking a detectable label can comprise a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to the first attachment position in the barcode domain.

[0046] The second complementary nucleic acid molecule and the second hybridizing nucleic acid molecule lacking a detectable label can comprise the same nucleic acid sequence. The second hybridizing nucleic acid molecule lacking a detectable label can comprise a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to the second attachment position in the barcode domain.

[0047] The present disclosure also provides a method for determining the nucleotide sequence of a nucleic acid, the method comprising: (1) hybridizing a first sequencing probe as disclosed herein to a target nucleic acid, optionally immobilized at one or more locations on a substrate; (2) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and a second detectable label to a first attachment location of at least three attachment locations of a barcode domain; (3) identifying the first detectable label and the second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment location; (4) removing the first detectable label and the second detectable label hybridized to the first attachment location; (5) hybridizing a second complementary nucleic acid molecule comprising a third detectable label and a fourth detectable label to a second attachment location of at least three attachment locations of a barcode domain; (6) hybridizing a second complementary nucleic acid molecule to the second attachment location. (7) removing the third detectable label and the fourth detectable label hybridized to the second attachment location; (8) hybridizing a third complementary nucleic acid molecule comprising a fifth detectable label and a sixth detectable label to a third attachment location of the at least three attachment locations of the barcode domain; (9) identifying the fifth detectable label and the sixth detectable label of the third complementary nucleic acid molecule hybridized to the third attachment location; and (10) identifying a linear order of at least six nucleotides of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe based on attributes of the first detectable label, the second detectable label, the third detectable label, the fourth detectable label, the fifth detectable label, and the sixth detectable label.

[0048] The method further includes (11) optionally removing at least a first sequencing probe from a first region of the immobilized target nucleic acid; (12) hybridizing at least a second sequencing probe disclosed herein to a second region of the target nucleic acid immobilized at one or more locations on the substrate, where the target binding domains of the first sequencing probe and the at least second sequencing probe are different; (13) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and a second detectable label to a first attachment location of the at least three attachment locations of the barcode domain; (14) detecting the first detectable label and the second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment location; and (15) hybridizing a second complementary nucleic acid molecule comprising a third detectable label and a fourth detectable label to a second attachment location of the at least three attachment locations of the barcode domain. (16) detecting the third detectable label and the fourth detectable label of the second complementary nucleic acid molecule hybridized to the second attachment location; (17) removing the third detectable label and the fourth detectable label hybridized to the second attachment location; (18) hybridizing a third complementary nucleic acid molecule comprising a fifth detectable label and a sixth detectable label to a third attachment location of the at least three attachment locations of the barcode domain; (19) identifying the fifth detectable label and the sixth detectable label of the third complementary nucleic acid molecule hybridized to the third attachment location; and (20) identifying a linear order of at least six nucleotides in a second region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the second sequencing probe based on attributes of the first detectable label, the second detectable label, the third detectable label, the fourth detectable label, the fifth detectable label, and the sixth detectable label.

[0049] The method can further include identifying a sequence of the immobilized target nucleic acid by reconstructing the linear order of nucleotides identified in at least the first region and at least the second region, respectively, of the immobilized target nucleic acid.

[0050] Steps (4) and (5) can be performed sequentially or simultaneously. The first detectable label and the at least second detectable label can have the same emission spectrum or different emission spectra. The third detectable label and the at least fourth detectable label can have the same emission spectrum or different emission spectra. The fifth detectable label and the at least sixth detectable label can have the same emission spectrum or different emission spectra.

[0051] The first complementary nucleic acid molecule may comprise a cleavable linker. The second complementary nucleic acid molecule may comprise a cleavable linker. The third complementary nucleic acid molecule may comprise a cleavable linker. The first complementary nucleic acid molecule, the second complementary nucleic acid molecule, and the at least third complementary nucleic acid molecule may each comprise a cleavable linker. The cleavable linker is preferably a light cleavable linker. The method of removing any one of the first complementary nucleic acid molecule, the second complementary nucleic acid molecule, and the at least third complementary nucleic acid molecule may comprise contact with light. UV light is preferred. The light may be provided by a light source selected from the group consisting of an arc lamp, a laser, a focused UV light source, and a light emitting diode.

[0052] In accordance with the present disclosure, a method for determining the nucleotide sequence of a nucleic acid includes: (1) hybridizing a first sequencing probe as disclosed herein to a first region of a target nucleic acid obtained from a gene of interest and optionally immobilized at one or more locations on a substrate; (2) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and a second detectable label to a first attachment location of the at least three attachment locations of a barcode domain; (3) detecting the first detectable label and the second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment location; (4) removing the first detectable label and the second detectable label hybridized to the first attachment location; (5) hybridizing a second complementary nucleic acid molecule comprising a third detectable label and a fourth detectable label to a second attachment location of the at least three attachment locations of the barcode domain; (6) detecting the second detectable label and the second detectable label hybridized to the second attachment location. (7) removing the third detectable label and the fourth detectable label hybridized to the second attachment location; (8) hybridizing a third complementary nucleic acid molecule comprising a fifth detectable label and a sixth detectable label to a third attachment location of the at least three attachment locations of the barcode domain; (9) identifying the fifth detectable label and the sixth detectable label of the third complementary nucleic acid molecule hybridized to the third attachment location; and (10) identifying a linear order of at least six nucleotides in a first region of an optionally immobilized target nucleic acid hybridized to the target binding domain of a first sequencing probe based on attributes of the first detectable label, the second detectable label, the third detectable label, the fourth detectable label, the fifth detectable label, and the sixth detectable label.

[0053] The method further includes (11) optionally removing at least a first sequencing probe from a first region of the immobilized target nucleic acid; (12) hybridizing at least a second sequencing probe disclosed herein to a second region of the target nucleic acid immobilized at one or more locations on the substrate, where the target binding domains of the first sequencing probe and the at least second sequencing probe are different; (13) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and a second detectable label to a first attachment location of the at least three attachment locations of the barcode domain; (14) detecting the first detectable label and the second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment location; and (15) hybridizing a second complementary nucleic acid molecule comprising a third detectable label and a fourth detectable label to a second attachment location of the at least three attachment locations of the barcode domain. (16) detecting the third detectable label and the fourth detectable label of the second complementary nucleic acid molecule hybridized to the second attachment location; (17) removing the third detectable label and the fourth detectable label hybridized to the second attachment location; (18) hybridizing a third complementary nucleic acid molecule comprising a fifth detectable label and a sixth detectable label to a third attachment location of the at least three attachment locations of the barcode domain; (19) identifying the fifth detectable label and the sixth detectable label of the third complementary nucleic acid molecule hybridized to the third attachment location; and (20) identifying a linear order of at least six nucleotides in a second region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the second sequencing probe based on attributes of the first detectable label, the second detectable label, the third detectable label, the fourth detectable label, the fifth detectable label, and the sixth detectable label.

[0054] The method can further include identifying a sequence of the immobilized target nucleic acid by reconstructing the linear order of nucleotides identified in at least the first region and at least the second region, respectively, of the immobilized target nucleic acid.

[0055] Steps (4) and (5) can be performed sequentially or simultaneously. The first detectable label and the at least second detectable label can have the same emission spectrum or different emission spectra. The third detectable label and the at least fourth detectable label can have the same emission spectrum or different emission spectra. The fifth detectable label and the at least sixth detectable label can have the same emission spectrum or different emission spectra.

[0056] The first complementary nucleic acid molecule may comprise a cleavable linker. The second complementary nucleic acid molecule may comprise a cleavable linker. The third complementary nucleic acid molecule may comprise a cleavable linker. The first complementary nucleic acid molecule, the second complementary nucleic acid molecule, and the at least third complementary nucleic acid molecule may each comprise a cleavable linker. The cleavable linker is preferably a light cleavable linker. The method of removing any one of the first complementary nucleic acid molecule, the second complementary nucleic acid molecule, and the at least third complementary nucleic acid molecule may comprise contact with light. UV light is preferred. The light may be provided by a light source selected from the group consisting of an arc lamp, a laser, a focused UV light source, and a light emitting diode.

[0057] The present disclosure also provides an apparatus for carrying out any of the methods disclosed herein.

[0058] The present disclosure also provides one or more kits that include a substrate, a population of sequencing probes as disclosed herein, at least three complementary nucleic acid molecules that include a first detectable label and at least two second detectable labels, and instructions for use. The one or more kits can further include at least one capture probe. The one or more kits can further include at least two capture probes.

[0059] Any one of the above aspects may be combined with any other aspect.

[0060] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the specification, the singular includes the plural, for example, the terms "a" and "the" are understood to be singular or plural, and the term "or" is understood to be inclusive, unless clearly indicated otherwise from the context. By way of example, "an element" means one or more elements. Throughout this specification, the word "comprising" or variations thereof ("comprises", "comprises") are understood to include the recited element, integer, step, group of elements, integers, or steps, but not to exclude any other element, integer, step, group of elements, integers, or steps. About is understood to be within 10%, or within 9%, or within 8%, or within 7%, or within 6%, or within 5%, or within 4%, or within 3%, or within 2%, or within 1%, or within 0.5%, or within 0.1%, or within 0.05%, or within 0.01% of the recited value. Unless clearly understood to the contrary from the context, all numerical values ​​presented herein are modified by the term "about."

[0061] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. References cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present specification, including definitions, will control. Other features and advantages of the present disclosure will become apparent from the following detailed description and claims. [Brief description of the drawings]

[0062] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0063] The above features and further features will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.

[0064] [Figure 1] FIG. 1 is a diagram of an example of a sequencing probe according to the present disclosure.

[0065] [Diagram 2] FIG. 2 shows the design of standard and three-part sequencing probes according to the present disclosure.

[0066] [Diagram 3] FIG. 3 is a diagram of an example of a reporter complex of the present disclosure hybridized to an example of a sequencing probe according to the present disclosure.

[0067] [Figure 4] FIG. 4 shows a schematic diagram of an example reporter probe according to the present disclosure.

[0068] [Diagram 5]FIG. 5 is a schematic diagram of some example reporter probes according to the present disclosure.

[0069] [Figure 6] FIG. 6 is a schematic diagram of an example reporter probe of the present disclosure that includes an "extra handle."

[0070] [Figure 7] FIG. 7 is a schematic diagram of several examples of reporter probes according to the present disclosure that contain different configurations of tertiary nucleic acids.

[0071] [Figure 8] FIG. 8 is a schematic diagram of some examples of reporter probes according to the present disclosure that include branched tertiary nucleic acids.

[0072] [Figure 9] FIG. 9 is a diagram of an example of a reporter probe according to the present disclosure that includes a cleavable linker modification.

[0073] [Figure 10] FIG. 10 shows possible locations for cleavable linker modifications in an exemplary reporter probe according to the present disclosure.

[0074] [Figure 11] FIG. 11 is a schematic diagram of capturing a single target nucleic acid using a two capture probe system according to the present disclosure.

[0075] [Figure 12-1] FIG. 12 shows results from an experiment utilizing the methods of the present invention to capture and detect a multi-cancer panel of 100 targets using FFPE samples. [Figure 12-2] Same as above explanation.

[0076] [Figure 13]FIG. 13 is a schematic diagram of two captured target DNA molecules hybridized to a capture probe, blocker oligo, and a sequencing probe for targeted sequencing of a large target nucleic acid molecule.

[0077] [Figure 14] FIG. 14 is a schematic diagram of one cycle of a sequencing method according to the present disclosure.

[0078] [Figure 15-1] FIG. 15 is a schematic diagram of one cycle of a sequencing method according to the present disclosure and the corresponding imaging data collected during this cycle. [Figure 15-2] Same as above explanation.

[0079] [Figure 16] FIG. 16 shows an example of the configuration of a pool of sequencing probes according to the present disclosure, in which eight different pools of sequencing probes are designed using eight different color combinations.

[0080] [Figure 17] FIG. 17 compares the barcode domain designs disclosed in US Patent Application Publication No. 2016 / 019470 with the barcode domain designs of the present disclosure.

[0081] [Figure 18] FIG. 18 is a schematic diagram of a sequencing probe or probes of the present disclosure hybridized to a captured target nucleic acid molecule.

[0082] [Figure 19] FIG. 19 shows a fluorescence image recorded during a sequencing method according to the present disclosure when a sequencing probe or multiple sequencing probes are hybridized to a target nucleic acid.

[0083] [Figure 20] FIG. 20 is a schematic diagram of multiple sequencing probes of the present disclosure bound along the length of a target nucleic acid and the corresponding recorded fluorescence image.

[0084] [Figure 21] FIG. 21 shows an example of imaging data recorded during a sequencing cycle according to the present disclosure, and the fluorescent signal intensity profile of a reporter probe according to the present disclosure.

[0085] [Figure 22] FIG. 22 is a schematic diagram of a sequencing cycle according to the present disclosure, in which a cleavable linker modification is used to darken one position of the barcode.

[0086] [Figure 23] FIG. 23 is an example illustrating a sequencing cycle according to the present disclosure, where one position in the barcode domain is darkened by substitution of the primary nucleic acid.

[0087] [Figure 24] FIG. 24 shows an example of combined capture of RNA and DNA from an FFPE sample.

[0088] [Diagram 25] FIG. 25 is a schematic diagram of how the sequencing methods of the present disclosure allow for sequencing of the same base of a target nucleic acid using different sequencing probes.

[0089] [Figure 26] FIG. 26 illustrates how multiple base calls at a particular nucleotide position on a target nucleic acid are recorded from one or more sequencing probes and combined to create a consensus sequence, thereby increasing the accuracy of the final base call.

[0090] [Figure 27] FIG. 27 shows a fluorescence image of the disclosed sequencing method recorded after capture, extension, and detection of a 33 kilobase DNA fragment.

[0091] [Figure 28-1] Figure 28 shows results from a sequencing experiment obtained using the sequencing method of the present disclosure and analyzed using the ShortStack™ algorithm. For the graph on the left, the sequences shown correspond clockwise starting from the top left graph to SEQ ID NOs: 3, 4, 6, 8, 7, 5. For the table on the right, the sequences correspond from top to bottom to SEQ ID NOs: 3, 4, 7, 8, 6, 5. [Figure 28-2] Same as above explanation.

[0092] [Figure 29] FIG. 29 shows a schematic of the experimental design for multiplex capture and sequencing of cancer gene targets from FFPE samples.

[0093] [Diagram 30] FIG. 30 shows a schematic of direct sequencing and results from an experiment using the sequencing methods of the present disclosure to test the integrity of RNA molecules.

[0094] [Diagram 31] FIG. 31 shows the results of sequencing RNA and DNA molecules having the same nucleotide sequence using the sequencing methods of the present disclosure.

[0095] [Diagram 32] FIG. 32 shows the results of multiplex target capture of an RNA panel.

[0096] [Diagram 33] FIG. 33 shows a schematic diagram of the overall process of the ShortStack™ software pipeline.

[0097] [Diagram 34] FIG. 34 shows the results of mutational analysis of the simulated dataset using the ShortStack™ software pipeline.

[0098] [Diagram 35] FIG. 35 shows the overall mutation read accuracy of the ShortStack™ software pipeline for different types of mutations.

[0099] [Diagram 36] FIG. 36 shows the intensity distribution for reporter complexes labeled with specific color combinations.

[0100] [Figure 37] FIG. 37 shows a typical deposition gradient of the present disclosure.

[0101] [Figure 38] FIG. 38 shows the capture efficiency of the present disclosure while titrating DNA mass inputs from 25 ng to 500 ng.

[0102] [Figure 39] FIG. 39 shows HLPC purification of an example of a reporter complex of the disclosure.

[0103] [Figure 40-1] FIG. 40 shows the hybridization efficiency and accuracy of reporter probes of the present disclosure in the presence of various buffer additives. [Figure 40-2] Same as above explanation.

[0104] [Diagram 41] FIG. 41 shows the rate of loss of target nucleic acid when utilizing the sequencing methods of the present disclosure in the presence of various buffer additives.

[0105] [Diagram 42]FIG. 42 shows the efficiency and error of a reporter probe of the present disclosure that includes a 12-mer complementary nucleic acid.

[0106] [Diagram 43] FIG. 43 shows reporter probe efficiency and error using an example 8×8×8 14-mer reporter set.

[0107] [Diagram 44] FIG. 44 shows reporter probe efficiency and error using an example 10×10×10 14-mer reporter set.

[0108] [Diagram 45] FIG. 45 shows a comparison of the performance of standard and three-part sequencing probes of the present disclosure.

[0109] [Diagram 46] FIG. 46 shows the effect of LNA substitutions in the target binding domain according to the present disclosure using individual probes.

[0110] [Figure 47] FIG. 47 shows the effect of LNA substitutions in the target binding domain according to the present disclosure using a pool of nine probes.

[0111] [Figure 48] FIG. 48 shows the effect of modified nucleotide and nucleic acid analog substitutions in a target binding domain according to the present disclosure.

[0112] [Figure 49] FIG. 49 shows results from an experiment quantifying the raw accuracy of a sequencing method according to the present disclosure.

[0113] [Figure 50]FIG. 50 shows results from an experiment to determine the accuracy of a sequencing method according to the present disclosure when nucleotides in a target nucleic acid are sequenced with two or more sequencing probes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0114] The present disclosure provides sequencing probes, reporter probes, methods, kits, and devices that provide rapid, enzyme-free, amplification-free, library-free nucleic acid sequencing with long read lengths and low error rates.

[0115] Compositions of the Disclosure

[0116] The present disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, at least six nucleotides in the target binding domain are capable of identifying corresponding (complementary) nucleotides in a target nucleic acid molecule and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in a target nucleic acid molecule; at least one or at least two nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs, and at least one or at least two nucleotides in the target binding domain are a universal base or a degenerate base; and a barcode domain comprising a synthetic backbone, the barcode domain comprising at least three attachment sites, each attachment site comprising at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, each attachment site of the at least three attachment sites corresponding to two nucleotides of the at least six nucleotides in the target binding domain, and each of the at least three attachment sites having a different nucleic acid sequence, wherein the nucleic acid sequence of each of the at least three attachment sites determines the position and identity of the corresponding two nucleotides of the at least six nucleotides in the target nucleic acid to which the target binding domain binds.

[0117] The present disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least 10 nucleotides and is capable of binding to a target nucleic acid, at least six nucleotides in the target binding domain are capable of identifying corresponding (complementary) nucleotides in a target nucleic acid molecule and at least four nucleotides in the target binding domain do not identify corresponding nucleotides in a target nucleic acid molecule; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment positions, each attachment position comprises at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, each attachment region of the at least three attachment positions corresponds to two nucleotides of the at least six nucleotides in the target binding domain, and each of the at least three attachment positions has a different nucleic acid sequence, and the nucleic acid sequence of each of the at least three attachment positions determines the position and attributes of the corresponding two nucleotides of the at least six nucleotides in the target nucleic acid to which the target binding domain binds.

[0118] The present disclosure also provides a population of sequencing probes comprising a plurality of any of the sequencing probes disclosed herein.

[0119] The target binding domains, barcode domains, and scaffolds of the disclosed sequencing probes, as well as complementary nucleic acid molecules (eg, reporter molecules or reporter complexes), are described in more detail below.

[0120] The sequencing probe of the present disclosure includes a target binding domain and a barcode domain. FIG. 1 is a schematic diagram of an example of a sequencing probe according to the present disclosure. FIG. 1 shows that the target binding domain can bind to a target nucleic acid. The target nucleic acid can be any nucleic acid to which the sequencing probe of the present disclosure can hybridize. The target nucleic acid can be DNA or RNA. The target nucleic acid can be obtained from a biological sample derived from a subject. The terms "target binding domain" and "sequencing domain" are used interchangeably herein.

[0121] The target binding domain can comprise a series of nucleotides (e.g., is a polynucleotide). The target binding domain can comprise DNA, or RNA, or a combination thereof. When the target binding domain is a polynucleotide, the target binding domain binds to the target nucleic acid by hybridizing to a portion of the target nucleic acid that is complementary to the target binding domain of the sequencing probe, as shown in FIG. 1.

[0122] The target binding domain of a sequencing probe can be designed to control the likelihood and rate at which hybridization and / or dehybridization of the sequencing probe occurs. In general, the lower the Tm of the probe, the greater the likelihood that the probe will dehybridize from the target nucleic acid. Thus, using a probe with a lower Tm will result in fewer probes binding to the target nucleic acid.

[0123] The length of the target binding domain partially influences the likelihood that a probe will hybridize to and remain hybridized to a target nucleic acid. In general, the longer the target binding domain (the more nucleotides it has), the less likely it is that a complementary sequence will exist in the target nucleotide. Conversely, the shorter the target binding domain, the more likely it will be that a complementary sequence will exist in the target nucleotide. For example, the probability that a tetramer sequence will be located in a target nucleic acid is 1 / 256, whereas the probability that a hexamer sequence will be located in a target nucleic acid is 1 / 4096. As a consequence of the above, a set of shorter probes will be more likely to bind to more positions in a given length of nucleic acid when compared to a set of longer probes.

[0124] In various cases, to increase the number of reads for a given length of nucleic acid, it is preferable to provide probes with shorter target binding domains, which allows for greater coverage of a target nucleic acid or a portion of said target nucleic acid, particularly a portion of special interest, for example when detecting mutations or SNP alleles.

[0125] The target binding domain can be any number of nucleotides in length: the target binding domain can be at least 12 nucleotides in length, at least 10 nucleotides in length, at least 8 nucleotides in length, at least 6 nucleotides in length, or at least 3 nucleotides in length.

[0126] Each nucleotide in the target binding domain can identify (or code for) a complementary nucleotide in the target molecule, or, alternatively, some nucleotides in the target binding domain identify (or code for) complementary nucleotides in the target molecule while some nucleotides in the target binding domain do not identify (or code for) a complementary nucleotide in the target molecule.

[0127] The target binding domain can include at least one natural base. The target binding domain can be free of natural bases. The target binding domain can include at least one modified nucleotide or nucleic acid analog. The target binding domain can be free of modified nucleotides or nucleic acid analogs. The target binding domain can include at least one universal base. The target binding domain can be free of universal bases. The target binding domain can include at least one degenerate base. The target binding domain can be free of degenerate bases.

[0128] A target binding domain can comprise any combination of natural bases (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural bases), modified nucleotides or nucleic acid analogs (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides or nucleic acid analogs), universal bases (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more universal bases), degenerate bases (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more degenerate bases). The natural bases, modified nucleotides or nucleic acid analogs, universal bases, degenerate bases of a particular target binding domain, when present in combination, can be arranged in any order.

[0129] Non-limiting examples of the term "modified nucleotide" or "nucleic acid analog" include bridged nucleic acid (LNA), bridged nucleic acid (BNA), propyne modified nucleic acid, zip nucleic acid (ZNA®), isoguanine, isocytosine. The target binding domain can include 0-6 (e.g., 0, 1, 2, 3, 4, 5, 6) modified nucleotides or nucleic acid analogs. The modified nucleotide or nucleic acid analog is preferably a bridged nucleic acid (LNA).

[0130] As used herein, non-limiting examples of the term "bridged nucleic acid (LNA)" include modified RNA nucleotides in which the ribose moiety contains a methylene bridge connecting the 2' oxygen and the 4' carbon. This methylene bridge locks the ribose into the 3'-endo conformation (also known as the north conformation) found in A-form RNA duplexes. The term inaccessible RNA can be used interchangeably with LNA. As used herein, non-limiting examples of the term "bridged nucleic acid (BNA)" include modified RNA molecules containing a 5- or 6-membered bridge structure with a fixed 3'-endo conformation (also known as the north conformation). The bridge structure connects the 2' oxygen of the ribose to the 4' carbon of the ribose. A variety of different bridge structures are possible, including carbon, nitrogen, and hydrogen atoms. As used herein, non-limiting examples of the term "propyne-modified nucleic acid" include pyrimidines, i.e., cytosine and thymine / uracil, containing propyne modifications at the C5 position of the nucleobase. As used herein, non-limiting examples of the term "Zip Nucleic Acid (ZNA®)" include oligonucleotides conjugated to a cationic spermine moiety.

[0131] As used herein, non-limiting examples of the term "universal base" include nucleotide bases that do not follow the Watson-Crick base pairing rules but can bind to any of the four basic bases (A, T / U, C, G) located on a target nucleic acid. As used herein, non-limiting examples of the term "degenerate base" include nucleotide bases that do not follow the Watson-Crick base pairing rules but can bind to at least two but not all four of the four canonical bases (A, T / U, C, G) located on a target nucleic acid. Degenerate bases can also be referred to as wobble bases, and these terms are used interchangeably herein.

[0132] The sequencing probe illustrated in FIG. 1 is a 6 nucleotide sequence (b) that specifically hybridizes to complementary nucleotides 1 to 6 of the target nucleic acid to be sequenced.1 -b 2 -b 3 -b 4 -b 5 -b 6 ) is shown. This hexameric portion of the target binding domain (b 1 -b 2 -b 3 -b 4 -b 5 -b 6 ) identifies (or codes for) a complementary nucleotide (1-2-3-4-5-6) in the target sequence. This hexamer sequence is flanked on each side by bases (N). The bases designated by (N) can independently be universal or degenerate bases. Typically, the bases designated by (N) are independently one of the canonical bases. The bases designated by (N) do not identify (or code for) the complementary nucleotide to which they bind in the target sequence, but rather are aligned along the (hexamer) sequence (b 1 -b 2 -b 3 -b 4 -b 5 -b 6 ) is independent of the nucleic acid sequence of

[0133] The sequencing probes shown in Figure 1 can be used in combination with the sequencing method of the present disclosure to sequence a target nucleic acid, which utilizes only a hybridization reaction and does not require covalent chemistry, enzymes, or amplification. A total of 4096 sequencing probes are required to sequence all possible hexamer sequences in a target nucleic acid molecule (4 6 =4096).

[0134] FIG. 1 is an example of one configuration of the target binding domain of a sequencing probe of the present disclosure. Table 1 shows several alternative configurations of the target binding domain of the present disclosure. One preferred target binding domain is referred to as a "6 LNA" target binding domain, which contains six LNAs at positions b1-b6 of the target binding domain. These six LNAs are flanked on each side by bases (N). Bases (N) are defined herein as having the (hexamer) sequence (b 1 -b 2 -b 3 -b 4 -b 5 -b 6 ) can be a universal / degenerate base independent of the nucleic acid sequence, or a regular base. In other words, base b 1 -b 2 -b 3 -b 4 -b 5 -b 6 may be specific for any given target sequence, whereas (N) bases are universal / degenerate bases or are the only bases that are unique to the four regular bases. 1 -b 2 -b 3 -b 4 -b 5 -b 6 For example, if the target sequence to be examined is CAGGCATA, then the bases b 1 -b 2 -b 3 -b 4 -b 5 -b 6 would be TCCGTA, whereas each of the (N) bases of the target binding domain could independently be A, C, T, or G, such that the resulting target binding domain could be any of the sequences ATCCGTAG, TTCCGTAC, GTCCGTAG, or any other of 16 possible sequences. Alternatively, it is contemplated that two (N) bases could precede the 6 LNAs. Alternatively, it is further contemplated that two (N) bases could follow the 6 LNAs.

[0135] [Table 1]

[0136] Table 1 also describes a "10-mer" target binding domain that contains 10 naturally occurring target-specific bases.

[0137] Table 1 further includes position b 1 ~b 6 Also described is a "Natural I" target binding domain that contains six natural bases in a nucleotide sequence. The six natural bases are flanked on each side by two (N) bases. Alternatively, all four (N) bases could precede the six natural bases. Alternatively, all four (N) bases could follow the six natural bases. Any number of the four (N) bases (i.e., 1, 2, 3, 4) could precede the six natural bases, and the remaining (N) base would follow the six natural bases.

[0138] Table 1 further includes position b 1 ~b 6 Also described is a "Natural II" target binding domain that contains six natural bases at 1. These six natural bases are flanked on each side by one (N) base. Alternatively, both (N) bases could be located before the six natural bases. Alternatively, both (N) bases could be located after the six natural bases.

[0139] Table 1 further includes the location of the target binding domain b 1 ~b 6 Also described is a "2 LNA" target binding domain that contains a combination of two LNAs and four natural bases at position b. The two LNAs and the four natural bases can appear in any order. For example, 3 and b 4 If it is possible to make it an LNA, position b 1 , b 2 , b 5 , b6 is a natural base. Base b 1 ~b 6 is flanked on each side by one (N) base, or base b 1 ~b 6 can be preceded by two (N) bases. Alternatively, base b 1 ~b 6 can be followed by two (N) bases.

[0140] Table 1 further includes the location of the target binding domain b 1 ~b 6 Also described is a "4 LNA" target binding domain that contains a combination of four LNAs and two natural bases at position b. The four LNAs and two natural bases can appear in any order. For example, at position b 2 ~b 5 If it is possible to make it an LNA, position b 1 and b 6 is a natural base. Base b 1 ~b 6 is flanked on each side by one (N) base, or base b 1 ~b 6 can be preceded by two (N) bases. Alternatively, base b 1 ~b 6 can be followed by two (N) bases.

[0141] The sequencing probe of the present disclosure includes a synthetic scaffold. The target binding domain and the barcode domain are operably linked. The target binding domain and the barcode domain can be covalently linked as part of one synthetic scaffold. The target binding domain and the barcode domain can be linked via a linker (e.g., a nucleic acid linker, a chemical linker). The synthetic scaffold can include any material (e.g., a polysaccharide, a polynucleotide, a polymer, a plastic, a fiber, a peptide, a peptide nucleic acid, a polypeptide). The synthetic scaffold is preferably rigid. The synthetic scaffold can include a single-stranded DNA molecule. The scaffold can include a "DNA origami" consisting of six DNA double helices (see, e.g., Lin et al., "Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA," Nature Chemistry; October 2012; Vol. 4(10): pp. 832-839). Barcodes can be made from DNA origami tiles (Jungmann et al., "Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT," Nature Methods, Vol. 11, No. 3, 2014).

[0142] The sequencing probes of the present disclosure can include a synthetic backbone that is partially double-stranded. The sequencing probes can include a single-stranded DNA synthetic backbone and a double-stranded DNA spacer between the target binding domain and the barcode domain. The sequencing probes can include a single-stranded DNA synthetic backbone and a polymer-based spacer between the target binding domain and the barcode domain that has mechanical properties similar to double-stranded DNA. Exemplary polymer-based spacers include polyethylene glycol (PEG)-based polymers.

[0143] The double-stranded DNA spacer can be about 1 to about 100 nucleotides in length, or about 2 to about 50 nucleotides in length, or about 20 to about 40 nucleotides in length. The double-stranded DNA spacer is preferably about 36 nucleotides in length.

[0144] One sequencing probe of the present disclosure is referred to as the "standard probe" and is shown in the left diagram of Figure 2. The standard probe of Figure 2 contains a barcode domain covalently linked to a target binding domain, such that the target binding domain and the barcode domain are in the same oligonucleotide strand. In the left diagram of Figure 2, a single-stranded oligonucleotide is attached to a stem oligonucleotide to create a double-stranded spacer region 36 nucleotides in length, called the stem sequence. With this structure, each sequencing probe in a pool of probes can hybridize to the same stem sequence.

[0145] Another sequencing probe of the present disclosure is called a "three-part probe" and is shown in the right diagram of FIG. 2. The three-part probe of FIG. 2 includes a barcode domain linked to a target binding domain via a linker. In this example, the linker is a single-stranded stem oligonucleotide that hybridizes to a single-stranded oligonucleotide containing the target binding domain and a single-stranded oligonucleotide containing the barcode domain, creating a double-stranded spacer region 36 nucleotides in length that bridges the barcode domain (18 nucleotides) and the target binding domain (18 nucleotides). With this example of a probe configuration, each barcode can be designed to hybridize to a single stem sequence in order to prevent exchange of barcode domains. Furthermore, different sequencing probes can be pooled together after each barcode domain is hybridized to its corresponding stem oligonucleotide.

[0146] The barcode domain comprises multiple attachment positions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more attachment positions). The number of attachment positions can be less than, equal to, or greater than the number of nucleotides in the target binding domain. The target binding domain can comprise more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides) than the number of attachment positions in the scaffold domain. The target binding domain can comprise 8 nucleotides and the barcode domain comprises 3 attachment positions. The target binding domain can comprise 10 nucleotides and the barcode domain comprises 3 attachment positions.

[0147] The barcode domain is not limited in length, so long as there is sufficient length for at least three attachment positions, as described below. The terms "attachment position," "position," and "spot" are used interchangeably herein. The terms "barcode domain" and "reporting domain" are used interchangeably herein.

[0148] Each attachment position in the barcode domain corresponds to two nucleotides (dinucleotides) in the target binding domain and thus to a complementary dinucleotide in the target binding domain that hybridizes to said dinucleotide in the target binding domain. As a non-limiting example, a first attachment position in the barcode domain corresponds to a first and second nucleotide in the target binding domain (e.g., in FIG. 1, R 1 is the first attachment position in the barcode domain, and R 1 is the dinucleotide b in the target binding domain 1 and b 2 , which identifies dinucleotides 1 and 2 of the target nucleic acid), and the second attachment position in the barcode domain corresponds to the third and fourth nucleotides in the target binding domain (e.g., in FIG. 1 , R 2 is the second attachment position in the barcode domain, and R 2is the dinucleotide b in the target binding domain 3 and b 4 , which identifies dinucleotides 3 and 4 of the target nucleic acid), the third attachment position in the barcode domain corresponds to the fifth and sixth nucleotides in the target binding domain (e.g., in FIG. 1 , R 3 is the third attachment position in the barcode domain, and R 3 is the dinucleotide b in the target binding domain 5 and b 6 , which identifies dinucleotides 5 and 6 of the target nucleic acid).

[0149] Each attachment location in the barcode domain includes at least one attachment region (e.g., 1 to 50, or more attachment regions). Some locations in the barcode domain can have more attachment regions than other locations (e.g., a first attachment location can have three attachment regions, whereas a second attachment location can have two attachment regions). Alternatively, each attachment location in the barcode domain has the same number of attachment regions. Each attachment location in the barcode domain can include one attachment region. Each attachment location in the barcode domain can include two or more attachment regions. At least one of the at least three attachment locations in the barcode domain can include a different number of attachment regions than the other two attachment locations in the barcode domain.

[0150] Each attachment region comprises at least one copy (i.e. 1-50, e.g. 10-30) of a nucleic acid sequence to which a complementary nucleic acid molecule (e.g. DNA or RNA) can reversibly bind. The nucleic acid sequences of the attachment regions at one attachment position can be the same, in which case the complementary nucleic acid molecules that bind to these attachment regions are the same. Alternatively, the nucleic acid sequences of the attachment regions at one position are not the same, in which case the complementary nucleic acid molecules that bind to these attachment regions are not the same.

[0151] The nucleic acid sequence comprising each attachment region in the barcode domain can be from about 8 to about 20 nucleotides in length. The nucleic acid sequence comprising each attachment region in the barcode domain can be from about 12 to about 14 nucleotides in length. Preferably, the nucleic acid sequence comprising each attachment region in the barcode domain is about 14 nucleotides in length.

[0152] Each of the nucleic acids comprising each attachment region in the barcode domain can be independently a canonical base, or a modified nucleotide, or a nucleic acid analog. At least one, or at least two, or at least three, or at least four, or at least five, or at least six nucleotides in the attachment region in the barcode domain can be modified nucleotides or nucleic acid analogs. A typical ratio of modified nucleotides or nucleic acid analogs to canonical bases in the barcode domain is 1:2 to 1:8. Exemplary modified nucleotides or nucleic acid analogs useful in attachment regions in the barcode domain are isoguanine and isocytosine. The use of modified nucleotides or nucleic acid analogs (e.g., isoguanine and isocytosine) can improve the efficiency and accuracy of binding of the reporter to the appropriate attachment region in the barcode domain while minimizing binding elsewhere, including to the target.

[0153] One or more attachment regions can be integrated with the polynucleotide backbone, i.e. the backbone is a single polynucleotide and the attachment regions are part of the sequence of said single polynucleotide. One or more attachment regions can be linked to modified monomers (e.g. modified nucleotides) in a synthetic backbone and said attachment regions can be branched off from the synthetic backbone. An attachment site can include two or more attachment regions, some of which branch off from the synthetic backbone while some of the attachment regions are integrated with the synthetic backbone. At least one attachment region in at least one attachment site can be integrated with the synthetic backbone. Each attachment region in each of said at least one attachment site can be integrated with the synthetic backbone. At least one attachment region in at least one attachment site can be branched off from the synthetic backbone. Each attachment region in each of at least three attachment sites can be branched off from the synthetic backbone.

[0154] Each attachment position in the barcode domain corresponds to one of 16 dinucleotides, namely adenine-adenine, adenine-thymine / uracil, adenine-cytosine, adenine-guanine, thymine / uracil-adenine, thymine / uracil-thymine / uracil, thymine / uracil-cytosine, thymine / uracil-guanine, cytosine-adenine, cytosine-thymine / uracil, cytosine-cytosine, cytosine-guanine, guanine-adenine, guanine-thymine / uracil, guanine-cytosine, and guanine-guanine. Thus, one or more attachment regions located in a single attachment position of the barcode domain correspond to one of 16 dinucleotides, and contain a nucleic acid sequence specific to the dinucleotide to which the attachment region corresponds. The attachment regions located in different attachment positions of the barcode domain contain unique nucleic acid sequences, even if those positions in the barcode domain correspond to the same dinucleotide. For example, given a sequencing probe of the present disclosure containing a target binding domain having a hexamer encoding the sequence AGAGAC, the barcode domain of the sequencing probe would contain three positions, with the first attachment position corresponding to the dinucleotide adenine-guanine, the second attachment position corresponding to the dinucleotide adenine-guanine, and the third attachment position corresponding to the dinucleotide adenine-cytosine. The attachment region located at position 1 of the probe in this example would contain a unique nucleic acid sequence that is different from the nucleic acid sequence of the attachment region located at position 2, even if both attachment positions 1 and 2 correspond to the dinucleotide adenine-guanine. The sequences of the specific attachment positions are designed to ensure that the complementary nucleic acids at each attachment position do not interact with the different attachment positions. In addition, there is no restriction on the nucleotide sequence of the complementary nucleic acid. It is preferred that the nucleotide sequence has no substantial homology (e.g., 50% to 99.9%) with known nucleotide sequences. This limits undesired hybridization of the complementary nucleic acid with the target nucleic acid.

[0155] FIG. 1 shows a diagram of an example of a sequencing probe of the present disclosure that includes an example barcode domain. The barcode domain illustrated in FIG. 1 has three attachment positions R 1 , R 2 , R 3 Each attachment site contains a hexameric sequence of a target binding domain (b 1 ~b 6 ) corresponds to a specific dinucleotide present in the 1 is position b 1 and b 2 Corresponding to R 2 is position b 3 and b 4 Corresponding to R 3 is position b 5 and b 6 Each position therefore encodes a specific dinucleotide present in the hexameric sequence of the target binding domain, thereby allowing the identification of the two specific bases (either A, C, G or T) present in each dinucleotide.

[0156] In the example barcode domain shown in FIG. 1, each attachment site contains a single attachment region integrated into a synthetic scaffold. Each attachment region of the three attachment sites contains a specific nucleotide sequence that corresponds to an individual dinucleotide encoded by each attachment site. For example, attachment site R 1 is the dinucleotide b 1 -b 2 Each of the antibodies contains an attachment region having a specific sequence corresponding to an attribute of the antibody.

[0157] The barcode domain can further comprise one or more binding regions. The barcode domain can comprise at least one single-stranded nucleic acid sequence adjacent to at least one attachment location. The barcode domain can comprise at least two single-stranded nucleic acid sequences adjacent to at least two attachment locations. The barcode domain can comprise at least three single-stranded nucleic acid sequences adjacent to at least three attachment locations. These adjacent portions are known as "toe-holds" and can be used to accelerate the exchange rate of oligonucleotides hybridized adjacent to the toe-holds by providing additional binding sites for single-stranded oligonucleotides ("toe-hold" probes; see, e.g., Seeling et al., "Catalyzed Relaxation of a Metastable DNA Fuel"; J. Am. Chem. Soc. 2006, 128(37), 12211-12220).

[0158] The sequencing probes of the present disclosure can have a total length of about 20 nanometers to about 50 nanometers (including the target binding domain, barcode domain, and any optional domains). The backbone of the sequencing probe can be a polynucleotide molecule containing about 120 nucleotides.

[0159] The sequencing probe may include a cleavable linker modification. Any cleavable linker modification known to those skilled in the art may be used. Non-limiting examples of cleavable linker modifications include UV light cleavable linkers, reducing agent cleavable linkers, and enzyme cleavable linkers. An example of an enzyme cleavable linker is the insertion of uracil for cleavage by USER™ enzyme. The cleavable linker modification may be located anywhere along the length of the sequencing probe, non-limiting examples of locations include the region between the target binding domain and the barcode domain. The right diagram of FIG. 10 shows cleavable linker modifications that may be incorporated into the probes of the present disclosure.

[0160] Reporter Probe

[0161] The nucleic acid molecule that binds (e.g., hybridizes) to a complementary nucleic acid sequence in at least one attachment region in at least one attachment location of the barcode domain of the sequencing probe according to the present disclosure comprises (directly or indirectly) a detectable label. The detectable label is referred to herein as a "reporter probe" or a "reporter probe complex," and these terms are used interchangeably herein. The reporter probe can be DNA, RNA, or PNA. The reporter probe is preferably DNA.

[0162] A reporter probe can include at least two domains, a first domain capable of binding to at least one complementary nucleic acid molecule and a second domain capable of binding to a first detectable label and at least a second detectable label. Figure 3 shows a schematic diagram of an example reporter probe according to the present disclosure attached to a first attachment position of a barcode domain of an example sequencing probe. In Figure 3, the first domain of the reporter probe (shown as a maroon checkerboard pattern) is attached to the attachment position R of the barcode domain. 1 and the second domain of the reporter probe (shown in grey) is linked to two detectable labels (one green label and one red label).

[0163] Alternatively, the reporter probe can comprise at least two domains, a first domain capable of binding to at least one first complementary nucleic acid molecule and a second domain capable of binding to at least one second complementary nucleic acid molecule, wherein the at least one first complementary nucleic acid molecule and the at least one second complementary nucleic acid molecule can be different (have different nucleic acid sequences).

[0164] A "primary nucleic acid molecule" is a reporter probe that comprises at least two domains, a first domain of which is capable of binding (hybridizing) to a complementary nucleic acid sequence in at least one attachment region in at least one attachment location of the barcode domain of the sequencing probe, and a second domain of which is capable of binding (hybridizing) to at least one additional complementary nucleic acid. The primary nucleic acid molecule can directly bind to a complementary nucleic acid sequence in at least one attachment region in at least one attachment location of the barcode domain of the sequencing probe. The primary nucleic acid molecule can indirectly bind to a complementary nucleic acid sequence in at least one attachment region in at least one attachment location of the barcode domain of the sequencing probe via a nucleic acid linker. The primary nucleic acid molecule can comprise a cleavable linker. The cleavable linker can be located between the first and second domains. The cleavable linker is preferably cleavable by light.

[0165] Each of the nucleic acids comprising the first domain of the primary nucleic acid molecule can be independently a regular base, or a modified nucleotide, or a nucleic acid analog. At least one, or at least two, or at least three, or at least four, or at least five, or at least six nucleotides in the first domain of the primary nucleic acid molecule can be a modified nucleotide or nucleotide analog. A typical ratio of modified nucleotides or nucleic acid analogs to regular bases in the barcode domain is 1:2 to 1:8. Exemplary modified nucleotides or nucleic acid analogs useful in the first domain of the primary nucleic acid molecule are isoguanine and isocytosine. The use of modified nucleotides or nucleic acid analogs (e.g., isoguanine and isocytosine) can improve the binding efficiency and binding accuracy of the first domain of the primary nucleic acid molecule to the appropriate complementary nucleic acid sequence in at least one attachment region in at least one attachment position of the barcode domain of the sequencing probe, while minimizing binding to other locations, including the target.

[0166] At least one additional complementary nucleic acid that is bound to a primary nucleic acid molecule is referred to herein as a "secondary nucleic acid molecule." A primary nucleic acid molecule can be bound to (e.g., hybridized with) at least one, or at least two, or at least three, or at least four, or at least five, or more secondary nucleic acid molecules. Preferably, a primary nucleic acid molecule is bound to (e.g., hybridized with) four secondary nucleic acid molecules.

[0167] The secondary nucleic acid molecule comprises at least two domains, a first domain capable of binding (e.g., hybridizing) to at least one complementary sequence in at least one primary nucleic acid molecule, and a second domain capable of binding (e.g., hybridizing) to (a) a first detectable label and at least a second detectable label, or (b) to at least one additional complementary nucleic acid molecule, or (c) to a combination thereof. The secondary nucleic acid molecule may comprise a cleavable linker. The cleavable linker may be located between the first domain and the second domain. The cleavable linker is preferably cleavable by light.

[0168] Each of the nucleic acids comprising the first domain of the secondary nucleic acid molecule can be independently a canonical base, or a modified nucleotide, or a nucleic acid analog. At least one, or at least two, or at least three, or at least four, or at least five, or at least six nucleotides in the first domain of the secondary nucleic acid molecule can be a modified nucleotide or nucleotide analog. A typical ratio of modified nucleotides or nucleic acid analogs to canonical bases in the barcode domain is 1:2 to 1:8. Exemplary modified nucleotides or nucleic acid analogs useful in the first domain of the secondary nucleic acid molecule are isoguanine and isocytosine. The use of modified nucleotides or nucleic acid analogs (e.g., isoguanine and isocytosine) can improve the efficiency and accuracy of binding of the first domain of the secondary nucleic acid molecule to the appropriate complementary nucleic acid sequence in the second domain of the primary nucleic acid molecule while minimizing binding to other sites.

[0169] At least one additional complementary nucleic acid that binds to a secondary nucleic acid molecule is referred to herein as a "tertiary nucleic acid molecule." A secondary nucleic acid molecule can bind (e.g., hybridize) to at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or more tertiary nucleic acid molecules. Preferably, the at least one secondary nucleic acid molecule binds (e.g., hybridizes) to one tertiary nucleic acid molecule.

[0170] The tertiary nucleic acid molecule comprises at least two domains, a first domain capable of binding (e.g., hybridizing) to at least one complementary sequence in at least one secondary nucleic acid molecule, and a second domain capable of binding (e.g., hybridizing) to a first detectable label and at least a second detectable label. Alternatively, the second domain can comprise a first detectable label and at least a second detectable label through direct or indirect attachment of the labels during oligonucleotide synthesis, for example, using phosphoramidite or NHS chemistry. The tertiary nucleic acid molecule can comprise a cleavable linker. The cleavable linker can be located between the first and second domains. The cleavable linker is preferably cleavable by light.

[0171] Each of the nucleic acids comprising the first domain of the tertiary nucleic acid molecule is independently a canonical base, or a modified nucleotide, or a nucleic acid analog. At least one, or at least two, or at least three, or at least four, or at least five, or at least six nucleotides in the first domain of the tertiary nucleic acid can be a modified nucleotide or nucleotide analog. A typical ratio of modified nucleotides or nucleic acid analogs to canonical bases in the first domain of the tertiary nucleic acid molecule is 1:2 to 1:8. Exemplary modified nucleotides or nucleic acid analogs useful in the first domain of the tertiary nucleic acid molecule are isoguanine and isocytosine. The use of modified nucleotides or nucleic acid analogs (e.g., isoguanine and isocytosine) can improve the efficiency and accuracy of binding of the first domain of the tertiary nucleic acid molecule to the appropriate complementary nucleic acid sequence in the second domain of the secondary nucleic acid molecule while minimizing binding to other sites.

[0172] The reporter probe binds a first detectable label and at least a second detectable label to produce a two-color combination. This dual combination of fluorescent dyes can include a single color overlap (e.g., blue-blue). As used herein, the term "label" includes a single moiety capable of generating a detectable signal, or multiple moieties capable of generating the same or substantially the same detectable signal. For example, the label can include a single yellow fluorescent dye (such as ALEXA FLUOR™ 532), or multiple yellow fluorescent dyes (such as ALEXA FLUOR™ 532, etc.).

[0173] The reporter probe can be conjugated to a first detectable label and at least a second detectable label, each detectable label being one of four fluorescent dyes: blue (B), green (G), yellow (Y), and red (R). Using these four dyes results in ten possible two-color combinations (BB; BG; BR; BY; GG; GR; GY; RR; RY; YY). In some aspects, the reporter probes of the present disclosure are labeled with one of eight possible color combinations, namely BB; BG; BR; BY; GG; GR; GY; YY, as shown in FIG. 3. The first detectable label and the at least second detectable label can have the same emission spectrum or different emission spectra.

[0174] In aspects involving a sequencing probe and a primary nucleic acid molecule, the disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, at least six nucleotides in the target binding domain are capable of identifying corresponding (complementary) nucleotides in a target nucleic acid molecule and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in a target nucleic acid molecule; at least one, or at least two, of the six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs; and the barcode domain comprises a synthetic backbone, the barcode domain comprising at least three attachment positions. Also provided is a sequencing probe, wherein each attachment location comprises at least one attachment region comprising at least one nucleic acid sequence to which at least one complementary primary nucleic acid molecule binds, said complementary primary nucleic acid molecule comprising a first detectable label and at least a second detectable label, each of said at least three attachment locations corresponds to two nucleotides of said at least six nucleotides in the target binding domain, and each of said at least three attachment locations has a different nucleic acid sequence, and the at least first detectable label and the at least second detectable label of each complementary primary nucleic acid molecule bound to each of said at least three attachment locations determine the position and identity of the corresponding two nucleotides of said at least six nucleotides in the target nucleic acid to which the target binding domain binds. The at least two nucleotides in the target binding domain that do not identify corresponding nucleotides in the target nucleic acid molecule can be any of four regular bases that are not specific for a target not specified by the at least six nucleotides in the target binding domain, or universal bases, or degenerate bases.

[0175] In aspects involving a sequencing probe and a primary nucleic acid molecule, the disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least 10 nucleotides and is capable of binding to a target nucleic acid, at least 6 nucleotides in the target binding domain are capable of identifying corresponding (complementary) nucleotides in the target nucleic acid molecule and at least 4 nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site being at least one complementary primary nucleic acid molecule to which at least one complementary primary nucleic acid molecule binds. Also provided is a sequencing probe comprising at least one attachment region comprising a nucleic acid sequence, the complementary primary nucleic acid molecule comprising a first detectable label and at least a second detectable label, each of the at least three attachment positions corresponding to two of the at least six nucleotides in the target binding domain, each of the at least three attachment positions having a different nucleic acid sequence, and the at least first detectable label and the at least second detectable label of each complementary primary nucleic acid molecule bound to each of the at least three attachment positions determine the position and identity of the corresponding two of the at least six nucleotides in the target nucleic acid bound by the target binding domain.

[0176] In aspects involving a sequencing probe, a primary nucleic acid molecule, and a secondary nucleic acid molecule, the disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, at least six nucleotides in the target binding domain are capable of identifying corresponding (complementary) nucleotides in a target nucleic acid molecule, and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in a target nucleic acid molecule, and at least one, or at least two, of the six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs, and the at least one, or at least two, of the target binding domain are any of four canonical bases that are not specific for a target not specified by other bases in the target binding domain, or a universal base, or a degenerate base. Also provided is a sequencing probe, wherein the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment locations, each attachment location comprising at least one attachment region comprising at least one nucleic acid sequence to which at least one complementary primary nucleic acid molecule is bound, the complementary primary nucleic acid molecule further comprising at least one complementary secondary nucleic acid molecule bound thereto comprising a first detectable label and at least a second detectable label, each of the at least three attachment locations corresponding to two of the at least six nucleotides in the target binding domain, each of the at least three attachment locations having a different nucleic acid sequence, and wherein the at least first detectable label and the at least second detectable label of each complementary secondary nucleic acid molecule bound to each of the at least three attachment locations determine the position and identity of the corresponding two of the at least six nucleotides in the target nucleic acid to which the target binding domain is bound.

[0177] In aspects involving a sequencing probe, a primary nucleic acid molecule, and a secondary nucleic acid molecule, the disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least 10 nucleotides and is capable of binding to a target nucleic acid, at least 6 nucleotides in the target binding domain are capable of identifying corresponding (complementary) nucleotides in the target nucleic acid molecule and at least 4 nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; and the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprising at least one nucleic acid sequence to which at least one complementary primary nucleic acid molecule binds. and wherein the complementary primary nucleic acid molecule further comprises at least one complementary secondary nucleic acid molecule bound to the target binding domain, the complementary primary nucleic acid molecule comprising a first detectable label and at least a second detectable label, each of the at least three attachment positions corresponding to two of the at least six nucleotides in the target binding domain, each of the at least three attachment positions having a different nucleic acid sequence, and wherein the at least first detectable label and the at least second detectable label of each complementary secondary nucleic acid molecule bound to each of the at least three attachment positions determine the positions and identities of the corresponding two of the at least six nucleotides in the target nucleic acid bound to the target binding domain.

[0178] In aspects involving sequencing probes, primary nucleic acid molecules, secondary nucleic acid molecules, and tertiary nucleic acid molecules, the disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, at least six nucleotides in the target binding domain are capable of identifying corresponding (complementary) nucleotides in a target nucleic acid molecule, and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; and at least one, or at least two, of the six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs, and the at least two nucleotides in the target binding domain that do not identify corresponding nucleotides in a target nucleic acid molecule can be any of four canonical bases that are not specific for a target not specified by the at least six nucleotides in the target binding domain, or a universal base, or a degenerate base. and a barcode domain comprising a synthetic backbone, the barcode domain comprising at least three attachment sites, each attachment site comprising at least one attachment region comprising at least one nucleic acid sequence to which at least one complementary primary nucleic acid molecule is bound, the complementary primary nucleic acid molecule further comprising at least one complementary secondary nucleic acid molecule bound to the at least one complementary secondary nucleic acid molecule further comprising at least one complementary tertiary nucleic acid molecule comprising a first detectable label and at least a second detectable label, each of the at least three attachment sites corresponding to two of the at least six nucleotides in the target binding domain, each of the at least three attachment sites having a different nucleic acid sequence, and wherein the first detectable label and the at least a second detectable label of each complementary tertiary nucleic acid molecule bound to each of the at least three attachment sites determine the position and identity of the corresponding two of the at least six nucleotides in the target nucleic acid to which the target binding domain is bound.

[0179] In an aspect including a sequencing probe, a primary nucleic acid molecule, a secondary nucleic acid molecule, and a tertiary nucleic acid molecule, the disclosure provides a sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least 10 nucleotides and is capable of binding to a target nucleic acid, at least 6 nucleotides in the target binding domain are capable of identifying corresponding (complementary) nucleotides in the target nucleic acid molecule and at least 4 nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprising at least one attachment region comprising at least one nucleic acid sequence to which at least one complementary primary nucleic acid molecule binds; Also provided is a sequencing probe having at least one complementary secondary nucleic acid molecule bound to the complementary primary nucleic acid molecule, the at least one complementary secondary nucleic acid molecule bound to the complementary tertiary nucleic acid molecule comprising a first detectable label and at least a second detectable label, each of the at least three attachment positions corresponding to two of the at least six nucleotides in the target binding domain, each of the at least three attachment positions having a different nucleic acid sequence, and the first detectable label and the at least a second detectable label of each complementary tertiary nucleic acid molecule bound to each of the at least three attachment positions determine the position and identity of the corresponding two of the at least six nucleotides in the target nucleic acid bound to the target binding domain.

[0180] The present disclosure also provides a sequencing probe and a reporter probe having detectable labels on both the surface of the secondary and tertiary nucleic acid molecules. For example, the secondary nucleic acid molecule can be bound to the primary nucleic acid molecule, and the secondary nucleic acid molecule can include both a first detectable label and at least a second detectable label, and can also be bound to at least one tertiary nucleic acid molecule that includes a first detectable label and at least a second detectable label. The first detectable label and at least a second detectable label located on the surface of the secondary nucleic acid molecule can have the same emission spectrum or different emission spectra. The first detectable label and at least a second detectable label located on the surface of the tertiary nucleic acid molecule can have the same emission spectrum or different emission spectra. The emission spectra of the detectable labels located on the surface of the secondary nucleic acid molecule can be the same as each other or different from the emission spectrum of the detectable labels located on the surface of the tertiary nucleic acid molecule.

[0181] 4 is a schematic diagram of an example of a reporter probe of the present disclosure, including an example of a primary nucleic acid molecule, a secondary nucleic acid molecule, and a tertiary nucleic acid molecule. The primary nucleic acid molecule includes a first domain at the 3' end, which includes a 12-nucleotide sequence that hybridizes to a complementary attachment region in one attachment position of the barcode domain of the sequencing probe. At the 5' end is a second domain that hybridizes to six secondary nucleic acid molecules. The illustrated secondary nucleic acid molecule, in turn, includes a first domain at the 5' end that hybridizes to the primary nucleic acid molecule and a domain at the 3' portion that hybridizes to five tertiary nucleic acid molecules.

[0182] Tertiary nucleic acid molecules contain at least two domains. The first domain is capable of binding to a secondary nucleic acid molecule. The second domain of the tertiary nucleic acid is capable of binding to a first detectable label and at least a second detectable label. The second domain of the tertiary nucleic acid is capable of binding to a first detectable label and at least a second detectable label by directly incorporating one or more fluorescently labeled nucleotide monomers into the sequence of the second domain of the tertiary nucleic acid. The second domain of the secondary nucleic acid molecule is capable of binding to a first detectable label and at least a second detectable label by hybridizing a labeled short polynucleotide to the second domain of the secondary nucleic acid molecule. These short polynucleotides (referred to as "labeled oligos") can be labeled by directly incorporating fluorescently labeled nucleotide monomers or by other nucleic acid labeling methods known to those skilled in the art. The tertiary nucleic acid illustrated in Figure 4 can be considered a "labeled oligo" and includes a first domain that hybridizes to a secondary nucleic acid molecule and includes a second domain that is fluorescently labeled, for example, by indirectly attaching a label during oligonucleotide synthesis using NHS chemistry or by incorporating one or more fluorescently labeled nucleotide monomers during synthesis of the tertiary nucleic acid molecule. The labeled oligo can be DNA, RNA, or PNA.

[0183] In an alternative aspect, the second domain of the secondary nucleic acid molecule can be bound to a first detectable label and at least a second detectable label. The second domain of the secondary nucleic acid molecule can be bound to a first detectable label and at least a second detectable label by directly incorporating one or more fluorescently labeled nucleotide monomers into the sequence of the second domain of the secondary nucleic acid. The second domain of the secondary nucleic acid molecule can be bound to a first detectable label and at least a second detectable label by hybridizing a labeled short polynucleotide to the second domain of the secondary nucleic acid. These short polynucleotides (referred to as "labeled oligos") can be labeled by directly incorporating fluorescently labeled nucleotide monomers or by other nucleic acid labeling methods known to those skilled in the art.

[0184] The primary nucleic acid molecule can comprise about 100, or about 95, or about 90, or about 85, or about 80, or about 75 nucleotides. The primary nucleic acid molecule can comprise about 100 to about 80 nucleotides. The primary nucleic acid molecule can comprise about 90 nucleotides. The secondary nucleic acid molecule can comprise about 90, or about 85, or about 80, or about 75, or about 70 nucleotides. The secondary nucleic acid molecule can comprise about 90 to about 80 nucleotides. The secondary nucleic acid molecule can comprise about 87 nucleotides. The secondary nucleic acid molecule can comprise about 25, or about 20, or about 15, or about 10 nucleotides. The tertiary nucleic acid molecule can comprise about 20 to about 10 nucleotides. The tertiary nucleic acid molecule can comprise about 15 nucleotides.

[0185] The reporter probes of the present disclosure can be designed in various ways. For example, a primary nucleic acid molecule can be hybridized to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) secondary nucleic acid molecules. Each secondary nucleic acid molecule can be hybridized to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tertiary nucleic acid molecule. To generate a reporter probe labeled with a specific two-color combination, a reporter probe is designed that includes secondary nucleic acid molecules, or tertiary nucleic acid molecules, or labeled oligos, or any combination of secondary nucleic acid molecules, tertiary nucleic acid molecules, and labeled oligos, labeled with each color of the specific two-color combination. For example, FIG. 4 shows a reporter probe of the present disclosure that includes a total of 30 dyes (15 dyes in color 1 and 15 dyes in color 2). To avoid color exchange or cross-hybridization between different fluorescent dyes, each tertiary nucleic acid or labeled oligo bound to a particular label or fluorescent dye contains a unique nucleotide sequence.

[0186] Figure 5 shows four examples of reporter probes of the present disclosure. The top left diagram of Figure 5 shows a 5x5 reporter probe. The 5x5 probe comprises a primary nucleic acid, the primary nucleic acid comprising a first domain of 12 nucleotides. The primary nucleic acid also comprises a second domain, the second domain comprising a nucleotide sequence capable of hybridizing to five secondary nucleic acid molecules. Each secondary nucleic acid molecule comprises a nucleotide sequence that allows five tertiary nucleic acids having detectable labels attached thereto to hybridize to each secondary nucleic acid.

[0187] The top right diagram of Figure 5 shows a 4x3 reporter probe. The 4x3 reporter probe comprises a primary nucleic acid, the primary nucleic acid comprising a first domain of 12 nucleotides. The primary nucleic acid also comprises a second domain, the second domain comprising a nucleotide sequence capable of hybridizing to four secondary nucleic acid molecules, each of which comprises a nucleotide sequence that allows three tertiary nucleic acid molecules having detectable labels attached thereto to hybridize to each of the secondary nucleic acid molecules.

[0188] The bottom left diagram of Figure 5 shows a 3x4 reporter probe. The 3x4 reporter probe comprises a primary nucleic acid, the primary nucleic acid comprising a first domain of 12 nucleotides. The primary nucleic acid also comprises a second domain, the second domain comprising a nucleotide sequence capable of hybridizing to three secondary nucleic acid molecules, each of which comprises a nucleotide sequence that allows four tertiary nucleic acid molecules having detectable labels attached thereto to hybridize to each of the secondary nucleic acid molecules.

[0189] The bottom right diagram of FIG. 5 shows a spacer 3×4 reporter probe. The spacer 3×4 reporter probe comprises a primary nucleic acid, the primary nucleic acid comprising a first domain of 12 nucleotides. Between the first and second domains of the primary nucleic acid is a spacer region of 20-40 nucleotides. The spacer is said to be 20-40 nucleotides in length, however, there is no limit to the length of the spacer and it can be less than 20 nucleotides or more than 40 nucleotides. The second domain of the primary nucleic acid comprises a nucleotide sequence capable of hybridizing to three secondary nucleic acid molecules. Each secondary nucleic acid comprises a nucleotide sequence that allows four tertiary nucleic acid molecules having detectable labels attached thereto to hybridize to each secondary nucleic acid molecule.

[0190] In Figure 5, each primary nucleic acid comprises a first domain that is 12 nucleotides in length. However, there is no limit to the length of the first domain of the primary nucleic acid and it can be less than or more than 12 nucleotides. Preferably, the first domain of the primary nucleic acid is 14 nucleotides.

[0191] Any feature of one particular reporter probe design shown in an individual figure in Figure 5 can be combined with any feature of a reporter probe design shown in another figure in Figure 5 or described elsewhere herein. For example, a 5x5 reporter probe can be modified to include a spacer region of about 20-40 nucleotides between the complementary nucleic acid and the primary nucleic acid. In another example, a 4x3 reporter probe can be modified to create a 4x5 reporter probe by modifying the four secondary nucleic acids to include nucleotide sequences that allow five tertiary nucleic acids linked to detectable labels to hybridize to the respective secondary nucleic acids.

[0192] Referring to FIG. 5, the 5×5 reporter will have a higher fluorescence intensity because it contains more fluorescent labels (25) than the 4×3 reporter (12). The fluorescence detected in a given field of view (FOV) is a function of a variety of variables, including the fluorescence intensity of a given reporter probe and the number of potentially bound target molecules within the FOV. The number of potentially bound target molecules per field of view (FOV) can be between 1 million and 2.5 million targets per FOV. Typical numbers of bound target molecules per FOV are 20,000-40,000, 220,000-440,000, and 1 million-2 million target molecules. A typical FOV is 0.05 mm. 2 ~1 mm 2 Yet another example of a typical FOV is 0.05 mm 2 ~0.65 mm 2 It is.

[0193] FIG. 6 shows a reporter probe design in which the secondary nucleic acid molecule includes an "additional handle." This "additional handle" does not hybridize to the tertiary nucleic acid molecule and is distal to the primary nucleic acid molecule. In FIG. 6, each "additional handle" is 12 nucleotides in length ("12-mer"), but the length is not limited and can be less than 12 nucleotides or more than 12 nucleotides. Each "additional handle" can include a nucleotide sequence of a first domain of a primary nucleic acid molecule to which the secondary nucleic acid hybridizes. Thus, when the reporter probe includes an "additional handle," the reporter probe can hybridize to the sequencing probe through the first domain of the primary nucleic acid molecule or through the "additional handle." Thus, the reporter probe is more likely to bind to the sequencing probe. This design of the "additional handle" can also improve the kinetics of hybridization. Without being bound to any theory, the "additional handle" can increase the effective concentration of the complementary nucleic acid of the reporter probe. Using the 5x4 "extra handle" reporter probe, it is expected that there will be approximately 4750 fluorescence counts per standard FOV. Using the 5x3 "extra handle" reporter probe, 4x4 "extra handle" reporter probe, 4x3 "extra handle" reporter probe, and 3x4 "extra handle" reporter probe, it is expected that there will be approximately 6000 fluorescence counts per standard FOV. The example reporter probe design shown in Figure 5 can also be modified to include an "extra handle".

[0194] Each secondary nucleic acid molecule of a reporter probe can be hybridized to multiple tertiary nucleic acid molecules that are all labeled with the same detectable label. For example, the left diagram of FIG. 7 shows a "5×6" reporter probe. The 5×6 reporter probe includes one primary nucleic acid that includes a second domain, the second domain including a nucleotide sequence that hybridizes to six secondary nucleic acid molecules. Each secondary nucleic acid includes a nucleotide sequence that allows five tertiary nucleic acid molecules with detectable labels attached thereto to hybridize to each secondary nucleic acid molecule. Each of the five tertiary nucleic acid molecules that are bound to a particular secondary nucleic acid molecule are labeled with the same detectable label. For example, three of the secondary nucleic acid molecules are bound to tertiary nucleic acid molecules labeled with a yellow fluorescent dye, and the remaining three secondary nucleic acid molecules are bound to tertiary nucleic acid molecules labeled with a red fluorescent dye.

[0195] Each secondary nucleic acid molecule of a reporter probe can hybridize to a tertiary nucleic acid molecule labeled with a different detectable label. For example, the center diagram of FIG. 7 shows a design of a "3×2×6" reporter probe. The "3×2×6" reporter probe includes one primary nucleic acid that includes a second domain, the second domain including a nucleotide sequence that hybridizes to six secondary nucleic acid molecules. Each secondary nucleic acid includes a nucleotide sequence that allows five tertiary nucleic acid molecules that are linked to a detectable label to hybridize to each secondary nucleic acid molecule. Each secondary nucleic acid binds to both a tertiary nucleic acid molecule labeled with a yellow fluorescent dye and a tertiary nucleic acid molecule labeled with a red fluorescent dye. In this particular example, three secondary nucleic acid molecules bind to two red tertiary nucleic acid molecules and three yellow tertiary nucleic acid molecules, while the remaining three secondary nucleic acid molecules bind to two red tertiary nucleic acid molecules and three yellow tertiary nucleic acid molecules. Each secondary nucleic acid molecule can bind to any number of tertiary nucleic acid molecules labeled with different detectable labels. In the central diagram of Figure 7, tertiary nucleic acid molecules bound to individual secondary nucleic acid molecules are arranged in alternating label colors (i.e., red-yellow-red-yellow-red or yellow-red-yellow-red-yellow).

[0196] In any of the described reporter probe designs, tertiary nucleic acids labeled with different detectable labels can be arranged in any order along the secondary nucleic acid. For example, the right diagram in Figure 7 shows a "FRET-resistant 3x2x6" reporter probe design that is similar to the 3x2x6 reporter probe design, differing only in the arrangement (e.g., linear order or grouping) of the red and yellow tertiary nucleic acid molecules along each secondary nucleic acid molecule.

[0197] FIG. 8 shows yet another reporter probe design of the present disclosure that includes individual secondary nucleic acid molecules that are bound to different tertiary nucleic acid molecules. The left diagram shows a "6x1x4.5" reporter probe that includes one primary nucleic acid molecule that includes a second domain that includes a nucleotide sequence that is hybridized to six secondary nucleic acid molecules. Each secondary nucleic acid molecule is hybridized to five tertiary nucleic acid molecules. Four of the five tertiary nucleic acid molecules hybridized to each secondary nucleic acid molecule are directly labeled with a detectable label of the same color. The fifth tertiary nucleic acid molecule (referred to as a branched tertiary nucleic acid) is bound to five labeled oligos that are the other color of the two color combination. Three of the six secondary nucleic acids are bound to a branched tertiary nucleic acid labeled with one color of the two color combination (red in this example), while the remaining three secondary nucleic acids are bound to a branched tertiary nucleic acid labeled with the other color of the two color combination (yellow in this example). Taken together, the 6x1x4.5 reporter probe is labeled with a total of 54 dyes, 27 dyes for each color. The center diagram in Figure 8 shows the "4x1x4.5" reporter probe, which shares the same overall architecture as the 6x1x4.5 reporter probe, except that the primary nucleic acid only binds to four secondary nucleic acids, resulting in a total of 36 dyes, 18 dyes for each color.

[0198] A reporter probe can contain the same number of dyes for each color of the two-color combination. A reporter probe can contain a different number of dyes for each color of the two-color combination. The choice of which color dye to have more in a reporter probe can be made based on the energy level of light absorbed by the two dyes. For example, the right diagram in Figure 8 shows a "5x5 energy optimized" reporter probe design. This reporter probe design contains 15 yellow dyes (higher energy) and 10 red dyes (lower energy). In this example, the 15 yellow dyes can make up the first label and the 10 red dyes can make up the second label.

[0199] Detectable moieties, labels, or reporters can be attached to secondary nucleic acid molecules, tertiary nucleic acid molecules, or labeled oligos in a variety of ways, including direct or indirect attachment of detectable moieties (fluorescent moieties, colorimetric moieties, etc.). Those skilled in the art can refer to references on labeling nucleic acids. Non-limiting examples of fluorescent moieties include yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanine, dansyl chloride, phycocyanin, phyoerythrin, and the like.

[0200] Fluorescent labels and their attachment to nucleotides and / or oligonucleotides have been described in many publications, including Haugland, Handbook of Fluorescent Probes and Research Chemicals, 9th Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Wetmur, Critical Reviews in Biochemistry and Molecular Biology, vol. 26:227-259 (1991). Specific methods applicable to the present disclosure are disclosed in the following sample literature: U.S. Patent Nos. 4,757,141; 5,151,507; and 5,091,519. One or more fluorescent dyes can be used as labels for the labeled target sequences. No. 5,188,934 (4,7-dichlorofluorescein dyes); No. 5,366,860 (spectrally resolvable rhodamine dyes); No. 5,847,162 (4,7-dichlororhodamine dyes); No. 4,318,846 (ether-substituted fluorescein dyes); No. 5,800,996 (energy transfer dyes); No. Lee et al. No. 5,066,580 (xanthine dyes); No. 5,688,648 (energy transfer dyes), etc. Labeling can also be done using quantum dots. It is disclosed, for example, in U.S. Patent Nos. 6,322,901; 6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143; 5,990,479; 6,207,392; U.S. Patent Application Publication Nos. 2002 / 0045045; and 2003 / 0017264.As used herein, the term "fluorescent label" includes signaling moieties that convey information through one or more molecular fluorescent absorption and / or emission properties, including fluorescence intensity, fluorescence lifetime, emission spectral properties, energy transfer, and the like.

[0201] Non-limiting examples of commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and / or oligonucleotide sequences include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINE GREEN™-5-dUTP, OREGON GREEN™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY TM 630 / 650-14-dUTP, BODIPY™ 650 / 665-14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXA FLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP, ALEXA FLUOR™ 546-14-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADE BLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR-14-UTP, BODIPY™ TR-14-UTP, RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546-14-UTP (Molecular Probes, Inc., Eugene, Oreg.), etc. Alternatively, the above fluorophores and those mentioned herein can be added during oligonucleotide synthesis, for example, using phosphoramidite or NHS chemistry. Protocols for the custom synthesis of nucleotides bearing other fluorophores are known in the art (see Henegariu et al. (2000) Nature Biotechnol. 18:345).2-Aminopurine is a fluorescent base that can be directly incorporated into oligonucleotide sequences during synthesis. Nucleic acids may also be pre-stained with intercalating dyes (DAPI, YOYO-1, ethidium bromide, cyanine dyes (e.g., SYBR Green), etc.).

[0202] Non-limiting examples of other fluorophores available for post-synthesis attachment include ALEXA FLUOR™ 350, ALEXA FLUOR™ 405, ALEXA FLUOR™ 430, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY TR, BODIPY Examples of suitable rhodamine dyes include rhodamine 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Pacific Orange, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, OR), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, NJ), and the like. FRET tandem fluorophores can also be used, non-limiting examples of which include PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexa dyes, and the like.

[0203] Metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or oligonucleotide sequences (Lakowicz et al. (2003) BioTechniques 34:62).

[0204] Other suitable labels for oligonucleotide sequences may include fluorescein (FAM, FITC), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), phospho-amino acids (e.g. P-tyr, P-ser, P-thr), etc. The following hapten / antibody pairs can be used for detection: biotin / a-biotin, digoxigenin / a-digoxigenin, dinitrophenol (DNP) / a-DNP, 5-carboxyfluorescein (FAM) / a-FAM. Each of the antibodies in the pair is derivatized with a detectable label.

[0205] The detectable labels described herein are spectrally resolvable. "Spectrally resolvable" in reference to multiple fluorescent labels means that the fluorescent emission bands of the labels are sufficiently distinct, i.e., non-overlapping, that the molecular tags to which each label is attached can be distinguished based on the fluorescent signal generated by each label by a standard optical detection system using, for example, a system of bandpass filters and photomultipliers. Examples of such systems are described in U.S. Pat. Nos. 4,230,558; 4,811,218, and the like, or in Wheeless et al., Flow Cytometry: Instrumentation and Data Analysis, Academic Press, New York, 1985, pp. 21-76. Spectrally resolvable organic dyes (fluorescein, rhodamine, and the like) mean that the emission maxima are at least 20 nm apart, and in another aspect, at least 40 nm apart. In reference to chelated lanthanide compounds, quantum dots, and the like, spectrally resolvable means that the emission maxima are at least 10 nm apart, or at least 15 nm apart.

[0206] The reporter probe may include one or more cleavable linker modifications. The one or more cleavable linker modifications may be located anywhere in the reporter probe. The cleavable linker modifications may be located between the first and second domains of the primary nucleic acid molecule of the reporter probe. FIG. 9 shows an example of a reporter probe of the present disclosure that includes a linker modification between the first and second domains of the primary nucleic acid molecule. The cleavable linker modification may be present between the first and second domains of the secondary nucleic acid molecule of the reporter probe. The cleavable linker modification may be present between the first and second domains of the primary and secondary nucleic acid molecules of the reporter probe. The left diagram of FIG. 10 shows an example of a reporter probe of the present disclosure that includes a cleavable linker modification between the first and second domains of the primary nucleic acid and between the first and second domains of the secondary nucleic acid.

[0207] As a cleavable linker modification, a compound of formula (I): [ka] or a stereoisomer or salt thereof, wherein in this formula R 1 is hydrogen, halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Each alkynyl independently has at least one substituent R 10 and optionally substituted with; R 2 is O, NH, N(C 1-6 alkyl); R 3 is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which may optionally be substituted with at least one substituent R 10 Each R 4 and R 7 are independent, C 1-6 Alkyl, C2-6 Alkenyl, C 2-6 Alkynyl, 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Each alkynyl may independently optionally be substituted with at least one substituent R 10 is substituted with;R 5 and R 9 are each independently cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which may optionally be substituted with at least one substituent R 10 is substituted with;R 6 is O, NH, N(C 1-6 alkyl); R 8 is O, NH, N(C 1-6 alkyl); each R 10 are independently hydrogen, halogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 Alkynyl, HaloC 1-6 Alkyl, haloC 2-6 Alkenyl, HaloC 2-6 Alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -CN, -NO 2 , oxo, -OR 11 , -SO 2 R 11 , -SO 3 - , -COR 11 , -CO 2 R 11 , -CONR 11 R 12 , -C(=NR 11 )NR 12 R 13 , -NR 11 R 12 , -NR 12 COR 12 , -NR 11 CONR 12 R 13 , -NR 11 CO 2 R 12 , -NR 11 SONR 12 R13 , -NR 11 SO 2 NR 12 R 13 , -NR 11 SO 2 R 12 R 11 , R 12 , R 13 may be the same or different, and each independently represents hydrogen, -C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 Alkynyl, HaloC 1-6 Alkyl, haloC 2-6 Alkenyl, HaloC 2-6 Alkynyl, C 1-6 Alkyloxy C 1-6 It is either alkyl-, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0208] In one aspect, R 1 is C 1-6 Alkyl, C 1-3 Alkyl (methyl, ethyl, propyl, isopropyl, etc.) is preferred; R 2 is NH or N; R 3 is a 5- or 6-membered cycloalkyl, preferably cyclohexyl; R 4 is C 1-6 Alkyl, C 1-3 Alkylene (methylene, ethylene, propylene, isopropylene, etc.) is preferred; R 5 is a 5- or 6-membered heterocyclyl containing one nitrogen atom and zero or one additional heteroatom selected from N, O, and S, said heterocyclyl optionally containing one or two R 10 is substituted with;R 6 is O;R 7 is C 1-6 Alkyl, C 1-3 Alkylene (methylene, ethylene, propylene, isopropylene, etc.) is preferred; R 8 is O;R 9is a 5- or 6-membered heterocyclyl containing one nitrogen atom and zero or one additional heteroatom selected from N, O, and S, said heterocyclyl optionally containing one or two R 10 Each R 10 are independently halogen, C 1-6 Alkyl, haloC 1-6 Alkyl, oxo, -SO 2 H, -SO 3 - Either:

[0209] In one aspect, R 3 is cyclohexyl, and R 4 is methylene, R 5 is 1H-pyrrole-2,5-dione, and R 9 is pyrrolidine-2,5-dione, and in some cases SO 3 - has been replaced with.

[0210] As a linker compound, [ka] or a stereoisomer or salt thereof.

[0211] As a linker compound, [ka] or a stereoisomer or salt thereof.

[0212] As linker compounds or linker modifications, [ka] Either one is possible.

[0213] As linker compounds or linker modifications, [ka] Either one is possible.

[0214] Reporter probes can be constructed by mixing three stock solutions together with water. One stock solution contains the primary nucleic acid molecule, one stock solution contains the secondary nucleic acid molecule, and the last stock solution contains the tertiary nucleic acid molecule. Table 2 shows examples of the amounts of each stock solution that can be mixed to construct reporter probes of each design. [Table 2]

[0215] target nucleic acid

[0216] The present disclosure provides a method for sequencing a nucleic acid using the sequencing probes of the present disclosure. The nucleic acid to be sequenced using the methods of the present disclosure is referred to herein as a "target nucleic acid." The term "target nucleic acid" refers to a nucleic acid molecule (DNA, RNA, PNA) whose sequence is sought by the probes, methods, and devices of the present disclosure. In general, the terms "target nucleic acid," "target nucleic acid molecule," "target nucleic acid sequence," "target nucleic acid fragment," "target oligonucleotide," and "target polynucleotide" are used interchangeably, and it is intended that the non-limiting examples include polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides, or analogs thereof) that can have various lengths. Non-limiting examples of nucleic acids include genes, gene fragments, exons, introns, intergenic DNA (non-limiting examples of which include heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, small interfering RNA (siRNA), non-coding RNA (ncRNA), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of a sequence, isolated RNA of a sequence, nucleic acid probes, and primers. Prior to sequencing using the disclosed method, the identity and / or sequence of the target nucleic acid is known. Alternatively, the identity and / or sequence is unknown. It is also possible that a portion of the sequence of the target nucleic acid is known prior to sequencing using the disclosed method. For example, the method can be used to determine point mutations in a known target nucleic acid molecule.

[0217] The method of the present disclosure directly sequences nucleic acid molecules obtained from a sample (e.g., a sample from an organism), and preferably there is no conversion (or amplification) step. As an example, to perform direct RNA-based sequencing, the method of the present disclosure does not require conversion of RNA molecules to DNA molecules (i.e., through synthesis of cDNA) before the sequence can be obtained. Because no amplification or conversion is required, the nucleic acid sequenced in the present disclosure will retain any unique bases and / or epigenetic markers present in the nucleic acid when it is present in or obtained from the sample. Such unique bases and / or epigenetic markers are lost in sequencing methods known in the art.

[0218] The disclosed methods allow sequencing at single molecule resolution, in other words, they allow the user to generate a final sequence based on data recovered from a single target nucleic acid molecule, without the need to combine data from different target nucleic acid molecules, thus preserving all the unique characteristics of the particular target.

[0219] The target nucleic acid can be obtained from any sample or source of nucleic acid (e.g., any cell, tissue, organism, in vitro, chemical synthesis, etc.). The target nucleic acid can be obtained by any method recognized in the art. The target nucleic acid can be obtained from a blood sample of a clinical subject. The target nucleic acid can be extracted or isolated or purified from a source or sample using methods and kits well known in the art.

[0220] The target nucleic acid can be fragmented by any means known in the art. Fragmentation is preferably performed by enzymatic or mechanical means. Mechanical means can be sonication or physical shearing. Enzymatic means can be performed by digestion with a nuclease (e.g., deoxyribonuclease I (DNase I)) or one or more restriction endonucleases.

[0221] When the nucleic acid molecule comprising the target nucleic acid is one complete chromosome, steps should be taken to avoid fragmenting said chromosome.

[0222] As is well known in the art, a target nucleic acid can contain naturally occurring or non-naturally occurring nucleotides, including modified nucleotides or nucleic acid analogs.

[0223] Target nucleic acid molecules can include DNA, RNA, or PNA molecules up to several hundred kilobases in length (e.g., 1 kilobase, 2 kilobases, 3 kilobases, 4 kilobases, 5 kilobases, 10 kilobases, 20 kilobases, 30 kilobases, 40 kilobases, 50 kilobases, 100 kilobases, 200 kilobases, 500 kilobases, or greater).

[0224] Capture Probe

[0225] The target nucleic acid can be immobilized on the substrate (eg, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more positions).

[0226] Examples of useful substrates include those that include a binding moiety, the binding moiety being selected from the group consisting of ligand, light source, carbohydrate, nucleic acid, receptor, lectin, and antibody. The capture probe includes a substrate binding moiety that can bind to the binding moiety of the substrate. Non-limiting examples of useful substrates that include reactive moieties include surfaces that include any of the following: epoxy, aldehyde, gold, hydrazide, sulfhydryl, NHS-ester, amine, alkyne, azide, thiol, carboxylate, maleimide, hydroxymethylphosphine, imidoester, isocyanate, hydroxyl, pentafluorophenyl-ester, psoralen, pyridyl disulfide or vinyl sulfone, polyethylene glycol (PEG), hydrogel, or mixtures thereof. Such surfaces can be obtained from commercial sources or prepared according to standard techniques. Non-limiting examples of useful substrates that include reactive moieties include OptArray-DNA NHS group (Accler8), Nexterion Slide AL (Schott), Nexterion Slide E (Schott).

[0227] The substrate can be any solid support known in the art that can immobilize the target nucleic acid, such as a coated slide or a microfluidic device. The substrate can be a surface, a membrane, a bead, a porous material, an electrode, an array. The substrate can be, for example, a polymeric material, a metal, silicon, glass, or quartz. The target nucleic acid can be immobilized on the surface of any substrate apparent to one of skill in the art.

[0228] When the substrate is an array, the substrate can include wells, the size and spacing of which varies depending on the target nucleic acid molecules to be attached. In one example, the substrate is configured to attach an array of target nucleic acids ordered at a very high density. Examples of densities of target nucleic acid arrays on a substrate include wells of 1 mm 2 In one embodiment, the number of target nucleic acid molecules per sample is 500,000 to 10,000,000, or 1,000,000 to 4,000,000, or 850,000 to 3,500,000 target nucleic acid molecules per sample.

[0229] The wells in the substrate are the locations for attachment of target nucleic acid molecules. The surface of the wells is functionalized with reactive moieties as described above to attract, immobilize and bind the target nucleic acid molecules by attracting and binding specific chemical groups present on the surface of the target nucleic acid molecules or on the surface of capture probes bound to the target nucleic acid molecules. It is well known that these functional groups can specifically attract and bind biomolecules through a variety of conjugation chemistries.

[0230] To sequence a single nucleic acid molecule on a substrate (such as an array), a universal capture probe, or a universal sequence complementary to the substrate binding portion of the capture probe, is attached to each well. Sequencing can then begin when a single target nucleic acid molecule is bound to the universal capture probe, or to a universal sequence complementary to the substrate binding portion of the capture probe and bound to said capture probe.

[0231] A target nucleic acid molecule can have one or more capture probes (i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more capture probes) attached to it. A capture probe includes a domain that is complementary to a portion of the target nucleic acid and a domain that includes a substrate binding portion. The portion of the target nucleic acid that is complementary to a capture probe can be at or near the end of the target nucleic acid.

[0232] The substrate binding portion of the capture probe can be biotin, and the substrate can be avidin (e.g., streptavidin). Useful substrates containing avidin are commercially available, including TB0200 (Accler8), SAD6, SAD20, SAD100, SAD500, SAD2000 (Xantec), SuperAvidin (Array-It), Streptavidin Slide (Cat. No. MPC 000, Xenopore), and STREPTAVIDINnslide (Cat. No. 439003, Greiner Bio-one). The substrate binding portion of the capture probe can be avidin (e.g., streptavidin), and the substrate can be biotin. Non-limiting examples of useful substrates containing biotin that are commercially available include Optiarray-Biotin (Accler8), BD6, BD20, BD100, BD500, BD2000 (Xantec).

[0233] The substrate binding portion of the capture probe can be a reactive moiety that can be bound to the substrate by photoactivation. The substrate can include a photoreactive moiety. Alternatively, the first portion of the nanoreporter can include a photoreactive moiety. Some examples of photoreactive moieties include aryl azides (such as N((2-pyridyldithio)ethyl)-4-azidosalicylamide); fluorinated aryl azides (such as 4-azido-2,3,5,6-tetrafluorobenzoic acid); benzophenone-based reagents (such as 4-benzoylbenzoic acid succinimidyl ester); and 5-bromo-deoxyuridine.

[0234] The substrate binding portion of the capture probe can be a nucleic acid capable of hybridizing to a complementary binding portion of the substrate. Each of the nucleic acids comprising the substrate binding portion of the capture probe can be independently a regular base, or a modified nucleotide, or a nucleic acid analog. At least one, or at least two, or at least three, or at least four, or at least five, or at least six nucleotides in the substrate binding portion of the capture probe can be modified nucleotides or nucleic acid analogs. A typical ratio of modified nucleotides or nucleic acid analogs to regular bases in the substrate binding portion of the capture probe is 1:2 to 1:8. Exemplary modified nucleotides or nucleic acid analogs useful in the substrate binding portion of the capture probe are isoguanine and isocytosine.

[0235] The substrate-binding portion of the capture probe can be immobilized to the substrate via other binding pairs that will be apparent to those skilled in the art. After binding to the substrate, the target nucleic acid can be elongated by applying a force sufficient to elongate the target nucleic acid (e.g., gravity, hydrodynamic force, electromagnetic force "electrostretching", flow-stretching, receding meniscus techniques, or combinations thereof). The capture probe can include or be associated with a detectable label (i.e., a reference spot).

[0236] The target nucleic acid can have a second capture probe bound to it, the second capture probe comprising a domain complementary to a second portion of the target nucleic acid. The second portion of the target nucleic acid bound to the second capture probe is different from the first portion of the target nucleic acid bound to the first capture probe. This portion can be at or near the end of the target nucleic acid. Binding of the second capture probe can occur after or during extension of the target nucleic acid, or to a target nucleic acid that has not yet been extended. The second capture probe can have a bond as described above.

[0237] The target nucleic acid can be bound by a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth capture probe that comprises a domain complementary to a third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth portion of the target nucleic acid. This portion can be at or near the end of the target nucleic acid. Binding of the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth capture probe can occur after or during extension of the target nucleic acid, or to a target nucleic acid that has not yet been extended. The third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth capture probe can have a binding as described above.

[0238] A capture probe can isolate a target nucleic acid from a sample by adding the capture probe to a sample containing the target nucleic acid. The capture probe binds to the target nucleic acid through a region of the capture probe that is complementary to a region of the target nucleic acid. When the target nucleic acid is contacted with a substrate that contains a portion that binds to the substrate binding portion of the capture probe, the nucleic acid is immobilized on the surface of the substrate.

[0239] FIG. 11 illustrates the capture of a single target nucleic acid using a two capture probe system according to the present disclosure. Genomic DNA is denatured at 95° C. and hybridized to a pool of capture reagents. The pool of capture reagents includes oligonucleotides, probe A, probe B, and an antisense block probe. Probe A includes a biotin moiety at the 3′ end of the probe and a sequence complementary to the 5′ end of the target nucleic acid. Probe B includes a purification binding sequence that allows a paramagnetic bead to be attached to the 5′ end of the probe and a nucleotide sequence complementary to the 3′ end of the target nucleic acid. The antisense block probe includes a nucleotide sequence complementary to the antisense strand of the portion of the target nucleic acid to be sequenced. After hybridization with the capture reagents, a sequencing window is created in the target nucleic acid between the hybridized probes A and B. The target nucleic acid is purified using paramagnetic beads that bind to the 5′ sequence of probe B. Any excess capture reagent or complementary antisense DNA strand is washed away, resulting in the purification of the desired target nucleic acid. The purified target nucleic acid is then passed through a flow cell that contains a surface (such as streptavidin) that can bind to the biotin moiety on the hybridized probe A. One end of the target nucleic acid then becomes attached to the surface of the flow cell. To capture the other end, the target nucleic acid is flow-stretched and a biotinylated probe that is complementary to the purified binding sequence of probe B is added. The biotinylated probe is capable of binding to the surface of the flow cell upon hybridization to the purified binding sequence of probe B, resulting in a captured target nucleic acid that is elongated and bound at both ends to the surface of the flow cell.

[0240] To ensure that the user "captures" as many target nucleic acid molecules as possible from a highly fragmented sample, it is useful to include multiple capture probes, each complementary to a different region of the target nucleic acid. For example, there can be three pools of capture probes, the first of which is complementary to a region of the target nucleic acid near the 5' end, the second of which is complementary to a region in the middle of the target nucleic acid, and the third of which is complementary to a region of the target nucleic acid near the 3' end. This can be generalized to "n regions of interest" per target nucleic acid. In this example, each pool of fragmented target nucleic acid binds to a capture probe that contains or is bound to a biotin tag. 1 / n of the input sample (n being the number of different regions in the target nucleic acid) is isolated in each pool chamber. The capture probe binds to the target nucleic acid of interest. The target nucleic acid is then immobilized via the biotin on the capture probe to an avidin molecule attached to a substrate. In some cases, the target nucleic acid is elongated, for example, through hydrodynamic or electrostatic forces. All n pools can be extended and ligated simultaneously, or to maximize the number of fully extended molecules, pool 1 (capturing the 5'-most region) can be extended and ligated first, followed by pool 2 (capturing the central region of the target) and finally pool 3.

[0241] The present disclosure also allows users to simultaneously capture and sequence multiple target nucleic acids, or to hybridize multiple capture probes to a sample containing a mixture of multiple target nucleic acids. The multiple target nucleic acids can include a group of two or more nucleic acids, each of which contains the same sequence, or a group of two or more nucleic acids, each of which does not necessarily contain the same sequence. Similarly, the multiple target nucleic acids can include a group of two or more capture probes, each of which contains the same sequence, or a group of two or more capture probes, each of which does not necessarily contain the same sequence. For example, using multiple capture probes, each of which contains the same sequence, allows users to capture multiple target nucleic acids, each of which contains the same sequence. Sequencing these multiple target nucleic acids containing the same sequence can achieve a high level of sequencing accuracy due to data redundancy. In another example, using a group of capture probes, each of which contains a complementary capture probe for each gene of interest, allows users to simultaneously capture and sequence two or more specific genes of interest. In this way, users can perform multiplex sequencing of specific genes. FIG. 12 shows results from an experiment in which a 100-target multi-cancer panel was captured and detected using the disclosed method using FFPE samples.

[0242] When full coverage sequencing is desired, the number of separate capture probes required is inversely related to the size of the target nucleic acid fragments. In other words, more capture probes will be required for highly fragmented target nucleic acids. For sample types that contain highly fragmented and degraded target nucleic acids (e.g., formalin-fixed paraffin-embedded tissues), it may be useful to include multiple pools of capture probes. Conversely, for samples with long target nucleic acid fragments (e.g., isolated nucleic acids obtained in vitro), one capture probe at the 5' end may be sufficient.

[0243] The region of the target nucleic acid between two capture probes, or the region after one capture probe and before the end of the target nucleic acid, is called the "sequencing window." In FIG. 11, the sequencing window that results when two capture probes are used to capture one target nucleic acid is labeled. The sequencing window is the portion of the target nucleic acid available for binding to the sequencing probes. The minimum sequencing window is the length of the target binding domain (e.g., 4-10 nucleotides), and the maximum sequencing window is most of an entire chromosome.

[0244] When sequencing large target nucleic acid molecules using the methods of the present disclosure, one or more "blocker oligos" can be hybridized along the length of the target nucleic acid to control the size of the sequencing window. The blocker oligos hybridize to specific locations on the target nucleic acid, thereby preventing the sequencing probes from binding to those locations, creating smaller sequencing windows of interest. Figure 13 shows a schematic of two captured target DNA molecules hybridized to a capture probe, a blocker oligo, and a sequencing probe. By creating smaller sequencing windows, the sequencing reaction is limited to specific regions of interest on the target DNA molecule, improving the speed and accuracy of sequencing. The use of blocker oligos is particularly useful when sequencing specific mutations at known locations in the target nucleic acid, since it is not necessary to sequence the entire target nucleic acid. The example in Figure 13 shows targeted sequencing at two heterologous sites to distinguish between two different haplotypes.

[0245] Methods of the Disclosure

[0246] The sequencing methods of the present disclosure involve reversibly hybridizing at least one sequencing probe disclosed herein to a target nucleic acid.

[0247] A method for sequencing a nucleic acid can include (1) hybridizing a sequencing probe as described herein to a target nucleic acid. The target nucleic acid can optionally be immobilized at one or more locations on a substrate. An example of a sequencing probe can include a target binding domain and a barcode domain; the target binding domain includes at least eight nucleotides hybridized to the target nucleic acid, where at least six nucleotides in the target binding domain can identify corresponding nucleotides in a target nucleic acid molecule (e.g., when the sequence of the target binding domain is exactly six nucleotides, these six nucleotides identify six nucleotides complementary to the target molecule to which the target binding domain is hybridized), and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in a target nucleic acid molecule (e.g., these at least two nucleotides do not identify two nucleotides complementary to the target molecule to which the target binding domain is hybridized); the target binding domain can include at least eight nucleotides hybridized to the target nucleic acid, where at least six nucleotides in the target binding domain can identify corresponding nucleotides in a target nucleic acid molecule (e.g., when the sequence of the target binding domain is exactly six nucleotides, these six nucleotides identify six nucleotides complementary to the target molecule to which the target binding domain is hybridized); and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in a target nucleic acid molecule (e.g., these at least two nucleotides do not identify two nucleotides complementary to the target molecule to which the target binding domain is hybridized); At least two nucleotides of the at least six nucleotides in the binding domain are modified nucleotides or nucleotide analogs; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment positions, each attachment position comprises at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, each of the at least three attachment positions corresponds to two nucleotides of the at least six nucleotides in the target binding domain, and each of the at least three attachment positions has a different nucleic acid sequence, and the nucleic acid sequence of each of the at least three attachment positions determines the position and attributes of the corresponding two nucleotides of the at least six nucleotides in the target nucleic acid to which the target binding domain binds.

[0248] The method includes hybridizing the sequencing probe to the target nucleic acid, (2) binding a first complementary nucleic acid molecule comprising a first detectable label and at least a second detectable label to a first of the at least three attachment positions of the barcode domain; (3) detecting the first detectable label and at least a second detectable label of the bound first complementary nucleic acid molecule; and (4) identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid. For example, when the first complementary nucleic acid molecule comprises two detectable labels, the two detectable labels identify the at least two nucleotides in the immobilized nucleic acid molecule.

[0249] After detecting the at least two detectable labels, the at least two detectable labels are removed from the first complementary nucleic acid molecule. Thus, the method further includes (5) freeing the first complementary nucleic acid molecule containing a detectable label by binding the first hybridizing nucleic acid molecule lacking a detectable label to the first attachment location, or contacting the first complementary nucleic acid molecule containing a detectable label with a force sufficient to release the first detectable label and at least a second detectable label. Thus, after step (5), the first detectable label is not bound to the first attachment location. The method further includes (6) binding a second complementary nucleic acid molecule comprising a third detectable label and at least a fourth detectable label to a second of the at least three attachment locations of the barcode domain; (7) detecting the third detectable label and the at least fourth detectable label of the bound second complementary nucleic acid molecule; (8) optionally identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid; (9) identifying a linear order of at least six nucleotides for at least a first region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe by repeating steps (5)-(8) until a complementary nucleic acid molecule comprising two detectable labels is bound to each of the at least three attachment locations in the barcode domain and the two detectable labels of the bound complementary nucleic acid molecule are detected; and (10) optionally removing the sequencing probe from the immobilized target nucleic acid.

[0250] The method further includes (11) hybridizing a second sequencing probe to the target nucleic acid immobilized at one or more locations on the substrate, where the target binding domains of the first and second sequencing probes are different; (12) binding a first complementary nucleic acid molecule comprising a first detectable label and at least a second detectable label to a first of the at least three attachment locations of the barcode domain; (13) detecting the first detectable label and at least a second detectable label of the bound first complementary nucleic acid molecule; (14) optionally identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid; and (15) freeing the first complementary nucleic acid molecule or complex comprising the detectable label by binding the first hybridizing nucleic acid molecule lacking the detectable label to the first attachment location, or releasing the first complementary nucleic acid molecule or complex comprising the detectable label by releasing the first detectable label and at least a second detectable label. (16) binding a second complementary nucleic acid molecule comprising a third detectable label and at least a fourth detectable label to a second of the at least three attachment locations of the barcode domain; (17) detecting the third detectable label and the at least a fourth detectable label of the bound second complementary nucleic acid molecule; (18) optionally identifying a location and identity of at least two nucleotides in the immobilized target nucleic acid; (19) identifying a linear order of at least six nucleotides for at least a second region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe by repeating steps (15)-(18) until a complementary nucleic acid molecule comprising two detectable labels is bound to each of the at least three attachment locations of the barcode domain and the two detectable labels of the bound complementary nucleic acid molecule are detected; and (20) optionally removing the second sequencing probe from the immobilized target nucleic acid.

[0251] The method can further include identifying a sequence of the immobilized target nucleic acid by reconstructing the linear order of nucleotides identified in at least the first region and at least the second region, respectively, of the immobilized target nucleic acid.

[0252] Steps (5) and (6) can be performed sequentially or simultaneously. The first detectable label and the at least second detectable label can have the same emission spectrum or different emission spectra. The third detectable label and the at least fourth detectable label can have the same emission spectrum or different emission spectra.

[0253] The first complementary nucleic acid can include a cleavable linker. The second complementary nucleic acid can include a cleavable linker. The first complementary nucleic acid and the second complementary nucleic acid can each include a cleavable linker. The cleavable linker is preferably cleavable by light. The releasing force can be light. UV light is preferred. The light can be provided by a light source selected from the group consisting of an arc lamp, a laser, a focused UV light source, and a light emitting diode.

[0254] The first complementary nucleic acid and the first hybridizing nucleic acid lacking a detectable label can comprise the same nucleic acid sequence. For example, the first hybridizing nucleic acid lacking a detectable label can comprise the same nucleic acid sequence as the portion of the first complementary nucleic acid molecule that binds to the first attachment position of the at least three positions of the barcode domain. The first hybridizing nucleic acid lacking a detectable label can comprise a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to the first attachment position in the barcode domain.

[0255] The second complementary nucleic acid and the second hybridizing nucleic acid lacking a detectable label can comprise the same nucleic acid sequence. The second hybridizing nucleic acid lacking a detectable label can comprise a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to the second attachment position in the barcode domain.

[0256] The invention further provides a method for sequencing a nucleic acid using a plurality of the sequencing probes disclosed herein, for example, a target nucleic acid is hybridized to two or more sequencing probes, each of which is capable of sequencing the portion of the target nucleic acid to which it is hybridized.

[0257] In accordance with the present invention, a method for sequencing a nucleic acid includes: (1) hybridizing at least one first population of first sequencing probes, the first population comprising a plurality of sequencing probes as described herein, to a target nucleic acid, optionally immobilized at one or more locations on a substrate; (2) binding a first complementary nucleic acid molecule comprising a first detectable label and at least a second detectable label to a first attachment location of the at least three attachment locations of a barcode domain; (3) detecting the first detectable label and the at least a second detectable label of the bound first complementary nucleic acid molecule; (4) identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid; and (5) freeing the first complementary nucleic acid molecule comprising the detectable label by binding a first hybridizing nucleic acid molecule lacking the detectable label to the first attachment location, or freeing the first complementary nucleic acid molecule comprising the detectable label by binding the first hybridizing nucleic acid molecule lacking the detectable label to the first attachment location. and a force sufficient to release at least a second detectable label; (6) binding a second complementary nucleic acid molecule comprising a third detectable label and at least a fourth detectable label to a second of the at least three attachment locations of the barcode domain; (7) detecting the third detectable label and the at least a fourth detectable label of the bound second complementary nucleic acid molecule; (8) optionally identifying a location and identity of at least two nucleotides in the immobilized target nucleic acid; (9) identifying a linear order of at least six nucleotides for at least a first region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe by repeating steps (5)-(8) until each of the at least three attachment locations in the barcode domain is bound by a complementary nucleic acid molecule comprising two detectable labels and the two detectable labels of the bound complementary nucleic acid molecule are detected; and (10) optionally removing at least a first population of the first sequencing probe from the immobilized target nucleic acid.

[0258] The method further includes (11) hybridizing at least one second population of a second sequencing probe comprising a plurality of sequencing probes described herein to a target nucleic acid optionally immobilized on a substrate at one or more locations, wherein the target binding domains of the first and second sequencing probes are different; and (12) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and at least a second detectable label to a first of the at least three attachment locations of the barcode domain. (13) detecting the first detectable label and at least a second detectable label of the bound first complementary nucleic acid molecule; (14) optionally identifying the location and identity of at least two nucleotides in the immobilized target nucleic acid; and (15) freeing the first complementary nucleic acid molecule or complex containing the detectable label by binding a first hybridizing nucleic acid molecule lacking a detectable label to the first attachment site, or freeing the first complementary nucleic acid molecule or complex containing the detectable label by binding a first hybridizing nucleic acid molecule lacking a detectable label to the first attachment site and at least a second detectable label. (16) binding a second complementary nucleic acid molecule comprising a third detectable label and at least a fourth detectable label to a second of the at least three attachment locations of the barcode domain; (17) detecting the third detectable label and the at least a fourth detectable label of the bound second complementary nucleic acid molecule; (18) optionally identifying a location and identity of at least two nucleotides in the immobilized target nucleic acid; (19) identifying a linear order of at least six nucleotides for at least a second region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe by repeating steps (15)-(18) until a complementary nucleic acid molecule comprising two detectable labels is bound to each of the at least three attachment locations of the barcode domain and the two detectable labels of the bound complementary nucleic acid molecule are detected; and (20) optionally removing at least a second population of second sequencing probes from the immobilized target nucleic acid.

[0259] The method can further include identifying a sequence of the immobilized target nucleic acid by reconstructing the linear order of nucleotides identified in at least the first region and at least the second region, respectively, of the immobilized target nucleic acid.

[0260] Steps (5) and (6) can be performed sequentially or simultaneously. The first detectable label and the at least second detectable label can have the same emission spectrum or different emission spectra. The third detectable label and the at least fourth detectable label can have the same emission spectrum or different emission spectra.

[0261] The first complementary nucleic acid molecule can include a cleavable linker. The second complementary nucleic acid molecule can include a cleavable linker. The first complementary nucleic acid molecule and the second complementary nucleic acid molecule can each include a cleavable linker. The cleavable linker is preferably cleavable by light. The releasing force can be light. UV light is preferred. The light can be provided by a light source selected from the group consisting of an arc lamp, a laser, a focused UV light source, and a light emitting diode.

[0262] The first complementary nucleic acid molecule and the first hybridizing nucleic acid molecule lacking a detectable label can comprise the same nucleic acid sequence. The first hybridizing nucleic acid molecule lacking a detectable label can comprise a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to the first attachment position in the barcode domain.

[0263] The second complementary nucleic acid molecule and the second hybridizing nucleic acid molecule lacking a detectable label can comprise the same nucleic acid sequence. The second hybridizing nucleic acid molecule lacking a detectable label can comprise a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to the second attachment position in the barcode domain.

[0264] This sequencing method is further described herein.

[0265] FIG. 14 shows a schematic of one complete cycle of the disclosed sequencing method. While the disclosed method does not require immobilization of the target nucleic acid prior to sequencing, in this example, the method begins with the target nucleic acid captured using a capture probe and bound to the surface of a flow cell, as shown in the top left diagram. A pool of sequencing probes is then flowed into the flow cell to allow the sequencing probes to hybridize to the target nucleic acid. In this example, the sequencing probes are those shown in FIG. 1. These sequencing probes contain a hexamer sequence in the target binding domain that hybridizes to the target nucleic acid. The hexamer is flanked on each side by (N) bases. The (N) bases can be universal / degenerate bases or can be any combination of bases b 1 -b 2 -b 3 -b 4 -b 5 -b 6 The hexamer sequence can be composed of any of the four canonical bases that are not specific for a target not specified by 6 ) allows for sequencing of any target nucleic acid. For this example, a set of 4096 sequencing probes hybridizes to the target nucleic acid in each of 8 pools of 512 sequencing probes. The hexamer sequences in the target binding domains of the sequencing probes will hybridize at positions along the length of the target nucleic acid where there is a perfect match of complementarity between the hexamer and the target nucleic acid, as shown in the top center diagram of FIG. 14. In this example, a single sequencing probe hybridizes to the target nucleic acid. Any unbound sequencing probes are washed out of the flow cell.

[0266] These sequencing probes have three attachment sites R1 , R 2 , R 3 It also contains a barcode domain having an attachment position R 1 The attachment region in comprises one or more nucleotide sequences corresponding to the first dinucleotide of the hexamer of the sequencing probe. Thus, only reporter probes that contain a complementary nucleic acid that corresponds to the attribute of the first dinucleotide present in the target binding domain of the sequencing probe will be able to bind at attachment position R. 1 Similarly, the sequencing probe will hybridize to the attachment position R 2 The attachment region in corresponds to a second dinucleotide present in the target binding domain and corresponds to the attachment position R of the sequencing probe. 3 The attachment region in corresponds to the third dinucleotide present in the target binding domain.

[0267] The method is continued in the top right diagram of Figure 14. A pool of reporter probes is flowed into a flow cell. Each reporter probe in the pool of reporter probes contains a detectable label in the form of a two-color combination and an attachment site for the sequencing probe, R. 1 Each reporter probe pool contains a complementary nucleic acid that can hybridize to a corresponding attachment region in the reporter probe. The two-color combination and complementary nucleic acid of a particular reporter probe corresponds to one of 16 possible dinucleotides, as explained above. Each pool of reporter probes is designed such that the two-color combination that corresponds to a particular dinucleotide is established prior to sequencing. For example, in the sequencing experiment shown in FIG. 14, the attachment position R 1 For the first pool of reporter probes hybridizing to the , the two color combinations yellow-red can be associated with the dinucleotide adenine-thymine. 1 After hybridization to the nucleotide sequence, any unbound reporter probe is washed from the flow cell and the detectable label of the bound reporter probe is recorded to determine the identity of the first dinucleotide of the hexamer.

[0268] The detectable label belonging to the reporter probe hybridized to the attachment position is removed. To remove the detectable label, the reporter probe may contain a cleavable linker, which may be supplemented by the addition of a suitable cleavage reagent. Alternatively, a complementary nucleic acid lacking a detectable label may be hybridized to the attachment position R of the sequencing probe. 1 By hybridizing the reporter probe to the attachment site R, the reporter probe is replaced with a reporter probe having a detectable label. 1 The process of causing a barcode domain attachment location that previously produced a detectable signal to no longer produce a detectable signal is referred to herein as "darkening."

[0269] A second pool of reporter probes is flowed into the flow cell, each reporter probe in the pool of reporter probes containing a detectable label in the form of a two-color combination and a sequencing probe attachment position R. 2 Each reporter probe contains a complementary nucleic acid that can hybridize to a corresponding attachment region in the reporter probe. The complementary nucleic acid of the two-color combination and a particular reporter probe corresponds to one of 16 possible dinucleotides. It is possible that a particular two-color combination corresponds to one dinucleotide in the context of a first pool of reporter probes and to a different dinucleotide in the context of a second pool of reporter probes. As shown in the lower right diagram of Figure 14, the reporter probe is attached to an attachment position R 2 After hybridization to the 6-mer, any unbound reporter probe is washed from the flow cell and the detectable label of the bound reporter probe is recorded to determine the identity of the second dinucleotide of the hexamer.

[0270] position R 2 To remove the detectable label of the reporter probe, the reporter probe may include a cleavable linker, which may be cleaved by the addition of an appropriate cleavage reagent. Alternatively, a complementary nucleic acid lacking a detectable label may be cleaved at the attachment site R of the sequencing probe. 2By hybridizing the reporter probe to the attachment site R, the reporter probe is replaced with a reporter probe having a detectable label. 2 no longer produces a detectable signal.

[0271] A third pool of reporter probes is then flowed into the flow cell, each reporter probe in the third pool containing a detectable label in the form of a two-color combination and a sequencing probe attachment site R. 3 Each reporter probe contains a complementary nucleic acid that can hybridize to a corresponding attachment region in the 2-color combination and the complementary nucleic acid of a particular reporter probe corresponds to one of 16 possible dinucleotides. As shown in the lower center diagram of Figure 14, the reporter probe is located at position R 3 After hybridization to the target nucleic acid, any unbound reporter probe is washed from the flow cell and the detectable label of the bound reporter probe is recorded to determine the identity of the third dinucleotide of the hexamer. In this manner, all three dinucleotides of the target binding domain are identified and can be reconstructed together to reveal the sequence of the target binding domain and, therefore, the sequence of the target nucleic acid.

[0272] To continue sequencing the target nucleic acid, any bound sequencing probes can be removed from the target nucleic acid. 3 The sequencing probe can be removed from the target nucleic acid even if it remains hybridized to position R. 3 The reporter probe hybridized to the target binding domain may be removed, for example, at position R 1 and R 2 The reporters can be removed from the barcode domain using the darkening procedure described above.

[0273] The sequencing cycle depicted in Figure 14 can be repeated any number of times, with each sequencing cycle beginning with hybridizing the same pool of sequencing probes to the target nucleic acid molecule, or beginning with hybridizing a different pool of sequencing probes to the target nucleic acid molecule. A first sequencing probe, or a second pool of sequencing probes, can bind to the target nucleic acid at a position that overlaps with the position at which the first pool of sequencing probes bound to the target nucleic acid during the first sequencing cycle. In this way, some nucleotides in the target nucleic acid can be sequenced more than once, and more than one sequencing probe can be used.

[0274] Figure 15 shows a schematic diagram of one complete cycle of the disclosed sequencing method and the corresponding imaging data collected during this cycle. In this example, the sequencing probe used is that shown in Figure 1, and the sequencing process is the same as that shown in Figure 14 and described above. After the sequencing domain of the sequencing probe is hybridized to the target nucleic acid, a reporter probe is attached to the first attachment site (R 1 ) is hybridized to the barcode domain. An image of the first reporter probe is then acquired to record a color dot. In FIG. 15, the color dot is shown as a dotted circle. A color dot corresponds to a single sequencing probe that is recorded during one complete cycle. In this example, seven sequencing probes are recorded (1-7). The first attachment position of the barcode domain is then darkened and a dual fluorescent reporter probe is hybridized to the second attachment position of the sequencing probe (R 2 ) on the sequencing probe. An image of the second reporter probe is then acquired to record a color dot. The second attachment site of the barcode domain is then darkened, and the dual fluorescent reporter probe is hybridized to the third attachment site of the sequencing probe (R 3) is hybridized to the third reporter probe. An image of the third reporter probe is then acquired to record the color dots. The three color dots from each of sequencing probes 1-7 are then sequenced. A decoding matrix is ​​then used to map each color dot to a specific dinucleotide to reveal the sequence of the target binding domain of sequencing probes 1-7.

[0275] The number of pools of reporter probes required to determine the sequence of the target binding domain of any sequencing probe bound to the target nucleic acid during one sequencing cycle is equal to the number of attachment positions in the barcode domain, so a barcode domain with three positions would cycle three reporter probes with the sequencing probe.

[0276] A pool of sequencing probes can contain multiple sequencing probes that are all the same sequence, or multiple sequencing probes that are not all the same sequence. When a pool of sequencing probes contains multiple sequencing probes that are not all the same sequence, there can be equal numbers of each of the different sequencing probes, or there can be different numbers of the different sequencing probes.

[0277] FIG. 16 illustrates an example of one configuration of a pool of sequencing probes according to the present disclosure, where the sequencing probes have (a) a target binding domain that contains six nucleotides (a hexamer) and specifically binds to a target nucleic acid, and (b) three attachment sites (R 1 , R 2 , R 3), eight different pools of sequencing probes are designed using the eight color combinations shown above. There are 4096 possible different hexamer sequences (4 x 4 x 4 x 4 x 4 = 4096). Since each of the three attachment sites in the barcode domain can hybridize to a complementary nucleic acid bound by one of the eight different color combinations, there are 512 different sets of three possible color combinations (8 x 8 x 8 = 512). For example, R 1 hybridizes to the complementary nucleic acid bound to the color combination GG, and R 2 hybridizes to the complementary nucleic acid bound to the color combination BG, 3 For a probe that hybridizes to a complementary nucleic acid bound to the color combination YR, the set of three color combinations is GG-BG-YR. Within a pool of sequencing probes, each different set of three color combinations will correspond to a different hexamer in the target binding domain. Since each pool contains 512 different hexamers, for a total of 4096 possible hexamers, eight pools are required to sequence all possible hexamers (4096 / 512=8). The specific sequencing probes in each of the eight pools are determined so that each sequencing probe optimally hybridizes to the target nucleic acid. There are several precautions to ensure optimal hybridization. These precautions include (a) separating the perfect complement of hexamers into different pools; (b) separating hexamers with high and low Tm into different pools; and (c) separating hexamers into different pools based on empirically learned hybridization patterns.

[0278] FIG. 17 illustrates the difference between the sequencing probes described in US Patent Application Publication No. 2016 / 0194701 and the sequencing probes of the present disclosure. As shown in the left diagram of FIG. 17, US Patent Application Publication No. 2016 / 0194701 describes a sequencing probe having a barcode domain that includes six attachment sites that hybridize to complementary nucleic acids. Each complementary nucleic acid is bound to one of four different fluorescent dyes. In this configuration, each color (red, blue, green, yellow) corresponds to one nucleotide (A, T, C, G) in the target binding domain. By designing the probes in this manner, 4096 different probes (4 6 ) is generated. As shown in the right diagram of FIG. 17, in one example of the present disclosure, the barcode domain of each sequencing probe contains three attachment sites that hybridize to complementary nucleic acids. Unlike US Patent Application Publication No. 2016 / 0194701, these complementary nucleic acids are bound by one of eight color combinations (GG, RR, GY, RY, YY, RG, BB, RB). Each color combination corresponds to a specific dinucleotide in the target binding domain. In this configuration, 512 combinations (8 3 ) different probes. To cover all possible hexamer combinations (4096) in a target nucleic acid domain, eight separate pools of these 512 different probes are required to sequence an entire target nucleic acid. Because eight color combinations are used to label the complementary nucleic acids, but there are 16 possible dinucleotides, some color combinations will correspond to different dinucleotides depending on which pool of sequencing probes is used. For example, in FIG. 17, in the first, second, third, and fourth pools of sequencing probes, the color combination BB corresponds to the dinucleotide AA, and the color combination GG corresponds to the dinucleotide AT. In the fifth, sixth, seventh, and eighth pools of sequencing probes, the color combination BB corresponds to the dinucleotide CA, and the color combination CT corresponds to the dinucleotide AT.

[0279] Multiple sequencing probes (i.e., two or more sequencing probes) can be hybridized within a sequencing window. During sequencing, the identity and spatial location of the detectable label attached to each sequencing probe among the hybridized multiple sequencing probes are recorded. This allows both the location and identity of multiple dinucleotides to be subsequently identified. In other words, by simultaneously hybridizing multiple sequencing probes to a single target nucleic acid molecule, multiple locations along the target nucleic acid can be sequenced simultaneously, thereby increasing the speed of sequencing.

[0280] FIG. 18 shows a schematic diagram of a single and multiple sequencing probes hybridized to a captured target nucleic acid molecule. A sequencing window between two hybridized 5' and 3' capture probes allows a single sequencing probe (left) or multiple sequencing probes (right) to be hybridized along the length of the target nucleic acid molecule. Hybridizing multiple sequencing probes along the length of the target nucleic acid molecule allows more than one location on the target nucleic acid molecule to be sequenced simultaneously, thus increasing the speed of sequencing. FIG. 19 shows a fluorescent image recorded during the sequencing method of the present disclosure when a single sequencing probe (left) or multiple sequencing probes (right) are hybridized to a captured target nucleic acid. The right diagram of FIG. 19 shows that the fluorescent signals from the individual probes of multiple sequencing probes bound along the length of the target nucleic acid can be spatially separated.

[0281] FIG. 20 shows a schematic diagram of multiple sequencing probes according to the present disclosure bound along the length of a target nucleic acid that is 15 kilobases in length, and the corresponding recorded fluorescent image. As shown in the right diagram of FIG. 20, the sequencing probes can be evenly spaced along the length of the target nucleic acid. As shown in the left diagram of FIG. 20, the sequencing probes do not need to be evenly spaced along the length of the target nucleic acid. The fluorescent image shown in FIG. 20 shows that it is possible to spatially separate signals from multiple sequencing probes bound along the length of the target nucleic acid to simultaneously obtain sequencing information at multiple locations of the target nucleic acid.

[0282] The distribution of the probes along the length of the target nucleic acid is crucial for the resolution of the detectable signal. Too many probes in one area can cause overlap of detectable labels, thus preventing the separation of two adjacent probes. This is explained as follows: Since one nucleotide is 0.34 nm long and the lateral (xy) spatial resolution of the sequencing device is about 200 nm, the resolution limit of the sequencing device is about 588 base pairs (i.e., 1 nucleotide / 0.34 nm x 200 nm). That is, when two probes are within about 588 base pairs of each other, the sequencing device will not be able to separate the signals from two probes hybridized to the target nucleic acid. Therefore, depending on the resolution of the sequencing device, the two probes will need to be separated by about 600 base pairs to be able to separate the detectable labels as separate "spots". Therefore, the optimal spacing should be one probe for every 600 bp of the target nucleic acid. Preferably, each sequencing probe in the population of probes is bound no closer than 600 nucleotides to each other. Various software approaches (e.g., using fluorescence intensity values ​​and wavelength-dependent ratios) can be used to monitor, limit and potentially analyze the number of probes hybridizing in one separable region of the target nucleic acid, and the population of probes can be designed accordingly. Furthermore, detectable labels (e.g., fluorescent labels) that provide more distinct signals can be selected. Furthermore, methods described in the literature (Small and Parthasarthy: "Superresolution localization methods," Annu. Rev. Phys Chem., 2014; 65:107-125) describe structured illumination and various super-resolution approaches that reduce the resolution limit of sequencing microscopes to a few tens of nanometers. With sequencing devices with higher resolution, it becomes possible to use probes with shorter target binding domains.

[0283] As mentioned above, the design of the Tm of probe can affect the number of probes hybridized to target nucleic acid.Alternatively or in addition, the concentration of sequencing probe in a population can be increased to increase the coverage of the probe in a specific region of target nucleic acid.The concentration of sequencing probe can be decreased to reduce the coverage of the probe in a specific region of target nucleic acid, for example, above the resolution limit of sequencing device.

[0284] The resolution limit of the two detectable labels is approximately 600 nucleotides, but this does not prevent the robust sequencing method of the present disclosure. In some aspects, the sequencing probes in any population will not be 600 nucleotides apart on the target nucleic acid. However, statistically (following a Poisson distribution), there will be target nucleic acids with only one sequencing probe bound to them, and therefore the sequencing probes are optically separable. For target nucleic acids that have multiple probes within 600 nucleotides (and therefore are not optically separable), data for these non-separable sequencing probes can be discarded. Importantly, the method of the present disclosure allows multiple binding and detection operations of multiple sequencing probes. Thus, it is possible to detect signals from all sequencing probes in some operations, signals from only some of the sequencing probes in some operations, and signals from none of the sequencing probes in some operations. In some aspects, the distribution of sequencing probes bound to target nucleic acids can be controlled (e.g., by controlling concentration or dilution) so that only one sequencing probe binds to one target nucleic acid.

[0285] Randomly, but depending in part on the length of the target binding domain, the Tm of the probe, and the concentration of the probe applied, it is possible for two distinct sequencing probes in a population to bind within 600 nucleotides of each other.

[0286] Alternatively, or in addition, the concentration of sequencing probes in a population can be reduced to reduce the coverage of the probes in a particular region of the target nucleic acid, e.g., above the resolution limit of the sequencing instrument, thereby generating a single readout from a resolution-limited spot.

[0287] If the sequence, or a portion of the sequence, of a target nucleic acid is known prior to sequencing the target nucleic acid using the methods of the present disclosure, sequencing probes can be designed and selected such that no two sequencing probes bind within 600 nucleotides of each other.

[0288] Prior to hybridization of the sequencing probe to the target nucleic acid, one or more complementary nucleic acid molecules can be bound to a first detectable label and at least a second detectable label can be hybridized to one or more attachment positions in the barcode domain of the sequencing probe. For example, prior to hybridization to the target nucleic acid, one or more complementary nucleic acid molecules bound to a first detectable label and at least a second detectable label can be hybridized to the first attachment position of each sequencing probe. Thus, the sequencing probe can generate a detectable signal from the first attachment position when contacted with the target nucleic acid, and therefore it is not necessary to provide a first pool of complementary nucleic acids or reporter probes directed to the first position on the barcode domain. In another example, one or more complementary nucleic acid molecules bound to a first detectable label and at least a second detectable label can be hybridized to all attachment positions in the barcode domain of the sequencing probe. Thus, in this example, a sequence of six nucleotides can be read out without the need to exchange complementary nucleic acids in sequence. The use of this prehybridized sequencing probe-reporter probe complex would eliminate many of the steps of the described method, thereby reducing the time to obtain sequence information, but the probes would benefit from non-overlapping detectable labels, e.g., fluorophores that are excited by and emit light at non-overlapping wavelengths.

[0289] During sequencing, the signal intensity from the recorded color dots can be used to more accurately sequence the target nucleic acid. Figure 21 shows imaging data recorded during one sequencing cycle of the present disclosure. The right panel of Figure 21 shows a fluorescence microscope image recorded after the reporter probe hybridizes to the first attachment position of the sequencing probe. The individual color dots are highlighted to highlight the specific color combination recorded. This shows that the dual fluorescent signals are clearly detectable and distinguishable. The bright signal from the fiducial marker is indicated by an arrow. The left panel of Figure 21 shows that the individual color spot intensities within a color dot can be used to determine the probability that a particular color dot corresponds to a color combination with a single color overlap (i.e., BB, GG, YY, or RR).

[0290] Darkening of a position in a barcode domain can be achieved by strand cleavage at the position of a cleavable linker modification present in a reporter probe hybridized to said position. FIG. 22 illustrates the use of a cleavable linker modification to darken a barcode position during a sequencing cycle. The first step, shown in the leftmost diagram of FIG. 22, involves hybridizing a primary nucleic acid of a reporter probe to a first attachment position of a sequencing probe. The primary nucleic acid hybridizes to a specific complementary sequence in the attachment region of the first position of the barcode domain. The first and second domains of the primary nucleic acid are covalently linked by a cleavable linker modification. In a second step, a detectable label is subsequently recorded to determine the identity and position of a specific dinucleotide in the target binding domain of the sequencing probe. In a third step, the first position of the barcode domain is darkened by cleaving the reporter probe at the position of the cleavable linker modification. This releases the second domain of the primary nucleic acid, thereby releasing the detectable label. Because the first domain of the primary nucleic acid molecule now remains hybridized to the first attachment position of the barcode domain, devoid of any detectable label, the first position of the barcode domain will no longer generate a detectable signal and will not be able to hybridize to any other reporter probe in the subsequent sequencing step. In the final step shown in the right-most diagram of Figure 22, a reporter probe hybridizes to the second position of the barcode domain and sequencing continues.

[0291] The attachment position of the barcode domain can be darkened by displacing any secondary or tertiary nucleic acid in the reporter probe that is bound to a detectable label, while leaving the primary nucleic acid of the reporter probe hybridized to the sequencing probe. This displacement can be achieved by hybridizing a secondary or tertiary nucleic acid that does not have a detectable label bound to it to the primary nucleic acid. FIG. 23 illustrates an example of a sequencing cycle of the present disclosure, in which one position in the barcode domain is darkened by displacement of a secondary nucleic acid that has a label. The leftmost diagram in FIG. 23 shows the start of the sequencing cycle, in which the primary nucleic acid molecule of the reporter probe is hybridized to the first attachment position of the barcode domain of the sequencing probe. A secondary nucleic acid molecule bound to a detectable label is then hybridized to the primary nucleic acid molecule, and the detectable label is recorded. To darken the first position of the barcode domain, the secondary nucleic acid molecule bound to a detectable label is replaced with a secondary nucleic acid molecule that lacks a detectable label. In the next step of the sequencing cycle, a reporter probe containing a detectable label is hybridized to the second position of the barcode domain. The attachment positions of the barcode domain can be darkened by hybridizing a nucleic acid not bound to a detectable label to the sequencing probe at the corresponding attachment position of the barcode domain to replace any primary nucleic acid molecule of the reporter probe. When the barcode domain contains at least one single-stranded nucleic acid sequence adjacent to at least one attachment position, the nucleic acid not bound to a detectable label can replace the primary nucleic acid molecule by hybridizing to the flanking sequence and the part of the barcode domain occupied by the primary nucleic acid molecule.If necessary, the rate of exchange of the detectable label can be increased by incorporating small single-stranded oligonucleotides that increase the rate of exchange of the detectable label (e.g., "toehold" probes; see, e.g., Seeling et al., "Catalyzed Relaxation of a Metastable DNA Fuel"; J. Am. Chem. Soc. 2006, 128(37), 12211-12220).

[0292] The complementary nucleic acid containing a detectable label, i.e., the reporter probe, can be removed from the attachment region but cannot be replaced with a hybridizing nucleic acid lacking a detectable label. This can occur, for example, by adding a chaotropic agent, and / or by increasing the temperature, and / or by changing the salt concentration, and / or by adjusting the pH, and / or by applying hydrodynamic forces. In these instances, fewer reagents (i.e., hybridizing nucleic acids lacking a detectable label) are required.

[0293] The disclosed method can be used to simultaneously capture and sequence RNA and DNA molecules (including mRNA and gDNA) from the same sample. Both RNA and DNA molecules can be captured and sequenced from the same sample in the same flow cell. The left diagram of Figure 24 is a schematic diagram showing how the disclosed method can be used to simultaneously capture, detect, and sequence both mRNA and gDNA from an FFPE sample.

[0294] The disclosed sequencing method further includes identifying the sequence of the immobilized target nucleic acid by reconstructing the linear order of the identified nucleotides for each region of the immobilized target nucleic acid. The reconstructing step utilizes a non-transitory computer readable storage medium having an executable program stored thereon, the program instructing a microprocessor to arrange the linear order of the identified nucleotides for each region of the target nucleic acid to obtain the sequence of the nucleic acid. The reconstructing operation can be performed in "real time", i.e., while data is being collected from the sequencing probe, rather than after all data has been collected or after complete data acquisition.

[0295] The raw specificity of the disclosed sequencing method is approximately 94%. The accuracy of the disclosed sequencing method can be increased to approximately 99% by using two or more sequencing probes to sequence the same base in the target nucleic acid. FIG. 25 shows how the disclosed sequencing method allows for sequencing of the same base in the target nucleic acid using different sequencing probes. The target nucleic acid in this example is a fragment of NRAS exon 2 (SEQ ID NO:1). The particular base of interest is a cytosine (C) that is highlighted in the target nucleic acid. This base of interest will hybridize to two sequencing probes that have different hybridization footprints to the target nucleic acid. In this example, sequencing probes 1-4 (barcodes 1-4) bind three nucleotides to the left of the base of interest, whereas sequencing probes 5-8 (barcodes 5-8) bind five nucleotides to the left of the base of interest. The base of interest is then sequenced by two different probes, increasing the amount of base reads at that particular position, thereby increasing the overall accuracy at that particular position. Figure 26 shows how multiple different base reads at a particular nucleotide position on a target nucleotide are recorded from one or more sequencing probes and combined to create a consensus sequence (SEQ ID NO:2), thereby improving the accuracy of the final base read.

[0296] "Hyb & Seq chemistry," "Hyb & Seq sequencing," and "Hyb & Seq" refer to the methods of the present disclosure described above.

[0297] Any aspect described above may be combined with any other aspect disclosed herein.

[0298] definition

[0299] The terms "annealing" and "hybridization" are used interchangeably herein and refer to the formation of a stable duplex. In one aspect, a stable duplex means that the duplex structure is not disrupted by stringent washing at a temperature about 5°C below or about 5°C above the Tm of one of the strands of the duplex and at low monovalent salt concentrations (e.g., less than 0.2 M, or less than 0.1 M, or salt concentrations known to those skilled in the art). The term "perfectly matched", when used in reference to a duplex, means that the polynucleotide and / or oligonucleotide strands constituting the duplex form a duplex structure with each other, and all nucleotides in each strand form Watson-Crick base pairs with nucleotides in the other strand. The term "duplex" includes, but is not limited to, pairs of available nucleoside analogs (such as deoxyinosine), nucleosides with 2-aminopurine bases, PNAs, and the like. A "mismatch" in a duplex between two oligonucleotides means that a pair of nucleotides in the duplex cannot form a Watson-Crick bond.

[0300] As used herein, the term "hybridization conditions" will typically include salt concentrations less than about 1 M, more typically less than about 500 mM, and even more typically less than about 200 mM. Hybridization temperatures can be as low as 5°C, but are typically greater than 22°C, more typically greater than about 30°C, and often greater than about 37°C. Hybridization is usually performed under stringent conditions (e.g., conditions under which a probe will specifically hybridize to a corresponding subsequence of a target). Stringent conditions are sequence-dependent and will vary in different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. Other factors can affect the stringency of hybridization, including the base composition and length of the complementary strands, the presence of organic solvents, and the degree of base mismatching, so the combination of parameters is more important than the absolute indication of any one parameter alone.

[0301] In general, stringent conditions are selected to be about 5°C lower than the Tm of a particular sequence at a defined ionic strength and pH. Examples of stringent conditions include a Na ion concentration (or other salt) of at least 0.01 M to 1 M at a pH of 7.0 to 8.3 and a temperature of at least 25°C. For example, 5xSSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25 to 30°C are suitable for hybridization of allele-specific probes. For stringent conditions, see, for example, Sambrook, Fritsche, Maniatis, Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press (1989) and Anderson, Nucleic Acid Hybridization, 1st Edition, BIOS Scientific Publishers Limited (1999). As used herein, "specifically hybridizes to" or "specifically hybridizes to" and similar expressions mean that a molecule binds to, double-strands, or hybridizes to a substantially specific nucleotide sequence or sequences under stringent conditions.

[0302] The detectable label associated with a particular position of the probe can be "read out" (e.g., detect the fluorescence) one or more times. "Read out" can be used as a synonym for the term "base read". Multiple reads improve accuracy. A target nucleic acid sequence is "read out" when a continuous stretch of sequence information derived from an original single target molecule is detected. Typically, this is generated through a multi-pass consensus (defined below). As used herein, the term "coverage" or "depth of coverage" refers to the number of times a region of a target is sequenced (through separate reads) and aligned with a reference sequence. Read coverage is the total number of reads that map to a particular reference target sequence. Base coverage is the total number of base reads at a particular genomic location.

[0303] A "read" is one unit of sequence output. A contiguous stretch of sequence information derived from an original single target molecule. Each read has a quality index related to the confidence level of the base reads in that read. A unit of sequencer output. A contiguous stretch of sequence information derived from an original single target molecule. In Hyb & Seq, all reads are generated through a multi-pass consensus.

[0304] "Read length" is an index describing the length of the sequence from each read (in bp).

[0305] As used herein, a "Hyb & Seq cycle" refers to all the steps required to detect each attachment region on the surface of a particular probe or population of probes. For example, for a probe capable of detecting six locations on the surface of a target nucleic acid, one "Hyb & Seq cycle" would include at least hybridizing the probe to the target nucleic acid, hybridizing a complementary nucleic acid / reporter probe to the attachment region at each of the six locations on the surface of the barcode domain of the probe, and detecting the detectable label associated with each of the six locations.

[0306] The term "k-mer probe" is a synonym for sequencing probe according to the present disclosure. A k-mer read is the basic unit of data in Hyb&Seq. One k-mer read is obtained from one target molecule per Hyb&Seq cycle. When multiple Hyb&Seq cycles are performed to generate a sufficient number of separate k-mer reads from a single target molecule, the separate k-mers can be unambiguously aligned to obtain a continuous stretch of sequence.

[0307] When two or more sequences from separate reads are aligned, the overlaps can be combined to produce a single consensus sequence. At positions (one column of the alignment) where the overlaps have the same bases, those bases become the consensus. Various rules can be used to generate a consensus for positions where there is a mismatch between the overlapping sequences. The rule called simple majority uses the most commonly occurring base in the column as the consensus. A "multi-pass consensus" is an alignment of all the separate probe reads from a single target molecule. Depending on the population of probes / total number of cycles of interrogation, each base position in a single target molecule can be interrogated with different levels of redundancy or overlap. In general, redundancy increases the confidence level of the base read.

[0308] A "consensus" is when two or more DNA sequences from separate reads are aligned, and the overlapping portions can be combined to generate a single consensus sequence. At positions where the overlapping portions have the same bases (one row of the alignment), those bases become the consensus. Various rules can be used to generate a consensus for positions where there is a mismatch between the overlapping sequences. The rule called simple majority uses the most commonly occurring base in a row as the consensus.

[0309] "Raw accuracy" is a measure of the inherent ability of the system to correctly identify bases. Raw accuracy depends on the sequencing technology. "Consensus accuracy" is a measure of the ability of the system to correctly identify bases using additional reads and the power of statistics. "Specificity" refers to the percentage of all reads per trial that map to the intended target. "Uniformity" refers to the variability of sequence coverage between target regions. High uniformity correlates with low variability. This characteristic is commonly reported as the percentage of targeted regions that are covered with an average depth of coverage of 20% or greater among all targeted regions. Random errors (i.e. errors inherent to the sequencing chemistry) can be easily corrected by "multiple passes" sequencing of the same target nucleic acid. With a sufficient number of passes, one can achieve essentially "perfect consensus" or "error-free" sequencing.

[0310] The methods described herein can be implemented and / or the results can be recorded using any device capable of implementing the methods and / or recording the results. Non-limiting examples of devices that can be used include electronic computing devices, including any type of computer. When implementing and / or recording the methods described herein in a computer, a computer program that can be used to cause the computer to execute the steps of the method can be contained in any computer readable medium capable of containing the computer program. Non-limiting examples of computer readable medium that can be used include diskettes, CD-ROMs, DVDs, ROMs, RAM, non-transitory computer readable media, other memories, other computer storage devices. A computer program that can be used to cause a computer to execute the steps of the method and / or reconstruct sequence information and / or record the results can also be provided on an electronic network (e.g., the Internet, an intranet, or other networks).

[0311] The "disposable sequencing card" can be incorporated into any fluorescence imaging device known in the art. Any fluorescence microscope with many different features can perform this sequencing readout. For example, wide-field lamps, lasers, LEDs, multiphoton, confocal illumination, total internal reflection illumination can be used for excitation and / or detection. The emission detection channel of the fluorescence microscope can be equipped with a camera(s) and / or photomultiplier(s) capable of filter or grating-based spectral resolution (spectrally resolved one or more emission wavelengths). A standard computer can be used to control the disposable sequencing card, the reagents passing through the card, and the detection by the fluorescence microscope.

[0312] Sequencing data can be analyzed by any number of standard next-generation sequencing assemblers (see, for example, Wajid and Serpedin, "Review of general algorithmic features for genome assemblers for next generation sequencers," Genomics, proteomics & bioinformatics, vol. 10(2): 58-73, 2012). Sequencing data acquired within a single diffraction-limited region of the microscope are "locally reconstructed" to generate a consensus sequence from multiple reads within a single diffraction spot. The reconstructed reads from many diffraction spots are then mapped together to generate a contiguous sequence that represents a de novo assembly of an entire targeted gene set or entire genome.

[0313] Additional teachings related to the present disclosure may be found in U.S. Patent Nos. 8,148,512, 7,473,767, 7,919,237, 7,941,279, 8,415,102, 8,492,094, and 8,519,115; U.S. Patent Application Publication Nos. 2009 / 0220978, 2009 / 0299640, 2010 / 0015607, 2010 / 0261026, 2011 / 0086774, and 2012 / 0086775. 2011 / 0145176, 2011 / 0201515, 2011 / 0229888, 2013 / 0004482, 2013 / 0017971, 2013 / 0178372, 2013 / 0230851, 2013 / 0337444, 2013 / 0345161, 2014 / 0005067, 2014 / 0017688, 2014 / 0037620, 2014 / 0087959, 2014 / 0154681, 2014 / 0162251, 2016 / 0194701. each of which is incorporated herein by reference in its entirety. EXAMPLES

[0314] Example 1: Single molecule length readout using Hyb & Seq chemistry

[0315] The disclosed sequencing probes and sequencing methods using said sequencing probes are conveniently referred to as Hyb & Seq. This term is used throughout the specification to describe the disclosed sequencing probes and sequencing methods. Hyb & Seq is a library-free, amplification-free, single-molecule sequencing technology that utilizes nucleic acid hybridization cycles of fluorescent molecular barcodes on the surface of natural targets.

[0316] A long readout using Hyb&Seq is demonstrated for a single DNA target 33 kilobases (kb) in length. The key steps are: (1) a long DNA molecule is captured on the surface of a sequencing flow cell and elongated by hydrodynamic forces; and / or (2) multiple perfectly matched sequencing probes hybridize along the long single-molecule target; and / or (3) fluorescent reporters hybridize to barcode regions in the sequencing probes to identify all bound sequences; and / or (4) spatially resolved fluorescence data is used to determine the relative positions of sequences within the single-molecule target.

[0317] Non-limiting examples of key advantages of long reads using Hyb&Seq include: read length is not limited by the chemistry as determined by the length of the molecule; and / or simple and limited sample preparation resulting in less fragmentation; and / or positional information associated with the sequencing probes aids in reconstruction; and / or the ability to phase variants into long range haplotypes.

[0318] Hyb & Seq chemistry design: Sequencing probes consist of a target-binding domain that base pairs with a single molecule target and at least three positions (R 1 , R 2 , R 3 ) in the barcode domain. The 4096 possible sequencing probes allow sequencing of any target sequence. Reporter probe: Three reporter probes bind sequentially to the above positions in the barcode domain. Each reporter complex corresponds to a specific dinucleotide. Function is exerted by hybridization.

[0319] The disclosed long-read and short-read sequencing methods utilize the same simple probe hybridization workflow for targeted capture of nucleic acids, as shown in Figures 18 and 19. Multiple sequencing probes can be hybridized simultaneously to the target nucleic acid, as shown in the right diagram of Figure 18, and optical resolution allows for the individual identification of several spots per long target, as shown in the right diagram of Figure 19. By hybridizing and recording multiple sequencing probes simultaneously, the information content of a single read is increased. Long-range haplotypes are inherent to single molecule analysis and can be reconstructed by actual physical location rather than computational reconstruction. Using the disclosed sequencing methods, long sequencing reads of up to hundreds of kilobases are possible.

[0320] As shown in Figure 20, a pool of special sequencing probes hybridizes to a 15 kilobase target in the expected pattern. The sequencing probes hybridize to the extended target (preferably the hydrodynamically extended target) at the expected sequence-specific positions and relative physical distances. The disclosed sequencing method has increased information content compared to short read techniques, allowing more bases to be read in each cycle. The disclosed long read sequencing method also records the relative positions of the sequencing reads. This aids in the reconstruction of the long reads. When using the disclosed long read sequencing method, read length = consensus sequence length = length of captured target molecule.

[0321] Figure 27 shows the results of an experiment in which a 33 kilobase DNA fragment was captured, extended, hybridized to a sequencing probe and a reporter probe, and detected. The sequencing method of the present disclosure is compatible with DNA fragments up to and greater than 33 kilobases. The read length is limited only by the initial length of the target nucleic acid fragment, not by the enzyme or sequencing chemistry.

[0322] Figure 13 shows the additional capabilities of the disclosed sequencing method for targeted phased long reads. Phased long range haplotypes are unique to the data and easily identified for phasing variants. Sequencing of the entire long target molecule is not necessary because "blocker oligos" can be used to restrict cycling of sequences to the sequencing window of interest.

[0323] The results of Example 1 demonstrate that the disclosed sequencing method is capable of single-molecule sequencing with long read lengths. In particular, the results show that single-stranded DNA molecules of 15 kilobases and 33 kilobases can be successfully captured and hydrodynamically extended; spatially resolved fluorescence data accurately correspond to actual relative positions along long single molecules; and multiple sequences of more than 10 bases are simultaneously read out per sequencing cycle.

[0324] Example 2: ShortStack™ Technology: Reference-Guided Accurate Reconstruction of Hyb&Seq Reads for Targeted Sequencing Resolving Short Nucleotide Variants and InDels

[0325] ShortStack is an open-source algorithm designed to reconstruct differential hexamer readouts (hexamer spectra) from Hyb&Seq. The algorithm is single-molecule based, using hexamer readouts from each imaged feature to identify targets, and a statistical approach to reconstruct these hexamer readouts into a consensus sequence with error correction.

[0326] Single molecule sequencing using Hyb&Seq chemistry and ShortStack was performed as follows: hexamer readouts of single molecule targets were generated after each cycle of hybridization using Hyb&Seq chemistry; after multiple cycles of hybridization, hexamer spectra covering multiple regions of each single molecule target were generated; the hexamer spectra were used together with reference sequences for each of the target nucleic acid molecules to derive a consensus sequence for each single molecule target.

[0327] Results of targeted sequencing using Hyb&Seq technology with ShortStack show that: single molecule target identification algorithms using hexamer spectra had a 100% success rate; and / or reference-guided reconstruction algorithms achieved single molecule consensus accuracy of >99% (QV ≈32) at 5 rounds of coverage, with consistent somatic variant detection (R 2 Approximately 90% were verified using pre-characterized gDNA samples; and / or in silico experiments using all 6mers and ShortStack confirmed average QVs >90 for larger target panels. The ShortStack algorithm can accurately reconstruct Hyb&Seq data. Figure 28 shows results from a sequencing experiment obtained using the sequencing method of the present disclosure and then analyzed using the ShortStack algorithm. In this experiment, the sequenced target nucleic acid contains a fragment of the gene BRAF (SEQ ID NO:3), a fragment of the gene EGFRex18 (SEQ ID NO:4), a fragment of the gene KRAS (SEQ ID NO:5), a fragment of the gene PIK3CA (SEQ ID NO:6), a fragment of the gene EGFRex20 (SEQ ID NO:7), and a fragment of the gene NRAS (SEQ ID NO:8). Figure 28 shows both base coverage and variant reads. The coverage plot shows base coverage in FFPE (formalin-fixed paraffin-embedded) gDNA. Results show that most bases of the diverse targets are covered by the available sequencing probes. Error plots show the error rate versus coverage at the interrogated positions in FFPE gDNA samples for the diverse targets. Results show that the error rate is less than 1% at 8 rounds of coverage. Frequency plots show the correlation between expected and known frequencies of variants in sequenced Horizon gDNA samples. The table includes the sequenced Horizon genome reference gDNA and shows that a portion of the variant molecules match the known frequency in the reference samples.

[0328] The results of Example 2 demonstrate that ShortStack is an accurate algorithm for partial reconstruction of hexamer spectra obtained using the sequencing methods of the present disclosure. In particular, the results show: 100% accuracy of target identification with average quality score per base of >30 using simulated data; >99% accuracy of base reads in the Hyb&Seq experimental data at 5 coverages; and frequency of variant detection from genomic DNA matching known values ​​(R 2 90%); and computational performance is excellent and directly proportional to the number of hexamers reconstructed, allowing the reconstruction of 69k molecules in about 15 minutes on a personal computer.

[0329] Example 3: Library-free targeted sequencing of native gDNA from FFPE samples using Hyb&Seq™ technology - a hybridization-based single molecule sequencing system

[0330] Using the sequencing method disclosed herein (Hyb & Seq), targeted sequencing of a cancer panel was performed on native gDNA from FFPE samples and demonstrated: accurate base reads in single molecule sequencing targeting cancer gene targets; and / or accurate detection of known cancer gene single nucleotide variants (SNVs) and insertions / deletions (InDels); and / or multiplexed capture of cancer gene targets from gDNA extracted from FFPE (median DNA fragment size 200 bases); and / or automated end-to-end sequencing on an improved prototype instrument.

[0331] The Hyb & Seq chemistry and workflow was as follows: genomic targets of interest were captured directly onto the surface of a sequencing flow cell; a pool containing hundreds of 6-mer sequencing probes was flowed into the sequencing chamber; fluorescent reporter probes were sequentially hybridized to the barcode regions of the sequencing probes in three reporter exchange cycles to identify bases in the 6-mers; once a base was identified, the sequencing probes were washed away; the cycle was repeated with a new pool of sequencing probes until the target region had been read out to sufficient depth.

[0332] Key advantages of Hyb & Seq: Simple and fast FFPE workflow - clinical samples to start sequencing in under 60 minutes; and / or No enzyme or amplification / library-free configuration; and / or Total hands-on time of 15 minutes; and / or High accuracy - low chemistry error rate + inherent error correction; and / or Both long and short reads - read length is dictated by the input sample and not limited by the chemistry.

[0333] The design of the Hyb&Seq chemistry is as described in Example 1. Hyb&Seq sample preparation for processing FFPE tissues consists of three simple steps: (1) deparaffinization and lysis in a single tube; (2) particle removal using a syringe filter; and (3) optional DNA fragmentation and target capture. The process requires the use of one to three 10-micron FFPE sections per sample. The entire process can be completed within 60 minutes and requires only common laboratory equipment, i.e., a heat block, pipettes, filters, and reagents.

[0334] Figure 29 shows a schematic of the experimental design for multiplexing capture and sequencing of cancer gene targets from FFPE samples. A total of 425 sequencing probes were designed and constructed to sequence a subset of 11 cancer gene targets (sequence numbers 3-13). Loci of known variants for each gene target were covered by multiple sequencing probes (perfect match + single mismatch). Base coverage and per-base accuracy were measured in these regions. Accuracy of variant detection was obtained using pre-characterized reference samples. The top diagram in Figure 29 shows that sequencing probes (blue) are aligned to the target sequence (grey) surrounding the known variant positions (red). For each variant position (red), four probe sequences with the respective (A, G, C, T) base variants were included. Tracking one target DNA molecule through 800 barcode exchange cycles during sequencing yielded a large number of hexamer readouts, which were reconstructed by the ShortStack™ algorithm as described in Example 2.

[0335] Figure 28 shows the sequencing results, including the observed and expected mean coverage, single-base error rate, and variant frequency for each target. The results from Example 3 demonstrate that Hyb & Seq sequencing can be used to perform multiplexed sequencing of 11 target regions in FFPE and reference gDNA samples to detect single-nucleotide variations with small errors.

[0336] Example 4: Using Hyb & Seq™ chemistry to directly perform single molecule RNA sequencing without conversion to cDNA

[0337] Direct single-molecule RNA sequencing using Hyb & Seq chemistry was performed as follows: natural RNA molecules were directly captured without conversion to cDNA and immobilized on the surface of a sequencing flow cell; a pool containing hundreds of hexamer sequencing probes was flowed into the sequencing flow cell; perfectly matched sequencing probes were randomly hybridized to the surface of the single-molecule RNA target; fluorescent reporter probes were sequentially hybridized to the barcode regions of the sequencing probes to identify the bases of the hexamers; after the bases were identified, the sequencing probes were washed away; the cycle was repeated until the target region was read out to sufficient depth.

[0338] Key results: Sequencing of targeted single RNA molecules showed a coverage profile similar to DNA; and / or RNA molecules were stably maintained on the flow cell surface over 200+ Hyb & Seq cycles; and / or mRNA and genomic DNA were simultaneously captured and quantified from a single FFPE slice; and / or 8 transcripts were multiplexed and quantified using as little as 10 ng of total RNA.

[0339] The design of the Hyb & Seq chemistry is as described in Example 1. The top panel of Figure 30 shows a schematic diagram of the experimental steps involved in direct RNA sequencing compared to those involved in traditional RNA sequencing performed using cDNA conversion. The middle and bottom panels of Figure 30 show results from an experiment investigating the compatibility of RNA molecules with the sequencing method of the present disclosure. In this experiment, four target RNA molecules were sequenced (SEQ ID NOs: 14-17). The results show that the RNA molecules can be captured and detected over at least 200 sequencing cycles.

[0340] Figure 31 shows the results from an experiment using the sequencing method of the present disclosure to perform direct single-molecule RNA sequencing. A natural RNA molecule encoding a fragment of NRASex2 (SEQ ID NO: 18) was directly captured without conversion to cDNA, immobilized on the surface of a sequencing flow cell, and sequenced using the method of the present disclosure. This experiment was also repeated using a DNA molecule captured instead of RNA. Figure 31 shows that the sequencing coverage of DNA and RNA was comparable. This shows that the sequencing method of the present disclosure can be used to directly sequence RNA without conversion to cDNA.

[0341] FIG. 24 shows an example of combined capture of RNA and DNA from FFPE samples. The left diagram of FIG. 24 is a schematic diagram showing that both gDNA and mRNA can be simultaneously captured, detected, and sequenced from FFPE samples using the methods of the present disclosure. Samples were prepared using the same FFPE workflow described in Example 3. The same capture protocol was used, but with RNA-specific and DNA-specific capture probes. DNA and RNA molecules were simultaneously sequenced in the same flow cell using the same sequencing probes. The right diagram of FIG. 24 shows results from an experiment to simultaneously capture and detect NRAS RNA and DNA from tonsil FPPE samples. The fluorescent image shows that both RNA and DNA can be captured and detected. The bar graph shows that specific RNA and DNA capture probes are required to simultaneously capture RNA and DNA.

[0342] Figure 32 shows the results of multiplex target capture of an RNA panel. Multiplex capture of eight moderately to highly expressed transcripts with respect to the Human Universal Reference RNA was performed with various inputs of total RNA (0 ng, 1 ng, 10 ng, 100 ng, 1000 ng). The multiplex captured RNA molecules were immobilized on the surface of a flow cell and quantified by hybridizing specific sequencing and reporter probes to the immobilized RNA molecules. The bottom panel of Figure 32 shows a fluorescent image of the captured RNA molecules with an input of 100 ng. An example of each RNA is circled, along with the transcript name and the corresponding color combination of the reporter complex used for identification. The top panel of Figure 32 shows the quantification of the counts for each specific RNA target.

[0343] The results of Example 4 demonstrate that single molecule RNA sequencing is feasible with Hyb & Seq chemistry. In particular, the results demonstrate: (1) direct RNA sequencing is possible without conversion to cDNA; (2) RNA molecules are stable throughout the Hyb & Seq cycling process; (3) both RNA and DNA molecules can be captured and sequenced in one Hyb & Seq workflow; and (4) targeted capture of mRNA panels can be performed with as little as 10 ng total RNA input.

[0344] Example 5: Integrated bioinformatics algorithms for high-throughput short molecular reads generated from the Hyb&Seq sequencing platform

[0345] The ShortStack™ software was designed to perform standard sequencing-based bioinformatics analysis tasks (alignment, error correction, mutant reads, read reconstruction, etc.). Figure 33 shows a schematic of the overall steps of the ShortStack™ software pipeline, including hexamer alignment and coverage estimation; and / or identification of mutant sequences; and / or construction of a graph data structure; and / or sequence reconstruction and error correction at the molecular level.

[0346] All algorithms were performed strictly within the scope of information obtained from single molecules, ensuring that the final mutation readout was not affected by the mutation frequency of the sample. The hexamers were grouped into different molecules according to their panel binding positions. To assign molecules to targets, the hexamers were aligned to all the different target regions for each molecule, and the best matched gene target was selected.

[0347] One statistical metric was measured to assess the quality of molecular identification. Alignments against N different target regions generate a distribution of N sums of coverage values ​​for each target. The match score with the highest sum of coverage values ​​was selected as the correct match. The z-score statistic of the selected top match score over the score distribution of all N different targets was measured. Molecular attributes with low confidence (z-score less than 2.5σ) were filtered out.

[0348] Key advantages of the ShortStack™ algorithm include its implementation of a hierarchical hash index design that accurately handles possible sequence ambiguities; and / or advanced algorithm design structures that ensure mapping quality through prioritization and prevent overestimation of mutations.

[0349] In addition, the mutation graph data structure allows computational modeling of different types of mutations (substitutions, insertions, deletions) and generates outputs for sequence reconstruction and variant reading (substitution variants are represented as additional nodes in a graph of the same length as the original sequence; insertions can be modeled by adding connected nodes of any length; deletions are modeled as adding empty base pair strings to artificial nodes in the graph); and / or in a blind mutation search (i.e. a search in a mutation-tolerant sequence alignment), the Hamming distance from every position in the reference sequence is measured and a new node is added to the graph representing the searched mutation; and / or coverage estimation for mutated hexamers is performed using a hierarchical hash table.

[0350] The constructed graph data structure allows for sequence reconstruction at the molecular level and the correction of instrumental errors. In the constructed graph, a dynamic programming algorithm is applied to find the best scoring pathway, where the score is defined as the normalized base coverage. The best scoring pathway in the graph represented the sequence reconstruction at the molecular level. The correct variants were included, while the instrumental errors in the hexamers were discarded.

[0351] Simulation data sets confirmed that the software can provide highly accurate sequence reconstruction and variant call results at the molecular level. Figure 34 shows the results of mutation analysis of a simulation data set using the ShortStack™ software pipeline. These results show the accuracy of variant call for 10 random mutations. For data sets with moderate instrumental errors, the accuracy averaged 99.39% (targeted search) and 98.02% (blind search). Under simulations with high instrumental errors, the performance averaged 97.19% (targeted search) and 93.53% (blind search). When the base coverage threshold at the molecular level was doubled, the results improved to 99.5% (2x coverage) and 99.9% (3x coverage).

[0352] The ShortStack™ software is able to handle a wide range of different mutations. Figure 35 shows the overall mutation reading accuracy, where each bar represents the average value from 10 different targets mutated for different types of mutations. The insertion and deletion lengths were selected between 1 bp and 15 bp. These results show that the mutation reading accuracy was 94.4% (single coverage), 97.2% (two-fold coverage), and 98.5% (three-fold coverage).

[0353] Example 6: Sample preparation for processing FFPE tissues for Hyb & Seq

[0354] Formalin-fixed paraffin-embedded (FFPE) tissue is a challenging type of sample input for traditional sequencing platforms. Hyb & Seq sample preparation methodology successfully handles FFPE tissue input for downstream sequencing. First, nucleic acids to be sequenced are extracted from formalin-fixed paraffin-embedded (FFPE) tissue in a one-step process. One or more FFPE sections of 10 μm thickness are heated in an aqueous nucleic acid extraction buffer to simultaneously melt the paraffin wax, degrade the tissue, and release the nucleic acids from the cells. Suitable extraction buffers are known in the art, and representative examples include proteinases, detergents (e.g., Triton-100), chelating agents (e.g., EDTA), and ammonium ions. The FFPE sections and extraction buffer are incubated at 56°C for 30 minutes to separate the paraffin from the tissue and allow proteinase K to digest the tissue structure, exposing the embedded cells to the detergent, allowing cell lysis. The solution is inverted three times at 8-minute intervals to facilitate mixing of the reagents during the tissue deparaffinization and digestion process, and after this step, the solution is heated to 98°C to facilitate reversal of formaldehyde crosslinks, making it easier to extract nucleic acids.

[0355] Once nucleic acids are extracted from FFPE tissues, the solution is filtered using 2.7 μm pore size glass fiber filters (Whatman) to remove tissue debris and frozen paraffin. The resulting solution is a homogenous translucent solution containing nucleic acids. The nucleic acids are highly fragmented due to the formalin fixation process and storage conditions. If further fragmentation is required, DNA can be physically sheared using a Covaris focused beam sonicator. Due to buffer conditions, extended sonication is required to shear the nucleic acids. Sonication is performed for 600 seconds using standard settings of 50 W peak input power, 20% duty factor, and 200 cycles / burst to maximize target capture. To achieve shorter fragment lengths, emulsified paraffin can be precipitated from the filtered solution by centrifugation at 21,000 g for 15 minutes at 4°C. This allows DNA to be sheared to approximately 225 bp.

[0356] Target capture is then performed by binding the capture probe pair to the target nucleic acid molecule during a rapid hybridization process. The 5' capture probe contains a 3' biotin moiety, allowing the target to bind to the streptavidin-coated surface of a flow cell during the target deposition process. The 3' capture probe contains a 5' tag sequence (G sequence) that allows it to bind to beads during the purification process. The reaction rate is governed by the concentration of the capture probe, which is added in the low nanomolar range to maximize the reaction rate. The capture probe hybridizes to the target adjacent to the region of interest to generate a sequencing window. For each DNA target, the capture probe set also contains an oligo with the same sequence configuration as the sequencing window to hybridize to the antisense strand of the target and prevent reannealing. The solution containing the capture probes is heated to 98°C for 3 minutes to denature the genomic DNA, followed by incubation at 65°C for 15 minutes. NaCl concentrations ranging from 400 mM to 600 mM are used in the hybridization reaction. A panel of over 100 experimentally validated targets is listed in Table 3, which details the genes and exons of the targeted DNA regions. [Table 3]

[0357] The capture probes are allowed to bind to the targeted DNA regions and then purified from the remainder of the genomic DNA, resulting in a target-enriched solution. Beads coated with antisense oligos (anti-G sequences) against the binding sequence of the 3' capture probe are incubated with the capture reaction mixture for 15 minutes at room temperature. After this binding step, the beads are washed three times with 0.1x SSPE to remove non-target DNA and the biotin-containing 5' capture probe. After washing, the beads are resuspended in 14 μl of 0.1x SSPE and then heated to 45°C for 10 minutes to elute the purified DNA target from the beads. After elution, 1 μl of 5 M NaCl is added to ensure that the capture probe remains bound to the DNA target.

[0358] The final step in the sample preparation process is to deposit the DNA targets onto the surface of the flow cell where they can be analyzed using the probes of the present disclosure as disclosed herein. A syringe pump is used to control the rate at which the targets are loaded into the fluidic channels of the flow cell, so that all targets have time to diffuse across the height of the channel and bind to the streptavidin surface. This loading method creates a density gradient of targets, with the number of molecules per unit area being greatest at the inlet of the fluidic channel and decreasing along the length of the channel in the direction of fluid flow toward the outlet. A flow rate of 0.35 μl / sec provides a large amount of capture within a channel length of approximately 10 mm for a channel that is 1.6 mm wide and 40 μm tall. Once the targets are bound to the surface by the biotinylated 5′ capture probe, a solution of biotinylated oligo(G hook), which is the reverse complement of the binding sequence of the 3′ capture probe, is injected to dangle the free ends of the targets, creating a cross-linked structure. The ssDNA region within the central region is the sequencing window of interest. A solution of G-sequence oligos is then added to hybridize to the excess G-hooks on the surface, reducing the amount of ssDNA on the surface. Figure 11 shows the capture of a single target nucleic acid using a two capture probe system according to the present disclosure.

[0359] Example 7: Multicolor reporter imaging for Hyb & Seq

[0360] The steps included in the image processing pipeline are background subtraction, registration, feature detection, and classification. For background subtraction, the average background for any given channel is a function of shot noise and exposure. In our system, the blue channel has the highest level of background, coupled with larger variations. A simple top-hat filter with a circular structuring element of radius 7 pixels is applied to subtract the localized background. For registration, perfect alignment of the features of interest is essential for multi-color and multi-cycle feature analysis. The system requires two forms of registration. For the first form, a local affine transformation is applied to every image channel in a single acquisition stack. This transformation is a function of the optics and therefore adapted to a given instrument. This function is pre-computed for every trial and applied to every acquired image. For the second form, a global transformation in the form of a fixed shift is calculated using normalized cross-correlation to capture the drift of the mechanical gantry during the trial. The next step is feature detection.

[0361] Once all images are registered, the matched filters are used to detect features through a Laplacian of Gaussian (LoG) filter. The filter is applied with a fixed kernel size (matched to the diffraction limit of the feature) and varying standard deviation (matched to the wavelength of the corresponding channel) to match the enhancement of the spot response. Local maxima are used to identify potential reporter locations. The intensity values ​​associated with each identified feature are collected and classified. The final step is classification. A Gaussian-based Naive Bayes model is used to classify the intensities of the multicolor reporters. This model assumes that the reporter intensities are independent of each other and follow a normal distribution. The model then uses the maximum a posteriori rule, or MAP rule, to find the optimal reporter (for all channels x l Calculate the probability that a particular feature y (specified by its strength in ) belongs to a given class (Ck).

number

[0362] The intensity distribution of reporter complexes labeled with each color combination is shown in FIG. 36. FIG. 36 shows a coding scheme using two dyes, blue and red. In the two-color coding scenario, there are six classes (including background). In the implemented system, 14 classes are possible by selecting four colors. Note that there is some overlap between the distributions of the single half dye and the full dye. As a consequence of the above, classification between these classes shows a larger error rate, with the maximum misclassification rate between "xG" and "GG" being 11.8%. The misclassification rate in the 10-class model is less than 0.2%. Since each reporter requires a maximum of eight classes, it is easy to select the one with the smallest classification error. The detected color code is translated into the identified base pair based on a lookup table. The features are tracked over multiple cycles using the probes disclosed herein.

[0363] Example 8: Purification and deposition of target nucleic acid using capture probes

[0364] A two capture probe system is used to capture the target nucleic acid molecule, resulting in highly specific enrichment. The capture probes are designed to bind to the target nucleic acid at positions adjacent to the targeted region of interest, creating a "sequencing window." The 5' capture probe (designated CapB) contains a 3' biotin moiety. The 3' capture probe (designated CapA) contains a 5' affinity tag sequence (designated G sequence). On average, the capture probes are approximately 40 nucleotides in length and are designed based on Tm and sequence context. The sequencing window is approximately 70 nucleotides in length and is easily adjusted. Figure 11 shows a schematic of the two capture probe system.

[0365] A biotin moiety on CapB connects the target nucleic acid to the streptavidin-coated surface of a sequencing flow cell. An affinity tag on CapB allows the target nucleic acid molecule to be reversibly bound to magnetic beads during purification. The use of CapA and CapB allows for high stringency target enrichment because both probes remain bound to a single target nucleic acid molecule, and the target survives both the magnetic bead purification and surface deposition processes. Multiplex capture has been demonstrated for up to 100 targets at a time. To achieve efficient capture in a short time, capture probes are added at concentrations ranging from 1 nM to 10 nM.

[0366] In experimental tests, a panel of about 10 target nucleic acid molecules was purified using G-beads and the two-probe capture system. CapA and CapB probes were first hybridized to the target nucleic acid. The G-sequence portion of the bound CapA probe then hybridized to the G-hook on the G-bead, ligating the target nucleic acid molecule to the G-bead. A series of stringent washes with 0.1×SSPE were performed to remove non-target DNA and unbound CapB. To release the target nucleic acid molecules from the G-beads, they were eluted at 45°C with low salt concentration to denature the G-sequence, while CapA and CapB remained hybridized to the target nucleic acid.

[0367] Tests show that when purifying a panel of approximately 100 target nucleic acid molecules, the nonspecific / background signal increases significantly. This increase in background can be attributed to several factors: (1) increased interactions between CapA and CapB probe species, leading to increased amounts of free CapB probe carried over through purification, and (2) increased interactions between CapB probes and G-hooks or G-beads, leading to the purification of unwanted target nucleic acids. Furthermore, as the size of the panel increases, the possible interactions between CapB and CapA species and the sequencing probes become exponentially larger. These interactions can interfere with the ability to densely deposit targets, resulting in wasted sequencing readouts.

[0368] To reduce nonspecific and background signals resulting from purification of released probe species and unwanted target nucleic acid molecules, several modifications of the purification procedure can be made. First, including formamide at a concentration of 30% v / v in the buffer used to bind the target nucleic acid molecules to the G-beads reduces background counts by a factor of two (as measured by counts in controls lacking target molecules). This is likely due to interference with incomplete hybridization of released capture probes to the G-hooks, allowing excess probe to be washed away. Second, including four isodG bases in the G-hooks on the G-beads and a complementary isodC base in the CapA G sequence reduces background counts by a factor of three (as measured by counts in controls lacking target molecules). Iso-dC and isodG are isomeric variants of the natural dC and dG bases. Because isobases base pair with other isobases but not with natural bases, only incomplete hybridization between the capture probe and the isoG hook can exist between the non-isobases of the G sequence and the isoG hook. These incomplete interactions are more easily disrupted during stringent washes. Finally, the subsequent purification of the isoG beads with Ampure® XP (Agencourt Biosciences Company) beads reduces background counts by at least 20-fold (as measured by counts in controls lacking target molecules). During purification with Ampure® XP beads, the DNA sample is mixed with a suspension of carboxylated magnetic beads in a solution of polyethylene glycol (PEG) and NaCl. The concentrations of PEG and NaCl can be titrated so that only molecules above a molecular weight threshold are precipitated and bound to the beads. The Hyb&Seq targets hybridized to the capture probes are on the order of 81 kDa, whereas the released probes are on the order of 17 kDa or less.By mixing a suspension of Ampure® XP beads with IsoG bead elution solution in a volume ratio of 1.8:1, hybridized targets bind to the beads, allowing a significant portion of the released probe to be washed away before final elution.

[0369] Thus, the model purification workflow consisted of the following steps: (1) hybridization of capture probe-target nucleic acid complexes to IsoG beads in 5x SSPE / 30% formamide; (2) washing of IsoG beads with 0.1x SSPE; (3) elution of capture probe-target nucleic acid complexes in 0.1x SSPE at 45°C; (4) binding of IsoG bead eluate to 1.8 volumes of Ampure® XP beads; (5) washing of Ampure® XP beads with 75% ethanol; (6) elution of capture probe-target nucleic acid complexes in 0.1x SSPE resulting in elution of target in 7.5 μl followed by addition of 0.5 μl of 5 M NaCl.

[0370] After purification, the capture probe-target nucleic acid complex is deposited on the sequencing surface using an infusion syringe pump and the purified target is slowly injected into the flow cell. To determine the deposition gradient, the flow cell is imaged at various positions along the length of the channel. A typical deposition gradient is shown in Figure 37. With a channel height of 20 μm, loading the sample at a flow rate of 0.167 μl / min results in target enrichment, with 80% of all targets binding within 5.1 mm along the length of the channel. This is achieved with an FOV of 0.0357 mm. 2 , which represents approximately 240 FOV for the Gen2 imager with a flow cell channel width of 1.7 mm. The gradient can be varied by adjusting the flow rate during deposition.

[0371] Utilizing the procedure described above, purification and deposition of a 100-plex target nucleic acid panel with genomic DNA sheared to a size of approximately 300 base pairs was investigated. A series of experiments was performed in triplicate with a range of DNA inputs from 25 ng to 500 ng. The total number of targets on the flow cell was extrapolated by imaging the deposition gradient to obtain the average counts, as shown in Figure 38. The capture efficiency was 6.6% and was consistent across the range of DNA mass inputs.

[0372] Example 9: Design and characterization of sequencing probes

[0373] The sequencing probe hybridizes to the target nucleic acid molecule via a target binding domain. In this example, the target binding domain is 8 nucleotides in length and contains a bridged nucleic acid (LNA) hexamer flanked by (N) bases (where (N) bases can be universal / degenerate or canonical bases) (N 1 -B 1 -B 2 -B 3 -B 4 -B 5 -B 6 -N 2 , where B 1 ~B 6 is the LNA, and N 1 and N 2 is (hexamer) sequence B 1 -B 2 -B 3 -B 4 -B 5 -B 6 The complete set of 4,096 sequencing probes encodes all possible hexamers, allowing sequencing of any target nucleic acid. Each sequencing probe also contains a barcode domain that encodes the hexamer sequence present in the target binding domain. Each barcode domain contains three positions (R 1 , R 2 , R 3) in the barcode domain. Each position in the barcode domain contains a unique sequence that corresponds to a specific dinucleotide in the target-binding domain hexamer and can bind to a specific labeled reporter complex. A schematic of the entire sequencing probe is shown in Figure 1. Each position in the barcode domain encodes eight possible "color combinations" created using four fluorescent dyes: blue (B), green (G), yellow (Y), and red (R). During each cycle of sequencing, a reporter complex binds to one of three positions in the barcode domain, indicating the identity of the corresponding dinucleotide in the target-binding domain hexamer. During three consecutive sequencing cycles, three "color combinations," one for each position in the barcode domain, are recorded, allowing the identification of the entire target-binding domain hexamer. The 4,096 sequencing probes are divided into eight pools, each associated with one of the 512 possible barcodes.

[0374] Example 10: Reporter complex design, purification, and binding conditions

[0375] In this example, each reporter complex is a branched structure of 37 DNA oligomers designed to carry a total of 30 fluorescent dyes (15 of each color in the color combination). The 37 DNA oligomers that make up the reporter complex can be classified by size. The largest oligomer, called the primary nucleic acid, is covalently linked to a complementary nucleic acid that is 12 or 14 nucleotides in length. The primary nucleic acid is 96 nucleotides in length. The complementary primary nucleic acid is located at position R on the barcode domain of the sequencing probe. 1 , R 2 , R 3The next largest DNA oligomer is 89 nucleotides in length and is called the secondary nucleic acid. There are six secondary nucleic acids per reporter complex, three for each color in the color combination. Each secondary nucleic acid contains a sequence 14 nucleotides in length that allows the secondary nucleic acid to hybridize to the primary nucleic acid. The smallest DNA oligomer is 15 nucleotides in length and is called the tertiary nucleic acid. There are 30 tertiary nucleic acids per two-color probe, 15 per color. A schematic diagram of the branched structure of 37 DNA oligomers is shown in Figure 4.

[0376] The tertiary nucleic acids contain a detectable label in the form of a fluorescent dye. There are four fluorescent dyes: blue (B), green (G), yellow (Y), and red (R). The combination of dyes in one reporter complex allows ten possible two-color combinations (BB, BG, BR, BY, GG, GR, GY, RR, YR, YY). To prevent color swapping or cross-hybridization between different fluorescent dyes, each secondary and tertiary nucleic acid corresponding to a particular fluorescent dye contains a unique sequence. For example, each tertiary nucleic acid labeled with Alexa 288 fluorophore, i.e., blue, contains a sequence that is complementary only to the blue secondary nucleic acid. The blue secondary nucleic acid further has a different sequence that is complementary only to the primary nucleic acid molecule that corresponds to the color combination that includes blue.

[0377] Each complementary nucleic acid is located at position R of the barcode domain of the sequencing probe. 1 , R 2 , R 3 Therefore, the same barcode domain contains different sequences between position R 1 and R 2 Even if position R 1 A complementary nucleic acid molecule identifying said dinucleotide at position R 2 Similarly, position R 2 A complementary nucleic acid molecule identifying said dinucleotide at position R 1The complementary nucleic acid is designed such that it can be efficiently displaced from the sequencing probe using competitive toehold exchange (for complementary nucleic acids 12 nucleotides in length) or UV cleavage (for complementary nucleic acids 14 nucleotides in length).

[0378] Preparation of the reporter complex occurs in two sequential hybridization steps: (1) hybridization of the tertiary nucleic acid to the secondary nucleic acid, followed by (2) hybridization of the tertiary nucleic acid plus the secondary nucleic acid to the primary nucleic acid. Four separate reactions of tertiary nucleic acid to secondary nucleic acid are prepared by combining 100 μM secondary nucleic acid with 600 μM tertiary nucleic acid in 4.2× SSPE buffer for 30 min at room temperature. Then, 24 reporter probes are prepared separately with 2 μM primary nucleic acid, 7.2 μM secondary nucleic acid plus dye#1 tertiary nucleic acid, and 7.2 μM secondary nucleic acid plus dye#2 tertiary nucleic acid in 4.8× SSPE. These reactions are heated to 45° C. for 5 min and then cooled to room temperature for 30 min. These 24 reactions are then sorted into three different pools corresponding to the barcode domains (i.e., R 1 , R 2 , R 3 ). For example, R 1 Eight different reporter probes (2 μM each) conjugated to barcode domains are pooled together and diluted 10-fold to a final working concentration of 200 nM for each reporter complex. The reporter complexes can be purified using high pressure liquid chromatography (HPLC). Figure 39 shows that the free oligomers and unsuccessfully formed probes can be removed by HPLC purification to obtain the reporter probes.

[0379] After preparing the reporter complexes, a standard test for quality assurance is to mix each of the three pools of reporter probes with the corresponding barcode region (R 1 , R 2 , R 3The reporter probes are tested for binding to either the 12-mer or the 12-mer chromatin (Figure 1). Testing is performed with modified sequencing probe constructs with only one barcode domain present and immobilized on the surface of a flow cell. Multiplexing all eight 12-mers representing each color is expected to identify all eight reporter probes with large color counts.

[0380] To improve the efficiency and accuracy of hybridization of the reporter probe with the barcode domain of the sequencing probe, various buffer additives were tested. Figure 40 shows results from an experiment showing that a buffer containing 5% dextran sulfate (500K) and 15% formamide or 15% ethylene carbonate allows the reporter probe to be hybridized with the highest efficiency and accuracy in a short time. However, Figure 41 shows results from an experiment showing that ethylene carbonate has a negative effect on the surface of the sequencing slide, resulting in a large loss of target nucleic acid over time. Thus, a buffer containing 5% dextran sulfate (500K) and 15% formamide is better for efficient and accurate hybridization of the reporter probe with the sequencing probe.

[0381] Example 11: Design and validation of complementary nucleic acid sequences

[0382] The reporter probe is a reporter probe that is attached to a specific position (R 1 , R 2 , R 3The sequences contain complementary nucleic acids that bind to either the reporter probe or the sequencing probe (either 12-mer or 14-mer). Complementary nucleic acids containing 12 nucleotides (12-mer) or 14 nucleotides (14-mer) were designed and tested to determine the most suitable sequences for hybridization. For screening, the optimal sequences were determined using the following criteria: the sequence must show high binding efficiency between the reporter probe and the sequencing probe with binding efficiency of over 80% in 10 sequencing cycles; the sequence must show fast hybridization kinetics occurring within 15-30 seconds; and the sequence must show high specificity with less than 5% cross-hybridization errors in the reporter pool.

[0383] Table 4 shows the 24 12-mer sequences that were identified (SEQ ID NOs: 19-42). Because each barcode domain contains three positions, the 24 12-mer sequences can be divided into three groups to create an 8x8x8 12-mer reporter set. [Table 4]

[0384] 14-mer sequences were designed in a similar manner, but differed from the 12-mer sequences in three ways. First, the 14-mer sequences contain longer hybridization sequences because they contain 14 single-stranded nucleotides that bind to specific positions on the barcode domain instead of the 12 single-stranded nucleotides present in the 12-mer. Second, the 14-mer sequences have greater sequence diversity because they were not designed for toehold-mediated removal. The 14-mer sequences hybridize more strongly to the sequencing probe, reducing the efficiency of toehold-mediated removal. Therefore, we sought a sequence-independent removal strategy for the 14-mer sequences and relaxed the sequence constraints during screening. An algorithm that includes the following set of rules was used to design sequences for screening. The rules are: nucleotide composition lacking "G" or "C" (i.e., low complexity sequence); GC content 40%-60%; melting temperature (Tm) 35°C-37°C; hairpin folding energy (dG) >2; and compatibility with other sequencing probes (Hamming distance ≥7). To minimize hybridization of the 14-mer sequence to genomic sequences that may be present in the target nucleic acid, potential sequences were filtered using the External RNA Controls Consortium sequence as a guide. Third, the 14-mer sequence was designed to be removed from the barcode domain of the sequencing probe by strand cleavage using a cleavable linker modification at the position where the 14-mer complementary nucleic acid is attached to the primary nucleic acid of the reporter complex. Removal of the 14-mer sequence results in "darkening" of the reporter complex signal, allowing for the next screening cycle and detection of the signal. A variety of cleavable linker modifications were examined, including linkers cleavable by UV light, linkers cleavable by reducing agents (such as TCEP), and linkers cleavable by enzymes (such as uracil, which is cleaved by USER™ enzyme). All of these cleavable linker modifications were found to promote efficient darkening of the reporter complex. Darkening was further enhanced by introducing the cleavable linker modification into the secondary nucleic acid.These cleavable linker modifications were positioned between the sequence that hybridizes to the primary nucleic acid and the sequence that hybridizes to the tertiary nucleic acid. Figure 10 shows possible locations within the reporter probe for the cleavable linker modifications.

[0385] Screening of potential 14-mer sequences identified two groups of acceptable sequences. Table 5 shows the first group, which contained 24 sequences (SEQ ID NOs: 43-66). These 24 sequences could be divided into three groups to create an 8x8x8 14-mer reporter set. [Table 5]

[0386] Table 6 shows the other group, which contained 30 sequences (SEQ ID NOs: 67-96). These 30 sequences were divided into three groups to generate a 10 x 10 x 10 14-mer reporter set. [Table 6]

[0387] After screening, the 8x8x8 12-mer reporter set, 8x8x8 14-mer reporter set, and 10x10x10 14-mer reporter set were experimentally validated. For the 8x8x8 12-mer binding scheme, validation was performed using the Hyb&Seq prototype and 10 sequencing cycles were recorded. Three pools of reporter probes were used in both the long and short workflow methods. All 512 possible barcode domains of the sequencing probes were explored. Table 7 shows the experimental steps for the long and short workflow methods. [Table 7]

[0388] The long workflow experiment resulted in a darkening efficiency of over 97%. We estimated that darkening was similarly efficient for the short workflow experiments, but anticipated that a small number of undarkened reporters may remain in each image and be erroneously referred to as new reporters. Indeed, the highest number of barcode counts in the short workflow experiment was YYYYYY. This was most likely due to undarkened artifacts and background. Figure 42 shows that the performance of the 8x8x8 12-mer reporter set is generally poorer in the short workflow compared to the long workflow. (The position of the barcode domain R 1 (binding to the position of the barcode domain R) and reporter complex 1 (binding to the position of the barcode domain R) 3 Reporter complex 3 (binding to ) was less efficient in the short workflow compared to the long workflow. This is expected for reporter complex 3 because it contains eight additional toehold oligonucleotides at a high concentration of 2.5 μM each, which may interfere with reporter hybridization. Reporter complex 1 should perform similarly in the two workflows because no toehold was used to remove the first reporter complex in either the short or long workflow. Total errors were also higher (1.3- to 2-fold) in the short workflow than in the long workflow for all three reporter probes.

[0389] We validated the 8x8x8 14-mer reporter set by examining the efficiency, specificity, and hybridization kinetics for all 512 possible sequencing probe barcode domains. The sequencing probe barcode domains were directly immobilized on the glass surface of a Hyb&Seq sequencing cartridge. Figure 43 shows that the 8x8x8 14-mer reporter probes hybridized in just 15 seconds with an average efficiency of 88% and an average error rate of 5.1%. The majority of this error is due to inaccurate identification of the reporter, not due to inaccurate hybridization. Reporter misclassification remains the largest source of reporter error.

[0390] The 10x10x10 14-mer reporter set was validated by examining the efficiency, specificity, and kinetics of hybridization to the barcode domains of 30 complementary truncated sequencing probes. Each barcode domain contained only one reporter binding site. The barcode domains were directly immobilized on the glass surface of a Hyb&Seq sequencing cartridge. Figure 44 shows that the 10x10x10 14-mer reporter set hybridized with an average efficiency of 90% in just 15 seconds, with an average error rate of 5.0%. Again, the majority of errors were due to inaccurate identification of the reporter, not due to inaccurate hybridization.

[0391] Example 12: Design and testing of standard and three-part sequencing probes

[0392] The target-binding and barcode domains of a sequencing probe are separated by a double-stranded "stem." Figure 2 shows two architectures of sequencing probes that were examined in the experiments. In a standard sequencing probe, the target-binding and barcode domains are on the same oligonucleotide, which is attached to a stem oligonucleotide to create a double-stranded region that is 36 nucleotides long. With this architecture, each sequencing probe in a pool of probes utilizes the same stem sequence. In a tripartite probe, the target-binding and barcode domains are separate DNA oligonucleotides that are attached to each other by a stem oligonucleotide of 36 nucleotides. To prevent possible exchange of barcode domains, each barcode has a unique stem sequence and is hybridized separately before pooling the sequencing probes.

[0393] Figure 45 shows the results of a series of experiments comparing the three-part sequencing probe with the standard sequencing probe. These experiments confirmed that the three-part sequencing probe survived one full sequencing cycle in about 80% of all readouts for both configurations, including detection of the third reporter probe. The three-part probe shows about 12% fewer counts compared to the standard sequencing probe. To study the tendency of barcode domain oligos to exchange, a high concentration of a short alternative oligonucleotide containing the same stem sequence was added to the reaction. The results showed that about 13% of the detected three-part sequencing probes had exchanged barcode oligos. Oligonucleotide exchange would need to be mitigated by incorporating a unique stem sequence. Despite slightly lower performance, the three-part probe offers the advantages of design flexibility, rapid oligo synthesis, and reduced cost.

[0394] Example 13: Effect of bridged nucleic acid substitutions in the target binding domain

[0395] The effect of placing bridged nucleic acids (LNA) by substitution into the target binding domain of the sequencing probe was investigated as follows: The sequencing probe was hybridized to a reporter probe in solution, and the appropriately formed sequencing probe-reporter probe was purified. The sequencing probe-reporter probe was then hybridized to a synthetic target nucleic acid in solution and loaded onto the surface of a prototype sequencing cartridge. The synthetic target nucleic acid consisted of 50 nucleotides and was biotinylated. The sequencing probes were tested individually or in pools of nine sequencing probes, where the probes were designed to bind along the length of the target nucleic acid. For analysis, the entire reaction was deposited on the surface of a streptavidin-coated cover slide using a circuit board device and then stretched by hydrodynamic forces. The reporter probes were then imaged and counted using appropriate devices and software (e.g., NanoString nCounter® device and software).

[0396] Each sequencing probe contained a target binding domain of 10 nucleotides (SEQ ID NO: 97). LNA substitutions within the target binding domain were performed to include 2, 3, or 4 LNA bases at the positions shown in FIG. 46. FIG. 46 shows that the binding affinity of each sequencing probe to the target nucleic acid increased with increasing number of LNA bases. Importantly, FIG. 46 shows that the incorporation of LNA bases did not decrease the specificity of the binding of the sequencing probe. A pool of nine sequencing probes was tested to determine the base coverage at which the probes were able to complete binding to the target. FIG. 47 shows that the introduction of a single LNA probe into the pool increased the coverage of the affected bases but had little effect on the binding of the surrounding probes. These results demonstrated that substitution with LNA bases can improve base sensitivity without decreasing specificity.

[0397] Example 14: Effect of modified nucleotide and nucleic acid analog substitutions in the target binding domain

[0398] The effect of introducing various modified nucleotide and nucleic acid analogue substitutions into the target binding domain of the sequencing probe, including bridged nucleic acid (LNA), bridged nucleic acid (BNA), propyne modified nucleic acid, zip nucleic acid (ZNA®), isoguanine, and isocytosine, was investigated as follows: Biotinylated target nucleic acids, 50 nucleotides in length, were loaded onto the surface of a streptavidin cover slide of a prototype sequencing cartridge. The sequencing probe and reporter probe were then introduced sequentially into the sample chamber, and images were acquired using a Hyb & Seq prototype instrument. Images were processed and counts were compared for each of the different sequencing probes. Substitutions were made in the target binding domain of the 10 nucleotide (SEQ ID NO: 99) of the sequencing probe to include LNA, BNA, propyne, and ZNA® bases at the positions shown in FIG. 48. FIG. 48 shows that the probes containing LNA and BNA showed the greatest increase in binding affinity, while specificity was maintained. This is shown by the number of counts detected for matched and mismatched targets. These results demonstrated that substitution with LNA or BNA bases can improve base sensitivity without compromising specificity.

[0399] Example 15: Determining the accuracy of the sequencing method according to the present disclosure

[0400] Figure 49 shows results from an experiment quantifying the raw specificity of a sequencing method according to the present disclosure. In this experiment, a sequencing reaction was performed in which a pool of four different sequencing probes was hybridized to a target nucleic acid containing a fragment of NRAS exon 2 (SEQ ID NO: 1). Each sequencing probe (barcodes 1-4) hybridized to the hexamer position b 5In this example, barcode 4 is the correct sequencing probe. After the sequencing probes were hybridized, the three positions (R 1 , R 2 , R 3 ) were sequentially hybridized with reporter probes and the corresponding fluorescence data were recorded. The central figure in Figure 49 shows the number of times each color combination was recorded and the percentage of times the correct combination was recorded for the positions of the three barcode domains. R 1 The color combinations in were correctly identified 96% of the time, and R 2 The color combinations in were correctly identified 97% of the time, and R 3 The color combination in was correctly identified 94% of the time. This resulted in an overall raw specificity of 94%, as shown in the bottom panel of Figure 49. Potential sources of error that could explain the misreading of the barcode domain locations include (a) nonspecific binding of the reporter probe to the surface of the flow cell and (b) inaccurate hybridization of the reporter probe. Estimates of reporter hybridization error were approximately 2-4%.

[0401] FIG. 50 shows results from an experiment to determine the accuracy of a sequencing method according to the present disclosure when nucleotides in a target nucleic acid are sequenced by two or more sequencing probes. As shown in the top diagram of FIG. 50, the target nucleic acid in this example is a fragment of NRAS exon 2 (SEQ ID NO: 1). The particular base of interest is a cytosine (C) which is highlighted in the target nucleic acid. This base of interest hybridizes to two different sequencing probes, each resulting in a different hybridization footprint to the target nucleic acid. In this example, sequencing probes 1-4 (barcodes 1-4) attach three nucleotides to the left of the base of interest, while sequencing probes 5-8 (barcodes 5-8) attach five nucleotides to the left of the base of interest. The center diagram of FIG. 50 shows the number of times a particular color combination was recorded at each position of the barcode domain of the sequencing probes. After quantification of the images, the base reading technique shown in Figure 26 can be used to record an average accuracy of approximately 98.98%.

Claims

1. A sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, wherein at least six nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; and at least two nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs; A sequencing probe, wherein the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprising at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, the nucleic acid sequences of the at least three attachment sites determine the positions and attributes of the at least six nucleotides in the target nucleic acid to which the target binding domain binds, and each of the at least three attachment sites has a different nucleic acid sequence.

2. A sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least eight nucleotides and is capable of binding to a target nucleic acid, wherein at least six nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least two nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; and at least two nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprising at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind; each of the at least three attachment positions corresponds to two of the at least six nucleotides in the target binding domain, and each of the at least three attachment positions has a different nucleic acid sequence; A sequencing probe, wherein the nucleic acid sequence at each of the at least three attachment positions determines the positions and identities of corresponding two of the at least six nucleotides in the target nucleic acid to which the target binding domain binds.

3. A sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least 10 nucleotides and is capable of binding to a target nucleic acid, wherein at least 6 nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least 4 nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; A sequencing probe, wherein the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprising at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, the nucleic acid sequences of the at least three attachment sites determine the positions and attributes of the at least six nucleotides in the target nucleic acid to which the target binding domain binds, and each of the at least three attachment sites has a different nucleic acid sequence.

4. A sequencing probe comprising a target binding domain and a barcode domain; the target binding domain comprises at least 10 nucleotides and is capable of binding to a target nucleic acid, wherein at least 6 nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule and at least 4 nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprising at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind; each of the at least three attachment positions corresponds to two of the at least six nucleotides in the target binding domain, and each of the at least three attachment positions has a different nucleic acid sequence; A sequencing probe, wherein the nucleic acid sequence at each of the at least three attachment positions determines the positions and identities of corresponding two of the at least six nucleotides in the target nucleic acid to which the target binding domain binds.

5. a) A composition comprising a target binding domain and a barcode domain wherein the target binding domain comprises at least 8 nucleotides and is capable of binding to a target nucleic acid, at least 6 nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule, and at least 2 nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; at least two nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprises at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, the nucleic acid sequences of the at least three attachment sites determine the positions and identities of the at least six nucleotides in the target nucleic acid to which the target binding domain binds, and each of the at least three attachment sites has a different nucleic acid sequence; a first complementary primary nucleic acid molecule that hybridizes to a first attachment position of said at least three attachment positions; wherein said first complementary primary nucleic acid molecule comprises at least two domains and a linker modification; a first domain of which hybridizes to a first attachment position of said barcode domain and a second domain of which is capable of hybridizing to at least one second complementary secondary nucleic acid molecule; The linker modification is 【Chemical 1】 wherein the linker modification is located between the first domain and the second domain.

6. a) A composition comprising a target binding domain and a barcode domain wherein the target binding domain comprises at least 8 nucleotides and is capable of binding to a target nucleic acid, at least 6 nucleotides in the target binding domain are capable of identifying corresponding nucleotides in the target nucleic acid molecule, and at least 2 nucleotides in the target binding domain do not identify corresponding nucleotides in the target nucleic acid molecule; at least two nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs; the barcode domain comprises a synthetic backbone, the barcode domain comprises at least three attachment sites, each attachment site comprising at least one attachment region comprising at least one nucleic acid sequence to which a complementary nucleic acid molecule can bind, the nucleic acid sequences of the at least three attachment sites defining the positions and identities of the at least six nucleotides in the target nucleic acid to which the target binding domain binds; each of the at least three attachment positions corresponds to two of the at least six nucleotides in the target binding domain, and each of the at least three attachment positions has a different nucleic acid sequence; the nucleic acid sequence at each of the at least three attachment positions defines the position and identity of corresponding two of the at least six nucleotides in the target nucleic acid to which the target binding domain binds; a first complementary primary nucleic acid molecule that hybridizes to a first attachment position of said at least three attachment positions; wherein said first complementary primary nucleic acid molecule comprises at least two domains and a linker modification; a first domain of which hybridizes to a first attachment position of said barcode domain and a second domain of which is capable of hybridizing to at least one second complementary secondary nucleic acid molecule; The linker modification is 【Chemistry 2】 wherein the linker modification is located between the first domain and the second domain.

7. The sequencing probe according to any one of claims 1 to 4, wherein the synthetic backbone comprises any one of a polysaccharide, a polynucleotide, a peptide, a peptide nucleic acid, and a polypeptide.

8. The sequencing probe of any one of claims 1 to 4, wherein the synthetic backbone comprises single-stranded DNA.

9. 5. The sequencing probe of claim 1, comprising a single-stranded DNA synthetic backbone and a double-stranded DNA spacer between the target binding domain and the barcode domain.

10. 10. The sequencing probe of claim 9, wherein the double-stranded DNA spacer is about 1 to about 100 nucleotides in length.

11. 10. The sequencing probe of claim 9, wherein the double-stranded DNA spacer is about 36 nucleotides in length.

12. 5. The sequencing probe of claim 1, wherein the number of nucleotides in the target binding domain is greater than the number of attachment positions in the barcode domain.

13. 5. The sequencing probe of claim 1, wherein the number of nucleotides in the target binding domain is at least 5 more than the number of attachment positions in the barcode domain.

14. 5. The sequencing probe of claim 1, wherein the target binding domain comprises 8 nucleotides and the barcode domain comprises 3 attachment positions.

15. 5. The sequencing probe of claim 1, wherein at least four of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs.

16. 5. The sequencing probe of claim 1, wherein at least six of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogs.

17. The sequencing probe according to any one of claims 1 to 4, wherein the modified nucleotide or nucleotide analogue is a bridged nucleic acid (LNA).

18. 5. The sequencing probe of claim 1, wherein at least one of the at least two nucleotides in the target binding domain that do not identify a corresponding nucleotide in the target nucleic acid molecule is located before the at least six nucleotides in the target binding domain, and at least one of the at least two nucleotides in the target binding domain that do not identify a corresponding nucleotide in the target nucleic acid molecule is located after the at least six nucleotides in the target binding domain.

19. The sequencing probe of any one of claims 1 to 4, wherein each attachment position in the barcode domain comprises the same number of attachment regions.

20. A sequencing probe according to any one of claims 1 to 4, wherein at least one of the at least three attachment positions in the barcode domain contains a different number of attachment regions than the other two.

21. The sequencing probe of any one of claims 1 to 4, wherein each attachment position in the barcode domain comprises one attachment region.

22. The sequencing probe of any one of claims 1 to 4, wherein each attachment position in the barcode domain comprises two or more attachment regions.

23. The sequencing probe according to any one of claims 1 to 4, wherein when the attachment position in the barcode domain comprises two or more attachment regions, the attachment regions are the same.

24. The sequencing probe according to any one of claims 1 to 4, wherein when the attachment position in the barcode domain comprises two or more attachment regions, the attachment regions are different.

25. 5. The sequencing probe of claim 1, wherein each nucleic acid sequence comprising each attachment position in the barcode domain is about 8 to about 20 nucleotides in length.

26. 5. The sequencing probe of claim 1, wherein each nucleic acid sequence comprising each attachment position in the barcode domain is about 12 nucleotides in length.

27. 5. The sequencing probe of claim 1, wherein each nucleic acid sequence comprising each attachment position in the barcode domain is about 14 nucleotides in length.

28. 5. The sequencing probe of claim 1, wherein at least one of the at least three attachment positions in the barcode domain is adjacent to at least one flanking single-stranded polynucleotide.

29. 5. The sequencing probe of claim 1, wherein each of the at least three attachment positions in the barcode domain is adjacent to at least one flanking single-stranded polynucleotide.

30. 5. The sequencing probe of claim 1, wherein at least one attachment region of the at least one attachment site is integrated into the synthetic backbone.

31. 5. The sequencing probe of claim 1, wherein each attachment region in each of said at least three attachment locations is integrated into said synthetic scaffold.

32. The sequencing probe according to any one of claims 1 to 4, wherein the complementary nucleic acid molecule is RNA, DNA, or PNA.

33. 33. The sequencing probe of claim 32, wherein the complementary nucleic acid molecule is DNA.

34. 5. The sequencing probe of claim 1, wherein the complementary nucleic acid molecule is a primary nucleic acid molecule, and the primary nucleic acid molecule directly binds to at least one attachment region within at least one attachment position of the barcode domain.

35. the primary nucleic acid molecule comprises at least two domains, a first domain capable of binding to at least one attachment region within at least one attachment location of said barcode domain; 35. The sequencing probe of claim 34, wherein the second domain is capable of binding to at least one complementary secondary nucleic acid molecule.

36. 36. The sequencing probe of claim 35, wherein the primary nucleic acid molecule comprises a cleavable linker.

37. 37. The sequencing probe of claim 36, wherein the cleavable linker is located between the first domain and the second domain.

38. 37. The sequencing probe of claim 36, wherein the linker is photocleavable.

39. 35. The sequencing probe of claim 34, wherein the primary nucleic acid molecule hybridizes to at least one attachment region within at least one attachment position of the barcode domain and comprises a first detectable label and at least a second detectable label.

40. 36. The sequencing probe of claim 35, wherein the primary nucleic acid molecule hybridizes to at least one attachment region within at least one attachment location of the barcode domain and to at least one second nucleic acid molecule.

41. 41. The sequencing probe of claim 40, wherein the primary nucleic acid molecule hybridizes to at least one, or two, or three, or four, or five, or more secondary nucleic acid molecules.

42. 41. The sequencing probe of claim 40, wherein the primary nucleic acid molecule hybridizes to four secondary nucleic acid molecules.

43. 40. The sequencing probe of claim 39, wherein the first detectable label and the at least second detectable label have the same emission spectrum or different emission spectra.

44. the secondary nucleic acid molecule comprises at least two domains; the first domain is capable of binding to a complementary sequence in at least one primary nucleic acid molecule; 36. The sequencing probe of claim 35, wherein the second domain is capable of binding to (a) the first detectable label and at least a second detectable label, or (b) at least one complementary third nucleic acid molecule, or (c) a combination thereof.

45. 45. The sequencing probe of claim 44, wherein the secondary nucleic acid molecule comprises a cleavable linker.

46. 46. ​​The sequencing probe of claim 45, wherein the cleavable linker is located between the first domain and the second domain.

47. 46. ​​The sequencing probe of claim 45, wherein the linker is photocleavable.

48. 45. The sequencing probe of claim 44, wherein the secondary nucleic acid molecule hybridizes to at least one primary nucleic acid molecule and comprises a first detectable label and at least a second detectable label.

49. 45. The sequencing probe of claim 44, wherein the secondary nucleic acid molecule hybridizes to at least one primary nucleic acid molecule and to at least one tertiary nucleic acid molecule.

50. 45. The sequencing probe of Claim 44, wherein the secondary nucleic acid molecule hybridizes to (a) at least one primary nucleic acid molecule, (b) at least one tertiary nucleic acid molecule, and (c) a first detectable label and at least a second detectable label.

51. 50. The sequencing probe of claim 49, wherein the secondary nucleic acid molecule hybridizes to at least 1, or 2, or 3, or 4, or 5, or 6, or 7, or more tertiary nucleic acid molecules.

52. 50. The sequencing probe of claim 49, wherein the secondary nucleic acid molecule hybridizes to one tertiary nucleic acid molecule.

53. 49. The sequencing probe of claim 48, wherein the first detectable label and the at least second detectable label have the same emission spectrum or different emission spectra.

54. the tertiary nucleic acid molecule comprises at least two domains; the first domain is capable of binding to a complementary sequence in a secondary nucleic acid molecule; 45. The sequencing probe of claim 44, wherein the second domain is capable of binding to a first detectable label and at least a second detectable label.

55. 55. The sequencing probe of claim 54, wherein the tertiary nucleic acid molecule comprises a cleavable linker.

56. 56. The sequencing probe of claim 55, wherein the cleavable linker is located between the first domain and the second domain.

57. 56. The sequencing probe of claim 55, wherein the linker is photocleavable.

58. 55. The sequencing probe of claim 54, wherein the tertiary nucleic acid molecule hybridizes to at least one secondary nucleic acid molecule and comprises a first detectable label and at least a second detectable label.

59. 55. The sequencing probe of claim 54, wherein the first detectable label and the at least second detectable label have the same emission spectrum or different emission spectra.

60. 58. The sequencing probe of claim 57, wherein the first detectable label and the at least second detectable label located on the surface of the secondary nucleic acid have the same emission spectrum, the at least first detectable label and the second detectable label located on the surface of the tertiary nucleic acid have the same emission spectrum, and the emission spectrum of the detectable label on the surface of the secondary nucleic acid molecule is different from the emission spectrum of the detectable label on the surface of the tertiary nucleic acid.

61. A population of sequencing probes comprising a plurality of sequencing probes according to any one of claims 1 to 4.

62. 1. A method for determining the nucleotide sequence of a nucleic acid, comprising: (1) hybridizing a first sequencing probe according to any one of claims 1 to 4 to a target nucleic acid, optionally immobilized at one or more locations on a substrate; (2) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and a second detectable label to a first attachment position of the at least three attachment positions of the barcode domain; (3) identifying the first detectable label and the second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment location; (4) removing the first detectable label and the second detectable label hybridized to the first attachment location; (5) hybridizing a second complementary nucleic acid molecule comprising a third detectable label and a fourth detectable label to a second attachment position of the at least three attachment positions of the barcode domain; (6) identifying the third detectable label and the fourth detectable label of the second complementary nucleic acid molecule hybridized to the second attachment location; (7) removing the third detectable label and the fourth detectable label hybridized to the second attachment location; (8) hybridizing a third complementary nucleic acid molecule comprising a fifth detectable label and a sixth detectable label to a third attachment position of the at least three attachment positions of the barcode domain; (9) identifying the fifth detectable label and the sixth detectable label of the third complementary nucleic acid molecule hybridized to the third attachment location; (10) Identifying the linear order of the at least six nucleotides of the optionally immobilized target nucleic acid hybridized to the target binding domain of the sequencing probe based on attributes of the first detectable label, the second detectable label, the third detectable label, the fourth detectable label, the fifth detectable label, and the sixth detectable label.

63. 1. A method for determining the nucleotide sequence of a nucleic acid, comprising: (1) hybridizing a first sequencing probe according to any one of claims 1 to 4 to a first region of a target nucleic acid obtained from a predetermined gene and optionally immobilized at one or more locations on a substrate; (2) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and a second detectable label to a first attachment position of the at least three attachment positions of the barcode domain; (3) detecting the first detectable label and the second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment site; (4) removing the first detectable label and the second detectable label hybridized to the first attachment location; (5) hybridizing a second complementary nucleic acid molecule comprising a third detectable label and a fourth detectable label to a second attachment position of the at least three attachment positions of the barcode domain; (6) detecting the third detectable label and the fourth detectable label of the second complementary nucleic acid molecule hybridized to the second attachment site; (7) removing the third detectable label and the fourth detectable label hybridized to the second attachment location; (8) hybridizing a third complementary nucleic acid molecule comprising a fifth detectable label and a sixth detectable label to a third attachment position of the at least three attachment positions of the barcode domain; (9) identifying the fifth detectable label and the sixth detectable label of the third complementary nucleic acid molecule hybridized to the third attachment location; (10) Identifying the linear order of the at least six nucleotides in a first region of an optionally immobilized target nucleic acid hybridized to the target binding domain of the first sequencing probe based on attributes of the first detectable label, the second detectable label, the third detectable label, the fourth detectable label, the fifth detectable label, and the sixth detectable label.

64. 64. The method of claim 62 or 63, wherein steps (4) and (5) are carried out sequentially or simultaneously.

65. 64. The method of Claim 62 or 63, wherein the first detectable label and the second detectable label have the same emission spectrum or have different emission spectra.

66. 64. The method of Claim 62 or 63, wherein the third detectable label and the fourth detectable label have the same emission spectrum or have different emission spectra.

67. 64. The method of claim 62 or 63, wherein the fifth detectable label and the sixth detectable label have the same emission spectrum or different emission spectra.

68. 64. The method of Claim 62 or 63, wherein the first complementary nucleic acid molecule comprises a cleavable linker.

69. 64. The method of Claim 62 or 63, wherein the second complementary nucleic acid molecule comprises a cleavable linker.

70. 64. The method of Claim 62 or 63, wherein the third complementary nucleic acid molecule comprises a cleavable linker.

71. 64. The method of Claim 62 or 63, wherein the first complementary nucleic acid molecule, the second complementary nucleic acid molecule, and the third complementary nucleic acid molecule each comprise a cleavable linker.

72. 69. The method of claim 68, wherein the cleavable linker is cleavable with light.

73. 73. The method of Claim 72, wherein removing the first complementary nucleic acid molecule comprises contacting the first complementary nucleic acid molecule with light.

74. 74. The method of claim 73, wherein the light is provided by a light source selected from the group consisting of an arc lamp, a laser, a focused UV light source, and a light emitting diode.

75. (11) optionally removing the at least a first sequencing probe from a first region of the immobilized target nucleic acid; (12) Hybridizing at least a second sequencing probe according to claim 1 or 2 to a second region of the target nucleic acid, optionally immobilized at one or more locations on a substrate, wherein the target binding domains of the first sequencing probe and the at least second sequencing probe are different; (13) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and a second detectable label to a first attachment position of the at least three attachment positions of the barcode domain; (14) detecting the first detectable label and the second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment site; (15) hybridizing a second complementary nucleic acid molecule comprising a third detectable label and a fourth detectable label to a second attachment position of the at least three attachment positions of the barcode domain; (16) detecting the third detectable label and the fourth detectable label of the second complementary nucleic acid molecule hybridized to the second attachment site; (17) removing the third detectable label and the fourth detectable label hybridized to the second attachment location; (18) hybridizing a third complementary nucleic acid molecule comprising a fifth detectable label and a sixth detectable label to a third attachment position of the at least three attachment positions of the barcode domain; (19) identifying the fifth detectable label and the sixth detectable label of the third complementary nucleic acid molecule hybridized to the third attachment location; (20) The method of claim 62 or 63, further comprising identifying a linear order of at least six nucleotides in a second region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the at least a second sequencing probe based on attributes of the first detectable label, the second detectable label, the third detectable label, the fourth detectable label, the fifth detectable label, and the sixth detectable label.

76. 76. The method of claim 75, comprising identifying the sequence of the optionally immobilized target nucleic acid by reconstructing the linear order of nucleotides identified in the first region and the second region, respectively, of the optionally immobilized target nucleic acid.

77. Apparatus for carrying out the method of any one of claims 62 to 76.

78. 62. A kit comprising a substrate, the population of sequencing probes of claim 61, at least one capture probe, at least three complementary nucleic acid molecules comprising a first detectable label and at least two second detectable labels, and instructions for use.

Images