Methods and compositions for sequence detection

Abstract

The present disclosure provides methods, compositions and systems for detecting a nucleic acid sequence. In particular, detection oligonucleotides and detection polynucleotide complex configurations are described that can increase the complexity and multiplexing capabilities of an assay for detecting and decoding amplification products that correlate to multiple target molecules of interest from a sample.

Description

[0001] METHODS AND COMPOSITIONS FOR SEQUENCE DETECTION

[0002] CROSS-REFERENCE

[0003]

[0001] This application claims the benefit of United States Provisional Patent Application Serial No. 63 / 634,313, filed on April 15, 2024, and is incorporated herein by reference in its entirety.

[0004] BACKGROUND

[0005]

[0002] There are many assays and methods usedin research for detecting nucleic acid sequences from a sample. For example, microarrays, direct sequencing of nucleic acids, and labeling of nucleic acid sequences with known sequence probes are some of the assays and methods available. Some of these methods are direct types of assays, where the nucleic acid of interest is identified directly. However, the ability to multiplex samples is limited when practicing these methods, and the direct sequencing of nucleic acids can be expensive and time consuming.

[0006] SUMMARY

[0007]

[0003] The present disclosure provides methods and compositions for identifying the sequence of a target nucleic acid of interest from a sample. More than one target nucleic acid of interest may be identifiable simultaneously according to the methods herein. Methods and compositions described herein may be useful in providing a multiplexed assay where multiple target nucleic acids of interest can be identified in one assay.

[0008]

[0004] Provided herein, in certain embodiments, is a detection polynucleotide complex, comprising an anchor oligonucleotide comprising a sequence complementary to a target nucleic acid, and two sequences that are complementary to two detection oligonucleotides, the two detection oligonucleotides, each of which is hybridized to its respective complementary sequence of the two sequences of the anchor oligonucleotide, wherein each of the two detection oligonucleotides is linked to a detection moiety, thereby generating a detection polynucleotide complex, and the target nucleic acid that is hybridized to its complementary sequence of the anchor oligonucleotide. In some embodiments, the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides are downstream of the sequence of the anchor oligonucleotide that is complementary to the target nucleic acid. In some embodiments, the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides flank the sequence of the anchor oligonucleotide that is complementary to the target nucleic acid. In some embodiments, the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides flank the sequence of the anchor oligonucleotide that is complementary to the target nucleic acid. In some embodiments, the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides are the same and the sequences of the two detection oligonucleotides are the same. In some embodiments, the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides are different and the sequences of the two detection oligonucleotides are different. In some embodiments, the detection moiety that is linked to each of the two detection oligonucleotides is the same. In some embodiments, the detection moiety that is linked to each of the two detection oligonucleotides is different. In some embodiments, the detection moiety linked to a first detection oligonucleotides of the two detection oligonucleotides is a fluorescent moiety and the detection moiety linked to a second of the two detection oligonucleotides is a quencher moiety. In some embodiments, the detection moiety linked to a first of the two detection oligonucleotides is a donor fluorescent moiety and the detection moiety linked to a second of the two detection oligonucleotides is an acceptor fluorescence moiety. In some embodiments, the detection moieties linked to the two detection oligonucleotides are fluorescent molecules that are detectab ly distinguishable. In some embodiments, the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides are separated by a spacer sequence. In some embodiments, the spacer sequence of the anchor oligonucleotide is a homopolymer sequence. In some embodiments, the spacer sequence of the anchor oligonucleotide is a random non-homopolymer sequence.

[0009]

[0005] Provided herein, in some embodiments, is a method of identifying the presence of a target molecule of interest from a sample, comprising providing an amplification product, wherein the amplification product comprises a target nucleic acid sequence of interest, or a complement thereof, and a code sequence, or a complement thereof, wherein the code sequence uniquely identifies the target nucleic acid sequence of interest, providing a detection polynucleotide complex, wherein the detection polynucleotide complex comprises an anchor oligonucleotide hybridized to two or more detection oligonucleotides, wherein each detection oligonucleotide of the two or more detection oligonucleotides is linked to a detection moiety, hybridizing the anchor oligonucleotide to the code sequence of the amplification product, imaging the amplification product that is hybridized to the anchor oligonucleotide of the detection polynucleotide complex, thereby generating signal intensity data from the detection moiety that is linked to the two or more detection oligonucleotides, decoding the signal intensity data, and identifying the presence of the target molecule of interest from the sample based on the decoding. In some embodiments, the code sequence comprises two or more different nucleic acid segments. In some embodiments, the method further comprises hybridizing the detection polynucleotide complex to one of the two or more different nucleic acid segments. In some embodiments, the decoding comprises soft decision decoding. In some embodiments, each detection moiety that is linked to the two or more detection oligonucleotides is the same. In some embodiments, each detection moiety that is linked to the two or more detection oligonucleotides is different. In some embodiments, the two or more detection oligonucleotides are hybridized to the anchor oligonucleotide in a stair step configuration. In some embodiments, the two or more detection oligonucleotides are hybridized to the anchor oligonucleotide in a U- shaped configuration. In some embodiments, the anchor oligonucleotide further comprises one or more spacer sequences between the two or more hybridized detection oligonucleotides. In some embodiments, the one or more spacer sequences are homopolymer sequences. In some embodiments, the one or more spacer sequences are random non -homopolymer sequences. In some embodiments, the one or more spacer sequences comprises homopolymer sequences and random non-homopolymer sequences. In some embodiments, the anchor oligonucleotide is hybridized to two detection oligonucleotides, wherein the detection moiety that is linked to a first detection oligonucleotide of the two detection oligonucleotides is a fluorescent moiety and the detection moiety that is linked to a second detection oligonucleotide of the two detection oligonucleotides is a quencher moiety, and wherein the imaging is performed after the first detection oligonucleotide is hybridized to the anchor oligonucleotide, and wherein the imaging is further performed after the second detection oligonucleotide is hybridized to the anchor oligonucleotide. In some embodiments, the anchor oligonucleotide is hybridized to two detection oligonucleotides, wherein the detection moiety that is linked to a first detection oligonucleotide of the two detection oligonucleotides is a fluorescent donor moiety and the detection moiety linked to a second detection oligonucleotide of the two detection oligonucleotides is a fluorescent accepter moiety, and wherein the imaging is performed after the first detection oligonucleotide is hybridized to the anchor oligonucleotide, and wherein the imaging is further performed after the second detection oligonucleotide is hybridized to the anchor oligonucleotide. In some embodiments, each detection moiety linked to the two or more detection oligonucleotides is a fluorescent molecule that is detectab ly distinguishable from each other.

[0006] Provided herein, in some embodiments, is a method of identifying the presence of a target molecule of interest from a sample, comprising providing an amplification product, wherein the amplification product comprises a target nucleic acid sequence of interest, or a complement thereof, and a code sequence, or a complement thereof, wherein the code sequence uniquely identifies the target nucleic acid sequence of interest, providing a detection oligonucleotide comprising a fluorescent moiety on a 5 ’ end of the detection oligonucleotide and a quencher molecule on a 3 ’ end of the detection oligonucleotide, wherein when the detection oligonucleotide is in a stem-loop configuration there is no detectable signal, and wherein the loop of the stem-loop configuration comprises a sequence that is complementary to the code sequence, or the complement thereof, hybridizing the detection oligonucleotide to the code sequence, or the complement thereof, to the amplification product, wherein the hybridizing releases the stem-loop configuration of the detection oligonucleotide, thereby generating a concatemeric amplification product, imaging the fluorescent signal of the detection oligonucleotide as it is hybridized to the code sequence, or the complement thereof, and identifying, based at least in part on the imaging, the presence of the target molecule of interest from the sample. In some embodiments, the hybridizing further comprises heating a reaction mixture comprising the detection oligonucleotide and the concatemeric amplification product to release the stem -loop configuration of the detection oligonucleotide. In some embodiments, the heating further comprises heating the reaction mixture to a temperature of 55 °C or more. In some embodiments, the identifying further comprises decoding an image of the fluorescent signal. In some embodiments, the decoding comprises soft decision decoding.

[0010]

[0007] Provided herein, in some embodiments, is a computer-implemented system for imaging a plurality of detection polynucleotides comprising: a digital processing device comprising at least one processor, an operating system configured to perform executable instructions, a memory, and a computer program including instructions executable by the digital processing device to introduce a plurality of detection polynucleotides to one or more concatemeric amplification products, hybridize the plurality of detection polynucleotides to a code or a portion thereof, and image one or more signals of the hybridized detection polynucleotides to a code or a portion thereof, wherein the instructions are performed for each nucleic acid segment of a code one or more times.

[0011]

[0008] In some embodiments, a method of identifying the presence of a target molecule of interest from a sample, comprises an amplification product, wherein the amplification product comprises a target nucleic acid sequence of interest, or a complement thereof, and a code sequence, or a complement thereof, wherein the code sequence uniquely identifies the target nucleic acid of interest, a detection oligonucleotide comprising a fluorescent moiety on the 5 ’ end and a quencher molecule on the 3’ end, wherein the 5’ end and the 3’ end are in close proximity and the detection oligonucleotide is a stem -loop configuration wherein there is no detectable signal from the detection oligonucleotide, and wherein the loop of the stem -loop configuration comprises a sequence that is complementary to the code sequence, or a complement thereof, hybridizing the detection oligonucleotide to the code sequence, or a complement thereof, to the amplification product, wherein the hybridizing releases the stemloop configuration of the detection oligonucleotide, thereby generating a concatemeric amplification product, imaging the fluorescent signal of the detection oligonucleotide as it is hybridized to the code sequence, or a complement thereof, and identifying, based at least in part on the imaging, the presence of the target molecule of interest from the sample. In some embodiments, the method further comprises heating a reaction mixture to release the stem-loop configuration of the detection oligonucleotide. In some embodiments, the heating further comprises heating the reaction mixture to at 55°C or more. In some embodiments, the identifying further comprises decoding an image of the fluorescent signal. In some embodiments, the decoding comprises soft decision decoding.

[0012]

[0009] In some embodiments, the present disclosure provides kits for practicing the disclosed methods. In some embodiments, a kit comprises one or more of the disclosed detection polynucleotide complexes, one or more of the disclosed anchor oligonucleotides and / or one or more of the disclosed detection oligonucleotides. In some embodiments, a kit comprises one or more of buffers, reagents, dNTPs, and labeled dNTPs. In some embodiments, a kit comprises one or more enzymes such as one or more DNA polymerases, one or more ligases, and one or more exonucleases. In some embodiments, a kit comprises one or more 96 well plates. In some embodiments, a 96 well plate that is included in a kit comprises an optically clear glass bottom. In some embodiments, the 96 well plate with an optically clear glass bottom is coated with a DNA immobilization composition. In some embodiments, a kit comprises instructions for practicing the methods described herein.

[0013]

[0010] Provided herein, in some embodiments, is a kit comprising a plurality of recognition elements, a plurality of detection nucleotides, one or more buffers, one or more reagents, and one or more enzymes selected from a ligase, a DNA polymerase, and an exonuclease. In some embodiments, the one or more buffers comprises a first buffer and a second buffer. In some embodiments, the first buffer is configured to promote hybridization. In some embodiments, the second buffer is configured to promote de -hybridization, ligation, nucleic acid digestion, and / or storage of a purified molecule. In some embodiments, the kit further comprises instructions for use, a manual, a protocol, or a combination thereof. In some embodiments, the kit further comprises one or more 96 well plates. In some embodiments, one of the one or more 96 well plates is configured to be assayed by an optical imaging device described herein.

[0014] [OH] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative examples of the present disclosure are shown and disclosed. As will be realized, the present disclosure is capable of other and different examples, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.

[0015] Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0016] INCORPORATION BY REFERENCE

[0017]

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

[0013] A better understanding of the features and advantages of the present methods and systems will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the methods and systems are utilized, and the accompanying drawings Figures, of which:

[0020]

[0014] Fig. 1 is an example of a recognition element for use in methods of the present disclosure.

[0021]

[0015] Fig. 2 is an example of a workflow for use in the present disclosure.

[0022]

[0016] Fig. 3 A depicts an example of a detection polynucleotide complex comprising an anchor oligonucleotide and a detection oligonucleotide.

[0023]

[0017] Fig. 3B depicts an example of a detection polynucleotide complex hybridized to a complementary code sequence, or portion thereof.

[0024]

[0018] Fig. 4 is a schematic diagram of a soft decision decoding workflow for decoding a code of a recognition element.

[0025]

[0019] Fig. 5 is an example instrument that comprises a computer system for use with the methods described herein.

[0020] Fig. 6 is an example of an application provision system for use with the methods described herein.

[0026]

[0021] Fig. 7 is an example of an application provision system for use with the methods described herein.

[0027]

[0022] Fig. 8A depicts an example of a detection polynucleotide complex hybridized to a code sequence of a concatemeric amplification product without spacer sequences.

[0028]

[0023] Fig. 8B depicts an example of a detection polynucleotide complex hybridized to a code sequence of a concatemeric amplification product with spacer sequences.

[0029]

[0024] Fig. 9 shows examples of four different detection polynucleotides with four different fluorescent moieties.

[0030]

[0025] Fig. 10 is an example configuration for a fluorescent nucleotide addition to a detection polynucleotide.

[0031]

[0026] Fig. 11 shows examples of images of the labeled detection polynucleotides shown in Fig. 10 used in detecting a code sequence in concatemeric amplification products.

[0032]

[0027] Fig. 12 is an example of a stair detection polynucleotide configuration.

[0033]

[0028] Fig. 13 is an example of a U-shaped detection polynucleotide configuration.

[0034]

[0029] Fig. 14A shows an example of a graph comparing signal generation using the stair detection polynucleotide configuration compared to a control detection polynucleotide configuration, using detection oligonucleotide B2.

[0035]

[0030] Fig. 14B shows an example of a graph comparing signal generation using the stair detection polynucleotide configuration compared to a control detection polynucleotide configuration, using detection oligonucleotide B4.

[0036]

[0031] Fig. 15A shows an example of a graph comparing signal generation for detection oligonucleotide B2 for a control detection polynucleotide, stair detection polynucleotide configuration, and U-shaped detection polynucleotide configuration.

[0037]

[0032] Fig. 15B shows an example of a graph comparing signal generation for detection oligonucleotide B4 fora control detection polynucleotide shown, stair detection polynucleotide configuration, and U-shaped detection polynucleotide configuration.

[0038]

[0033] Fig. 16A shows an example of a graph comparing stair detection oligonucleotide configurations, wherein one of the detection oligonucleotides is labeled with Cy5 (Bl).

[0039]

[0034] Fig. 16B shows an example of a graph comparing stair detection oligonucleotide configurations, wherein one of the detection oligonucleotides is labeled with RFP (B3).

[0035] Fig. 17 is an example of a decoding scheme for decoding two code sequences with detection polynucleotide stair configurations incorporating a fluorescence quenching mechanism.

[0040]

[0036] Fig. 18 shows a non-limiting example of a graph using the fluorescence quenching mechanism shown in Fig. 17.

[0041]

[0037] Fig. 19A shows an example of a probe configuration for a detection polynucleotide scenario comprising a two-cycle signal configuration where the second cycle signal is a fluorescence shift.

[0042]

[0038] Fig. 19B shows an example of a signal output based on the two-cycle signal configuration shown in Fig. 19A.

[0043]

[0039] Fig. 20A shows an example of a probe configuration for a detection polynucleotide scenario comprising a labeled U-shaped anchor oligonucleotide and two differently labeled detection oligonucleotides for a three color, three cycle signal configuration.

[0044]

[0040] Fig. 20B shows an example of a signal output for the three-cycle signal configuration shown in Fig. 20A.

[0045]

[0041] Fig. 21A shows an example of a probe configuration for a detection polynucleotide scenario comprising a labeled U-shaped anchor oligonucleotide, two differently labeled detection oligonucleotides of different characteristics for a four-color signal configuration.

[0042] Fig. 21B shows an example of a signal output for the four-signal configuration of Fig.

[0046] 21A

[0047]

[0043] Fig. 22 shows an example of a scenariofor decoding using a molecular beacon detection oligonucleotide configuration.

[0048] DETAILED DESCRIPTION

[0049]

[0044] One method for identifying nucleic acid sequences from a sample is to do so indirectly. Indirect nucleic acid identification can include using another nucleic sequence as a proxy for the original nucleic acid sequence. Assays and methods that indirectly identify the presence of a nucleic acid of interest from a sample also have the potential of multiplexing many different target identifications in one assay.

[0050]

[0045] There are methods available for determining the sequence of a nucleic acid of interest, for example using next generation sequencing and PCR detection. Other options include using sequence specific probes and, either directly or indirectly, identifying the nucleic acid sequence of interest. These methods typically target one nucleic acid sequence at a time. However, the multiplexing of nucleic acid sequence identification simultaneously for multiple targets is a powerful tool.

[0051]

[0046] The present disclosure provides methods and compositions for determining the sequence of nucleic acids in a multiplex format by providing detection options. The target sequence detection options disclosed herein can lead to increasing the number of multiplexed targets which can be highly beneficial for drug discovery, disease detection, and scientific problem solving.

[0052]

[0047] The methods and compositions as disclosed herein for determining a nucleic acid sequence may comprise an assay. In some embodiments, the assay is a solution -based assay. In some embodiments, the assay is a surface-bound assay. In some embodiments, the assay is a hybrid assay that includes a surface-bound component and a solution -based component. In some embodiments, the assay is performed in tubes or in a plate-based format, such as a multi-well plate for example a 96 well plate. In some embodiments, a multi-well plate may include, for example, an array of wells. In some embodiments, the assay may be performed on a microfluidics device. In some embodiments, the assay is performed partially in tubes and partially in a multi-well plate.

[0053]

[0048] In some embodiments, a recognition element may be used in the assay described herein. A recognition element used in the assay includes sequences that are complementary to a target sequence of interest, wherein the complementary sequence can hybridize a target sequence of interest, a code sequence that can be used to identify the target sequence of interest that has hybridized to its complement on the recognition element, and one or more functional sequences such as sequencing primer binding sites, one or more amplification primer binding sites, unique molecular identifier sequences (UMIs), sample indexes, or combinations thereof. In some embodiments, an amplification primer binding site may be adjacent to the code in a recognition element. The amplification primer binding site(s) may, in some cases, be universal primer sequence(s) that are common to all recognition elements in a set of recognition elements.

[0054] Amplification primer binding site sequences may also be a code sequence or a portion thereof. A code sequence can be a combination of a number of subsequences, called nucleic acid segments, wherein their combination can identify a target sequence of interest that has hybridized to a recognition element. In some embodiments, an amplification primer can be a nucleic acid segment or a portion thereof. Unique identifier sequences (UMIs) and sample indexes, which oftentimes find utility in next generation sequencing reactions for counting, error correction and sample identification purposes, may also be part of code such as one or more nucleic acid segments.

[0049] Once a recognition element has recognized and hybridized to a target of interest, the recognition element may be circularized and ligated to generate a circular, ligated recognition element. The circular and ligated recognition element can thenbe amplified in anticipation of a decoding event to identify the code associated with the original target of interest that hybridized to the recognition element. Amplification may be by any method of amplification, including for example, nucleic acid extension, PCR, isothermal amplification, rolling circle amplification, and / or ultrarapid amplification. Surface based amplification may be performed using PCR with surface-anchored primers (e.g., bridge amplification technology), or recombinase polymerase amplification (RPA) (e.g., ExAmp technology).

[0055]

[0050] In one embodiment, the amplification operation may comprise a rolling circle amplification (RCA) reaction to generate a concatemeric amplification product.

[0056]

[0051] In one embodiment, a recognition element may include a sequence which may prevent RCA of the recognition element while allowing for linear double -stranded PCR products. The non-extendable sequence may, for example, be located between a pair of amplification primer binding site sequences present on the recognition element. In one embodiment, a recognition element may include a restriction enzyme site that may be cleaved to yield a linear DNA molecule.

[0057]

[0052] In some embodiments, a concatemeric amplification product may be sequenced to determine the nucleotide sequence of the code associated with the target molecule of interest. Any sequencing technology may be used to sequence the product. Examples of sequencing technologies that may be used include sequencing by synthesis, avidity sequencing, sequencing by hybridization, sequencing by ligation, and nanopore sequencing.

[0058]

[0053] In some embodiments, a sequencing library may be generated from a set of recognition elements or complements or amplicons thereof. The library may be sequenced to determine the code of the recognition element associated with a target molecule of interest. The code sequence may then be used as a digital count of the target molecule specific decoding event. In one embodiment, a sequencing library may be generated from a circularized recognition element. In another embodiment, a sequencing library may be generated from a concatemeric amplification product of a recognition element. In one embodiment, a concatemeric amplification product or a portion thereof that includes at least the code may be directly sequenced to determine the code associated with the target molecule of interest.

[0059]

[0054] Fig. 2 is a non-limiting example of an encoded assay for use with the detection polynucleotides disclosed herein. A linear recognition element 210 comprises a 5' end 220a which is complementary to a portion of a target nucleic acid interest 222, a 3 ' end 220b which is complementary to another portion of a target nucleic acid of interest 222 from a sample, a code 216, and additional functional sequences 212, 214, 218 such as, for example, amplification primer binding sites, capture sequences, cleavage sites, sequencing primer binding sites, unique molecular identifiers, and the like. A target nucleic acid of interest 222 which is complementary to the 5' 220a and 3' 220b ends of the linear recognition element 210 hybridizes to the linear recognition element, thereby bringing the ends into proximity for ligating to generate a circular and ligated recognition element 225. The circular and ligated recognition element 225 is subjected to, for example, extension amplification using one of the functional sequences 212, 214, 218, or the code 216 or a portion thereof, as a primer binding site. The result is a concatemeric amplification product 230 which can be decoded using detection polynucleotide complexes disclosed herein for identifying and determining the presence of the target nucleic acid of interest from a sample.

[0060]

[0055] Additional examples of encoded assays can be found in WO2022 / 109496 A2, which is incorporated herein by reference in its entirety.

[0061]

[0056] The methods and compositions described herein may include providing recognition elements to an encoded assay for identifying the presence of a target molecule of interest from a sample. In some embodiments, a plurality of recognition elements is provided. In some embodiments, each recognition element in the plurality of recognition elements comprises one or more target recognition regions. The target recognition regions of the recognition elements comprise one or more nucleic acid sequence(s) configured to hybridize to a target nucleic acid molecule. In some embodiments, the one or more nucleic acid sequences hybridize to one or more target nucleic acid sequences of the target nucleic acid molecule. In some embodiments, the target recognition region is configured to hybridize to one or more regions of the target nucleic acid molecule flanking a target of interest (e.g., SNP, indel, etc.). In some embodiments, the target recognition region is configured to hybridize to a variant nucleic acid of interest (e.g., the target recognition region base pairs with the SNP when the target of interest is a SNP).

[0062]

[0057] As shown in Fig. 1, in a non-limiting example of a recognition element used in encoded assays described herein comprises two target recognition regions, one at the 5’ end and another at the 3 ’ end. A recognition element further comprises a code. In Fig. 1, the example code is made up of four nucleic acid segments. However, the number of nucleic acid segments, be it one or more than one, is not limiting and the number of segments depends on the complexity of the assay (e.g., how many targets of interest are to be identified concurrently). In some embodiments, a target nucleic acid molecule of interest itself comprises the code, or an additional code such as a barcode that identifies another target molecule of interest such as a protein. In either embodiment, the code may be detected as a proxy for the target molecule, be it a nucleic acid or a protein, or both.

[0063]

[0058] In some embodiments, the structure of the recognition element may vary. In some embodiments, the structure of the recognition element may configure into a specific structure when hybridized to a target nucleic acid. Non-limiting examples of a recognition element configuration may include a padlock probe, a molecular inversion probe, a hairpin oligonucleotide, a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a combination thereof. In some embodiments, the recognition element is linear prior to hybridization to its complementary target nucleic acid of interest. In some embodiments, the linear recognition element is circularized once hybridized to the respective target nucleic acid of interest and ligated thereafter. In some embodiments, the recognition element is circular prior to hybridization to the target nucleic acid of interest. In one embodiment, the target nucleic acid of interest may serve as a primer for an extension reaction, for example to initiate rolling circle amplification of the recognition element.

[0064]

[0059] In some embodiments, the recognition element may be configured to be a padlock probe once the recognition element is hybridized to the target nucleic acid of interest. Padlock probes may be referred to as linear oligonucleotides whose ends are complementary to adjacent target sequences, or to non-adjacent target sequences thereby leaving a gap between the ends of the hybridized recognition element. Upon hybridization to a target nucleic acid, the two ends (e.g. , 5 ’ end and 3 ’ end) of the recognition element are adjacently located, generating a padlock probe configuration for subsequent ligation. Alternatively, the two ends of the recognition element are brought in proximity to, but not directly adjacent to, each other upon hybridization to a target nucleic acid. In this instance, a gap is left between the 5' and 3' hybridized ends of the recognition element which can be filled in several ways, for example by extension of the 5' end until it is adj acent to the 3 ' end, or by hybridizing a third oligonucleotide that fills the gap . In any scenario, the recognition element ends are ligated together if hybridization, or hybridization and gap fill, occurs thereby generating circular and ligated recognition elements that are indicative of the hybridization event. In the absence of a target of interest, there would be no or minimal hybridization and no or minimal circularization and subsequent ligation.

[0065]

[0060] In some embodiments, a recognition element may further comprise one or more functional sequences. Functional sequences may include, but are not limited to, primer binding sites, cleavage sites, unique molecular identifiers, capture sequences, sample indices or combinations thereof. In some embodiments, a primer binding site and / or a cleavage site are universal in nature, such that a plurality of recognition elements shares the same sequence(s). A unique molecular identifier and / or sample index may be included in a recognition element for error correction or to identify a source of material, respectively.

[0066]

[0061] The methods described herein may relate to the use of a code for identifying a target nucleic acid of interest from a sample that hybridized to a recognition element to initiate a ligation event. A code in a recognition element may be used to associate the recognition element 5' and 3' end regions with a target nucleic acid of interest, thereby determining the presence of a target nucleic acid of interest from a sample without having to directly assay the target molecule itself. As such, a code in a recognition element uniquely identifies the presence of a target molecule from a sample. Using codes, any number of recognition elements can be multiplexed in one encoded assay as each code is unique and correlates to the presence of one target molecule. In some embodiments, the code is selected from a set of codes wherein the set of codes make up a “code space”. In some embodiments, the code comprises a plurality of nucleic acid segments, where each nucleic acid segment corresponds to one or more computational symbols, or colors, that are used in a decoding process. Fig. 1 shows a non-limiting example where four nucleic acid segments make up the code of the recognition element, wherein each of the nucleic acid segments can be decoded using detection polynucleotides as disclosed herein and the combination of the decoded nucleic acid segments thereby builds the full code that is unique to the target nucleic acid of interest from a sample. The codes are detected as proxies, thereby serving as an indirect analysis of the presence of a target molecule from a sample as the code correlates with the presence of the target molecule that hybridized to the recognition element allowing ligation, amplification and decoding. If there is no hybridization of a target of interest to its complementary sequences of a recognition element, there is expected to be no ligation (e.g., as the 5’ and the 3 ’ ends of the recognition are not expected to be adjacent), no amplification and subsequently nothing to decode. As such, if there is no amplification product to decode, that would be an indication that the target molecule of interest was potentially absent from the sample, or at such a low incidence that hybridization resulted in too few amplification products to cross the threshold for detection by decoding.

[0067]

[0062] In some embodiments, each code from the set of codes may be from a predetermined set of codes. In some embodiments, each code from the set of codes may be selected to ensure that the selected code differs from other codes in the set of codes. As such, in some embodiments, several selection criteria may be implemented to generate a set of codes, wherein each code of a set of codes comprises from one to more than one nucleic acid segment. In some embodiments, selection of the codes, or nucleic acid segments that make up a code, may incorporate a Hamming distance criterion.

[0063] In some embodiments, to generate a code selected from a set of codes for use in a recognition element, a Hamming distance (HD) selection criterion may be implemented between any two codes of the set of codes, and also between any two nucleic acid segments that may be used in a code. A Hamming distance between two codes in a set of codes may refer to the number of symbols, or nucleotides, that differ between the two codes in the set of codes. In essence, the Hamming distance measures the number of changes that would need to be made to a first code sequence to change the string of symbols, in this case nucleotides, to the second code. As such, a Hamming distance criterion used to select a code cannot be greater than the length of the code. For example, if the length of a code is measured by the number of cycles or flows of decoding runs or queries and that number being eight cycles, and if each cycle corresponds to one symbol or color, therefore eight symbols or colors, then the maximum Hamming distance is eight. In some embodiments, the Hamming distance may be a minimum Hamming distance. In some embodiments, the Hamming distance may be a maximum Hamming distance. In some embodiments, a minimum Hamming distance may be from about 2-10. In some embodiments, the Hamming distance is between 2-7. In some embodiments, the Hamming distance is between 3-5. The Hamming distance increases as the number of codes that can be used decreases, as the purpose of the code is to impart a way to uniquely identify one target molecule from another target molecule.

[0068]

[0064] The code may have a certain length in nucleotides. In some embodiments, the code has a length of greater than or equal to about three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides. In some embodiments, the code has a length of fewer than or equal to about 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 contiguous nucleotides. In some embodiments, the length is about 5 to 200, 10 to 150, 15 to 100, 20 to 90, or 30 to 80 contiguous nucleotides. The length of the code may be further divided into a number of discrete nucleic acid segments, as exemplified in Fig. 1.

[0069]

[0065] In some embodiments, each code from a set of codes may be generated using a 4-ary nucleotide alphabet of A, C, G, and T. In some embodiments, each code of a set of codes is generated using a 3 -ary nucleotide alphabet of a set of three of A, C, G, and T. In some embodiments, the codes can be generated from arbitrary symbols, or colors, 1 to 4, corresponding to the fluorophores that are associated with a unique string of nucleotides. The numbers then serve as a mechanism to numerate colors or mixes of colors that are utilized to query the codes for decoding.

[0066] A code of a recognition element can comprise one or more nucleic acid segments. For example, as shown in Fig. 1, the example of the code comprises four nucleic acid segments.

[0070]

[0067] In some embodiments, the recognition elements provided herein may comprise a code comprising one or more nucleic acid segments. The one or more nucleic acid segments, or the complements thereof, within the code are used as a proxy for detection of the target molecule recognized by the recognition element.

[0071]

[0068] The number of nucleic acid segments present in a code of a recognition element may be considered in the design of the recognition element. The number of segments in a code helps to determine the nucleotide length of the recognition element. For example, a recognition element that includes a code comprising five segments may comprise a greater nucleotide length than a recognition element that includes a code of two segments. A recognition element with a larger nucleotide length may run up against synthesis limits and is at a greater risk of synthesis errors. Alternatively, a recognition element with a smaller nucleotide length may avoid synthesis limits and risks in synthesis errors. A recognition element with a larger nucleotide length may include less space for other portions of the recognition element, such as the target recognition regions, functional sequences, universal sequences, etc.

[0072]

[0069] In some embodiments, the code comprises 2 to 10 nucleic acid segments. In some embodiments, the code comprises 2 to 8 nucleic acid segments. In some embodiments, the code comprises 3 to 5 nucleic acid segments. In some embodiments, the code comprises at least 4 nucleic acid segments, at least 5 nucleic acid segments, at least 6 nucleic acid segments, at least 7 nucleic acid segments, at least 8 nucleic acid segments, at least 9 nucleic acid segments, at least 10 nucleic acid segments.

[0073]

[0070] In some embodiments, each nucleic acid segment may comprise a length in nucleotides. In some embodiments, each nucleic acid segment may comprise a length of 10 to 30 nucleotides. In some embodiments, each nucleic acid segment may comprise a length of 10 to 25 nucleotides. In some embodiments, each nucleic acid segment may comprise a length of 15 to 20 nucleotides. In some embodiments, each nucleic acid segment may comprise a length of 2 or more nucleotides, 4 or more nucleotides, 6 or more nucleotides, 8 or more nucleotides, 10 or more nucleotides, 12 or more nucleotides, 14 or more nucleotides, 16 or more nucleotides, 18 or more nucleotides, 20 or more nucleotides, 22 or more nucleotides. In some embodiments, the nucleic acid segments in a code are of the same length. In some embodiments, the nucleic acid segments in a code are not the same length.

[0074]

[0071] A Hamming distance selection criterion may be implemented between any two nucleic acid segments of a code. A Hamming distance between two nucleic acid segments in a code refers to the number of symbols that differ between the segments. In essence, the Hamming distance measures the number of changes that would need to be made to a first nucleic acid segment to change the string of symbols, or nucleotides, to a second nucleic acid segment. In some embodiments, the Hamming distance may be a minimum Hamming distance. In some embodiments, the Hamming distance may be a maximum Hamming distance. In some embodiments, a minimum Hamming distance maybe from about 2-20, about 3-19, about 4-18, about 5-17, about 6-16, about 7-15, about 8-14, about 9-13, or about 10-12. In some embodiments, a minimum Hamming distance may be greater than or equal to about 2, greater than or equal to about 3 , greater than or equal to about 4, greater than or equal to about 5, greater than or equal to about 6, greater than or equal to about 7, greater than or equal to about 8, greater than or equal to about 9, greater than or equal to about 10, greater than or equal to about 11, greater than or equal to about 12, greater than or equal to about 13, greater than or equal to about 14, greater than or equal to about 15, greater than or equal to about 16, greater than or equal to about 17, greater than or equal to about 18, greater than or equal to about 19, greater than or equal to about 20.

[0075]

[0072] In some embodiments, a nucleic acid segment may comprise a universal primer binding site for amplification. For example, a segment may comprise an amplification primer binding site for performing rolling circle amplification (RCA) for generating a plurality of concatemeric amplification products.

[0076]

[0073] In some embodiments, a nucleotide or nucleic acid sequence of each segment may correspond to one or more computational symbols, such as a detection color, for performing a decoding process. For example, one or more nucleic acid segments of a code may be detected with a first pool of detection polynucleotide complexes to produce one or more detectable binding complexes, for example by using a fluorescent label. In some embodiments, the one or more detectable binding complexes, once imaged, produce one or more optical signals such as fluorescence in a particular wavelength. When all or substantially all segments of the code are detected by iteratively applying additional pools of detection polynucleotide complexes to the amplification products, a series of optical signals are observed and collated, generating a code profile.

[0077]

[0074] The application of detection polynucleotides or detection polynucleotide complexes to amplification products for decoding can be called a “flow” or "cycle" or “query”, wherein a flow, cycle or query is the number of times a particular segment, or a portion thereof, of an amplification product is queried, or the number of times a detection polynucleotide is flowed over an amplification product in order to detect a nucleic acid segment sequence. If a nucleic acid segment is present, a detection polynucleotide that comprises a sequence complementary to that nucleic acid segment may hybridize to its complementary nucleic acid segment and the attached detectable label is detected, for example imaged. In some embodiments, one or more optical signals observed from querying an amplification product with detection polynucleotide complexes translates to one or more computational symbols such that each optical signal can be imaged and decoded. In some embodiments, a plurality of nucleic acid segments on a recognition element may correspond to at least three computational symbols. In some embodiments, the optical signal may be a color or a non-color. In some embodiments, the optical signal may be a combination of colors (e.g., when the detection polynucleotide complex comprises a plurality of detectable labels). In some embodiments, the computational symbols or colors can be referred to as numbers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.). In some embodiments, each detection polynucleotide complex comprises a detectable label, as such for example when four different fluorescent moieties are used as detectable labels there are four symbols, 1 to 4. However, the number of symbols can be larger depending on the combination of detectable labels with each unique detection polynucleotide complex. For example, for a set of 16 unique detection polynucleotide complexes wherein each has one of four fluorescent moieties there would be 16 computational symbols used for decoding if all 16 unique detection polynucleotide complexes are used to decode an amplification product. However, additional ways to increase the number of computational symbols for decoding include, but are not limited to, adding levels of identifiability associated with a particular detectable signal such as whether a detectable signal is brighter or dimmer compared to a normal level of signal, whether there is a combination of detectable colors that is used to identify a particular nucleotide. As such, the number of computational symbols that may be used may be limited by practicality for any given assay.

[0078]

[0075] In some embodiments, the methods described herein may use a number of computational symbols. The number of computational symbols used in the methods and systems described herein may be considered in the design of the recognition elements. For example, in some embodiments, a detection scheme using a larger number of computational symbols may lead to a larger code space and a greater number of codes that may be generated, which may allow for a greater amount of information that may be detected thereby allowing for a higher degree of assay target molecule multiplexing. In some embodiments, a detection scheme using a smaller number of computational symbols may be limited in the amount of information that can be detected. In some embodiments, using a larger number of computational symbols may result in a faster detection process (less time to determine a target molecule compared to using a smaller number of computational symbols). In some embodiments, a detection scheme using a larger number of computational symbols may require greater instrument complexity, which may lead to potential drawbacks such as color crosstalk, wherein the computational symbols used in the detection scheme may become difficult to distinguish from other computational symbols. In some embodiments, a greater number of computational symbols may require that a more complex detection tool be used.

[0079]

[0076] In some embodiments, each nucleic acid segment may correspond to a combination of computational symbols. In some embodiments, each nucleic acid segment may correspond to one or more computational symbols, two or more computational symbols, three or more computational symbols, four or more computational symbols, five or more computational symbols, six or more computational symbols, seven or more computational symbols, eight or more computational symbols, nine or more computational symbols, or 10 or more computational symbols. In some embodiments, each segment may correspond to 10 or less computational symbols, nine or less computational symbols, eight or less computational symbols, seven or less computational symbols, six or less computational symbols, five or less computational symbols, four or less computational symbols, three or less computational symbols, or two or less computational symbols.

[0080]

[0077] The methods described herein may include amplification of a circularized and ligated recognition element. In some embodiments, a target nucleic acid molecule is amplified. In some embodiments, the target nucleic acid molecule is a combination of a recognition element and a target nucleic acid molecule. In some embodiments, the amplification is selective amplification. For example, in some embodiments, amplification is able to occur if a target recognition region of a recognition element recognizes and binds to a complementary target nucleic acid of interest. In some embodiments, amplification occurs if a primer is used that is complementary to one or more of a portion of a recognition element, a portion of a nucleic acid segment of a code, or another sequence in the recognition element that is complementary to a primer used for amplification. In some embodiments, the amplification is non -selective. For example, in some embodiments randomers can be used to prime amplification from a recognition element. In some embodiments, a recognition element comprises PCR primer binding sites, for example PCR primer binding sites that flank the code of a recognition element.

[0081]

[0078] In some embodiments, the methods described herein may include selectively amplifying a subset of nucleic acids. For example, in some embodiments, a subset of a plurality of recognition elements hybridized to a plurality of target nucleic acid molecules may be amplified. The subset may comprise a percentage of the total amount of recognition elements hybridized to target nucleic acid molecules as described herein. In some embodiments, the sub set may include 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the total amount of recognition elements hybridized to target nucleic acid molecules. In some embodiments, the subset may include 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the total amount of recognition elements hybridized to target nucleic acid molecules.

[0082]

[0079] In some embodiments, the amplification may include rolling circle amplification (RCA). In some embodiments, the RCA may generate a concatemer as an amplification product, wherein the concatemer contains multiple copies of a circularized ligated recognition element, including associated codes, target recognition regions, and any other functional sequences that are included in the circularized and ligated recognition element. In some embodiments, RCA may be performed while the circularized and ligated recognition element is in solution. In some embodiments, RCA may be performed on a circularized recognition element while the circularized recognition element is immobilized, either reversibly or non -reversibly, on a substrate or surface. In some embodiments, the substrate or surface is a solid support and includes, but is not limited to, a bead, a flow cell, a microwell, a nanowell, a well, a slide. In some embodiments, the substrate is glass such as optical glass of imaging quality. In some embodiments, the substrate is plastic, polycarbonate, etc. In some embodiments, the substrate is positively charged or negatively charged. In some embodiments, the substrate is an anionic substrate. In some embodiments, the substrate is a cationic substrate. In some embodiments, the substrate comprises an immobilization composition, such as polyacrylamide, branched PEI, linear PEI, poly(P-aminoester) and poly(amidoamine), PEG, a gel, poly -L-ly sine, silane, agarose, muscle mimetic catecholamine polymer, and the like. In some embodiments, the substrate has no charge. In some embodiments, the substrate has no immobilization composition. In some embodiments, a substrate comprises a cationic polymer coated surface. An RCA reaction may be performed in the presence of a cationic polymer coated surface, resulting in simultaneous immobilization and amplification of a ligated recognition element. RCA primers may be supplied in solution or bound to the cationic polymer-coated surface prior to, or concurrent with, performing the RCA reaction.

[0083]

[0080] In some embodiments, amplification may include on-surface polymerase chain reaction (PCR), isothermal amplification, RCA, ultrarapid amplification, or a combination thereof. In some embodiments, amplification may include polymerase chain reaction (PCR). In some embodiments, PCR is multiplexed PCR. In some embodiments, PCR is ultrafast multiplexed PCR. The amplification methods disclosed herein may include isothermal amplification. Non- limiting examples of isothermal amplification include Nicking endonuclease amplification reaction (NEAR), Transcription mediated amplification (TMA), Loop -mediated isothermal amplification (LAMP), Helicase-dependent amplification (HD A), Nucleic Acid Sequence Based Amplification (NASBA), Strand displacement amplification (SDA), Multiple Displacement Amplification (MDA), Rolling Circle Amplification (RCA), bridge amplification, or Ramification (RAM) amplification method. In some embodiments, the amplification method is provided in Fakruddin M, Mannan KS, Chowdhury A, Mazumdar RM, Hossain MN, Islam S, Chowdhury MA. Nucleic acid amplification: Alternative methods of polymerase chain reaction. J Pharm Bioallied Sci. 2013 Oct;5(4):245-52, which is hereby incorporated by reference in its entirety.

[0084]

[0081] The methods described herein may include introducing detection polynucleotides, also known as detection polynucleotide complexes, to amplified recognition elements. A detection oligonucleotide may be a single stranded oligonucleotide, or it may be partially single stranded and partially double stranded as a detection polynucleotide. A detection oligonucleotide or a detection polynucleotide may comprise a detectable label.

[0085]

[0082] In some embodiments, the methods described herein may include introducing one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 15 or more, 20 or more, 25 or more, 50 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1,000 or more detection polynucleotides (or single stranded oligonucleotides) to an amplification product. In some embodiments, the methods described herein may include introducing 1,000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 50 or less, 25 or less, 20 or less, 15 or less, 10 or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, or two or less detection polynucleotides to an amplification product.

[0086]

[0083] In some embodiments, a detection polynucleotide may comprise a detectable label (e.g., fluorescent molecule). In some embodiments, a detection polynucleotide may be a single stranded oligonucleotide with a portion that is complementary to a code, or a portion of a code, and a detectable label. In some embodiments, a detection polynucleotide may comprise two oligonucleotides. Fig. 3A shows a non-limiting example of a structure of a detection polynucleotide 300 comprising two oligonucleotides. A first oligonucleotide 330 comprises a detectable label 340. A second oligonucleotide 350 comprises a portion that is complementary to the first oligonucleotide 310 and a second portion 320 that is complementary to a code or a portion of a code 360 (e.g., a nucleic acid segment of a code). The first oligonucleotide 330 hybridizes to the second oligonucleotide 350, thereby generating a detection polynucleotide. For decoding, a portion of the second oligonucleotide 320 hybridizes to its code complement 360 as seen in Fig- 3B, and a signal is detected from the detectable label, thereby identifying the presence of the code which in turn is correlated back to the presence of a target nucleic acid of interest from a sample.

[0087]

[0084] The detection polynucleotide may comprise various nucleotide lengths. In some embodiments, the detection polynucleotide may comprise a length of 5 to 25 nucleotides. In some embodiments, the detection polynucleotide may comprise a length of 5 to 20 nucleotides. In some embodiments, the detection polynucleotide may comprise a length of 5 to 15 nucleotides. In some embodiments, the detection polynucleotide may comprise a length of 5 to 10 nucleotides. In some embodiments, the detection polynucleotide may comprise a length of 5 to 8 nucleotides.

[0088]

[0085] In some embodiments, the detection polynucleotide may comprise a length of between about 5-100 nucleotides, between about 10-80 nucleotides, between about 20-60 nucleotides, between about 30-50 nucleotides, and between 15-30 nucleotides. In some embodiments, the detection polynucleotide may comprise one or more detectable labels. In some embodiments, the one or more detectable labels comprise a fluorescent moiety. The fluorescent moiety may emit in the red, far-red, near-red, yellow, green, or blue wavelengths. In some embodiments, the fluorescent moiety comprises one or more of 6-FAM (6 -carb oxy fluorescein), JOE (6-carboxy- 4',5'-dichloro-2',7'-dimethoxyfluorescein), TAMRA (6-carboxytetramethylrhodamine), 5-Cy5 (5 -carboxyrhodamine), 5-Cy5.5 (5 -carboxylic acid succinimidyl ester), 5 -Cyl (5- carboxyrhodamine), (hexachlorofluorescein), Alexa Fluor 488 (AF488), Alexa Fluor 514 (AF514), Texas Red, Cyanine 3, Cyanine 5, Pacific Blue, Tetramethyl rhodamine, Oxazole Yellow, Atto647N, and Rhodamine 6G (R6G). In some embodiments, the detection polynucleotide may comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or 10 or more fluorescent moieties. In some embodiments, the detection polynucleotide may comprise 10 or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, or two or less fluorescent moieties.

[0089]

[0086] In some embodiments, the fluorescent moiety may comprise an organic dye, a biological fluorophore, a quantum dot, or a combination thereof. In some embodiments, the organic dye may comprise an organic molecule. In some embodiments, the organic dye may comprise a coumarin, a cyanine, a benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a borapolyazaindacene, a xanthene, or a combination thereof. The organic dye may correspond to a color. For example, the organic dye may correspond to a green, a yellow, a blue, an indigo, a red, an orange a purple, a pink, a violet, or a combination thereof. In some embodiments, the organic dye may correspond to no color. In some embodiments, the organic dye may correspond to a black color. In some embodiments, the organic dye may correspond to a white color.

[0090]

[0087] In some embodiments, the detectable moiety can be identified by imaging. When the detectable label is a fluorophore, the fluorophore may emit a color in the visible light spectrum which can be captured by fluorescent imaging and associated filters. In some embodiments, the fluorophore may emit in a wavelength in the range between 400 nanometers (nm) and 900 nm. In some embodiments, the fluorophore may emit in a wavelength between 400 nm and 475 nm, 475 nm and 490 nm, 490 nm and 530 nm, 530 nm and 575 nm, 575 nm and 600 nm, 600 nm and 700 nm, or 700 nm and 800 nm. In some embodiments, the fluorophore may emit a wavelength of 400 nm or more, 425 nm or more, 450 nm or more, 475 nm or more, 500 nm or more, 525 nm or more, 550 nm or more, 575 nm or more, 600 nm or more, 625 nm or more, 650 nm or more, 675 nm or more, 700 nm or more, 725 nm or more, 750 nm or more, 775 nm or more, 800 nm or more, 825 nm or more, 850 nm or more, 875 nm or more, or 900 nm or more. In some embodiments, the fluorophore may emit a wavelength of 900 nm or less, 875 nm or less, 850 nm or less, 825 nm or less, 800 nm or less, 775 nm or less, 750 nm or less, 725 nm or less, 700 nm or less, 675 nm or less, 650 nm or less, 625 nm or less, 600 nm or less, 575 nm or less, 550 nm or less, 525 nm or less, 500 nm or less, 475 nm or less, 450 nm or less, 425 nm or less, or 400 nm or less.

[0091]

[0088] The detectable labels (e.g., fluorescent moieties) may be optically distinct. The number of optically distinct detectable labels used in the methods described herein can impact the amount of information that is detected. For example, a detection scheme using a larger number of optically distinct detectable labels may allow for a higher amount of multiplexing of codes, which may in turn allow for a greater amount of target molecule related information to be detected and captured. A detection scheme using a smaller number of optically distinct detectable labels would allow for a lesser amount of target molecule related information to be detected and captured. In some embodiments, using a larger number of optically distinct detectable labels may lead to a detection process that identifies a target molecule in less time as compared to using a fewer number of optically distinct detectable labels when querying a concatemeric amplification product. In some embodiments, a detection scheme using a larger number of optically distinct detectable labels may lead to greater instrument complexity, which may lead to fluorescence detection crosstalk, whereby the fluorescence emission spectra of the optically distinct fluorescent moieties may not yield distinct fluorescence signals. In some embodiments, a detection scheme using a greater number of optically distinct fluorescent moieties may require use of a more complex detection tool.

[0092]

[0089] In some embodiments, the detection polynucleotides may be provided in one or more detection pools. In some embodiments, the methods herein may use one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 45 or more, or 50 or more detection pools. In some embodiments, the methods herein may use 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, ten or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, or two or less detection pools.

[0093]

[0090] In some embodiments, each detection pool provided may comprise a number of detection polynucleotides. In some embodiments, each detection pool may comprise two or more, three or more, four or more, five or more, ten or more, 15 or more, 25 or more, 50 or more, 100 or more, 150 or more, 250 or more, 500 ormore, 1,000 or more, 1,500 or more, 2,500 or more, or 5,000 or more detection polynucleotides. In some embodiments, each detection pool may comprise 5,000 orless, 2,500 or less, 1,500 orless, 1,000 or less, 500 or less, 250 or less, 150 or less, 100 or less, 50 or less, 25 orless, 15 or less, ten or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, or two or less detection polynucleotides.

[0094]

[0091] The number of detection pools and the number of detection polynucleotides in each detection pool may be considered in the design of the recognition elements. For example, one advantage to using a smaller number of detection pools and detection polynucleotides in the methods described herein may be to lower design costs. Conversely, one advantage to using a larger number of detection pools and detection polynucleotides in the methods described herein may be the need for a higher degree of multiplexing for target molecule detection and larger amounts of information that may be detected.

[0095]

[0092] The methods described herein may include imaging a plurality of detection polynucleotides that have hybridized to their complementary code or a portion of a code in order to obtain identifiable signals which can be correlated back to the presence of a target molecule of interest. In some embodiments, the signals may be associated with one ormore segments of a code, or portions thereof, for each concatemeric amplification product. In some embodiments, the imaging is performed by an imaging system comprising a fluorescence detection system.

[0093] In some embodiments, the imaging may be conducted using an imaging system. The imaging system may comprise at the minimum a camera, a detector, an illuminator, a condenser, or a combination thereof. In some embodiments, the imaging may include images of fluorescence emission, luminescence, or a combination thereof. In some embodiments, the imaging systems comprise components or sub -systems of a larger system that may also include optics modules including when needed fluorescence filters, fluidics modules, temperature control modules, translation stages, robotic fluid dispensing and / or microplate handling, processors or computers, instrument control software, data analysis and display software, etc. In some embodiments, the imaging system is a fluorescence imaging system. In some embodiments, the imaging may include fluorescent images from the fluorescent moieties present on the labeled probes.

[0096]

[0094] In some embodiments, the image may comprise fluorescence information from one or more wavelengths. In some embodiments, the fluorescence information may comprise emission data from a wavelength from about 220-830 nanometers (nm), about 230-820 nm, about 240- 810 nm, about 250-800 nm, about 260-790 nm, about 270-780 nm, about 280-770 nm, about 290-760 nm, about 300-750 nm, about 310-740 nm, about 320-730 nm, about 330-720 nm, about 340-7 lO nm, about 350-700 nm, about 360-690 nm, about 370-680 nm, about 380-670 nm, about 390-660 nm, about 400-650 nm, about 410-640 nm, about 420-630 nm, about 430- 620 nm, about 440-610 nm, about 450-600 nm, about 460-590 nm, about 470-580 nm, about 480-570 nm, about 490-560 nm, about 500-550 nm, about 510-540 nm, about 520-530 nm, or a combination thereof.

[0097]

[0095] Image detection and capture may relate to iteratively repeating the operations of: (i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof. In some embodiments, the iterative repetition of the operations is performed for each nucleic acid segment of a code one or more times.

[0098]

[0096] In some embodiments, the iteratively repeating the operations of: (i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof comprises two or more iterative repetitions. For example, the methods described herein may comprise 2-50 iterative repetitions, 2-10 iterative repetitions, or 2-8 iterative repetitions. In some embodiments, the methods described herein may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more iterative repetitions of: (i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof.

[0097] In some embodiments, the number of iterative repetitions of the operations of (i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof corresponds to the number of nucleic acid segments present in a code of the recognition element. In some embodiments, each nucleic acid segment of the code of the recognition element may undergo a number of iterative repetitions of the operations of: (i) introducing detection polynucleotides to the concatemeric amplification products; (ii) hybridizing the detection polynucleotides to a code or a portion thereof; and (iii) imaging the signals of the hybridized detection polynucleotides to a code or a portion thereof. For example, the methods described herein may comprise iteratively repeating the operations two times per segment, three times per segment, or four times per segment. In some embodiments, the methods described herein may comprise iteratively repeatingthe operations two or more times per segment, three or more times per segment, four or more times per segment, five or more times per segment, six or more times per segment, seven or more times per segment, eight or more times per segment, nine or more times per segment, 10 or more times per segment, 11 or more times per segment, 12 or more times per segment, 13 or more times per segment, 14 or more times per segment, 15 or more times per segment, 16 or more times per segment, 17 or more times per segment, 18 or more times per segment, 19 or more times per segment, or 20 or more times per segment.

[0099]

[0098] Additional methods for imaging a detection polynucleotide can be found in WO2023 / 158993A2, which is incorporated herein by reference in its entirety.

[0100]

[0099] Several models may be used to identify a code that is associated with a target molecule based on the fluorescence signals generated and images captured from detection polynucleotide hybridization to code sequences. In one embodiment, decoding makes use of a hard decision decoding model. In another embodiment, decoding makes use of a soft decision decoding model.

[0101]

[0100] For soft decision decoding, it may not be necessary to identify each base specifically. For example, signals generated during each detection event may be detected and recorded to produce a data set that may be used as input into a model to calculate a probability that a specific code is present without requiring that each base of a code be determined. Although it may not be necessary in a soft decision decoding model to make a hard decision about the identity of each nucleotide, a model may nevertheless include assigning a probability or identity to each nucleotide in the sequence of a code, wherein each nucleotide in the sequence of a code may be sequenced. Data gathered includes intensity readings for signals produced by the hybridized detection polynucleotide fluorescent moiety in various spectral bands. A set of intensity readings are detected by imaging, stored and used as input into a soft decision decoding model for determining a probability that a particular code is present, and hence a target nucleic acid is present in the sample.

[0102]

[0101] A model may be developed or trained using data from known codes, such as signal intensity data across a predetermined spectrum. The model may be used to calculate a set of probabilities across a set of one or more codes, indicating, for example, for each code, a probability that it is present in a concatemeric amplification product.

[0103]

[0102] The probability that a particular code is present is indicative of the probability that a particular target molecule associated with the code is present in the sample of interest. Data indicating the probability that a particular target is present is, for example, to calculate probabilities relevant to diagnosis or screening of various medical conditions, or selection of drugs for treatment of various medical conditions.

[0104]

[0103] A soft decoding decision model may include using an algorithm to predict the presence of target molecules from a sample. In some embodiments, the algorithm is a soft-decision decoding algorithm. In some embodiments, the algorithm is applied to the codes of the concatemeric amplification products for predicting the presence of a target molecule from a sample.

[0105]

[0104] The methods disclosed herein may comprise soft decision decoding to predict the presence of the code in a recognition element or concatemeric amplification product thereof, wherein the presence of the code correlates and serves as a proxy for the presence of a target nucleic acid in a sample. In some embodiments, the methods described herein may use soft decision decoding. In some embodiments, the methods described herein may use hard decision decoding. For hard decision decoding, signals from queried concatemers are extracted from images. This is the same for soft decision decoding, in that signals that are generated and imaged are extracted from the images. For hard decision decoding, hard basecalls are generated from the intensities of the signals, whereas with soft decision decoding no hard basecalls are necessary as all of the signal range is retained. The code assignment for hard decision decoding is determined by matching nucleotide reads to codes, whereas with soft decision decoding, the signals are cross correlated against the expects signals and a likely code is assigned, as such a probabilistic methodology. When using soft decision decoding, it is not necessary for the model to identify each base specifically. For example, signals (e.g., fluorescent signals) generated during each cycle of a detection process may be detected and recorded to produce a data set that may be used as input into a model to calculate a probability that a specific code is present.

[0106]

[0105] The permutation space on a recognition element is the totality of factors that determines the number of unique nucleotide possibilities at each nucleic acid segment. Factors comprise the number of segments present on a recognition element, the number of incubation periods or times a segment is queried with a detection pool detection polynucleotides, and the number of computational symbols or colors.

[0107]

[0106] Fig. 4 details a non-limiting example of a soft decision decoding pipeline. The soft decision decoding pipeline may be used for determining the presence of a target molecule from a sample based on detection and decoding of a code associated with the target molecule that originally hybridized to a recognition element. Images of the sample are acquired, aligned, and processed to extract the intensity of the features, or signals of interest across the imaged field of view in multiple spectral channels, thereby generating a code profile. The corrected intensities of said features are fed through a series of algorithms that make up the soft decoder pipeline. At first, the intensity profiles of the codes are learned based on features of high confidence or high intensity. This trained model provides a template for each code from which the rest of the features of interest are compared to in the second operation. Third, a confidence score is computed from the difference between the intensity profile of each feature and the trained profiles. Several filters can be applied to remove outliers, duplicates, and low confidence decoded concatemers. The output is a table of decoded concatemers with associated filter status, confidence score, and a likely assignment to one of the codes of one or more concatemeric amplification products.

[0108]

[0107] In some embodiments, a recognition element comprises a larger code, for example a code with four segments instead of two or three. In some embodiments, a recognition element comprising a larger code may result in a detection scheme with better error correction. Additionally, in some embodiments, a larger code may result in a lower signal -to-noise ratio.

[0109]

[0108] In some embodiments, a recognition element comprises a smaller code, for example a code with two segments, or one segment. In some embodiments, a recognition element comprising a small code may result in a detection scheme with lower error correction abilities. Further, in some embodiments, a small code may result in a higher signal-to-noise ratio.

[0109] In some embodiments, the systems may comprise a solid substrate configured to immobilize one or more of a circularized and ligated recognition element, a concatemeric amplification product, a detection polynucleotide, and a hybridized complex of a concatemeric amplification product and a detection polynucleotide complex. In some embodiments, the systems comprise a welled plate or a flowcell. In some embodiments, the systems comprise a fluid flow controller, a temperature controller, an imaging system, a computer system, or any combination thereof.

[0110] [HO] In some embodiments, the systems disclosed herein may include a solid substrate or a solid surface. The solid substrates and surfaces disclosed herein may be referred to as a substrate, a support, a solid support, or a surface. The substrate may be modified for immobilizing circularized and ligated recognition elements or concatemeric amplification products, or both. Example solid substrates include, but are not limited to, glass, modified or functionalized glass, plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, inorganic glasses, plastics, optical fiber bundles, optically clear glass, and other polymers. In some embodiments, the plastic solid substrates may include acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, or polyurethanes. In some embodiments, the silica -based solid substrates may include silicon or modified silicon.

[0111] [Hl] In some embodiments, the substrate may be a welled plate. In some embodiments, the substrate may be a 96-well plate. In some embodiments, the substrate may be a 4-well plate, a 6- well plate, an 8-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 384-well plate, an 864-well plate, or a 1,536-well plate. In some embodiments, the substrate may have greater than or equal to 96 wells. In some embodiments, the substrate may have less than or equal to 96 wells.

[0112]

[0112] In some embodiments, the substrate may be a flowcell. In some embodiments, the flowcell may have two or more lanes. In some embodiments, the flowcell may have two or less lanes.

[0113]

[0113] In some embodiments, the substrate may be a microarray, a slide, a chip, a microwell, a tube, a column, a particle, a bead, or a paramagnetic bead.

[0114]

[0114] In some embodiments, the substrate may comprise a coating. In some embodiments, the coating may comprise a layer that may be charged. In some embodiments, the coating layer may be positively charged. In some embodiments, the coating layer may be negatively charged. In some embodiments, the coating may be non-charged. In some embodiments, the substrate may comprise a surface comprising a cation-coating layer. In some embodiments, the substrate may comprise a surface comprising an anion-coating layer. In some embodiments, the substrate may comprise a surface comprising a neutral-charged layer. In some embodiments, the substrate may be coated with streptavidin. In some embodiments, the substrate may be coated with avidin. In some embodiments, the substrate may be coated with one or more antibodies.

[0115]

[0115] The systems disclosed herein may comprise a fluidics system. The fluidics system may comprise a fluid flow controller. In some embodiments, the fluid flow controller may comprise one or more pumps, valves, mixing manifolds, reagent reservoirs, waste reservoirs, or any combination thereof. In some embodiments, the fluidic system and subcomponents of the fluidics system are fluidically connected to the reaction vessel of the present disclosure. In some embodiments, the fluidic system and subcomponents of the fluidics system iteratively flow in reagents (e.g., buffers, detector polynucleotides, anchor polynucleotides, detection oligonucleotide complexes, etc.) to the reaction vessel. In some embodiments, the reaction vessel comprises a solid substrate configured to immobilize the circularized and ligated recognition elements or concatemeric amplification products thereof.

[0116]

[0116] The systems disclosed herein may comprise a temperature system. The temperature system may comprise a temperature controller. The temperature controller may be incorporated into the systems described herein to facilitate accuracy of the methods and systems described herein. In some embodiments, the temperature controller may comprise temperature control components. Non-limiting examples of temperature control components include resistive heating elements, infrared light sources, heating or cooling devices, heat sinks, thermocouples, thermistors, or a combination thereof. In some embodiments, the temperature controller may provide changes in temperature over specified time intervals. In some embodiments, the temperature controller may provide an increase in temperature. In some embodiments, the temperature controller may provide a decrease in temperature. In some embodiments, the temperature controller may provide for cycling of temperatures between two or more set temperatures so that thermocycling or amplification may be performed. In some embodiments, the temperature controller may provide a constant temperature.

[0117]

[0117] The systems disclosed herein may comprise an imaging system. In some embodiments, signals produced by the labeled probes disclosed herein may be imaged by the imaging systems disclosed herein. The imaging system may comprise one or more light sources, one or more optical components, one or more filters, one or one or more imaging sensors for imaging and detection, or a combination thereof. In some embodiments, the one or more light sources may comprise light from a bulb. In some embodiments, the one or more optical components may comprise lenses, mirrors, digital mirror devices, prisms, optical filters, colored glass filters, narrowband interference filters, broadband interference filters, dichroic reflectors, diffraction gratings, apertures, optical fibers, optical waveguides, or a combination thereof. In some embodiments, the one or more imaging sensors may comprise a charge -coupled device (CCD) sensor or camera, a complementary metal-oxide-semiconductor (CMOS) imaging sensor or camera, a negative-channel metal-oxide semiconductor (NMOS) imaging sensor or camera, or a combination thereof.

[0118]

[0118] Various operations of the methods and systems disclosed herein may be performed by a computer system of the present disclosure. Referring to Fig. 5, a non-limiting example of a block diagram is shown depicting a non-limiting example of a machine that includes a computer system 500 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and / or methodologies for static code scheduling of the present disclosure. The components in Fig. 5 are examples and do not limit the scope of use or functionality of any hardware, software, embedded logic component, or a combination of two or more such components implementing particular embodiments.

[0119]

[0119] Computer system 500 may include one or more processors 501, a memory 503, and a storage 508 that communicate with each other, and with other components, via a bus (solid lines). The bus may also link a display 532, one or more input devices 533 (which may, for example, include a keypad, a keyboard, a mouse, a stylus, etc.), one or more output devices 534, one or more storage devices 535, and various tangible storage media 536. All of these elements may interface directly or via one or more interfaces or adaptors to the bus. For instance, the various tangible storage media 536 can interface with the bus via storage medium interface 526. Computer system 500 may have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers.

[0120]

[0120] Computer system 500 includes one or more processor(s) 501 (e.g., central processing units (CPUs), general purpose graphics processing units (GPGPUs), or quantum processing units (QPUs)) that carry out functions. Processor(s) 501 optionally contains a cache memory unit 502 for temporary local storage of instructions, data, or computer addresses. Processor(s) 501 are configured to assist in execution of computer readable instructions. Computer system 500 may provide functionality for the components depicted in Fig. 5 as a result of the processor(s) 501 executing non -transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory 503, storage 508, storage devices 535, and / or storage medium 536. The computer-readable media may store software that implements particular embodiments, and processor(s) 501 may execute the software. Memory 503 may read the software from one or more other computer-readable media (such as mass storage device(s) 535, 536) or from one or more other sources through a suitable interface, such as network interface 520. The software may cause processor(s) 501 to carry out one or more processes or one or more operations of one or more processes described or illustrated herein. Carrying out such processes or operations may include defining data structures stored in memory 503 and modifying the data structures as directed by the software.

[0121]

[0121] The memory 503 may include various components (e.g., machine readable media) including, but not limited to, a random-access memory component (e.g., RAM 504) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), phasechange random access memory (PRAM), etc.), a read-only memory component (e.g., ROM 505), and any combinations thereof. ROM 505 may act to communicate data and instructions unidirectionally to processor(s) 501, and RAM 504 may act to communicate data and instructions bidirectionally with processor(s) 501. ROM 505 and RAM 504 may include any suitable tangible computer-readable media described below. In one example, a basic input / output system 506 (BIOS), including basic routines that help to transfer information between elements within computer system 500, such as during start-up, may be stored in the memory 503.

[0122]

[0122] Fixed storage 508 is connected bidirectionally to processor(s) 501, optionally through storage control unit 507. Fixed storage 508 provides additional data storage capacity and may also include any suitable tangible computer-readable media described herein. Storage 508 may be used to store operating system 509, executable(s) 510, data 511, applications 512 (application programs), and the like. Storage 508 can also include an optical disk drive, a solid-state memory device (e.g., flash-based systems), or a combination of any of the above. Information in storage 508 may, in appropriate cases, be incorporated as virtual memory in memory 503.

[0123]

[0123] In one example, storage device(s) 535 may be removably interfaced with computer system 500 (e.g., via an external port connector (not shown)) via a storage device interface 525. Particularly, storage device(s) 535 and an associated machine-readable medium may provide non-volatile and / or volatile storage of machine-readable instructions, data structures, program modules, and / or other data for the computer system 500. In one example, software may reside, completely or partially, within a machine-readable medium on storage device(s) 535. In another example, software may reside, completely or partially, within processor(s) 501.

[0124] Bus connects a wide variety of subsystems. Herein, reference to a bus may encompass one or more digital signal lines serving a common function, where appropriate. Bus may be any of several types of bus structures including, but not limited to, a memory bus, a memory controller, a peripheral bus, a local bus, and any combinations thereof, using any of a variety of bus architectures. As an example, and not by way of limitation, such architectures include an Industry Standard Architecture (ISA) bus, an Enhanced ISA (EISA) bus, a Micro Channel Architecture (MCA) bus, a Video Electronics Standards Association local bus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI -Express (PCLX) bus, an Accelerated Graphics Port (AGP) bus, HyperTransport (HTX) bus, serial advanced technology attachment (SATA) bus, and any combinations thereof.

[0124]

[0125] Computer system 500 may also include an input device 533. In one example, a user of computer system 500 may enter commands and / or other information into computer system 500 via input device(s) 533. Examples of an input device(s) 533 include, but are not limited to, an alpha-numeric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, a touch screen, a multi-touch screen, a joystick, a stylus, a gamepad, an audio input device (e.g., a microphone, a voice response system, etc.), an optical scanner, a video or still image capture device (e.g., a camera), and any combinations thereof. In some embodiments, the input device is a Kinect, Leap Motion, or the like. Input device(s) 533 may be interfaced to bus via any of a variety of input interfaces 523 (e.g., input interface 523) including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above.

[0125]

[0126] In particular embodiments, when computer system 500 is connected to network 530, computer system 500 may communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 530. Communications to and from computer system 500 may be sent through network interface 520. For example, network interface 520 may receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 530, and computer system 500 may store the incoming communications in memory 503 for processing. Computer system 500 may similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 503 and communicated to network 530 from network interface 520. Processor(s) 501 may access these communication packets stored in memory 503 for processing.

[0127] Examples of the network interface 520 include, but are not limited to, a network interface card, a modem, and any combination thereof. Examples of a network 530 or network segment 530 include, but are not limited to, a distributed computing system, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, a peer-to-peer network, and any combinations thereof. A network, such as network 530, may employ a wired and / or a wireless mode of communication. In general, any network topology may be used.

[0126]

[0128] Information and data can be displayed through a display 532. Examples of a display 532 include, but are not limited to, a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid crystal display (OLED) such as a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display, a plasma display, and any combinations thereof. The display 532 can interface to the processor(s) 501, memory 503, and fixed storage 508, as well as other devices, such as input device(s) 533, via the bus. The display 532 is linked to the bus via a video interface 522, and transport of data between the display 532 and the bus can be controlled via the graphics control 521. In some embodiments, the display is a video projector. In some embodiments, the display is a headmounted display (HMD) such as a VR headset. In further embodiments, suitable VR headsets include, by way of non-limiting examples, HTC Vive, Oculus Rift, Samsung Gear VR, Microsoft HoloLens, Razer OSVR, FOVE VR, Zeiss VR One, Avegant Glyph, Freefly VR headset, and the like. In still further embodiments, the display is a combination of devices such as those disclosed herein.

[0127]

[0129] In addition to a display 532, computer system 500 may include one or more other peripheral output devices 534 including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof. Such peripheral output devices may be connected to the bus via an output interface 524. Examples of an output interface 524 include, but are not limited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof.

[0128]

[0130] In addition, or as an alternative, computer system 500 may provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which may operate in place of or together with software to execute one or more processes or one or more operations of one or more processes described or illustrated herein. Reference to software in this disclosure may encompass logic, and reference to logic may encompass software. Moreover, reference to a computer-readable medium may encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, or both, where appropriate. The present disclosure encompasses any suitable combination of hardware, software, or both.

[0129]

[0131] Those of skill in the art will appreciate that the various illustrative logical blocks, modules, circuits, and algorithm operations described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and operations have been described above generally in terms of their functionality.

[0130]

[0132] The various illustrative logical blocks, modules, and circuits described in connection with the embodiments disclosed herein may be implemented or performed with a general - purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general -purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

[0131]

[0133] The operations of a method or algorithm describedin connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium. An example of a storage medium is coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

[0132]

[0134] In accordance with the description herein, suitable computing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, notepad computers, set -top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles. Those of skill in the art will also recognize that select televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers, in various embodiments, include those with booklet, slate, and convertible configurations, known to those of skill in the art.

[0133]

[0135] In some embodiments, the computing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications. Those of skill in the art will recognize that suitable server operating systems include, by way of non -limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Those of skill in the art will recognize that suitable personal computer operating systems include, by way of non- limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX -like operating systems such as GNU / Linux®. In some embodiments, the operating system is provided by cloud computing. Those of skill in the art will also recognize that suitable mobile smartphone operating systems include, by way of non-limiting examples, Nokia® Symbian® OS, Apple® iOS®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®. Those of skill in the art will also recognize that suitable media streaming device operating systems include, by way of non-limiting examples, Apple TV®, Roku®, Boxee®, Google TV®, Google Chromecast®, Amazon Fire®, and Samsung® HomeSync®. Those of skill in the art will also recognize that suitable video game console operating systems include, by way of non -limiting examples, Sony® PS3®, Sony® PS4®, Microsoft® Xbox 360®, Microsoft Xbox One, Nintendo® Wii®, Nintendo® Wii U®, and Ouya®.

[0134]

[0136] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non -transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked computing device. In further embodiments, a computer readable storage medium is a tangible component of a computing device. In further embodiments, a computer readable storage medium is optionally removable from a computing device. In some embodiments, a computer readable storage medium includes, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, distributed computing systems including cloud computing systems and services, and the like. In some cases, the program and instructions are permanently, substantially permanently, semi -permanently, or non-transitorily encoded on the media.

[0135]

[0137] In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program, or use of the same. A computer program includes a sequence of instructions, executable by one or more processor(s) of the computing device’s CPU, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), computing data structures, and the like, which perform particular tasks or implement particular abstract data types. In light of the disclosure provided herein, those of skill in the art will recognize that a computer program may be written in various versions of various languages.

[0136]

[0138] The functionality of the computer readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program is provided from one location. In other embodiments, a computer program is provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more mobile applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof.

[0137]

[0139] In some embodiments, the computer programs described herein may be used to perform at least one function. The computer programs described herein may perform functions related to storing data, receiving data, analyzing data, exporting data, or a combination thereof. In some embodiments, the computer programs described herein may perform functions related to applying selection criteria, including in silico selection criteria, functional selection criteria, or a combination thereof. In some embodiments, the computer programs may receive sequence information, including sequence information for nucleic acid segments. The sequence information may be configured as an array, a table, a list, or combination thereof. The sequence information may be formatted in a variety of ways, including, but not limited to a .txt file, a FASTA file, an .xls file, or a combination thereof. The computer programs described herein may apply selection criterion or selection criteria to a set of nucleic acid segments. The computer programs may sort the nucleic acid segments, determine or compute characteristics of the nucleic acid segments, perform calculations, reorder the nucleic acid segments, or a combination thereof. In some embodiments, the computer programs described herein may store information related to the nucleic acid segments. In some embodiments, the computer program may use information stored related to the nucleic acid segments to apply selection criteria to the nucleic acid segments. In certain embodiments, the computer program may receive information and / or data related to nucleic acid segments, selection criteria, or a combination thereof. In some embodiments, the computer programs may perform functions related to analyzing data from functional assays, including, but not limited to functional assays described herein. In some embodiments, analyzing data from functional assays may comprise image analysis, image quantification, intensity quantification, feature identification, or a combination thereof. The computer programs described herein may also export information. In some embodiments, the exported information may comprise images, files, data tables, documents, folders, or a combination thereof.

[0138]

[0140] In some embodiments, a computer program includes a web application. In light of the disclosure provided herein, those of skill in the art will recognize that a web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, a web application is created upon a software framework such as Microsoft® .NET or Ruby on Rails (RoR). In some embodiments, a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, XML, and document oriented database systems. In further embodiments, suitable relational database systems include, by way of non -limiting examples, Microsoft® SQL Server, mySQL™, and Oracle®. Those of skill in the art will also recognize that a web application, in various embodiments, is written in one or more versions of one or more languages. A web application may be written in one or more markup languages, presentation definition languages, client-side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, a web application is written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or extensible Markup Language (XML). In some embodiments, a web application is written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, a web application is written to some extent in a client-side scripting language such as Asynchronous JavaScript and XML (AJAX), Flash® ActionScript, JavaScript, or Silverlight®. In some embodiments, a web application is written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tel, Smalltalk, WebDNA®, or Groovy. In some embodiments, a web application is written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, a web application integrates enterprise server products such as IBM® Lotus Domino®. In some embodiments, a web application includes a media player element. In various further embodiments, a media player element utilizes one or more of many suitable multimedia technologies including, by way of non -limiting examples, Adobe® Flash®, HTML 5, Apple® QuickTime®, Microsoft® Silverlight®, Java™, and Unity®.

[0139]

[0141] Referring to Fig. 6, in a non-limiting example, an application provision system comprises one or more databases 600 accessed by a relational database management system (RDBMS) 610. Suitable RDBMSs include Firebird, MySQL, PostgreSQL, SQLite, Oracle Database, Microsoft SQL Server, IBMDB2, IBM Informix, SAP Sybase, Teradata, and the like. In some embodiments, the application provision system further comprises one or more application severs 620 (e.g., such as Java servers, .NET servers, PHP servers, and the like) and one or more web servers 630 (e.g., such as Apache, IIS, GWS and the like). The web server(s) 630 optionally expose one or more web services via app application programming interfaces (APIs) 640. Via a network, such as the Internet, the system provides browser-based and / or mobile native user interfaces.

[0140]

[0142] Referring to Fig. 7, in a non-limiting example, an application provision system alternatively has a distributed, cloud-based architecture 700 and comprises elastically load balanced, auto-scaling web server resources 710 and application server resources 720 as well as synchronously replicated databases 730.

[0141]

[0143] In some embodiments, a computer program includes a mobile application provided to a mobile computing device. In some embodiments, the mobile application is provided to a mobile computing device at the time it is manufactured. In other embodiments, the mobile application is provided to a mobile computing device via the computer network described herein.

[0142]

[0144] In view of the disclosure provided herein, a mobile application is created by techniques known to those of skill in the art using hardware, languages, and development environments known to the art. Those of skill in the art will recognize that mobile applications are written in several languages. Suitable programming languages include, by way of non -limiting examples, C, C++, C#, Objective-C, Java™, JavaScript, Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML / HTML with or without CSS, or combinations thereof.

[0143]

[0145] Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting examples, Airplay SDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (iOS) SDK, Android™ SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK.

[0144]

[0146] Those of skill in the art will recognize that several commercial forums are available for distribution of mobile applications including, by way of non -limiting examples, Apple® App Store, Google® Play, Chrome WebStore, BlackBerry® App World, App Store for Palm devices, App Catalog for webOS, Windows® Marketplace for Mobile, Ovi Store for Nokia® devices, Samsung® Apps, and Nintendo® DSi Shop.

[0145]

[0147] In some embodiments, a computer program includes a standalone application, which is a program that is run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. Those of skill in the art will recognize that standalone applications are often compiled. A compiler is a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language or machine code. Suitable compiled programming languages include, by way of non -limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB .NET, or combinations thereof. Compilation is often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable complied applications.

[0146]

[0148] In some embodiments, the computer program includes a web browser plug-in (e.g., extension, etc.). In computing, a plug-in is one or more software components that add specific functionality to a larger software application. Makers of software applications support plug-ins to enable third-party developers to create abilities which extend an application, to support easily adding new features, and to reduce the size of an application. When supported, plug-ins enable customizing the functionality of a software application. For example, plug-ins are commonly used in web browsers to play video, generate interactivity, scan for viruses, and display particular file types. Those of skill in the art will be familiar with several web browser plug-ins including, Adobe® Flash® Player, Microsoft® Silverlight®, and Apple® QuickTime®. In some embodiments, the toolbar comprises one or more web browser extensions, add-ins, or add-ons. In some embodiments, the toolbar comprises one or more explorer bars, tool bands, or desk bands.

[0147]

[0149] In view of the disclosure provided herein, those of skill in the art will recognize that several plug-in frameworks are available that enable development of plug-ins in various programming languages, including, by way of non-limiting examples, C++, Delphi, Java™, PHP, Python™, and VB .NET, or combinations thereof.

[0150] Web browsers (also called Internet browsers) are software applications, designed for use with network-connected computing devices, for retrieving, presenting, and traversing information resources on the World Wide Web. Suitable web browsers include, by way of nonlimiting examples, Microsoft® Internet Explorer®, Mozilla® Firefox®, Google® Chrome, Apple® Safari®, Opera Software® Opera®, andKDEKonqueror. In some embodiments, the web browser is a mobile web browser. Mobile web browsers (also called microbrowsers, mini -browsers, and wireless browsers) are designed for use on mobile computing devices including, by way of non- limiting examples, handheld computers, tablet computers, netbook computers, subnotebook computers, smartphones, music players, personal digital assistants (PDAs), and handheld video game systems. Suitable mobile web browsers include, by way of non -limiting examples, Google® Android® browser, RIM BlackBerry® Browser, Apple® Safari®, Palm® Blazer, Palm® WebOS® Browser, Mozilla® Firefox® for mobile, Microsoft® Internet Explorer® Mobile, Amazon® Kindle® Basic Web, Nokia® Browser, Opera Software® Opera® Mobile, and Sony® PSP™ browser.

[0148]

[0151] In some embodiments, the platforms, systems, media, and methods disclosed herein include software, server, and / or database modules, or use of the same. In view of the disclosure provided herein, software modules are created by techniques known to those of skill in the art using machines, software, and languages known to the art. The software modules disclosed herein are implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, a distributed computing resource, a cloud computing resource, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, a plurality of distributed computing resources, a plurality of cloud computing resources, or combinations thereof. In various embodiments, the one or more software modules comprise, by way of non- limiting examples, a web application, a mobile application, a standalone application, and a distributed or cloud computing application. In some embodiments, software modules are in one computer program or application. In other embodiments, software modules are in more than one computer program or application. In some embodiments, software modules are hosted on one machine. In other embodiments, software modules are hosted on more than one machine. In further embodiments, software modules are hosted on a distributed computing platform such as a cloud computing platform. In some embodiments, software modules are hosted on one or more machines in one location. In other embodiments, software modules are hosted on one or more machines in more than one location.

[0152] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more databases, or use of the same. In view of the disclosure provided herein, those of skill in the art will recognize that many databases are suitable for storage and retrieval of nucleic acid segment sequences or analysis thereof information. In various embodiments, suitable databases include, byway of non -limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity -relationship model databases, associative databases, XML databases, document oriented databases, and graph databases. Further non-limiting examples include SQL, PostgreSQL, MySQL, Oracle, DB2, Sybase, and MongoDB. In some embodiments, a database is Internet-based. In further embodiments, a database is web-based. In still further embodiments, a database is cloud computing based. In a particular embodiment, a database is a distributed database. In other embodiments, a database is based on one or more local computer storage devices.

[0149] Kits

[0150]

[0153] Provided herein are kits related to the methods, compositions and systems described herein. In some embodiments, the kits may comprise a plurality of recognition elements, a plurality of detection polynucleotides, one or more buffers, one or more reagents, instructions for use, a manual, a protocol, or a combination thereof.

[0151]

[0154] In some embodiments, a kit may comprise one or more buffers. In some embodiments, a kit may comprise two buffers. In some embodiments, a first buffer of a kit may be configured to promote hybridization. In some embodiments, a second buffer of a kit may be configured to promote de-hybridization, ligation, nucleic acid digestion, storage of a purified molecule. In some embodiments, a kit may comprise one or more reagents. In some embodiments, a kit comprises one or more enzymes. In some embodiments, a kit comprises one or more of a ligase, a DNA polymerase, and an exonuclease. In some embodiments, a kit may comprise instructions for use, a manual, a protocol, or a combination thereof. In some embodiments, a kit comprises one or more 96 well plates. In some embodiments, one of the 96 well plates of a kit is configured to be assayed by an optical imaging device described herein .

[0152]

[0155] In some embodiments, decoding of concatemeric amplification products as disclosed herein comprises a detection polynucleotide, or detection polynucleotide complex. The detection polynucleotide comprises a complementary sequence to a code, or a portion of a code such as a nucleic acid segment or a portion thereof, that is part of a concatemeric amplification product, wherein the code correlates to the presence of a target of interest from a sample as described herein. In essence, a target molecule of interest from a sample, if it is present, is allowed to hybridize to its complementary sequences found in a recognition element. The recognition element comprises a code, oftentimes a code that comprises two or more nucleic acid segments which collectively serve as the code, wherein the code is unique to any given recognition element, wherein the recognition element hybridizes to a specific target molecule of interest, as such the unique code is unique to that hybridization event and to the target molecule of interest. As such, once decoded, the code can be correlated back to, and used as a proxy for, the presence of the target molecule of interest from the sample.

[0153]

[0156] In some embodiments, a detection polynucleotide comprises two oligonucleotides hybridized or otherwise bound together. A first oligonucleotide, the anchor oligonucleotide, comprises two sequences; one complementary to a code, a portion of a code, one or more nucleic acid segments that comprise the code or portions thereof, and one complementary region for hybridization to a second oligonucleotide. The second oligonucleotide, the detection oligonucleotide, comprises a detection moiety attached to an oligonucleotide that has complementarity to a portion of the anchor oligonucleotide. A detection moiety can be a fluorescent molecule, an organic dye, and the like as described herein. Figs. 8A-B show nonliming examples of two detection polynucleotides. In Fig. 8A, the anchor oligonucleotide may comprise a complementary code sequence 810 and a sequence 820 that hybridizes to a detection oligonucleotide 830, wherein the detection oligonucleotide comprises a detection moiety 840. The detection moiety 840, in this example, is a fluorescent molecule. Fig. 8B shows that there can be one or more spacer nucleotide sequences 850 between the complementary code sequence 810 and the complementary detection oligonucleotide sequence 820 of the anchor oligonucleotide, and further between the detection oligonucleotide sequence 830 and the fluorescent molecule 840. The spacers can be of any length and any sequence and can serve the purpose of generating space between the different parts of a detection polynucleotide to guard against any hinderance issues which may occur and impede the hybridization of the detection polynucleotide and the complementary code sequence for decoding. Fig. 9 shows examples of four different color configurations of four different detection polynucleotides from Figs. 8A-B. For example, the code complementary sequences 910 for each of the four anchor oligonucleotides can be the same or different (e.g., 911, 912, 913), such that different codes are targeted, or the same code is targeted (910). The detection oligonucleotide complementary region 920 of the anchor oligonucleotide can be the same or different (e.g., 921, 922, 923), depending on whether the detection oligonucleotide sequence 930 for each of the detection oligonucleotides is the same or different (e.g., 931, 932, 933). The detection moiety 940 of the detection oligonucleotides can be the same or different (e.g., 941, 942, 943). As such, the combination of all elements can be used to expand the decoding ability of an assay for multiple target molecules of interest in a multiplex assay by combining different anchor sequences with a multitude of different detection oligonucleotides comprising different detection moieties.

[0154] Additionally, the same code sequence can be queried multiple times using different fluorescent moieties, thereby allowing a pattern of detection that is unique to a particular code sequence that correlates with a specific concatemeric amplification product aligned and the presence of a target of interest.

[0155]

[0157] In some embodiments, a detection polynucleotide comprises an anchor oligonucleotide and a detection oligonucleotide. In some embodiments, a decoding assay comprises a plurality of detection polynucleotides. In some embodiments, the plurality of detection polynucleotides comprises a plurality of detection moieties. In some embodiments, the detection moieties are fluorescent molecules, wherein one or more of the fluorescent molecules has discernable emission spectra such that each fluorescent moiety can be identified independent of another fluorescent moiety. As seen in Figs. 8A-B and 9, a detection moiety can be appended, or linked, to the end of a detection oligonucleotide. For example, a detection oligonucleotide is linked or attached to a detection moiety prior to hybridizing with an anchor oligonucleotide to generate a detection polynucleotide.

[0156]

[0158] In some embodiments, a detection moiety can be added to the detection polynucleotide after hybridization or complexing of a template oligonucleotide to an anchor oligonucleotide. For example, adding a fluorescent nucleotide (e.g., reversibly terminated, irreversibly terminated or unblocked) to the 3 ’ end of an anchor nucleotide hybridized to a template oligonucleotide is shown in Fig. 10 This configuration may comprise an anchor oligonucleotide comprising a sequence complementary to a code or a portion thereof 1010 and a sequence complementary to a template oligonucleotide 1020. In some embodiments, the template oligonucleotide comprises a complementary anchor sequence 1030 and an overhang on the 5 ’ end of one or more nucleotides 1040. In some embodiments, a non-complementary, or partially non-complementary, blocking sequence 1050 is incorporated into the 3 ’ end of the template oligonucleotide to prevent extension from occurring. With the addition of a DNA polymerase and one or more labeled dNTPs, a fluorescent nucleotide 1060 can be added to the 3’ end of the anchor oligonucleotide using one or more nucleotides 1040 of the template oligonucleotide as a template. Benefits of such a configuration might include, but are not limited to, the ability to add fluorescent nucleotides that are specifically suited to a particular detection and imaging system, cost savings and reagent savings as additional purifications of the detection polynucleotides may not be necessary. As such, a detection workflow that may be more efficient, both in terms of time and cost savings.

[0159] In some embodiments, a stairstep detection polynucleotide configuration can be employed to decode a concatemeric amplification product. An example of a stairstep detection polynucleotide is shown in Fig. 12 and includes an anchor oligonucleotide comprising a sequence complementary to a code or a portion thereof 1210, a sequence complementary 1220 to a first detection oligonucleotide 1250, a spacer sequence 1230, and a sequence complementary 1240 to a second detection oligonucleotide 1260. Both of the detection oligonucleotides comprise a fluorescent moiety attached thereto. In some embodiments, the detection moieties attached to the detection oligonucleotides are the same. In some embodiments, the detection moieties attached to the detection oligonucleotides are different. Fig. 12 shows a non-limiting example of two detection oligonucleotides, however, in some embodiments, there may be more than two “steps” to accommodate more than two detection oligonucleotides depending on the complexity of the decoding. For example, in some cases, more than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 “steps” may be used. In some embodiments, the spacer sequence 1230 may be any sequence. For example, the space sequence 1230 is not complementary to a sequence in the rest of the anchor oligonucleotide, in the detection oligonucleotide, or any sequence in a concatemeric amplification product. One purpose of the spacer sequence 1230 includes, but is not limited to, providing additional space for two detection oligonucleotides including their fluorescent moieties to hybridize to their respective locations on a stairstep anchor oligonucleotide without interaction or hindrance. A spacer sequence 1230 may be the same nucleotide, for example a homopolymer such as TTTTTT, or a random nucleotide sequence that is not a homopolymeric sequence. In some embodiments, a decoding event as described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 stairstep detection polynucleotides that are detectably distinguishable.

[0157]

[0160] In some embodiments, a detection polynucleotide configuration for decoding a concatemeric amplification product is shown in Fig 13. An example of a U-shaped detection polynucleotide configuration as shown in Fig. 13 may include an anchor oligonucleotide comprising a sequence that is complementary to a code or portion of a code in a concatemeric amplification product 1310, two sequences 1320 and 1330 that are complementary to two detection oligonucleotides 1340 and 1350, respectively, and optionally spacer sequences 1360. In some embodiments, the complementary sequence pairs (e.g., 1320 / 1340 and 1330 / 1350) may be the same or may be different. In some embodiments, the detection moieties affixed to the two detection oligonucleotides may be the same or may be different. In some embodiments, the optional spacer sequences 1360 may be the same nucleotide, such as a homopolymer string of Ts or other nucleotide, or the spacer sequences maybe random nucleotide sequences that are not homopolymeric. However, it is preferred that any spacer sequence may not share homology with other sequences in the reaction including a concatemeric amplification product. In some embodiments, a decoding event as described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 U-shaped detection polynucleotides that are detectab ly distinguishable upon imaging.

[0161] Table 1 provides non-limiting examples of fluorescent moieties that may be useful in detection polynucleotides described here. The list is not exhaustive and is provided as nonlimiting examples.

[0158] Table 1 -Fluorescence moieties for use in detection polynucleotide complexes

[0159]

[0162] In some embodiments, a detection polynucleotide configuration for decoding a concatemeric amplification product is shown in Fig. 17. The exemplary detection polynucleotide of Fig. 17 may utilize fluorescence quenching event(s) to decode a concatemeric amplification product. In this particular fluorescence quenching example, there are two different detection polynucleotides shown to demonstrate how one might increase complexity in decoding. However, one detection polynucleotide can also be used, as can more than two detection polynucleotides, depending on the complexity of the decoding desired. In some embodiments, an anchor oligonucleotide may comprise a sequence complementary to a code or a portion of a code 1710a and 1710b, and two stairs 1720a, 1720b and 1730a, 1730b that are complementary to two detection oligonucleotides sequences 1740a, 1740b and 1750a, 1750b, respectively. In some embodiments, a first detection oligonucleotide 1740a and 1740b may be affixed or linked to a detection moiety 1760a and 1760b. In this example, the detection moiety is a fluorescence moiety whose emission spectrum is different for each detection polynucleotide, as such the second detection oligonucleotide 1750a and 1750b comprises a quenching moiety 1770a and 1770b that quenches the fluorescence of its paired fluorescent moiety 1760a and 1760b. In additional embodiments for fluorescence quenching systems, a first single stranded anchor detection oligonucleotide may be labeled with a fluorescent moiety, wherein a first single stranded anchor detection oligonucleotide comprises a sequence complementary to a code or a portion thereof and a second sequence that is complementary to a detection oligonucleotide that is labeled with a matched quencher molecule. As such, hybridization of the single stranded anchor detection oligonucleotide to its complementary code sequence is imaged to provide a first signal. A quencher labeled detection oligonucleotide is hybridized to its complementary sequence on the anchor oligonucleotide and the proximity of the quencher with the fluorescent partner quenches or greatly reduces the fluorescent intensity of the matched fluorophore, thereby providing a dark state image or greatly reduced fluorescence image which can be used to identify the presence of a code in a concatemeric amplification product and thereby the presence of a target molecule of interest from a sample.

[0160]

[0163] The non-limiting example of the detection configuration of Fig. 17, where there are two different detection polynucleotides with two different fluorescence quenching spectra and two different code sequence targets, demonstrates an example of how complexity for a decoding system described herein can be increased to identify a plurality of concatemeric amplification products in one experiment. The number of detection polynucleotides that leverage a fluorescence quenching event are multitude, only limited by the number of fluorescence quenching partners that can distinguished upon fluorescent imaging. As such, the number of detection polynucleotides practicing fluorescence quenching as exemplified in Fig. 17 can be at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least 10 detection polynucleotides that are detectably distinguishable. In some embodiments, a decoding event as described herein comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least ten detection polynucleotides that are detectably distinguishable, wherein two or more of the detection polynucleotides are complementary to the sample code sequence. In some embodiments, a decoding event as described herein comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least 10 detection polynucleotides that are detectably distinguishable, wherein two or more of the detection polynucleotides are complementary to different code sequences, or portions thereof. In some embodiments, the use of detection polynucleotides configured with a fluorescence quenching system eliminates the need for multiple dehybridization and / or washing operations in a decoding workflow, thereby generating efficiencies in time and cost for decoding a concatemeric amplification product. Table 2 list includes examples of fluorescent moiety / quencher moiety partners that may find utility in detection polynucleotide complexes as described herein. This list is not exhaustive and is provided as non-limiting examples.

[0161] Table 2-Fluorescent moieties and paired quenchers

[0162]

[0163]

[0164] In some embodiments, increasing complexity for a decoding event as described herein comprises a cyclic method of detection as depicted in Figs. 19A-B, 20A-B and 21A-B. By cycling a decoding event into two or more decoding events, a pattern of signals, such as fluorescent signals, can be generated wherein said pattern of signals is used to determine the presence of a code sequence and as such the presence of the target molecule from a sample. An example for decoding using a cycling scenario comprises setting up patterns of detection based on fluorescent shifts or multiple fluorescence detection events over two or more imaging cycles when using two or more detection oligonucleotides for any given anchor oligonucleotide in a detection polynucleotide complex. As shown in Fig. 19A, a first non-limiting example comprises using an anchor oligonucleotide comprising a complementary sequence 1910 to a code or a portion thereof, that is itself labeled with a fluorescent moiety 1920, as such a detection oligonucleotide in lieu of a detection polynucleotide complex. In this example, Fig. 19A shows a first fluorescently labeled detection oligonucleotide is hybridized to its complementary code sequence (cycle 1) and a fluorescent image is captured of the cycle 1 hybridization event, in this instance four differently labeled detection oligonucleotides are hybridized and imaged revealing four different fluorescent signals. A second cycle of applying detection oligonucleotides that are fluorescently labeled are added to the detection reaction, whereby the labeled detection oligonucleotides are complementary to a sequence of the anchor oligonucleotide, thereby generating detection polynucleotide complexes which are imaged for providing a pattern of fluorescence that, in combination with the cycle 1 pattern of fluorescence, can be used to detection and subsequently decode the concatemeric amplification product. The second cycle fluorescent moieties affixed to the detection oligonucleotide 1930 can be any of the original fluorescent moieties, a subset of the original moieties or new fluorescent moieties.

[0165] Fig. 19B demonstrates non-limiting examples of signal patterns when performing cycle 1 and 2 detection as shown in Fig. 19A. Based on a four-flu oroph ore labeling system of detection oligonucleotides N and combined oligonucleotides with either the same fluorophore, a subset thereof or additional new fluorophores Y, there may be N x Y combinations for the signal patterns emerging from a second cycle. As such, two cycle detection scheme may greatly increase assay complexity by utilizing the same base detection oligonucleotides in a two-image cycle scenario. The second cycle may include a Forster resonance energy transfer, or FRET, partner for the original fluorophore attached to the anchor detection oligonucleotide, such that the anchor attached donor fluorescent moiety may, via energy transfer, excite an acceptor fluorescent moiety of a detection oligonucleotide that has hybridized to that anchor oligonucleotide, thereby generating a detectable shift in fluorescence emission. Alternatively, a detection oligonucleotide labeled with a fluorescent moiety that emits at a different, discernible wavelength than the label attached to the anchor detection oligonucleotide may hybridize to the anchor detection oligonucleotide thereby providing a second signal distinct from the first. These two scenarios are depicted in Fig. 19B. Further, a detection oligonucleotide maybe attached to a quencher which is a quenching partner of the fluorophore of the anchor detection oligonucleotide, such that hybridization may quench the expected fluorescence yielding a dark state, such that the absence of fluorescence or greatly decreased fluorescence is indicative of the presence of a code sequence.

[0164]

[0166] Table 3 lists FRET donor and acceptor molecules that may be used in the disclosure detection polynucleotide complexes. The list is not exhaustive and is provided as non-limiting examples.

[0165] Table 3-F6rster resonance energy transfer donor and acceptor molecules

[0166]

[0167] Figs. 20A-B show non-limiting examples of a cycle scheme using a U-shaped detection polynucleotide as described herein. In this cycling scenario, an anchor oligonucleotide comprising a sequence complementary to a code, or a portion of a code 2010 is flanked by two non-complementary sequences, one of which is linked to a fluorescent moiety 2020 for a first detection event. In this example, a first image of one color for cycle 1 (Fig. 20B) and two consecutive cycles wherein cycle 2a images the hybridization of a detection oligonucleotide 2030 to a first arm of an anchor detection oligonucleotide and cycle 2b images the hybridization of a second detection oligonucleotide 2040 to a second arm of an anchor detection oligonucleotide provides an image signal pattern such that, combined, the color patterns from cycle 1, 2a and 2b (Fig. 20B) are used to detect and decode a code of a concatemeric amplification product. The U-shaped detection polynucleotide cycling example may include four or more differently labeled anchor detection oligonucleotides and the two detection oligonucleotides for hybridizing to the anchor detection oligonucleotides may be labeled with the same fluorescent moiety, a subset of the four different fluorescent moieties attached to the anchor detection oligonucleotide, or additional new discernible fluorescent moieties. As such, by using multiple cycles of hybridization and imaging of a base anchor detection oligonucleotide combined with the various additional detection options, the complexity of a detection and decoding assay may be greatly increased for identifying the presence of multiple target molecules of interest from a sample.

[0167]

[0168] Figs. 21A-B present another non-limiting cyclic scenario for detecting and decoding a concatemeric amplification product. This example is similar to that of Figs. 20A-B, for example a single stranded anchor oligonucleotide is fluorescently labeled and comprises a sequence complementary 2110 to a code sequence and two sequences, 2120 and 2130, wherein one of the arms 2120 is linked to a fluorescent moiety. The second arm 2130 can be shorter in length than the other arm. The difference in length provides another option to differentiate multiple signals in a complex signaling scenario. In this example, a single stranded fluorescently labeled anchor detection oligonucleotide is added to concatemeric amplification products for hybridization to its complementary code sequence or a portion thereof, the reaction is imaged and one or more of the second and / or third labeled detection oligonucleotides 2140 and 2150 can be added and a second fluorescent image recorded. A third event, wherein temperature is increased to remove the smaller detection oligonucleotide 2150 followed by a third image for detection and decoding. Each of the first and second detection oligonucleotides 2140 and 2150 may be separately added and hybridized to an anchor oligonucleotide, wherein each addition is imaged, temperature adjusted and a fourth image capture, such that a base cycle 1 first image, a cycle 2a second image, a cycle 2b third image and a temperature event image captured (Fig. 21B) wherein the patterns in their combination can be used to decode a concatemeric amplification product. In some embodiments, a single stranded anchor detection oligonucleotide can hybridize to one or more code sequences, wherein each anchor oligonucleotide that identifies a different code sequence also has a different, distinguishable fluorescent moiety attached.

[0168]

[0169] In some embodiments, a single stranded anchor detection oligonucleotide can hybridize to one or more code sequences or portions thereof, however the same fluorescent moiety is linked to the anchor detection oligonucleotide regardless of target. In some embodiments, a first and second detection oligonucleotide have the same fluorescent moiety. In some embodiments, a first and second detection oligonucleotide each have different fluorescent moieties that are distinguishable upon imaging.

[0169]

[0170] In some embodiments, a detection oligonucleotide configuration is that of a molecular beacon (1996, Tyagi and Kramer, Nat Biotech 14(3): 303 -308). Some advantages of using a molecular beacon-based detection oligonucleotide include, but are not limited to, alleviating the need for multiple hybridizations and washings during an assay based on the on / off states of an open and closed molecular beacon configuration. A molecular beacon typically comprises a linear oligonucleotide with a fluorescent moiety at its 5 ’ end and a quencher moiety at its 3 ’ end, wherein at lower temperatures the linear oligonucleotide forms a stem -loop configuration bringing the two ends in proximity such that there is no detectable fluorescent signal, the closed state of the molecular beacon. The closed state occurs below the melting temperature of the stem-loop structure (e.g., the length of the stem sequence, the GC content of the stem sequence), for example if a reaction condition temperature is below 55 °C and the melting temperature of the molecular beacon is 60°C, the molecular beacon may be closed until the reaction condition temperature is raised above 60°C at which point the stem-loop structure is relieved and the fluorophore and quencher are separated and fluorescence is restored. Similarly, the ends of the molecular beacon may be FRET partners, such that in the closed state energy transfer from the donor fluorophore to the acceptor fluorophore is imaged or recorded at the acceptor fluorophore wavelength and in the open state there are two fluorescent signals that can imaged or recorded. For example, as shown in Fig. 22, a detection oligonucleotide that is labeled on a first end with a fluorescent moiety and on a second end with a fluorescent quencher 2210 may form a hairpin structure, which may result in no detectable signal. The detection oligonucleotide molecular beacon configuration may be linearized 2220 and allowed to hybridize to its complementary code sequence, quenching is relieved, for example the reaction can be heated to relax and linearize the molecular beacon hairpin structure, and the fluorescent signal can be imaged. In some embodiments, there are a plurality of detection oligonucleotides of molecular beacon configuration used simultaneously for decoding a concatemeric amplification product. In some embodiments, each of the detection oligonucleotide molecule beacon configurations in a plurality have different fluorescent quencher pairs on either end of the oligonucleotide. In some embodiments, a subset of detection oligonucleotide molecular beacons has the sample complementary code sequence in common, however with either the same or two or more different fluorescent quencher pairs. The degree of code sequence complementarity of the detection oligonucleotides in combination with the number of different fluorescent quencher pairs can dictate the complexity and multiplex options for decoding concatemeric amplification products to identify a plurality of targets of interest present from a sample. Table 2 as previuosly presented lists fluorescent quencher pairs that may be useful for decoding using the methodsand compositions described herein.

[0170] EXAMPLES

[0171]

[0171] The following examples are not meant to be limiting to the present disclosure. A skilled artisan would understand that molecular biological techniques such as ligation, exonuclease digestion, and the like can be performed differently depending on the enzyme used and its environmental activity requirements.

[0172]

[0172] The sequences of the source target recognition elements, anchor oligonucleotides and detection oligonucleotides used in one or more of the following Examples can be predicated on whether detection of the code sequences of the amplification products from the target recognition elements were previously known to provide high intensity signal, low intensity signal or a variable intensity signal based on previously performed experiments using typical detection polynucleotide conditions as described herein. Two amplification products being detected, P3-S2 and P4-S7, were known to result in high intensity fluorescence when detected using a typical detection polynucleotide, amplification product P3 -S8 was known to result in low intensity fluorescence, while amplification product Pl -S5 was known to result in a variable signal intensity. While the mechanism is not totally understood, it is contemplated the high versus low versus variable with regards to a given amplification product signal intensity may be due to one or more of, or additional, issues comprising the detection polynucleotides, issues with an anchor oligonucleotide or a detection oligonucleotide, issues with the recognition element, for example unwanted generation of secondary structures or suboptimal hybridization between one, two or three of the anchor, detection and recognition element sequences. Additionally, the signal intensity variability may be due to concatemeric amplification product structure such that the hybridization of detection polynucleotides is impeded or compromised due to the way the concatemeric amplification product was structurally configured post rolling circle amplification. However, the present disclosure is not limited to any degree of signal intensity that might be generated from a concatemeric amplification product detection event, as the three signal intensities serve as templates for practicing the different ways detection can be performed. The three different products with different levels of expression provided the opportunity to evaluate the different detection polynucleotides options for known different intensity scenarios. Table d provides sequences of the code sequence targeted, the anchor oligonucleotide sequences and the detection oligonucleotide sequences used in the Examples herein. Table 4-Sequences of codes, anchor oligonucleotides and detection polynucleotides

[0173]

[0174] Example 1- Incorporation of a labeled nucleotide into a detection polynucleotide

[0175]

[0173] This example relates to experiments where single labeled nucleotides were incorporated into detection polynucleotides for detecting concatemeric amplification products.

[0176]

[0174] Two single stranded oligonucleotides, a first oligonucleotide which hybridizes to a sequence of a code and the second which hybridizes to a portion of the first oligonucleotide, are pre-annealed in a 1 :1.1 ratio in a buffer (lOmM Tris, pH8, 50mM NaCl and ImM EDTA), heated to 95°C for 5 min., and slowly allowed the two probes to hybridize to each other by allowing the reaction tube to come to room temperature to generate unlabeled detection polynucleotides.

[0177]

[0175] To generate fluorescently labeled detection polynucleotides, fluorescently labeled dNTPs (Fl-dATP, Fl-dTTP, Fl-dCTP, Fl-dGTP, from x) and a DNA polymerase are combined to generate a labeling reaction mixture. The different labeling reaction mixtures for each of the fluorescently labeled dNTPs are added to four different tubes, where each tube had lOOnM of unlabeled detection polynucleotides (total volume lOOpl), the labeling reactions are incubated at 45°C for 15 min. The reactions are stopped by the addition of 5 pl 0.5M EDTA and 5 l 20x TBS. Fig. 10 shows a schematic of the unlabeled and labeled detection polynucleotide complexes.

[0178]

[0176] Circularized and ligated recognition elements that included the code that was complementary to the first oligonucleotide are amplified by rolling circle amplification to generate concatemeric amplification products. The concatemeric amplification products are aliquoted into wells of a 96 well plate coated to immobilize nucleic acids. The four different labeled detection polynucleotide complexes are added to different wells of concatemeric amplification products, the labeled detection polynucleotides are allowed to hybridize to their target sequence in the amplification products, and the wells are imaged using a BioTek Lionheart fluorescent automated microscope (Agilent).

[0179]

[0177] Non-limiting examples of images are shown in Fig. 11. Results demonstrate specific incorporation of the intended fluorescently labeled dNTPs into unlabeled detection polynucleotides and successful hybridization of the labeled detection polynucleotides to the target sequence in concatemeric amplification products. The labeled dATP and dCTP are visible in the Cy5 fluorescent channel while the labeled dTTP and dGTP are visible in the RFP fluorescent channel. As such, fluorescently labeling a detection polynucleotide with a labeled dNTP is a viable option for use in methods of detecting the code of a recognition element for indirectly identifying the presence of a target molecule from a sample.

[0180] Example 2-Detecting using stair and U-shaped detection polynucleotide configurations

[0181]

[0178] This example details experiments where an anchor oligonucleotide was configured to include two complementary sequences for the hybridization of two fluorescently labeled oligonucleotides for detecting a code sequence in a stair configuration (Fig. 12) or a U-shaped configuration (Fig. 13).

[0179] Linear recognition elements comprising code sequences (10 nM) are combined with a ligation reaction comprising 5 nM synthetic oligonucleotides that are complementary to recognition target regions to effect linear recognition element circularization, AmpLigase enzyme, AmpLigase buffer and water. The reactions are incubated at 95°C for 1 min, 65°C for 20 min, cycled through denaturation and ligation ten times, and held at 4 °C. Exonuclease digestion to remove any linear recognition elements is performed by adding Exo I, Exo III, Lambda exonuclease and CutSmart (NEB) to the ligation reactions. The reactions are incubated at 37°C for 30 min, followed by exonuclease inactivation at 80°C for 30 min. The resulting circularized and ligated recognition elements are purified.

[0182]

[0180] Amplification of the purified circularized and ligated recognition elements is performed by adding 1 pM of the circularized and ligated recognition elements, along with 333 nM of an amplification primer to wells of a 96 well coated plate. EquiPhi 29 DNA polymerase, enzyme buffer, dNTPs, DTT and water was added to the wells and the samples are incubated at 42°C for 2-3 hours, the wells are washed with TE, an incubation with 0.1 M NaOH for 1 min. is performed, the samples are washed with TETS and stored in lx TBS. The resulting concatemeric amplification products are detected.

[0183]

[0181] Table 5 details the combinations used for decoding the concatemeric amplification product targets.

[0184] Table 5-Detection polynucleotide stair and U-shaped configurations for code detection

[0185]

[0182] To the sample wells are added 0.5 uM of the anchor oligonucleotide and 0.5 uM of the fluorescently labeled oligonucleotides. Hybridization of the anchor oligonucleotide with its specific fluorescently labeled detection oligonucleotide, and the detection polynucleotide with its target code sequence in the concatemeric amplification product is allowed to proceed, after which imaging is performed with the BioTek Lionheart fluorescent automated microscope. Control experiments, where one fluorescently labeled detection oligonucleotide was hybridized to and anchor oligonucleotide instead of two, was performed. Anchor oligonucleotide sequences used for this experiment included SEQ ID NOs: 7-26.

[0186]

[0183] Example results are detailed in Figs. 14A-B and Figs. 15A-B. Figs. 14A-B details paired controls with two detection oligonucleotide sequences (e.g., B2 and B4). The target concatemeric amplification product P1-S5 was known to give a variable signal intensity depending on the fluorescently labeled oligonucleotide sequence used for detction. The stair configuration boosted the Pl -S5 signal intensity over two times that of the control, regardless of detection oligonucleotide B2 or B4. The boost in signal also holds true for those samples that were known to provide good signal intensity, P3-S2 and P4-S7, in that the stair configuration boosted signal for detection two or more times that of the matched control. The products P3 -S8 was known to provide poor signal intensity, and while there was some boost in signal using the stair configuration it appears that using the detection oligonucleotide B4 was preferential to B2.

[0187]

[0184] The boost in signal did not hold true for the U-shaped detection polynucleotide configuration as seen in Figs. 15A-B, when applied to samples known to provide a good signal with B2 and B4. As such, the stair configuration with two detection oligonucleotides was more effective at increasing signal intensity for detecting a code sequence than the U-shaped configuration with two detection oligonucleotides. It was determined that the stair configuration increased signal intensity when detecting all code sequences queried, regardless of whether a sample was known to be good, variable or even bad even though the signal for those known to have bad signal with B2 and B4 were still significantly less intense.

[0188] Example 3- Detecting using fluorescence / quencher detection polynucleotide configuration

[0189]

[0185] This example details experiments where a fluorescent labeled oligonucleotide was hybridized to an anchor oligonucleotide to generate a detection polynucleotide for decoding a code sequence, followed by incorporation of a quencher oligonucleotide and performing the same process for a second code sequence in the same concatemeric amplification product. By fluorescently detecting a detection event, quenching that detection event, and querying another sequence without cycling through washes and dehybridization operations can save considerable time and money when performing an assay.

[0190]

[0186] Circularized and ligated recognition elements with target code sequences are generated following Example 2. Following exonuclease digestion to remove any linear DNA, IpM of circularized and ligated recognition elements is aliquoted into wells of a 96 well plate and 333nMof amplification primer is added to the wells. A master mix for performing rolling circle amplification is prepared with component concentrations of 0.5pM EquiPhi29™ buffer, 0.15 U / pl EquiPhi29™ DNA polymerase (ThermoFisher), 1 mM DTT, 1 mM each dNTP. Master mix (35 pl) is added to each well containing circularized and ligated recognition elements and amplification primers. Annealing of the amplification primer and rolling circle amplification is performed at 42°C for 2-3 hours, samples are washed with TE buffer, O.lMNaOH is added, and samples are incubated for 1 min. and washed with TETS. The resulting concatemeric amplification products in the wells are used as the test samples for detection.

[0191]

[0187] For detecting the targeted concatemeric amplification products, detection polynucleotides are generated by hybridization on the amplification products in a stairstep configuration (Fig. 17), where imaging occurred first after the fluorescently labeled oligonucleotide hybridized to its target code sequence and second after the associated quencher labeled oligonucleotide is added to the amplification products. Anchor oligonucleotides that included sequences complementary to a targeted code sequence, a fluorescently labeled oligonucleotide and a quencher labeled oligonucleotide are synthesized. Fluorescently labeled detection oligonucleotides and quencher labeled oligonucleotides are also synthesized, Table 6 shows the different fluorescently labeled detection oligonucleotides and quencher oligonucleotides and how they are paired during experiments. Anchor oligonucleotides used include SEQ ID NOs: 35-39.

[0192] Table 6-Detection oligonucleotide and quencher oligonucleotides

[0193]

[0188] A first cycle of detection (fluorescence detection) is performed by incubating aliquots of the concatemeric amplification products with a hybridization buffer, blocker DNA, 0.5uM of an anchor oligonucleotide, 0.5uM of a fluorescently labeled oligonucleotide followed by imaging in either a Cy5 channel or RFP channel on a BioTek Lionheart fluorescent automated microscope, depending on the fluorophore attached to the detection oligonucleotide. The second cycle of detection (fluorescence quenching) is performed by adding to the wells 0.5uM of a quencher labeled oligonucleotide that matches to the fluorophore on the detection oligonucleotide, followed by hybridization and fluorescence detection.

[0194]

[0189] For control sample wells, the sample process was performed using the fluorescently labeled oligonucleotides, with no quencher labeled oligonucleotides added for the first and second detection cycles, respectively.

[0195]

[0190] An example of the results is found in Fig. 18. In general, the addition of the quencher labeled oligonucleotide decreases the fluorescence of the matched fluorescently labeled oligonucleotide, but it is not a complete quench, and it is variable between the different fluorescent moieties and their detection oligonucleotide sequence. However, data demonstrates that fluorescence was significantly quenched upon addition of the matched quencher labeled oligonucleotide. This experiment demonstrates that a fluorescence quenching scenario may be utilized to increase the complexity of a multiplex assay as described herein.

[0196] Example 4-Decoding using the stair configuration and two different fluorescently labeled detection oligonucleotides

[0197]

[0191] This example details experiments that were performed for detection using the stair configuration and two different fluorescently labeled detection oligonucleotides.

[0198]

[0192] Rolling circle amplification of the four circularized and ligated target recognition elements is performed as previously described and aliquoted into a coated 96 well plate.

[0199]

[0193] Detection polynucleotides are generated for a first set of controls that followed the normal detection workflow (one fluorescently labeled detection oligonucleotide and one anchor oligonucleotide), a second set of controls that followed the stair configuration from Example 2 where two of the same fluorescently labeled detection oligonucleotides are hybridized onto one stair anchor oligonucleotide and the test sample using the stair configuration of Example 2, except the two fluorescently labeled detection oligonucleotides are labeled with two different fluorescent moieties. Hybridization of the anchor oligonucleotides to the detection oligonucleotides to generate the detection polynucleotide and concurrent hybridization of the detection polynucleotide to the target code sequences is performed as previously described.

[0200]

[0194] Tables 7 provides the details for anchors, detection oligonucleotides and targeted codes of the concatemeric amplification products. Different detection oligonucleotides are assayed. Anchor oligonucleotides used in this experiment include SEQ ID NOs: 27-34.

[0201] Table 7-Detection polynucleotides with multiple fluorescent moieties

[0202]

[0195] For the double labeled assay, there are two anchor oligonucleotides used, one anchor oligonucleotide comprised the complementary Bl detection oligonucleotide sequence for the first stair and the second stair was complementary to the B3 detection oligonucleotide. The second anchor oligonucleotide reversed the order of the two detection oligonucleotides. Results can be seen in Figs. 16A-B, demonstrating the viability of detecting a code sequence using two differently labeled detection oligonucleotides for a stair configuration detection polynucleotide. Fig. 16A shows the Bl fluorescently labeled detection oligonucleotide results detected in the Cy5 fluorescent channel and Fig. 16B the B3 fluorescently labeled detection oligonucleotide results detected in the RFP fluorescent channel. The experiment of using two differently labeled detection oligonucleotides demonstrated the utility of adding additional complexity to multiplexing of target detection using multiple fluorescent moieties for each anchor oligonucleotide using the methods described herein. The signal intensities were similar between the control and the dual labeled stair configuration regardless of the order of fluorescence detection or the position of the labeled detection oligonucleotides hybridized to the stair anchor oligonucleotide. Further, this experiment demonstrates that two labeled detection oligonucleotides were able to hybridize to one anchor oligonucleotide, ruling out that the signal intensity was not due to non-specific hybridization.

[0203]

[0196] While certain examples of methods and systems have been shown and disclosed herein, one of skill in the art will realize that these are provided by way of example only and not intended to be limiting within the specification. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the scope disclosed herein. Furthermore, it shall be understood that all aspects of the disclosed methods and systems are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables and the description is intended to include such alternatives, modifications, variations or equivalents.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A detection polynucleotide complex, comprising: a) an anchor oligonucleotide comprising a sequence complementary to a target nucleic acid, and two sequences that are complementary to two detection oligonucleotides; b) the two detection oligonucleotides, each of which is hybridized to its respective complementary sequence of the two sequences of the anchor oligonucleotide, wherein each of the two detection oligonucleotides is linked to a detection moiety, thereby generating a detection polynucleotide complex; and c) the target nucleic acid that is hybridized to its complementary sequence of the anchor oligonucleotide.

2. The detection polynucleotide complex of claim 1, wherein the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides are downstream of the sequence of the anchor oligonucleotide that is complementary to the target nucleic acid.

3. The detection polynucleotide complex of claim 1, wherein the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides flank the sequence of the anchor oligonucleotide that is complementary to the target nucleic acid.

4. The detection polynucleotide complex of claim 2 or 3, wherein the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides are the same and the sequences of the two detection oligonucleotides are the same.

5. The detection polynucleotide complex of claim 2 or 3, wherein the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides are different and the sequences of the two detection oligonucleotides are different.

6. The detection polynucleotide complex of any one of claims 1-5, wherein the detection moiety that is linked to each of the two detection oligonucleotides is the same.

7. The detection polynucleotide complex of any one of claims 1-5, wherein the detection moiety that is linked to each of the two detection oligonucleotides is different.

8. The detection polynucleotide complex of any one of claims 1-5, wherein the detection moiety linked to a first of the two detection oligonucleotides is a fluorescent moiety and the detection moiety linked to a second of the two detection oligonucleotides is a quencher moiety.

9. The detection polynucleotide complex of any one of claims 1-5, wherein the detection moiety linked to a first of the two detection oligonucleotides is a donor fluorescent moiety and the detection moiety linked to a second of the two detection oligonucleotides is an acceptor fluorescence moiety.

10. The detection polynucleotide complex of any one of claims 1-5, wherein the detection moieties linked to the two detection oligonucleotides are fluorescent molecules that are detectably distinguishable.

11. The detection polynucleotide complex of any one of claims 1-5, wherein the two sequences of the anchor oligonucleotide that are complementary to the two detection oligonucleotides are separated by a spacer sequence.

12. The detection polynucleotide complex of claim 11, wherein the spacer sequence of the anchor oligonucleotide is a homopolymer sequence.

13. The detection polynucleotide complex of claim 11, wherein the spacer sequence of the anchor oligonucleotide is a random non-homopolymer sequence.

14. A method of identifying the presence of a target molecule of interest from a sample, comprising: a) providing an amplification product, wherein the amplification product comprises a target nucleic acid sequence of interest, or a complement thereof, and a code sequence, or a complement thereof, wherein the code sequence uniquely identifies the target nucleic acid sequence of interest; b) providing a detection polynucleotide complex, wherein the detection polynucleotide complex comprises an anchor oligonucleotide hybridized to two or more detection oligonucleotides, wherein each detection oligonucleotide of the two or more detection oligonucleotides is linked to a detection moiety;c) hybridizing the anchor oligonucleotide to the code sequence of the amplification product; d) imaging the amplification product that is hybridized to the anchor oligonucleotide of the detection polynucleotide complex, thereby generating signal intensity data from the detection moiety that is linked to the two or more detection oligonucleotides; and e) decoding the signal intensity data and identifying the presence of the target molecule of interest from the sample based on the decoding.

15. The method of claim 14, wherein the code sequence comprises two or more different nucleic acid segments.

16. The method of claim 15, further comprising hybridizing the detection polynucleotide complex to one of the two or more different nucleic acid segments.

17. The method of any of claims 14-16, wherein the decoding comprises soft decision decoding.

18. The method of any of claims 14-17, wherein each detection moiety that is linked to the two or more detection oligonucleotides is the same.

19. The method of any of claims 14-17, wherein each detection moiety that is linked to the two or more detection oligonucleotides is different.

20. The method of any of claims 14-19, wherein the two or more detection oligonucleotides are hybridized to the anchor oligonucleotide in a stair step configuration.

21. The method of any of claims 14-19, wherein the two or more detection oligonucleotides are hybridized to the anchor oligonucleotide in a U-shaped configuration.

22. The method of any of claims 14-21, wherein the anchor oligonucleotide further comprises one ormore spacer sequences between the two or more hybridized detection oligonucleotides.

23. The method of claim 22, wherein the one or more spacer sequences are homopolymer sequences.

24. The method of claim 22, wherein the one or more spacer sequences are random non- homopolymer sequences.

25. The method of claim 22, wherein the one or more spacer sequences comprises homopolymer sequences and random non-homopolymer sequences.

26. The method of any of claims 14-25, wherein the anchor oligonucleotide is hybridized to two detection oligonucleotides, wherein the detection moiety that is linked to a first detection oligonucleotide of the two detection oligonucleotides is a fluorescent moiety and the detection moiety that is linked to a second detection oligonucleotide of the two detection oligonucleotides is a quencher moiety, and wherein the imaging is performed after the first detection oligonucleotide is hybridized to the anchor oligonucleotide, and wherein the imaging is further performed after the second detection oligonucleotide is hybridized to the anchor oligonucleotide.

27. The method of any of claims 14-25, wherein the anchor oligonucleotide is hybridized to two detection oligonucleotides, wherein the detection moiety that is linked to a first detection oligonucleotide of the two detection oligonucleotides is a fluorescent donor moiety and the detection moiety linked to a second detection oligonucleotide of the two detection oligonucleotides is a fluorescent accepter moiety, and wherein the imaging is performed after the first detection oligonucleotide is hybridized to the anchor oligonucleotide, and wherein the imaging is further performed after the second detection oligonucleotide is hybridized to the anchor oligonucleotide.

28. The method of any of claims 14-17, wherein each detection moiety linked to the two or more detection oligonucleotides is a fluorescent molecule that is detectab ly distinguishable from each other.

29. A method of identifying the presence of a target molecule of interest from a sample, comprising: a) providing an amplification product, wherein the amplification product comprises a target nucleic acid sequence of interest, or a complement thereof, and a code sequence, or a complement thereof, wherein the code sequence uniquely identifies the target nucleic acid sequence of interest;b) providing a detection oligonucleotide comprising a fluorescent moiety on a 5 ’ end of the detection oligonucleotide and a quencher molecule on a 3 ’ end of the detection oligonucleotide, wherein when the detection oligonucleotide is in a stem-loop configuration there is no detectable signal, and wherein the loop of the stem -loop configuration comprises a sequence that is complementary to the code sequence, or the complement thereof; c) hybridizing the detection oligonucleotide to the code sequence, or the complement thereof, to the amplification product, wherein the hybridizing releases the stem-loop configuration of the detection oligonucleotide, thereby generating a concatemeric amplification product; d) imaging the fluorescent signal of the detection oligonucleotide as it is hybridized to the code sequence, or the complement thereof; and e) identifying, based at least in part on the imaging, the presence of the target molecule of interest from the sample.

30. The method of claim 29, wherein c) further comprises heating a reaction mixture comprising the detection oligonucleotide and the concatemeric amplification product to release the stemloop configuration of the detection oligonucleotide.

31. The method of claim 30, wherein the heating further comprises heating the reaction mixture to a temperature of 55°C or more.

32. The method of any of claims 29-31, wherein the identifying further comprises decoding an image of the fluorescent signal.

33. The method of claim 32, wherein the decoding comprises soft decision decoding.

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