Detection and digital quantitation of multiple targets, with protein detection and gene therapy applications

Abstract

The disclosure provides compositions, methods, and systems for implementation of highly sensitive molecular diagnostic assays, with applications in cell and gene therapy.

Description

Atty. DocketNo.: 43161-65181 / WO (004WO)DETECTION AND DIGITAL QUANTITATION OF MULTIPLE TARGETS, WITH PROTEIN DETECTION AND GENE THERAPY APPLICATIONS1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application Nos.: 63 / 729,166, filed December 6. 2024, and 63 / 802,978, filed May 9, 2025, the entire disclosures of which are hereby incorporated by reference in their entireties.2. SEQUENCE LISTING

[0002] The instant application contains a sequence listing which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, entitled 43161-65181WO_SEQLISTING, was created on December 5, 2025, is 20,409 bytes in size.3. TECHNICAL FIELD

[0003] The disclosure generally relates to systems, methods, and compositions for detection of targets in fields related to protein detection and gene therapy.4. BACKGROUND

[0004] High-sensitivity detection of target material of a sample has applications in many fields. Cunent approaches typically involve next generation sequencing (NGS); however, NGS-based approaches can be slow and / or are limited in sensitivity’, depending upon the nature of the target(s) being detected.

[0005] In one exemplary situation, as an unintended byproduct of gene therapy vector manufacturing. DNA from host cells in which viral particles propagate can make their way into final stock preparations. If not removed, residual host DNA in therapeutic vectors or introduced cells can pose several risks. The residual host DNA may trigger an immune response, causing inflammation and tissue damage by being recognized as foreign. Theresidual host DNA could also interfere with expression of the therapeutic gene, potentially leading to insufficient or unstable protein production. Residual host genes then might engage in pathways that dilute the intended therapeutic effect. Finally, the integration of residual host DNA into the genome raises ethical concerns about unforeseen long-term consequences for patients. Assessing the purity of a preparation prior to administration is therefore a critical step for quality control and safety.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0006] In another exemplary use case, proximity' ligation assays (PL As) enhance traditional immunoassays by enabling detection of interacting proteins through amplification. PLA technologies have been applied to detection of capsid proteins of recombinant adeno- associated virus (rAAV) vectors, which are widely used for in vivo gene therapy. Accurate assessment of the full versus empty7capsid ratio and AAV genome integrity7is vital for determining doses and conducting dose-escalation studies. While PLA has been combined with other digital PCR platforms to quantify rAAV particle titer and evaluate full versus empty capsid ratios, these platforms have limitations in their dynamic range, limiting the load concentration of detectable targets. Furthermore, when assessing genome integrity by detecting multiple targets, these platforms struggle to distinguish between physically-linked targets and unlinked targets that coincidentally occupy the same partition.

[0007] Detection and digital quantitation of targets using partition-based systems (e.g., as in digital PCR) has, thus, been traditionally limited by the number of partitions available, partition format (e.g., 3D format vs. ID or 2D formats), number of colors available for detection and / or analysis (e.g.. in relation to dye / probe tagging limitations, in relation to detection limitations of imaging systems, etc.), high apparatus costs, operation in a high- occupancy regime requiring statistical error correction factors for assessment of results, material costs, and other factors. For instance, in traditional PCR multiplexing reactions, targets are differentiated using one probe per target conjugated with dyes of different excitation and emission spectra, which restricts multiplexing to systems that can cope with multiple emission spectra for detection of fluorescence from the different probe dyes. Furthermore, signal overlap associated with operation in a high-occupancy regime ty pically results in reduced precision in quantitation and other aspects of sample characterization. Such traditional approaches include methods to compensate for deficiencies (e.g., with use of computational tools involving virtual partitioning). Limitations of existing approaches can, however, be overcome by the invention(s) disclosed herein.

[0008] As such, there is a need for innovation in fields related to protein detection and gene therapy.5. SUMMARY

[0009] Currently, platforms, methods, and compositions for high sensitivity' detection of protein targets and / or targets associated with gene therapy and / or cell therapy applications are limited in several ways. Available technologies have limitations in their dynamic range performance (e.g., with respect to dynamic counting range for detection of low targetAtty. DocketNo.: 43161-65181 / WO (004WO) numbers through high target numbers), limiting the load concentration of detectable targets. Furthermore, when assessing genome integrity by detecting multiple targets, available technologies struggle to distinguish between physically linked targets and unlinked targets that coincidentally occupy the same partition. With respect to gene therapy vector manufacturing, DNA from host cells in which viral particles propagate during the manufacturing process can be passed to final stock preparations, and can pose several risks. The residual host DNA may trigger an immune response, could interfere with expression of the therapeutic gene, could engage in pathways that dilute the intended therapeutic effect, and / or raises ethical concerns about unforeseen long-term consequences for patients with respect to residual DNA-genome integration. Assessing the purity of a preparation prior to administration is therefore a critical step for quality control and safety.

[0010] Accordingly, this disclosure discloses embodiments, variations, and examples of systems, methods, and compositions for providing high-sensitivity detection of targets (e.g., involving high input samples), as well as for providing suitable dynamic range performance and multiplexing performance for applications in fields related to protein detection, cell therapy, and / or gene therapy. Such systems, methods, and compositions are able to achieve goals of digital PCR, qPCR, and NGS technologies, in an efficient, and accurate manner, and with less complex instrumentation.

[0011] In one aspect, by ensuring a single linked target or set of targets per partition and the capacity for multiplexed analyses with multi-channel and / or multicolor detection, unlinked (i.e., fragmented vector) co-occupancy concerns are mitigated and / or otherwise eliminated using embodiments, variations, and examples of systems, methods, and compositions disclosed herein. This enables and significantly simplifies the analysis of target linkage and provides more reliable results for vector characterization (e.g., AAV vector characterization).

[0012] An aspect of the disclosure thus provides compositions, methods, and systems for implementation of highly multiplexed molecular diagnostic assays as disclosed herein. In some embodiments, the systems, methods, and compositions as disclosed herein enable transgene integrity and viral titer characterization, including long-read amplicons, within a single assay, with high sensitivity and low variability. The exemplary systems, methods, and compositions disclosed herein demonstrates the value of direct molecule counting by designing a novel multiplex assay that interrogates different regions of the gene-of-interest (Gol) or region-of-interest (Rol) to accurately quantify the ratio of partial versus complete transgene while simultaneously measuring titer in the same sample. Unlike dPCR, this singlemolecule approach reduces and / or prevents false co-occupancy of fragmented, unlinkedAtty. DocketNo.: 43161-65181 / WO (004WO) targets, ensuring accurate genome integrity data. Complete transgene integration is further validated with the ability to analyze long-read amplicons up to 2000 bp. The systems, methods, and compositions disclosed herein make gene therapy viral characterization workflows more versatile and approachable compared to other methods. An aspect of the technology that powers performance of the systems, methods, and compositions is a structured gel matrix within a standard PCR tube that enables true single-occupancy suspension of individual gene-of-interest (Gol) or region-of-interest (Rol) fragment(s).

[0013] An aspect of the disclosure thus provides compositions, methods, and systems for high sensitivity detection of target proteins of a sample. In an aspect, the present disclosure provides a method for high sensitivity detection of target proteins of a sample, the method comprising: generating a plurality of partitions comprising at least 500.000 partitions within a single closed container, wherein the plurality of partitions comprises: a set of target proteins of the sample, and a set of processing materials, wherein each partition of the plurality of partitions contains at most one target protein of the set of target proteins; reacting the set of processing materials with the set of target proteins within the plurality of partitions; detecting signals indicative of target proteins of the set of target proteins from at least a subset of the plurality of partitions upon scanning the single closed container with a detection system; and returning a characterization of detected target proteins of the set of target proteins of the sample from the detected signals. In some embodiments, the plurality of partitions comprises at least 1 million partitions. In some embodiments, the plurality of partitions is characterized by less than 15% occupancy of partitions by the set of target proteins. In some embodiments, the plurality of partitions is stabilized in position as a partition matrix in a close-packed format. In some embodiments, the set of target proteins comprises a capsid protein. In some embodiments, the capsid protein is a VP 1. VP2. or VP3 capsid protein. In some embodiments, the capsid protein is selected from a wild-type or modified capsid protein selected from: AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-B; hu.45-B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh, 19-B; rh.49-B; rh.52-B; rh, 13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu. 9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.l-C; hu.!8-C; hu.3-C; hu.25-C; hu.!5-C; hu. !6-C; hu. l l-C; hu. lO-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D: AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu.17-Atty. DocketNo.: 43161-65181 / WO (004WO)E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; And 13; Ancl26; Ancl27; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; Anc80L44; Anc80Ll; Ancl lO; and Anc8ODI. In some embodiments, the set of processing materials comprises, for a first target protein of the set of target proteins, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the first target protein, the set of targetspecific primers comprising: a target-specific primer comprising a first adapter sequence, and a first fluorophore-labeled oligonucleotide corresponding to the first adapter sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second target protein of the set of target proteins, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the second target protein, the set of targetspecific primers comprising: a target-specific primer comprising a second adapter sequence, and a second fluorophore-labeled oligonucleotide corresponding to the second adapter sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, the set of processing materials comprises, for a first target protein and a second target protein of the set of target proteins, a primer set comprising: at least one primer configured to tag the first target protein with a first probe having a first fluorophore, and at least one primer configured to tag the second target protein with a second probe having a second fluorophore. In some embodiments, the set of processing materials comprises a set of probes configured to: associate with target proteins of the set of target proteins upon reacting the set of processing materials with the set of target proteins, and emit fluorescent signals upon associating with target proteins of the set of target proteins. In some embodiments, reacting the set of processing materials with the set of target proteins comprises binding the set of target proteins with probes of a set of probes. In some embodiments, detecting signals indicative of target proteins of the set of target proteins comprises detecting signals emitted from probes of a set of probes associated with target proteins of the set of target proteins. In some embodiments, the method further comprises performing a proximity ligation assay (PLA) with contents of the plurality of partitions. In some embodiments, the method provides at least a 6-log dynamic range that enables simultaneous quantification of one or more target proteins of the set of target proteins.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0014] In another aspect, the present disclosure provides a method for high sensitivity detection of vector targets of a sample, the method comprising: generating a plurality’ of partitions comprising at least 500,000 partitions within a single closed container, wherein the plurality of partitions comprises: a set of vector targets of the sample, and a set of processing materials, wherein each partition of the plurality of partitions contains at most one vector target of the set of vector targets or one linked set of vector targets of the set of vector targets; reacting the set of processing materials with the set of vector targets within the plurality of partitions; detecting signals indicative of vector targets of the set of vector targets from at least a subset of the plurality' of partitions upon scanning the single closed container with a detection system; and returning a characterization of detected vector targets of the set of vector targets of the sample from the detected signals. In some embodiments, the set of vector targets comprises an adeno-associated virus (AAV) viral vector, an adenovirus (AdV) viral vector, a lentivirus viral vector, or a retrovirus viral vector. In some embodiments, the adeno-associated virus (AAV) viral vector comprises a vector genome, wherein the full- length vector genome comprises a linked set of vector targets of the set of vector targets, the linked set of vector targets comprising at least a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the full-length vector genome has a length of up to 5 kb. In some embodiments, the plurality of partitions comprises at least 1 million partitions. In some embodiments, the plurality of partitions is characterized by less than 15% occupancy of partitions by the set of vector targets. In some embodiments, the plurality' of partitions is stabilized in position as a partition matrix in a close-packed format. In some embodiments, the set of vector targets comprises a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a Rep target, and a helper target. In some embodiments, the linked set of vector targets comprises two or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises at least a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the cargo target is a gene therapy cargo target. In some embodiments, the promoter target is selected from CMV, SV40, EFla, CAG, PGK, Ubc, human beta actin, CBh, CaMKIIa, TEF1, Hl, p5, p40, and p41. In some embodiments, the transcription terminator target is selected from SV40, hGH. BGH, and rbGlob. In some embodiments, the transcription terminator target further comprises a polyA signal, wherein the polyA signal comprises a AAUAAA nucleotideAtty. DocketNo.: 43161-65181 / WO (004WO) sequence motif. In some embodiments, the inverted terminal repeat (ITR) target comprises an inverted terminal repeat (ITR) from wild-type adeno-associated virus (AAV) or a variant thereof. In some embodiments, the set of vector targets comprises one or more Rep targets. In some embodiments, the one or more Rep targets comprise a rep gene encoding a Rep protein. In some embodiments, the Rep protein is selected from a Rep 78 protein, a Rep 68 protein, a Rep 52 protein, and a Rep 40 protein. In some embodiments, the Rep protein is from an AAV serotype selected from AAV1. AAV2. AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, and AAV13. In some embodiments, the one or more Rep targets comprise at least one promoter. In some embodiments, the linked set of vector targets comprises two or more Rep targets. In some embodiments, the set of vector targets comprises a helper target. In some embodiments, the linked set of vector targets comprises two or more helper targets. In some embodiments, the helper target comprises a helper virus gene. In some embodiments, the helper virus gene is selected from one or more of: Adenovirus 5 or Adenovirus 2. In some embodiments, the set of processing materials comprises, for a first vector target of the set of vector targets, a primer set comprising: a common primer, and a set of target-specific pnmers configured to interact with a target region of the first vector target, the set of target-specific primers comprising: a target-specific primer comprising a first adapter sequence, and a first fluorophore-labeled oligonucleotide corresponding to the first adapter sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second vector target of the set of vector targets, a primer set comprising: a common primer, and a set of targetspecific primers configured to interact with a target region of the second vector target, the set of target-specific primers comprising: a target-specific primer comprising a second adapter sequence, and a second fluorophore-labeled oligonucleotide corresponding to the second adapter sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, the set of processing materials comprises, for a first vector target and a second vector target of the set of vector targets, a primer set comprising: at least one primer configured to tag the first vector target with a first probe having a first fluorophore, and at least one primer configured to tag the second vector target with a second probe having a second fluorophore. In some embodiments, the set of processing materials comprises a set of probes configured to: associate with vector targets of the set of vector targets upon reacting the set of processing materials with the set of vector targets, and emit fluorescent signalsAtty. DocketNo.: 43161-65181 / WO (004WO) upon associating with vector targets of the set of vector targets. In some embodiments, reacting the set of processing materials with the set of vector targets comprises binding the set of vector targets with probes of a set of probes. In some embodiments, detecting signals indicative of vector targets of the set of vector targets comprises detecting signals emitted from probes of a set of probes associated with vector targets of the set of vector targets. In some embodiments, returning the characterization comprises a linkage analysis of a full- length vector genome comprising a linked set of vector targets characteristic of the full-length vector genome, the linked set of vector targets characteristic of the full-length vector genome comprising two or more vector targets of the set of vector targets. In some embodiments, the linkage analysis provides a metric characterizing integrity of the full-length vector genome. In some embodiments, the metric characterizing the integrity of the full-length vector genome is determined from counts of the linked set of vector targets characteristic of the full-length vector genome, counts of fragments of the linked set of vector targets characteristic of the full-length vector genome, counts of unlinked vector targets of the set of vector targets, or a combination thereof. In some embodiments, the linked set of vector targets characteristic of the full-length vector genome, the fragments of the linked set of vector targets characteristic of the full-length vector genome, the unlinked vector targets of the set of vector targets, and amplicons thereof, comprise a length of up to 2000 base pairs. In some embodiments, returning the characterization comprises identification of a fraction of virions of the sample comprising a full-length vector genome. In some embodiments, returning the characterization provides a metric characterizing full capsid values associated with virions of the sample, a metric characterizing partially filled capsid values associated with virions of the sample, a metric characterizing empty capsid values associated with virions of the sample, a metric characterizing full versus empty capsid ratios associated with virions of the sample, a metric characterizing full versus partially filled capsid ratios associated with virions of the sample, or a combination thereof. In some embodiments, returning the characterization comprises identification of a concentration of a full-length vector genome of the sample. In some embodiments, returning the characterization provides a count of the full-length vector genomes of the sample. In some embodiments, returning the characterization comprises two or more of: a linkage analysis of a full-length vector genome comprising a linked set of vector targets characteristic of the full-length vector genome; identification of a fraction of virions of the sample comprising a full-length vector genome; and identification of a concentration of a full-length vector genome of the sample. In some embodiments, the method further comprises performing a proximity ligation assay (PLA) with contents of theAtty. DocketNo.: 43161-65181 / WO (004WO) plurality of partitions. In some embodiments, the method provides at least a 6-log dynamic range that enables simultaneous quantification of one or more vector targets of the set of vector targets.

[0015] An aspect of the disclosure thus provides compositions, methods, and systems for implementation of highly multiplexed molecular diagnostic assays involving color combinatorics, stimulus-responsive probes, tandem probes, conjugated polymer probes, and other mechanisms for increasing the number of targets (e.g., protein targets, nucleic acid targets, vector targets, targets associated with gene therapy and / or cell therapy vector manufacturing, etc.) that can be simultaneously detected in a digital assay. As disclosed in more detail herein, combinations of mechanisms can provide a number of targets that can be differentially detected according to n! / [r! (n-r) ! ], where n represents the number of available colors, and r represents the number of selected colors from the number of available colors. Permutations of mechanisms can provide a number of targets that can be differentially detected according to n! / [ (n-r)!] + n, where n represents the number of available colors, and r represents the number of selected colors from the number of available colors. In examples, the numbers of targets that can be differentially tagged and detected from a single sample and within a single assay run can be greater than 10 targets, greater than 15 targets, greater than 20 targets, greater than 25 targets, greater than 30 targets, greater than 35 targets, greater than 40 targets, greater than 45 targets greater than 50 targets, greater than 55 targets, greater than 60 targets, greater than 65 targets, greater than 70 targets, greater than 75 targets, greater than 80 targets, greater than 85 targets, greater than 90 targets, or greater than 100 targets, with optical detection of signals from targets.

[0016] Differential detection is achieved in part due to the high number of partitions involved when using the technologies disclosed, where distribution of sample targets across partitions results in low occupancy of partitions by targets, and large partition numbers contribute to significantly low or zero percentages of doublets (e.g., single partitions occupied by two targets), triplets (e.g., single partitions occupied by three targets), or other forms of multiplets (single partitions occupied by multiple targets). In particular, successful multiplexing at this level is attributed to the high degree of partitioning (with achievable numbers of generated partitions disclosed herein) and extremely low occupancy (with achievable percent occupancies disclosed herein), such that multiple molecules from the target molecules of interest have a minimal (or zero) probability of occupying the same partition as another target molecule. In such a high-partition and low-occupancy regime, there is no competitionAtty. DocketNo.: 43161-65181 / WO (004WO) associated with multiple target molecules per partition, and the platform is not subject to problems related to differences in PCR efficiency between different target molecules.

[0017] In the context of digital multiplexed analyses, the disclosure also provides systems, methods, and compositions that can achieve a high dynamic range, due to the number of partitions involved and occupancy of the partitions by targets of the sample. In examples, the systems, methods, and compositions can provide a dynamic counting range of: over 4 orders of magnitude from a lower count capability to a higher count capability (e.g., at least 104), over 5 orders of magnitude from a lower count capability to a higher count capability (e.g., at least 105), over 6 orders of magnitude from a lower count capability to a higher count capability (e.g., at least 106), over 7 orders of magnitude from a lower count capability to a higher count capability (e.g.. at least 107), or greater (e.g.. for sample volumes disclosed herein). In examples, the systems, methods, and compositions can achieve quantification of targets over a 4-log dynamic range, over a 5-log dynamic range, over a 6-log dynamic range, over a 7-log dynamic range, or greater (e.g., for sample volumes disclosed herein).

[0018] For partitions arranged and / or stabilized in position as a partition matrix (e.g., in close-packed format,) within a closed container, the systems, methods, and compositions disclosed herein can provide discernable signals from individual partitions, with readout performed using multiple color channels (e.g., 2 color channels, 3 color channels, 4 color channels. 5 color channels, 6 color channels, 7 color channels, 8 color channels, 9 color channels. 10 color channels, etc.) corresponding to light sources and optics involved in detection, with suitable signal-to-noise (SNR) characteristics in relation to background fluorescence.

[0019] For multiplexed analyses, methods disclosed herein involve detection of signals from a large number of partitions, where detected signals correspond to a set of signal combinatorics paired with targets of a set of targets potentially represented in the sample and contained within partitions of the set or plurality of partitions, and wherein the set of targets has a total number greater than the number of channels used to detect signals corresponding to the set of signals combinatorics. In examples, the set of signal combinatorics involves color combinatorics with combinations of up to 3 colors, up to 4 colors, up to 5 colors, up to 6 colors, up to 7 colors, up to 8 colors, up to 9 colors, up to 10 colors, up to 15 colors, up to 20 colors, or greater (from each of the set or plurality of partitions), where each combination of colors has a corresponding target associated with the respective combination.

[0020] In one embodiment, the set or plurality of partitions involves partitions of a partition matrix within a closed container, and the set of combinatorics involves combinations of up toAtty. DocketNo.: 43161-65181 / WO (004WO)3 colors, up to 4 colors, up to 5 colors, up to 6 colors, up to 7 colors, etc. detectable from each of the set or plurality of partitions. Additionally or alternatively, multiplexing involving stimulus-responsive materials can expand the number of targets that can be differentially tagged and detected by a factor equal to the number of states through which probes used to tag targets can transition. Additionally or alternatively, multiplexing involving materials that exhibit Foerster resonance energy transfer (FRET) behavior can expand the number of targets that can be differentially tagged and detected by a factor equal to the number of FRET capable probes used.

[0021] The disclosure also provides compositions that produce significantly improved signal- to-noise (SNR) values with reduced background, in relation to detection techniques disclosed herein (e.g., based on lightsheet imaging, etc.) for partitions arranged in bulk in 3D. In examples, target signals can be at least 102greater than background noise signals, 103greater than background noise signals, 104greater than background noise signals, 105greater than background noise signals, 106greater than background noise signals, 107greater than background noise signals, or better. Background noise can be attributed to fluorescence from adjacent partitions and adjacent planes of the set of planes of partitions in the context of noise sources with closely-positioned partitions.

[0022] In examples associated with reaction materials disclosed herein and used for partition digital PCR, determining the target signal value can include: for each plane of a set of planes of partitions under interrogation (e.g., by lightsheet detection, by another method of detection, etc.): determining a categorization based upon a profde of positive partitions represented in a respective plane, determining a target signal distribution and a noise signal distribution specific to the profile, and determining a target signal intensity and a noise signal intensity for the respective plane. Here, the target signal value can be an average value (or other representative value) of the target signal intensities determined from the set of planes, and the background noise signal value can be an average value (or other representative value) of the noise signal intensities determined from the set of planes.

[0023] The disclosure also provides oligonucleotide compositions and designs for multiplexed assays (e.g., locked nucleic acid (LNA) assays, KASP assays, Taqman assays, etc.). Such improved oligonucleotides improve sample processing, with respect to primer cleanup / removal, reduction of background, implementation of compatible forward and reverse primers for direct multiplexed assays (e.g., PCR), implementation of checks for complementarity of amplicons to non-self probes (i.e., in both sense and antisense strands), implementation of checks for complementarity of primers to probes (i.e., in both sense andAtty. DocketNo.: 43161-65181 / WO (004WO) antisense strands), generation of positive and negative controls for a clinical workflow, establishment of limits of detection (LoDs) and other metrics for ultraPCR assays performed using methods, systems, and compositions disclosed herein, and / or other improvements.

[0024] Examples of partition generation methods can include generating a high number of partitions (e.g., at least 100,000 partitions, at least 500,000 partitions, at least 1 million partitions, at least 2 million partitions, at least 3 million partitions, at least 4 million partitions, at least 5 million partitions, at least 6 million partitions, at least 7 million partitions, at least 8 million partitions, at least 9 million partitions, at least 10 million partitions, at least 15 million partitions, at least 20 million partitions, at least 25 million partitions, at least 30 million partitions, at least 40 million partitions, at least 50 million partitions, at least 100 million partitions, greater than 100,000 partitions, greater than 500,000 partitions, greater than 1 million partitions, greater than 2 million partitions, greater than 3 million partitions, greater than 4 million partitions, greater than 5 million partitions, greater than 6 million partitions, greater than 7 million partitions, greater than 8 million partitions, greater than 9 million partitions, greater than 10 million partitions, greater than 15 million partitions, greater than 20 million partitions, greater than 25 million partitions, greater than 30 million partitions, greater than 40 million partitions, greater than 50 million partitions, greater than 100 million partitions, etc.) within a collecting container having a volumetric capacity (e.g., less than 50 microliters, from 50 through 100 microliters and greater, etc.), where partitions have a characteristic dimension (e.g., from 1-50 micrometers, from 10-50 micrometers, from 10-30 micrometers, etc.) that is relevant for digital analyses, target detection, individual molecule partitioning, or other applications.

[0025] In relation to occupancy, embodiments, variations, and examples of partitioning are conducted in a manner such that each partition has one or zero targets (i.e., at most one target), such that the partitions are characterized as having low occupancy (e.g., less than 15% occupancy of partitions by individual targets, less than 14% occupancy of partitions by individual targets, less than 13% occupancy of partitions by individual targets, less than 12% occupancy of partitions by individual targets, less than 11% occupancy of partitions by individual targets, less than 10% occupancy of partitions by individual targets, less than 9% occupancy of partitions by individual targets, less than 8% occupancy of partitions by individual targets, less than 7% occupancy of partitions by individual targets, less than 6% occupancy of partitions by individual targets, less than 5% occupancy of partitions by individual targets, less than 4% occupancy of partitions by individual targets, less than 3%Atty. DocketNo.: 43161-65181 / WO (004WO) occupancy of partitions by individual targets, less than 2% occupancy of partitions by individual targets, less than 1% occupancy of partitions by individual targets, etc.).

[0026] Compositions, methods, and systems disclosed herein can further involve use of a single primer with tandem adapters or multiple primers used to tag targets with probes. Multiplexed primers configured to flank target-specific probes that encode for different targets can be used. Multiplexed primer compositions can be configured for 20-plex amplification of loci of interest for each a set of targets being analyzed, 30-plex amplification of loci of interest for each a set of targets being analyzed, 40-plex amplification of loci of interest for each a set of targets being analyzed, 50-plex amplification of loci of interest for each a set of targets being analyzed, 60-plex amplification of loci of interest for each a set of targets being analyzed. 70-plex amplification of loci of interest for each a set of targets being analyzed, 80-plex amplification of loci of interest for each a set of targets being analyzed, 90- plex amplification of loci of interest for each a set of targets being analyzed, 100-plex amplification of loci of interest for each a set of targets being analyzed, or greater.

[0027] Relatedly, an aspect of the disclosure provides embodiments, variations, and examples of devices and methods for rapidly generating partitions of a partition matrix and distributing targets (e.g., multiplexed target detection) / target material (e.g., nucleic acid material, protein / polypeptide material) across partitions, where, the device includes: a first substrate defining a reservoir comprising a reservoir inlet and a reservoir outlet; a membrane coupled to the reservoir outlet and comprising a distribution of holes; and a supporting body comprising an opening configured to retain a collecting container in alignment with the reservoir outlet. During operation, the first substrate can be coupled with the supporting body and enclose the collecting container, with the reservoir outlet aligned with and / or seated within the collecting container. During operation, the reservoir can contain a sample fluid (e.g., a mixture of targets, such as nucleic acids and / or proteins, of the sample and materials for an amplification reaction), where application of a force to the device or sample fluid generates a plurality of partitions within the collecting container at a high rate (e.g., of at least 50,000 partitions / minute, of at least 100,000 partitions / minute, of at least 200,000 partitions / minute, of at least 300,000 partitions / minute, of at least 400,000 partitions / minute, of at least 500,000 partitions / minute, of at least 600,000 partitions / minute, of at least 700,000 partitions / minute, of at least 800,000 partitions / minute, of at least 900,000 partitions / minute, of at least 1 million partitions / minute, of at least 2 million partitions / minute, of at least 3 million partitions / minute, of at least 4 million partitions / minute, of at least 5 million partitions / minute, of at least 6 million partitions / minute, etc.), where the partitions areAtty. DocketNo.: 43161-65181 / WO (004WO) stabilized in position as a partition matrix (e.g., in a close-packed format, in equilibrium stationary positions) within the collecting container.

[0028] An aspect of the disclosure provides embodiments, variations, and examples of a method for rapidly generating partitions of a partition matrix within a collecting container at a high rate, each of the plurality of partitions including an aqueous mixture for a digital analysis, wherein upon generation, the plurality of partitions is stabilized in position (e.g., in a close-packed format, at equilibrium stationary positions, etc.) within a continuous phase. In some embodiments, the plurality of partitions is stabilized in position as a partition matrix in a close-packed format. In aspects, partition generation can be executed by driving the sample fluid through a distribution of holes of a membrane, where the applied force can be one or more of centrifugal (e.g., under centrifugal force), associated with applied pressure, magnetic, or otherwise physically applied.

[0029] In relation to a single-tube workflow in which the collecting container remains closed (e.g., the collecting container has no outlet, there is no flow out of the collecting container, to avoid sample contamination), method(s) can further include transmitting heat to and from the plurality of partitions within the closed collecting container according to an assay protocol. In relation to generation of emulsions having suitable clarity (e.g., with or without refractive index matching), method(s) can further include transmission of signals from individual partitions from within the closed collecting container, for readout (e.g., by a detection system, such as an optical detection platform, or by another suitable detection platform).

[0030] Where method(s) include transmitting heat to and from the plurality’ of partitions, within the closed container, the partitions are stable across a wide range of temperatures (e.g., 1 °C through 95 °C, greater than 95 °C, less than 1 °C) relevant to various digital analyses and other bioassays, where the partitions remain consistent in morphology and remain unmerged with partitions.

[0031] The disclosure generally provides mechanisms for efficient capture, distribution, and labeling of targets / target material (e.g., DNA, RNA, miRNA, nucleic acids, polynucleotides, oligonucleotides, proteins, small molecules, single analytes, multianalytes, etc.) in order to enable genomic, proteomic, and / or other multi-omic characterization of materials, in parallel and in a multiplexed manner, for various applications.

[0032] In examples, the approach discussed is designed around a simple workflow to enable deployment to local and decentralized laboratories. First, samples are carried end-to-end in the same PCR tube for user convenience and to minimize sample contamination. Second, ultra-partitioning and amplification can be performed in standard laboratory equipment suchAtty. DocketNo.: 43161-65181 / WO (004WO) as a swing bucket centrifuge and thermal cycler, lowering the infrastructure cost for adoption. However, compositions of the disclosure can also be utilized in coordination with various technologies for isolating material in single-molecule format (e.g., by use of wells, by use of droplets, by use of other partitioning elements, using non-microfluidic devices, using microfluidic devices, etc.).

[0033] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0034] The disclosure provides compositions, methods, and systems for multiplexed detection of targets that can provide value in research or other non-clinical settings, with or without evaluation and processing of live human or mammalian biological material, and without the immediate purpose of obtaining a diagnostic result of a disease or health condition.

[0035] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0036] 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 embodiments of the present disclosure are shown and described. The present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.6. INCORPORATION BY REFERENCE

[0037] All publications, patents, and patent applications mentioned in this specification, including U.S. Provisional Application Nos. 63 / 729,166 and 63 / 802,978, are herein incorporated by reference in their entireties for all purposes and 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.Atty. DocketNo.: 43161-65181 / WO (004WO)7. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also "figure” and ‘"FIG.” herein), of which:

[0039] FIG. 1A depicts a flowchart of an embodiment of a method for multiplexed detection and digital quantitation of targets.

[0040] FIG. IB depicts a flowchart of a variation of a method for detection and digital quantitation of targets, with applications in characterizing protein targets of a sample.

[0041] FIG. 1C depicts a flowchart of a variation of a method for detection and digital quantitation of targets, with applications in characterizing vector targets of a sample.

[0042] FIG. ID depicts a flowchart of a variation of a method for detection and digital quantitation of targets, with applications in characterizing undesired DNA targets of a sample.

[0043] FIG. IE depicts a flow chart of portions of an embodiment of a method for multiplexed detection and digital quantitation of targets.

[0044] FIG. IF depicts a flowchart of a variation of a method for detection and digital quantitation of targets.

[0045] FIG. 1G depicts a flow chart of a variation of a method for detection and digital quantitation of targets, with applications in characterizing protein targets of a sample.

[0046] FIG. 1H depicts a flow chart of a variation of a method for detection and digital quantitation of targets, with applications in characterizing vector targets of a sample.

[0047] FIG. IK depicts a flow chart of a variation of a method for detection and digital quantitation of targets, with applications in characterizing undesired DNA targets of a sample.

[0048] FIG. 2 depicts a schematic of components implemented in an embodiment of a method for multiplexed detection of targets.

[0049] FIG. 3A depicts a schematic of differences in multiplexing with low-partition systems in comparison to high-partition systems.

[0050] FIG. 3B depicts a schematic of different exemplary strategies for labeling a target with one or more probes.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0051] FIG. 3C depicts a schematic of example color combinations for differential detection and quantitation of targets.

[0052] FIG. 3D depicts a schematic of example color combinations for differential detection and quantitation of targets.

[0053] FIG. 3E depicts a schematic of an exemplary7emulsion where targets within partitions are tagged in a manner involving color combinatorics.

[0054] FIG. 3F depicts variations of tagging a target with tandem probes, in relation to positioning of fluorophores and quenchers.

[0055] FIG. 3G depicts exemplary' scenarios involving use of tandem probes and amplitudebased differentiation of signals involving two colors.

[0056] FIG. 3H depicts variations of tagging different targets of a sample with single probes or tandem probes.

[0057] FIG. 31 depicts a variation of multiplexing involving probes capable of FRET behavior.

[0058] FIG. 3J depicts a schematic of an example of a FRET-capable probe.

[0059] FIG. 3K depicts a schematic of an example of stimulus-responsive probes used for tagging and detection of different targets.

[0060] FIG. 4A depicts a schematic of achievable levels of multiplexing, with combinations and permutations of probes and detected colors.

[0061] FIG. 4B depicts a schematic demonstrating expansion of multiplexing ability for an assay, with combinations of multiplexing strategies.

[0062] FIG. 5A depicts alternative assay chemistry for performing differential detection and quantitation of targets in a multiplexed manner.

[0063] FIG. 5B depicts an embodiment of a process for determining a signal to noise ratio (SNR) for an embodiment of a digital multiplexed analysis.

[0064] FIG. 6 depicts a schematic of an embodiment of a system for partitioning samples.

[0065] FIG. 7 depicts schematics of portions of an embodiment of a method for detection of one of the targets in a multiplex panel.

[0066] FIG. 8 depicts a schematic related to digital quantitation of targets (e.g., where the number of targets is greater than the number of fluorescent channels).

[0067] FIG. 9 illustrates a computer system that is programmed or otherwise configured to implement methods provided herein.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0068] FIG. 10 provides data demonstrating a performance comparison of the exemplary' platform vs. qPCR and dPCR methods. Data generated from same DNA samples to showcase platform dynamic range and variability.

[0069] FIGs. 11A-11B depict an exemplary' experimental design where the protocol involved: Starting from 0.8 pg (ca. 500,000 molecules) of target AAV ssDNA, primers F / R at 04:20 molar ratio. UM probe kit from Countable PCR. PCR: 2-step 30 cycles, annealing at 60 °C and 1 minute elongation time.

[0070] FIG. 12 shows a schematic representing an exemplary 4-way multiplex.

[0071] FIG. 13 depicts images from an exemplary' four-pl ex assay using Universal Multiplex on four different channels (A) show the power of direct counting of molecules. One-tube preparation (B) allows for powerful direct visualization of different targets in the same prep (close-up, C).

[0072] FIG. 14 depicts results from a series of dilutions for each of three targeted areas of transgene. N=4 replicates are reported. The table provides R2 values for the linearity of signal along the dilution series. Even with a broad signal range, linearity and quality’ remain strong. Dilution factor of 1 corresponds to 0.0008 ng of input ssDNA from AAV.

[0073] FIG. 15 provides representative images from an exemplary single reaction sample for linkage analysis. The schematic reflects the visualization of compartments containing either partial or complete transgene.

[0074] FIG. 16 depicts results of different ratios of complete vs artificially digested vector after analysis. The x-axis shows the ratio of complete:partial transgene, while the y-axis shows the % of complete transgene in the sample determined from linkage analysis.

[0075] FIG. 17 depicts an exemplary’ design for a long-read protocol executed using the exemplary platform: 0.5 pg (ca. 85,000 molecules) of target dsDNA, primers F / R / TM / PA at 10: 10:05: 15 molar ratio. TM® probe for FMA fluorophore. PCR: 2-step, 40 cycles, annealing at 60 °C and 2 minute elongation time. Single replicates are reported.

[0076] FIG. 18 illustrates that despite no assay -specific optimization, all amplicons produced results with good signal-to-noise ratio (SNR).

[0077] FIG. 19A-19C illustrate the exemplary steps of plasmid manufacturing, viral manufacturing, and cell processing. The figures also illustrate exemplary single and / or multiplex assays which may be used to characterize purity', genome titer, integrity', functional titer, and vector copy number (VCN).

[0078] FIG. 20 illustrates an exemplary 1-plex design for genomic titer.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0079] FIG. 21 illustrates an exemplary workflow design for characterizing purity, genome titer, integrity, and / or functional titer.

[0080] FIG. 22 illustrates an exemplary workflow design for characterizing vector copy number (VCN).

[0081] FIG. 23 illustrates an exemplary 2-plex design for vector copy number (VCN).

[0082] FIG. 24 illustrates an exemplary 3-plex design for purity.

[0083] FIG. 25 illustrates exemplary samples to be sampled for purity.

[0084] FIGs. 26A-26B illustrate challenges surrounding analyses of full versus partial vectors (i.e., single molecule vs. co-occupancy; linked vs. unlinked vectors / targets).

[0085] FIG. 27 illustrates an exemplary adeno-associated virus (AAV) vector and exemplary' targets which may be used to confirm completeness of said AAV vector.

[0086] FIG. 28 illustrates restriction endonuclease (RE) sites and Universal Multiplexing (UMA) primer sites for an exemplary linkage analysis.

[0087] FIG. 29 illustrates exemplary' targets / components of a viral vector.

[0088] FIG. 30 illustrates exemplary (UMA) primers designed for primer walking. The sequences shown in the figure (in order from top to bottom) correspond to SEQ ID NOs: 13- 21.

[0089] FIG. 31 illustrates the principle of Universal Multiplexing chemistry'. (A) During initial cycles of PCR, the UM primer (a forward primer with UM adapter) binds to the template and extends. (B) In subsequent PCR cycles, the non-UM primer (a reverse primer, unmodified) binds to the sense forward template (now with UM adapter) and extends to create an UM probe complementary sequence. (C) The detection of the target amplicon occurs via hybridization of the UM probe to the target amplicon.

[0090] FIG. 32 depicts results of a comparison between counts per target obtained from a 4- plex UM assay versus a 4-plex HP assay targeting the same genes. Counts averaged across four replicates per condition are show n. Standard deviations were used for the error bars. The difference betw een HP and UM in each assay was less than 2 % in all targets.

[0091] FIG. 33 depicts results of a comparison of 1-plex, 2-plex, 3-plex, and 4-plex designs for Countable PCR assays targeting four different human genes. The counts for all four targets remained consistent, regardless of whether other targets w ere present or not. Counts averaged across three replicates per condition are shown. The error bars represent standard deviations. The difference between 1-plex and 4-plex was -0.13%, 0.35%, -0.84% and 1.03% for RPP30. JAK2, RAD51 and MET. respectively.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0092] FIG. 34 illustrates the dynamic range of UM with 4-plex assay (N = 8). Four different targets were quantified using UM probes across dilution series from 0 to 1,000,000 of template. The error bars represent standard deviations. Even in multiplexed conditions, UM showed a linear increase in counts per target across a 6-log range.

[0093] FIG. 35 depicts results of probe permutation in high / medium / low expression targets (N = 4). Three targets with varying expression levels were quantified using different combinations of UM probes as show n in the table above. The error bars represent standard deviations. The assay performance remained consistent regardless of the UM adapter choice or expression level. %CV was calculated across samples with the same targets, regardless of probe combinations.

[0094] FIG. 36 depicts results of a combination of a 4-plex assay utilizing HP chemistry for two targets and utilizing UM (N = 4). The error bars represent standard deviations. Each target can be quantified without interference, either by HP or UM chemistry.8. DETAILED DESCRIPTION OF THE INVENTION(S)

[0095] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by w ay of example only. Numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention disclosed herein can be employed.8.1. Definitions

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods, systems, and compositions belong.

[0097] “AAV” is adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes, serot pes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.

[0098] The term “AAV capsid protein” or simply “capsid protein” refers to a VP1, VP2, or VP3 capsid protein. In some embodiments, the AAV capsid protein is a wild type or modified capsid protein of AAV9; AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-Atty. DocketNo.: 43161-65181 / WO (004WO)B; hu.45-B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh,19-B; rh.49-B; rh.52-B; rh.l3-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B: hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C: hu. 9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu. l-C; hu. l8-C; hu.3-C; hu.25-C; hu,15-C; hu,16-C; hu.l l-C; hu,10-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu,17-E; hu.6-E; hu.66-E; rh.38- E; hu.32-F: AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83: Anc84; Anc94; Ancl l3;Ancl26; Ancl27; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33;Anc80L36; Anc80L44; Anc80Ll; And 10; and Anc80DI. The modified capsid protein can be a VP1, VP2, or VP3 capsid protein with a targeting moiety (e.g. targeting peptide).

[0099] The term “inverted terminal repeat' ’ (or “ITR”) refers to a polynucleotide sequence found at the ends of AAV genomes that form a hairpin, which contributes to the genome’s ability to self-prime (allowing for primase-independent synthesis of the complementary second DNA strand) and provides for encapsidation of the genome into an AAV particle. An ITR can be a wild-type ITR or a variant thereof.

[0100] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2. or greater than or equal to 3.

[0101] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3. 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0102] Furthermore, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.Atty. DocketNo.: 43161-65181 / WO (004WO)8.2. General Overview

[0103] The invention(s) disclosed herein can confer several benefits over conventional systems, methods, and compositions, as disclosed in the summary and herein.

[0104] The invention(s) enable high-sensiti vity detection of targets (e.g., involving high input samples), as well as provide for suitable dynamic range performance and multiplexing performance for applications in fields related to protein detection, cell therapy, and / or gene therapy. Such systems, methods, and compositions are able to achieve goals of digital PCR, qPCR, and NGS technologies, in an efficient, and accurate manner, and with less complex instrumentation.

[0105] By ensuring a single linked target or set of targets per partition and the capacity for multiplexed analyses with multi-channel and / or multicolor detection, unlinked (i.e., fragmented vector) co-occupancy concerns are mitigated and / or otherwise eliminated using embodiments, variations, and examples of inventions disclosed herein. This enables and significantly simplifies the analysis of target linkage and provides more reliable results for vector characterization (e.g., AAV vector characterization).

[0106] In particular, the invention(s) enable detection and digital quantitation of a set of targets having a number much greater than the number of channels (e.g., color channels, fluorescence detection channels) available for detection. Multiplexed detection involving a greater number of targets than available color channels for detection is based upon one or more of: color combinatorics, stimulus-responsive probes, tandem probes, conjugated polymer probes, and other mechanisms for increasing the number of targets that can be simultaneously detected in a digital assay. Such functionality is attributed to operation in a regime involving low occupancy of a large number of partitions, such that there is an extremely low probability of overlap between target template molecules within individual partitions. Large partition numbers contribute to significantly low' percentages of doublets (e.g., single partitions occupied by two targets), triplets (e.g., single partitions occupied by three targets), or other forms of multi-plets (single partitions occupied by multiple targets). As such, signals from different amplified target templates distributed across individual partitions can be differentially detected and analyzed in relation to performance of digital assays.

[0107] Additionally or alternatively, the invention(s) can provide functionality for detection of other target analytes in a differentiable and multiplexed manner. In examples, analytes / targets can include one or more of: DNA, RNA, miRNA, nucleic acids,Atty. DocketNo.: 43161-65181 / WO (004WO) polynucleotides, oligonucleotides, proteins, small molecules, single analytes, multianalytes, chemicals, and / or other analytes. For nucleic acid targets, capture probes of compositions and / or processing materials disclosed herein can include complementary molecules to the nucleic acid targets. For protein targets or small molecule targets, capture probes of the compositions and / or processing materials disclosed herein can include antibodies or aptamers conjugated with specific sequences (e.g., nucleic acid sequences) for detection.

[0108] In another specific use case, the invention(s) can provide functionality for detecting and quantifying a panel of synthetic targets, for instance, in order to provide quality controls for biological standards used to standardize assay performance with multiple synthetic targets in different concentrations. In another specific use case, nucleic acids exogenously introduced into cells (e.g.. such as in genome editing applications, such as in CRISPR applications) or viral vectors (e.g., unintentional incorporation of host DNA into manufactured viral vectors) can be measured using the invention(s) disclosed herein (e.g., for genome editing applications, for gene therapy applications).

[0109] The invention(s) can be applied to samples from human organisms, other multicellular animals, plants, fungi, unicellular organisms, viruses, and / or other material, with respect to evaluating presence or absence of sets of targets in parallel. Characterizations of the sets of targets can then be used for diagnostic purposes and / or for generation of targeted therapies to improve states of organisms from which the samples were sourced. The invention(s) can also provide value in research or other non-clinical settings, with or without evaluation and processing of live human or mammalian biological material, and without the immediate purpose of obtaining a diagnostic result of a disease or health condition.

[0110] In combination with a higher number of colors / dyes used for detection, the invention(s) can further improve the number of targets that can be detected from a sample wi thin a single container, in a single-tube workflow.

[0111] The invention(s) confer(s) the benefit of providing non-naturally occurring compositions for facilitating interactions with and amplification of a large set of target analytes from a sample in parallel, with improved efficiency, without utilizing complex microliuidic setups, and in a manner that reduces overall costs. As such, the invention(s) provide a alternative to other methods for detection and digital quantitation of a large number of target analytes in a multiplexed manner.

[0112] Additionally or alternatively, the invention(s) can confer any other suitable benefit.Atty. DocketNo.: 43161-65181 / WO (004WO)8.3. Methods and Materials

[0113] As show n in FIG. 1A, embodiments of a method 100 for multiplexed detection and quantitation of targets include: detecting signals indicative of a profile of a set of targets, from a sample distributed across a set or plurality of partitions (e.g., a high number of partitions at low occupancy) SI 10, and returning a characterization of the sample based upon the profile S120. As shown in FIG. IF, embodiments of a method 200 for multiplexed detection and quantitation of targets include: generating a plurality7of partitions (e.g., at least 500,000 partitions) within a single closed container, wherein the plurality of partitions comprises: a set of targets of a sample, and a set of processing materials S210; reacting the set of processing materials with the set of targets within the plurality of partitions S220; detecting signals indicative of targets of the set of targets from at least a subset of the plurality of partitions S230; and returning a characterization of detected targets of the set of targets of the sample S240. In embodiments, said signals correspond to a set of signal combinatorics and / or other differentiable signals resulting from probes used to tag targets, wherein signal combinatorics of the set of signal combinatorics are paired with targets of the set of targets, and where the set of targets has a total number greater than the number of channels used to detect signals (e.g.. color signals, fluorescent signals) corresponding to the set of signal combinatorics. As such, the methods can provide unique labeling for multiplexed characterization of a panel of targets based upon signal combinatorics, without requiring traditional multiplexing based solely upon signal amplitudes.

[0114] In a variation, as shown in FIG. IB, a method 100b for high sensitivity detection of target proteins of a sample can include: detecting signals indicative of a profile of a set of protein targets from a sample distributed across a set or plurality of partitions SI 10b, and returning a characterization of detected proteins of the set of protein targets SI 20b. In a variation, as shown in FIG. 1G, a method 200b for high sensitivity detection of target proteins of a sample can include: generating a plurality of partitions (e.g., at least 500,000 partitions) w ithin a single closed container, w herein the plurality of partitions comprises: a set of target proteins of the sample, and a set of processing materials S210b; reacting the set of processing materials w ith the set of target proteins w ithin the plurality7of partitions S220b; detecting signals indicative of target proteins of the set of target proteins from at least a subset of the plurality of partitions S230b; and returning a characterization of detected target proteins of the set of target proteins of the sample S240b. Exemplary protein targets can include capsid proteins, proteins associated with viral vectors (e.g., AAV vectors), and / or other suitableAtty. DocketNo.: 43161-65181 / WO (004WO) proteins. Exemplary aspects of the characterization can include one or more of: full / partially filled / empty capsid ratios, viral and / or vector genome integrity (e.g., AAV genome integrity for determining doses and / or conducting dose-escalation studies), particle titer characteristics, fragmented vector aspects, target linkage characteristics, and / or other suitable characteristics. In variations and examples, the methods 100b and / or 200b can be applied to generation of genome titer characterizations (e.g., viral titer measurements) for viral stocks and samples including extracted DNA, with analyses of one or more target regions. In variations and examples, the methods 100b and / or 200b can be applied to generation of vector copy number characterizations (e.g., VCN measurements) for clones and samples including promoter components, cargo components, terminator components, and host components. In variations and examples, the methods 100b and / or 200b can be applied to generation of purity characterizations for cell and / or gene therapy applications, with analysis of viral stocks and samples including extracted DNA, involving analyses of one or more target regions, host DNA targets, and mycoplasma targets. In variations and examples, the methods 100b and / or 200b can be applied to generation of integrity measurements for digested plasmid samples, with analyses of one or more targets of samples, including promoter targets, cargo targets, terminator targets, and inverted terminal repeat sequences.

[0115] In exemplary workflows, plasmid manufacturing can involve: fermentation, plasmid recovery, purification, and formulation. In exemplary workflows, viral manufacturing can involve: expansion, transfection (e.g., with manufactured plasmids), clarification, concentration, purification, and sterilization (with characterization of purify, genome titer, and integrity aspects, as disclosed herein). In exemplary' workflows, cell processing can involve: isolation and enrichment, activation and modification (e.g., with outputs of sterilization from viral manufacturing), expansion, harvesting, and formulation, with analysis of viral copy number and functional titer. Exemplary workflows involving embodiments, variations, and examples of methods 100b, 100c, lOOd, 200b, 200c, and 200d disclosed herein can thus support qualify control in relation to steps for: Viral Stock Concentration (Genome Titer) with measuring total genomes in a sample (e.g., 1 x 1012vg / mL); detecting full versus partial integration (e.g., with determination of the proportion of full genomes) and calculating the concentration of full genomes (e g., vector genomes or other genomes); performing purify check steps; performing viral titer (i.e., Functional Titer) steps by performing a transduction assay to measure infectivity; performing transduction of target cells; performing VCN testing; and preparing transduced cells for in-vivo administration by expand cells.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0116] In a variation, as shown in FIG. 1C, a method 100c for high sensitivity detection of vector targets of a sample can include: detecting signals indicative of a profile of a set of vector targets from a sample distributed across a set or plurality of partitions SI 10c, and returning a characterization of detected vector targets of the set of vector targets S 120c. In a variation, as show n in FIG. 1H, a method 200c for high sensitivity7detection of vector targets of a sample can include: generating a plurality of partitions (e.g., at least 500,000 partitions) within a single closed container, wherein the plurality of partitions comprises: a set of vector targets of the sample, and a set of processing materials S210c; reacting the set of processing materials with the set of vector targets within the plurality7of partitions S220c; detecting signals indicative of vector targets of the set of vector targets from at least a subset of the plurality of partitions S230c: and returning a characterization of detected vector targets of the set of vector targets of the sample S240c. In specific examples, the set of vector targets can include: a gene therapy cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. Exemplary aspects of the characterization can include one or more of: full / partially filled / empty capsid ratios, viral and / or vector genome integrity (e.g., AAV genome integrity for determining doses and / or conducting doseescalation studies), particle titer characteristics, fragmented vector aspects, target linkage characteristics, and / or other suitable characteristics. In variations and examples, the methods 100b and / or 200b can be applied to generation of genome titer characterizations (e.g., viral titer measurements) for viral stocks and samples including extracted DNA. with analyses of one or more target regions. In variations and examples, the methods 100c and / or 200c can be applied to generation of vector copy number characterizations (e.g., VCN measurements) for clones and samples including promoter components, cargo components, terminator components, and host components. In variations and examples, the methods 100c and / or 200c can be applied to generation of purity characterizations for cell and / or gene therapy applications, w ith analysis of viral stocks and samples including extracted DNA, involving analyses of one or more target regions, host DNA targets, and mycoplasma targets. In variations and examples, the methods 100c and / or 200c can be applied to generation of integrity measurements for digested plasmid samples, w ith analyses of one or more targets of samples, including promoter targets, cargo targets, terminator targets, inverted terminal repeat (ITR) targets, long terminal repeat (LTR) targets, Rep targets, and helper targets.

[0117] The characterization can entail a gene therapy vector analysis. In more detail, by examining the co-localization of all targets (e.g., four or more vector targets disclosed herein) within the full-length vector, the invention(s) can enable identification of the fraction ofAtty. DocketNo.: 43161-65181 / WO (004WO) virions (e.g., AAV virions) within the stock that have full length DNA and / or a full-length vector genome (e.g., as an evaluation of vector integrity). The presence of less than the anticipated signals for the targets (e.g., four or more co-localized signals) indicates partially filled vectors, offering a comprehensive assessment of manufacturing quality, and providing a metric characterizing full capsid values, a metric characterizing partially filled capsid values, a metric characterizing empty capsid values, a metric characterizing full versus empty capsid ratios, a metric characterizing full versus partially filled capsid ratios, a combination thereof, and / or other suitable metrics. Systems, methods, and compositions, such as those disclosed herein, that support 4+ detection channels, enable this assay design to work for gene therapy vector analysis.

[0118] In some embodiments of the methods 100, 100b. 100c. lOOd, 200, 200b. 200c, and 200d, returning the characterization comprises a linkage analysis of a full-length vector genome comprising a linked set of vector targets characteristic of the full-length vector genome, the linked set of vector targets characteristic of the full-length vector genome comprising two or more vector targets of the set of vector targets. In some embodiments, the linkage analysis provides a metric characterizing integrity of the full-length vector genome. In some embodiments, the metric characterizing the integrity of the full-length vector genome is determined from counts of the linked set of vector targets characteristic of the full-length vector genome, counts of fragments of the linked set of vector targets characteristic of the full-length vector genome, counts of unlinked vector targets of the set of vector targets, or a combination thereof. In some embodiments, the linked set of vector targets characteristic of the full-length vector genome, the fragments of the linked set of vector targets characteristic of the full-length vector genome, the unlinked vector targets of the set of vector targets, and amplicons thereof, comprise a length of up to 2000 base pairs. In some embodiments, returning the characterization comprises identification of a fraction of virions of the sample comprising a full-length vector genome. In some embodiments, returning the characterization provides a metric characterizing full capsid values associated with virions of the sample, a metric characterizing partially filled capsid values associated with virions of the sample, a metric characterizing empty capsid values associated with virions of the sample, a metric characterizing full versus empty capsid ratios associated with virions of the sample, a metric characterizing full versus partially filled capsid ratios associated with virions of the sample, or a combination thereof. In some embodiments, returning the characterization comprises identification of a concentration of a full-length vector genome of the sample. In some embodiments, returning the characterization provides a count of the full-length vectorAtty. DocketNo.: 43161-65181 / WO (004WO) genomes of the sample. In some embodiments, returning the characterization comprises two or more of: a linkage analysis of a full-length vector genome comprising a linked set of vector targets characteristic of the full-length vector genome; identification of a fraction of virions of the sample comprising a full-length vector genome; and identification of a concentration of a full-length vector genome of the sample.

[0119] The methods 100c and / or 200c can be combined with proximity ligation assay (PLA) steps for capsid-dependent (e.g., AAV capsid-dependent) signal detection. Traditionally, enzyme-linked immunosorbent assays (ELISAs) for protein detection suffer from low sensitivity and high background noise; however, PLAs can achieve a high specificity by utilizing multiple recognition with multiple types of antibodies, thereby achieving high sensitivity by DNA amplification. Thus far, PLAs demonstrated in the context of qPCR have failed to provide suitable absolute quantification performance. The systems, methods, and compositions disclosed herein can incorporate PLAs with provision of unprecedented dynamic range, and avoidance of unlinked targets that unintentionally enter a single partition (obviating the need for linkage analysis). As such, using the PLA approach in combination with partitioning and material aspects disclosed herein, and measuring proteins (e.g., such as capsids and DNA inside AAVs, simultaneously) in partitions, it is possible to identify whether AAVs are full capsids or empty capsids over high dynamic ranges. In an example, the PLA approach can involve an AAV vector component as target protein, one or more antibodies (e.g., a rabbit antibody and a mouse antibody) for the AAV vector, and a kit that can link antibodies through a PLA approach, for AAV capsid detection.

[0120] As such, embodiments, variations, and examples of methods 100c and / or 200c leverage partitioning performance and format aspects disclosed herein, for detection of particles / components (e.g., AAV particles / components) across a wide dynamic range, with the ability to interrogate vector integrity (e.g., in relation to full vs. empty' capsid ratios, in relation to full vs. partially filled capsid ratios) in an unprecedented manner.

[0121] In a variation, as show n in FIG. ID, a method lOOd for high sensitivity detection of undesired DNA (e.g., contaminating DNA, residual DNA, residual host DNA, etc.) during gene therapy vector manufacturing can include: detecting signals indicative of an undesired DNA target component from a sample distributed across a set or plurality’ of partitions SI lOd, and returning a characterization of the undesired DNA target component S120d. In a variation, as shown in FIG. IK, a method 200d for high sensitivity detection of undesired DNA targets (e.g., contaminating DNA. residual DNA, residual host DNA. etc.) during gene therapy and / or cell therapy vector manufacturing can include: generating a plurality ofAtty. DocketNo.: 43161-65181 / WO (004WO) partitions (e.g., at least 500,000 partitions) within a single closed container, wherein the plurality of partitions comprises: a set of undesired DNA targets of a sample, and a set of processing materials S210d; reacting the set of processing materials with the set of undesired DNA targets within the plurality7of partitions S220d; detecting signals indicative of undesired DNA targets of the set of undesired DNA targets from at least a subset of the plurality of partitions S230d; and returning a characterization of detected undesired DNA targets of the set of undesired DNA targets of the sample S240d.

[0122] In one example of methods lOOd and / or 200d, the undesired DNA target component can involve a panel of targets that span all or a portion of the human genome, where the panel of targets can include a set of discrete target regions of the human genome. In relation to this example, the method can include steps for amplification of host DNA within partitions of a partition matrix, using method steps and materials disclosed herein (e.g., with implementation of targeted PCR). The panel of targets can be designed as a panel of primer sets that cover selected regions of the host human genome. All targets of the panel of targets can be tagged with probes that can be detected using a single channel (or alternatively, a set of channels). The set of processing materials used for amplification and detection can include materials disclosed in relation to Step SI 30, Step 210d, Step 220d, or any other step(s) disclosed herein. Alternatively, the set of processing materials used for amplification and detection can omit Taqman chemistry materials.

[0123] In another example of methods lOOd and / or 200d, the undesired DNA target component can include a panel of targets specific for each chromosome of interest in relation to the undesired DNA target component. The panel of targets can be designed as a panel of primer sets that cover a given chromosome or chromosomes. All targets of the panel of targets can be tagged with probes that can be detected using a set of channels, for differential detection based upon signal combinatorics (e.g., with targets belonging to particular chromosome being tagged and amplified in order to produce a signal corresponding to the particular chromosome), as disclosed in relation to Steps S130, Step 210d, Step 220d, subsequent steps, or any other step(s) provided herein. The set of processing materials used for amplification and detection can include materials disclosed in relation to Step SI 30, Step 210d, Step 220d, or any other step(s) disclosed herein. Alternatively, the set of processing materials used for amplification and detection can omit Taqman chemistry materials. In relation to this example, differential tagging of targets for each chromosome can be used to produce analyses of host contamination enriched for specific chromosomes versus host contamination that is uniform across chromosomes.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0124] In another example of methods lOOd and / or 200d, the undesired DNA target component can include a single target that exists multiple times in the human genome. The single target can include, for example, Alu / LINE / SINE elements (e.g., ancient transposons), ribosomal RNA (rRNA) genes, transfer RNA (tRNA) genes, or other repeat elements. The human genome contains approximately 1.5 million copies of Alu elements (-300 bp each) and make up about 10% of the total DNA in the human genome, and such Alu targets can be detected in relation to methods lOOd and / or 200d, for detection of residual host DNA during gene therapy viral vector manufacturing. The target can be tagged with one or more probes that can be detected using a channel, and using processing materials, as disclosed in relation to Steps S130, Step 21 Od, Step 220d, subsequent steps, or any other step(s) provided herein. The set of processing materials used for amplification and detection can include materials disclosed in relation to Step SI 30, Step 210d, Step 220d, or any other step(s) disclosed herein.

[0125] In another example of methods lOOd and / or 200d, detection of the undesired DNA target component can leverage human DNA methylation patterns. In this example, methyldependent restriction endonucleases can be used to cut human DNA at specific methyl positions, which create ligation-ready overhangs that can be tagged and detected. In a specific example, MspJI is a methyl-dependent restriction endonuclease, which only cuts human DNA at specific methyl-C positions, creating ligation-ready overhangs. After digestion, ligation of adapter DNA with known primer sites can be amplified and detected, as disclosed in relation to Step SI 30, Step 21 Od, Step 220d, subsequent steps, or any other step(s) provided herein. Use of MspJI can have an advantage to more un-biased approaches. For instance, use of MspJI can capture more host DNA compared to PCR targeted amplification. However, other restriction endonucleases can be used (e.g., isoschizomers of MspJI).

[0126] For PCR target amplification and detection methods associated with examples of methods lOOd and / or 200d disclosed herein, samples of viral vector preparations can be mixed with primers / probes as disclosed in relation to Step SI 30, Step 21 Od, Step 220d, or any other step(s) herein. Single molecules are then distributed across generated partitions, where the distribution of single molecules includes the vector DNA (e.g., AAV vector DNA) plus any host contaminating DNA (e.g., residual DNA). Contents of partitions of the partition matrices are then amplified (e.g., using PCR), and scanned, according to steps subsequent to step S130, Step 210d, or Step 220d. In relation to returned analyses, counts of host targets relative to viral counts equals the observed fraction of contamination, which is used to guide manufacturing quality control decisions.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0127] For methylation-leveraging methods associated w ith examples of methods lOOd and / or 200d disclosed herein, samples are digested (e.g., with MspJI restriction endonuclease, an isoschizomer of MspJI restriction endonuclease, or any other suitable restriction endonuclease), followed by ligation w ith an adapter pool covering all combinations of overhangs resulting from the restriction endonuclease that adds known primer sites. The ligated sample library is purified (e.g.. using SPRI purification) to remove buffer and unused adapters. Samples of viral vector preparations are then mixed with primers and probes as disclosed in relation to Step SI 30, Step 210d, Step 220d, or any other step(s) herein. Single molecules are then distributed across generated partitions, w here the distribution of single molecules includes the vector DNA (e.g., AAV vector DNA) plus any host contaminating DNA (e.g.. residual DNA). Contents of partitions of the partition matrices are then amplified (e.g., using PCR), and scanned, according to steps subsequent to step SI 30, Step 21 Od, or Step 220d. In relation to returned analyses, counts of host targets relative to viral counts equals the observed fraction of contamination, which is used to guide manufacturing qualitycontrol decisions.

[0128] In variations of the methods lOOd and / or 200d, other undesired target components that represent residual or otherwise contaminating factors that should not or are not desired to pass forw ard during cell therapy and / or gene therapy vector manufacturing can be detected (e.g., nucleic acid components, protein components, other biological components, other contaminating components, etc.). Embodiments, variations, and examples of methods lOOd and / or 200d can achieve greater detection sensitivity in relation to the high mass input capacity of the inventions disclosed herein. Embodiments, variations, and examples of methods lOOd and / or 200d can achieve faster time to results because the methods lOOd and / or 200d are not limited by NGA sequencing time and analysis. Embodiments, variations, and examples of methods lOOd and / or 200d also solve the issue of quality- control for gene therapy vector manufacture, to determine the fraction of host DNA contamination (i.e. human DNA from cell cultures), without the need for NGS and following analyses. As such, methods lOOd and / or 200d can support cell and gene therapy development in an unprecedented way, by leveraging unique molecular approaches for detection of host DNA. The high mass input capacity of the systems, methods, and compositions disclosed herein also allows for more sensitive residual host DNA detection, in part because of the wide dynamic range.

[0129] Methods disclosed herein can further include steps for processing a sample or providing an environment for producing the processed sample such that it can produceAtty. DocketNo.: 43161-65181 / WO (004WO) signals indicative of the targets and / or profde(s), where processing the sample can include (e.g., as show n in FIG. IE): combining a sample with a set of processing materials, the set of processing materials comprising a) a set of primers (e.g., for each of the set of targets, a set of target-specific forward primers corresponding to different targets of the set of targets, and a common reverse primer for the set of target-specific forward primers; or other primer designs), and b) a master mixture including amplification reagents SI 30; distributing the sample with the set of processing materials, across a set or plurality of partitions of a partition matrix (e.g., a high number of partitions at low occupancy, such that different targets of the set of targets do not co-inhabit a single partition) SI 40; performing target-specific tagging and amplification, with the set of processing materials, for target regions associated with the set of targets across a set of stages SI 50; and detecting signals indicative of a profile of the set of targets (e.g., detecting signals from each of the set or plurality of partitions) S160.

[0130] The methods function to enable detection of genetic variations in biological sample material, in a multiplexed manner. In more detail, the methods enable performance of ultra- high multiplexed target detection by implementing a high number of partitions (e.g., at least 50,000 partitions, at least 100,000 partitions, at least 200,000 partitions, at least 500,000 partitions, at least 1 million partitions, at least 10 million partitions, at least 20 million partitions, at least 30 million partitions, at least 50 million partitions, at least 100 million partitions, more than 50,000 partitions, more than 100,000 partitions, more than 200,000 partitions, more than 500,000 partitions, more than 1 million partitions, more than 10 million partitions, more than 20 million partitions, more than 30 million partitions, more than 50 million partitions, more than 100 million partitions, etc.) with low-occupancy (e.g., less than 15% occupancy, less than 10% occupancy, less than 8% occupancy, less than 5% occupancy, etc.) of partitions by targets. In particular, use of a low-occupancy platform involving high numbers of partitions provides a regime where the probability of encountering more than one target in a partition is very low such that a unique color combination can be inferred from the particular target color-coded by the unique color combination.

[0131] In relation to detection and effective use of sample processing materials, the invention(s) involve detection of signals from targets of interest of a processed sample, where the signals correspond to different color combinatorics of a set of color combinatorics, alone or in combination with other types of differentiable signals, where color combinatorics of the set of color combinatorics are paired with targets of the set of targets, and where the set of targets has a total number greater than the number of color channels used to detect colors corresponding to the set of color combinatorics. In particular, due to the high-degree ofAtty. DocketNo.: 43161-65181 / WO (004WO) partitioning described, any positive partition (e.g., partition of an emulsion generated from the sample and containing a target) will contain only one color combination corresponding to fluorescent materials used during processing of the sample, thereby providing an accurate mechanism for multiplexed detection.

[0132] In specific examples, the method(s) can provide a multi-color combinatoric scheme with 5-color assay. As disclosed in more detail herein, the methods can provide mechanisms for multi-color combinatorics using competitive target-specific or allele-specific assays (e.g., Kompetitiv allele-specific PCR (KASP), PCR allele competitive extension (PACE), etc.) and / or other assay chemistries, because they involve no additional probe sequence within generated amplicons, provide a low degree of assay complexity, and thus result in significantly reduced assay cost for a panel of targets. In variations, such assays can be based upon target-specific (e.g., allele-specific) oligonucleotide extension and fluorescence resonance energy' transfer for signal generation. In alternative variations, such assays can be based upon generation and detection of other types of signals.

[0133] The methods can further provide functionality for multiplexed detection of genetic variants in a sample by optimizing the amount of information obtained using lower-cost and / or a reduced set of sample processing materials compared to traditional assays based upon fluorescent detection, involving a higher number of primer ty pes, probe ty pes, quencher types, and probe additives. In combination with a higher number of colors / dyes used for detection, the methods can further improve the number of targets that can be detected from a sample within a single container.

[0134] The method(s) can be implemented by embodiments, variations, and examples of system components described in U.S. Application number 17 / 230,907 filed on 14-APR-2021 and / or U.S. Application number 17 / 687.080 filed 04-MAR-2022. which are each hereby incorporated in its entirety by this reference. Additionally or alternatively, the method(s) can be implemented by other system elements.8.3.1. Method - Assay Materials and Compositions8.3.I.I. Sample Types and Targets

[0135] In variations, the methods 100 and / or 200 can be used to process sample types including biological fluids including or derived from one or more of: blood (e.g., whole blood, peripheral blood, non-peripheral blood, blood lysate, etc ), plasma, serum, saliva, reproductive fluids, mucus, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluids,Atty. DocketNo.: 43161-65181 / WO (004WO) otic fluid, sweat, interstitial fluid, synovial fluid, cerebral-spinal fluid, urine, gastric fluids, biological waste, other biological fluids; tissues (e.g., homogenized tissue samples); food samples; liquid consumable samples; and / or other sample materials. Samples can be derived from human organisms, other multicellular animals, plants, fungi, unicellular organisms, viruses, and / or other material. In specific examples, samples processed can include maternal samples (e.g., blood, plasma, serum, urine, chorionic villus, etc.) including maternal and fetal material (e.g., cellular material, cell-free nucleic acid material, other nucleic acid material, etc.) from which prenatal detection or diagnosis of genetic disorders (e.g., aneuploidies, genetically inherited diseases, other chromosomal issues, etc.) can be performed.

[0136] In another specific example, samples processed can include samples from cell and / or gene therapy development processes, including samples from one or more of: a plasmid manufacturing process, a viral manufacturing process, a cell processing process, or any other process for the development of cell and / or gene therapies. In some embodiments, samples can include those from, or be derived from, a step associated with a plasmid manufacturing process, including one or more of: a fermentation step, a plasmid recovery step, a purification step, a formulation step, or any other step of a plasmid manufacturing process. In some embodiments, samples can include those from, or be derived from, a step associated with a viral manufacturing process, including one or more of: an expansion step, a transfection step, a clarification step, a concentration step, a purification step, a sterilization step, or any other step of a viral manufacturing process. In some embodiments, samples can include those from, or be derived from, a step associated with a cell processing process, including one or more of: an isolation step, an enrichment step, an activation step, a modification step, an expansion step, a harvesting step, a formulation step, or any other step of a cell processing process.

[0137] In embodiments, targets detected in a multiplexed manner according to embodiments, variations, and examples of the methods 100 and / or 200 can include: nucleic acids (e.g., DNA, RNA, miRNA, polynucleotides, oligonucleotides, etc.), proteins, amino acids, peptides, small molecules, single analytes, multianalytes, chemicals, and / or other target material. Detection of such targets can enable genomic, proteomic, and / or other multi-omic characterizations and diagnoses for vanous applications, including: genome editing, plasmid manufacturing (e.g., fermentation steps, plasmid recovery steps, purification steps, formulation steps, or any other steps of plasmid manufacturing), viral and / or vector manufacturing (e.g., expansion steps, transfection steps, clarification steps, concentration steps, purification steps, sterilization steps, or any other steps of viral and / or vector manufacturing), cell processing (e.g., isolation steps, enrichment steps, activation steps.Atty. DocketNo.: 43161-65181 / WO (004WO) modification steps, expansion steps, harvesting steps, formulation steps, or any other steps of cell processing), gene therapy development, cell therapy development, and quality control aspects of these and other applications. Characterization and diagnoses can include: purity (e.g., with respect to residual host DNA, mycoplasma, viral concentration), genome titer (e.g. viral concentration in sample), genome and / or transgene integrity (e.g., assessing proportion of viral particles containing a full vector genome and / or transgene, for example by distinguishing physically linked targets versus unlinked targets), functional titer (e.g., assessing viral infectivity, for example to calculate input dose of viral vector), and vector copy number (e.g., assessing the average number of copies of viral vector within a target sell), full / empty capsid ratios and full / partially filled capsid ratios). Genetic targets can include one or more of: single nucleotide polymorphisms (SNPs), copy number variations (CNVs), insertions, deletions, genes, methylated loci, and / or other loci of interest.

[0138] In variations, SNPs tagged in a massively parallel manner and detected in a multiplexed manner according to methods disclosed herein can include SNPs associated with any chromosome having a minor allele fraction (MAF) greater than 0.4. SNPs evaluated can alternatively be characterized by MAF above another suitable threshold (e.g., MAF > 0.2, MAF > 0.3, etc.). SNPs evaluated can be for coding regions (e.g., synonymous, non- synonymous, missense, nonsense) and / or non-coding regions. SNPs evaluated can be biallelic or multiallelic, with more than two alleles per SNP.

[0139] In examples, SNPs can be associated with chromosomes 13, 18, 21, X, Y. and / or other chromosomes, at various loci (e.g., from 10 to 20,000 polymorphic loci); however, SNPs evaluated can additionally or alternatively be associated with other chromosomes and / or loci. Furthermore, the size of the panel of targets can be determined based upon the likelihood of detecting at least one SNP that is homozygous in the mother and heterozygous in the fetus, such that it can be used as a marker for estimation of FF.

[0140] The size of the SNP panel being evaluated, threshold MAF for each SNP, and chromosomal distribution can thus be selected to optimize or otherwise increase the probability of returning an accurate estimate of FF or other characterization, based upon the methods disclosed herein.

[0141] Furthermore, SNPs selected for evaluation can have allele pairs that are well- discriminated (e.g., with respect to stabilizing-destabilizing characteristics). For instance, SNPs can be selected with prioritization of G / T, C / A, and T / A SNPs having high destabilization strength characteristics.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0142] In examples, SNPs can include one or more of: rs2737653 with G / T alleles, rs2737654 with T / G alleles, rsl 160680 with C / T alleles, rs701232 with C / T alleles, rsl736442 with A / G alleles, rs7232004 with G / T alleles, rsl498553 with C / T alleles, and other suitable SNPs.

[0143] Additionally or alternatively, in other specific applications, target material tagged in a multiplexed manner and evaluated according to methods disclosed herein can provide diagnostics and / or characterizations in relation to one or more of: monitonng or detection of products (e.g., proteins, chemicals) released from single cells (e.g., interleukins or other compounds released from immune cells); monitoring cell survival and / or division for single cells; monitoring or detection of enzymatic reactions involving single cells; antibiotic resistance screening for bacteria; characterization of pathogens in a sample (e.g., in relation to infections, sepsis, in relation to environmental and food samples, etc.); microbiome characterizations (e.g., based upon detection of hypervariable regions of rRNA); characterization of heterogeneous cell populations in a sample; characterization of individual cells or viral particles; monitoring of viral infections of a single host cell; liquid biopsies and companion diagnostics; detection of cancer forms from various biological samples (e.g., from cell-free nucleic acids, tissue biopsies, biological fluids, feces, etc.) based upon characterization of target panels; detection and / or monitoring of minimal residual diseases; monitoring responses to therapies; detection or prediction of rejection events of transplanted organs; other diagnostics associated with other health conditions; other characterizations of statuses of other organisms; and other suitable applications.8.3.1.1.1. Target Proteins

[0144] In a variation of the methods 100 and / or 200, the methods 100b and / or 200b provide for high sensitivity detection of target proteins of a sample. In some embodiments, the sample comprises a set of target proteins. In some embodiments, the sample comprises a viral vector. In some embodiments, the viral vector is selected from an adeno-associated virus (AAV) viral vector, an adenovirus (AdV) viral vector, a lentivirus viral vector, a retrovirus viral vector, and any other suitable viral vector. In some embodiments, the set of target proteins comprises a protein associated with the viral vector. In some embodiments, the set of target proteins comprises a capsid protein.

[0145] In variations, the viral vector is an adeno-associated virus (AAV) viral vector. In some embodiments, the capsid protein is a VP1, VP2, or VP3 capsid protein. In someAtty. DocketNo.: 43161-65181 / WO (004WO) embodiments, the capsid protein is a naturally occurring VP1, VP2. or VP3 capsid protein. In some embodiments, the capsid protein is a non-naturally occurring VP1, VP2, or VP3 capsid protein. In some embodiments, the non-naturally occurring VP1, VP2, or VP3 capsid protein includes a capsid protein generated by biological or chemical alteration or in silico design, or variation of a naturally occurring AAV capsid protein. In some embodiments, the non- naturally occurring VP1. VP2, or VP3 capsid protein differs in primary amino acid sequence from naturally occurring capsids. In some embodiments, the non-naturally occurring VP I, VP2, or VP3 capsid protein includes a biologic or chemical alteration or variation of a naturally occurring AAV capsid protein other than or in addition to a change in the primary amino acid sequence.

[0146] Accordingly, in some embodiments, the AAV capsid protein includes, but is not limited to, a capsid protein of various AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13) or a variant thereof. A non-naturally occurring VP1, VP2, or VP3 capsid protein can further include an artificial capsid protein created by in silico design or synthesis. An artificial capsid protein includes, but is not limited to, AAV capsid proteins disclosed inPCT / US2014 / 060163, USP9695220, PCT / US2016 / 044819, PCT / US2018 / 032166, PCT / US2019 / 031851, and PCT / US2019 / 047546, which are incorporated herein by reference in their entireties.

[0147] In some embodiments, the AAV capsid protein is the capsid protein of AAV9 (Genbank Ace. No: AAS99264.1), AAV1 (Genbank Ace. No: AAD27757.1), AAV2 (Genbank Ace. No: AAC03780.1), AAV3 (Genbank Ace. No: AAC55049.1), AAV3b (Genbank Ace. No: AF028705.1), AAV4 (Genbank Ace. No: AAC58045.1), AAV5 (Genbank Ace. No: AAD13756.1), AAV6 (Genbank Ace. No: AF028704.1). AAV7 (Genbank Ace. No: AAN03855.1), AAV 8 (Genbank Ace. No: AAN03857.1), AAV10 (Genbank Ace. No: AAT46337.1), AAVrhlO (Genbank Ace. No: AY243015.1), AAV11 (Genbank Ace. No: AAT46339.1), AAV12 (Genbank Ace. No: ABI16639.1), AAV13 (Genbank Ace. No: ABZ10812. 1), or AAVpol (Genbank Ace. No: FJ688147.1).

[0148] In some embodiments, the AAV capsid protein can be VP1 capsid protein having a sequence selected from: (AAV1 (AAD27757)), (AAV2 (AAC03780)), (AAV3 (AAC55049)), (AAV5 (AAD13756)), (AAV6 (AAB95450)), (AAV7 (AF513851_2)), (AAV8 (AF513852_2)), (AAV9 (AAS 99264)), (AAV10 (AAT46337)), (AAV hu.68), Anc80L65. and AAV9. The AAV capsid protein can be a VP2 or VP3 protein having a part of one of the sequences. For example, VP2 protein can have a sequence corresponding toAtty. DocketNo.: 43161-65181 / WO (004WO) amino acids 138 to 736 of AAV9 VP1 and VP3 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1 protein.

[0149] In some embodiments, the AAV capsid protein can be VP1 capsid protein having any member sequence of the ancestral AAV library selected from (AAV1 (AAD27757)), (AAV2 (AAC03780)), (AAV3 (AAC55049)), (AAV5 (AAD13756)), (AAV6 (AAB95450)), (AAV7 (AF513851_2)), (AAV8 (AF513852_2)), (AAV9 (AAS99264)), (AAV10 (AAT46337)), (AAV hu.68), Anc80L65, and AAV9. The AAV capsid protein can be a VP2 or VP3 protein having a part of one of the sequences. For example, VP2 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1 and VP3 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1 protein.

[0150] In some embodiments, the AAV capsid protein is a liver-toggle mutant described in WO2019 / 217911, which is incorporated by reference in its entirety herein. In some embodiments, the AAV capsid protein comprises one or more modifications as described in WO2019 / 217911 or WO 2021 / 050614, which are both incorporated by reference herein in their entireties. In some embodiments, the AAV capsid protein comprises one or more modifications as described in PCT Application No. PCT / US2022 / 015842, which is herein incorporated by reference in its entirety.

[0151] In some embodiments, the capsid protein is selected from a wild-type or modified capsid protein selected from: AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9. AAV10, AAV11, AAV12, AAV13; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1 -A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-B; hu.45-B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh,19-B; rh.49-B; rh.52-B; rh, 13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu. 9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu. l-C; hu,18-C; hu.3-C; hu.25-C; hu, 15-C; hu, 16-C; hu. l l-C; hu.lO-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40- E; hu.67-E; hu,17-E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; And 13; Ancl26; Ancl27; Anc80L27; Anc80L59;Anc80E60; Anc80L62; Anc80L65; Anc80L33; Anc80E36; Anc80E44; Anc80Ll; And 10; and Anc80DI.

[0152] In some embodiments, the AAV capsid protein is a capsid protein (VP1, VP2 or VP3) of an AAV variant selected from: AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4;Atty. DocketNo.: 43161-65181 / WO (004WO)AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63- B; hu.56-B; hu.45-B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh,19-B; rh.49-B; rh.52-B; rh.l3-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu. 9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.l-C; hu.18-C; hu.3-C; hu.25-C; hu.l5-C; hu.l6-C; hu.l l-C; hu.lO-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.5O-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; hu.42-E; rh.57-E; rh.40-E; rh74; hu.67-E; hu,17-E; hu.6-E; hu.66- E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; Anc80L44; Anc80Ll; Anc80-55, Anc80-129, Anc80- 156, Anc80-751, Anc80-1029, Anc80-1712; AncllO; and Anc8ODL

[0153] In some embodiments, the capsid protein is a capsid protein of an AAV selected from: AAV9; Anc80L65; Anc80-55; Anc80-129; Anc80-156; Anc80-751; Anc80-1029; Anc80-1712; AAV2; AAV1; AAV6; AAV3; AAV4; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-B; hu.45- B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh,19-B; rh.49-B; rh.52-B; rh,13-B; AAV2- B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu. 9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.l-C; hu,18-C; hu.3-C; hu.25-C; hu,15-C; hu, 16-C; hu.ll-C; hu.lO-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu,17-E; hu.6-E; hu.66-E; rh.38-E; hu.32- F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; Ancll3; Ancl26; Ancl27; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L33; Anc80L36; Anc80L44: Anc80Ll; And 10; and Anc80DI. In some embodiments, the Capsid protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% ammo acid sequence identity to the sequence of a AAV capsid protein of an AAV selected from: AAV9;Anc80L65; Anc80-55; Anc80-129; Anc80-156; Anc80-751; Anc80-1029; Anc80-1712; AAV2; AAV1; AAV6; AAV3; AAV4; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44- A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-B; hu.45-B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh,19-B; rh.49-B; rh.52-B; rh,13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu. 9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu. l-C; hu,18-C; hu.3-C; hu.25-C; hu.15-C; hu,16-C; hu.l l-C; hu,10-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E;Atty. DocketNo.: 43161-65181 / WO (004WO) rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu, 17-E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; And 13; Ancl26; Ancl27; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L33; Anc80L36; Anc80L44; Anc80Ll; And 10; and Anc80DI.8.3.1.1.2. Vector Targets

[0154] In a variation of the methods 100 and / or 200. the methods 100c and / or 200c provide for high sensitivity detection of vector targets of a sample. In some embodiments, the vector target is a nucleotide sequence and / or gene. In some embodiments, the vector target is a nucleotide sequence and / or gene encoding genetic information (e.g., encoding for a protein).

[0155] In some embodiments, the sample comprises a set of vector targets. In some embodiments, the set of vector targets comprises a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR) target, a Rep target, a helper target, or any other suitable vector target. In some embodiments, the set of vector targets comprises a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR) target, a Rep target, and a helper target. In some embodiments, the set of vector targets comprises a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, and a long terminal repeat (LTR) target. In some embodiments, the set of vector targets comprises a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a Rep target, and a helper target. In some embodiments, the set of vector targets comprises a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target.

[0156] In some embodiments, the set of vector targets comprises a cargo target. In some embodiments, the cargo target is a gene therapy cargo target. In some embodiments, the gene therapy cargo target is a transgene. In some embodiments, the cargo target (e.g., transgene) is flanked by one or more inverted terminal repeat (ITR) targets (e.g., in a linked set of vector targets). In some embodiments, the cargo target (e.g., transgene) is flanked by two inverted terminal repeat (ITR) targets (e.g., in a linked set of vector targets). In some embodiments, the cargo target comprises a heterologous nucleic acid sequences encoding a reporter gene (e.g., a fluorescent or luminescent reporter). In some embodiments, the cargo target comprises a sequence encoding one or more biomarkers. In some embodiments, the cargoAtty. DocketNo.: 43161-65181 / WO (004WO) target (e.g., transgene) is a gene chosen to improve one or more signs and / or symptoms of a disease, disorder, or condition. In some embodiments, the cargo target and / or transgene may integrate into a host cell genome. In some embodiments, the cargo target and / or transgene is a functional version of a disease associated gene (i.e., gene isoform(s) which are associated with a disease, disorder or condition) found in a host cell. In some embodiments, the cargo target and / or transgene is an optimized version of disease-associated gene found in a host cell (e.g., codon optimized or expression-optimized variant). In some embodiments, the cargo target and / or transgene is a variant of a disease-associated gene found in a host cell (e g., functional gene fragment or variant thereof). In some embodiments, the cargo target and / or transgene is a gene that causes expression of a peptide that is normally expressed in one or more healthy tissues. In some embodiments, the cargo target and / or transgene is a gene that causes expression of a peptide that is normally expressed diseased tissue. In some embodiments, the cargo target and / or transgene comprises a gene encoding a functional protein. In some embodiments, the cargo target and / or transgene can be, for example, a reporter gene (e.g.. beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent polypeptide (GFP), red fluorescent polypeptide (RFP), chloramphenicol acetyltransferase (CAT), or luciferase, or fusion polypeptides that include an antigen tag domain such as hemagglutinin or Myc), or a therapeutic gene (e.g., genes encoding hormones or receptors thereof, growth factors or receptors thereof, differentiation factors or receptors thereof, immune system regulators (e.g., cytokines and interleukins) or receptors thereof, enzymes, RNAs (e.g., inhibitory RNAs or catalytic RNAs), or target antigens (e.g., oncogenic antigens, autoimmune antigens). In some embodiments, the cargo target and / or transgene comprises a nucleotide sequence of SEQ ID NO: 21.

[0157] In some embodiments, the cargo target and / or transgene can be selected depending, at least in part, on the particular disease or deficiency being treated. Simply by way of example, gene transfer or gene therapy can be applied to the treatment of a disease or disorder. The cargo target and / or transgene also can be, for example, an immunogen that is useful for immunizing a subject (e.g., a human, an animal (e.g., a companion animal, a farm animal, an endangered animal). For example, immunogens can be obtained from an organism (e.g., a pathogenic organism) or an immunogenic portion or component thereof.lt would be understood that the methods, systems, and compositions disclosed herein are not to be limited by any particular cargo target and / or transgene.

[0158] In some embodiments, the set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of cargo targets. InAtty. DocketNo.: 43161-65181 / WO (004WO) some embodiments, the set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of cargo targets. In some embodiments, the set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six cargo targets. In some embodiments, the set of vector targets comprises one cargo target. In some embodiments, the set of vector targets comprises two cargo targets. In some embodiments, the set of vector targets comprises three cargo targets. In some embodiments, the set of vector targets comprises four cargo targets. In some embodiments, the set of vector targets does not comprise a cargo target.

[0159] In some embodiments, the set of vector targets comprises a promoter target. In some embodiments, the promoter target is selected from CMV, SV40, EFla, CAG, PGK, Ubc, human beta actin, CBh, CaMKIIa. TEF1, Hl, p5, pl 9, p40. p41, and a combination thereof. In some embodiments, the promoter target is selected from CMV, SV40, EFla, CAG, PGK, UBC, human beta actin, CBh, CaMKIIa, TEF1, Hl, p5, p!9, p40, and p41.

[0160] In some embodiments, the promoter is from an AAV serotype selected from AAV1, AAV2. AAV3. AAV3B. AAV4. AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11. AAV 12, AAV 13, and a variant thereof. In some embodiments, the p5 promoter is an AAV2 p5, AAV4 p5, or AAV 10 p5 promoter. In some embodiments, the pl 9 promoter is a AAV2 p!9, AAV4 p!9, or AAV10 pl9 promoter. In some embodiments, the p40 promoter is an AAV2 p40, AAV4 p40, or AAV 10 p40 promoter.

[0161] In some embodiments, the promoter target has a length of up to 100 bp, up to 150 bp, up to 200 bp, up to 250 bp, up to 300 bp, up to 350 bp, up to 400 bp, up to 450 bp, up to 500 bp, up to 550 bp, up to 600 bp, up to 650 bp, up to 700 bp, up to 750 bp, up to 800 bp, up to 850 bp, up to 900 bp, up to 950 bp, up to 1000 bp, or greater.

[0162] In some embodiments, the promoter target has a length of at least 100 bp, at least150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, at least450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, at least 1000 bp, or greater.

[0163] In some embodiments, the promoter target has a length of about 100-200 bp, 200- 300 bp, 300-400 bp, 400-500 bp, 500-600 bp, 600-700 bp, 700-800 bp, 800-900 bp, 900-1000 bp, or any intermediate length.

[0164] In some embodiments, the set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of promoter targets. In some embodiments, the set of vector targets comprises at least one, at least two, atAtty. DocketNo.: 43161-65181 / WO (004WO) least three, at least four, at least five, at least six, or a greater number of promoter targets. In some embodiments, the set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six promoter targets. In some embodiments, the set of vector targets comprises one promoter target. In some embodiments, the set of vector targets comprises two promoter targets. In some embodiments, the set of vector targets comprises three promoter targets. In some embodiments, the set of vector targets comprises four promoter targets. In some embodiments, the set of vector targets does not comprise a promoter target.

[0165] In some embodiments, the set of vector targets comprises a transcription terminator target. In some embodiments, the transcription terminator target is selected from SV40, hGH, BGH, rbGlob, and a combination thereof. In some embodiments, the transcription terminator target is selected from SV40, hGH, BGH, and rbGlob. In some embodiments, the transcription terminator target comprises SV40. In some embodiments, the transcription terminator target is SV40. In some embodiments, the transcription terminator target comprises hGH. In some embodiments, the transcription terminator target is hGH. In some embodiments, the transcription terminator target comprises BGH. In some embodiments, the transcription terminator target is BGH. In some embodiments, the transcription terminator target comprises rbGlob. In some embodiments, the transcription terminator target is rbGlob. In some embodiments, the transcription terminator target further comprises a polyA transcription termination sequence. In some embodiments, the transcription terminator target further comprises a polyA signal. In some embodiments, the polyA signal comprises a AAUAAA nucleotide sequence motif.

[0166] In some embodiments, the set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of transcription terminator targets. In some embodiments, the set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of transcription terminator targets. In some embodiments, the set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six transcription terminator targets. In some embodiments, the set of vector targets comprises one transcription terminator target. In some embodiments, the set of vector targets comprises two transcription terminator targets. In some embodiments, the set of vector targets comprises three transcription terminator targets. In some embodiments, the set of vector targets comprises four transcription terminator targets. In some embodiments, the set of vector targets does not comprise a transcription terminator target.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0167] In some embodiments, the set of vector targets comprises an inverted terminal repeat (ITR) target. In some embodiments, the inverted terminal repeat (ITR) target comprises an inverted terminal repeat (ITR) from wild-type adeno-associated virus (AAV) or a variant thereof. In some embodiments, the inverted terminal repeat (ITR) target comprises an inverted terminal repeat (ITR) from wild-ty pe adeno-associated virus (AAV). In some embodiments, the inverted terminal repeat (ITR) target comprises an inverted terminal repeat (ITR) from a variant of wild-ty pe adeno-associated virus (AAV). In some embodiments, the inverted terminal repeat (ITR) target comprises an inverted terminal repeat (ITR) from wildtype adenovirus (AdV) or a variant thereof. In some embodiments, the inverted terminal repeat (ITR) target comprises an inverted terminal repeat (ITR) from wild-type adenovirus (AdV). In some embodiments, the inverted terminal repeat (ITR) target comprises an inverted terminal repeat (ITR) from a variant of wild-type adenovirus (AdV).

[0168] In some embodiments, the inverted terminal repeat (ITR) target has a length of up to 10 bp, up to 15 bp, up to 20 bp, up to 25 bp, up to 30 bp, up to 35 bp, up to 40 bp, up to 45 bp, up to 50 bp, up to 55 bp, up to 60 bp, up to 65 bp, up to 70 bp, up to 75 bp, up to 80 bp, up to 85 bp, up to 90 bp, up to 95 bp, up to 100 bp, up to 105 bp, up to 110 bp, up to 115 bp, up to 120 bp, up to 125 bp, up to 130 bp, up to 135 bp, up to 140 bp, up to 145 bp, up to 150 bp, up to 155 bp, up to 160 bp, up to 165 bp, up to 170 bp, up to 175 bp, up to 180 bp, up to 185 bp. up to 190 bp, up to 195 bp, up to 200 bp, up to 205 bp, up to 210 bp, up to 215 bp, up to 220 bp, up to 225 bp, up to 230 bp, up to 235 bp. up to 240 bp. up to 245 bp, up to 250 bp, up to 255 bp, up to 260 bp, up to 265 bp, up to 270 bp, up to 275 bp, up to 280 bp, up to 285 bp, up to 290 bp, up to 295 bp, up to 300 bp, up to 305 bp, up to 310 bp, up to 315 bp, up to 320 bp, up to 325 bp, up to 330 bp, up to 335 bp, up to 340 bp, up to 345 bp, up to 350 bp, up to 355 bp, up to 360 bp, up to 365 bp, up to 370 bp, up to 375 bp. up to 380 bp, up to 385 bp, up to 390 bp, up to 395 bp, up to 400 bp, or greater.

[0169] In some embodiments, the inverted terminal repeat (ITR) target has a length of at least 10 bp, at least 15 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, at least 40 bp, at least 45 bp, at least 50 bp, at least 55 bp, at least 60 bp, at least 65 bp, at least 70 bp, at least 75 bp, at least 80 bp, at least 85 bp, at least 90 bp, at least 95 bp, at least 100 bp, at least 105 bp, at least 110 bp, at least 115 bp, at least 120 bp, at least 125 bp, at least 130 bp, at least 135 bp, at least 140 bp, at least 145 bp, at least 150 bp, at least 155 bp, at least 160 bp, at least 165 bp, at least 170 bp, at least 175 bp, at least 180 bp, at least 185 bp, at least 190 bp, at least 195 bp, at least 200 bp. at least 205 bp. at least 210 bp, at least 215 bp, at least 220 bp, at least 225 bp, at least 230 bp, at least 235 bp, at least 240 bp, at least 245 bp, at least 250 bp, atAtty. DocketNo.: 43161-65181 / WO (004WO) least 255 bp, at least 260 bp, at least 265 bp, at least 270 bp, at least 275 bp, at least 280 bp, at least 285 bp, at least 290 bp. at least 295 bp. at least 300 bp, at least 305 bp, at least 310 bp, at least 315 bp, at least 320 bp, at least 325 bp, at least 330 bp, at least 335 bp, at least 340 bp, at least 345 bp, at least 350 bp, at least 355 bp, at least 360 bp, at least 365 bp, at least 370 bp, at least 375 bp, at least 380 bp, at least 385 bp, at least 390 bp, at least 395 bp, at least 400 bp, or greater.

[0170] In some embodiments, the inverted terminal repeat (ITR) target has a length of about 10-20 bp, 20-30 bp, 30-40 bp, 40-50 bp, 50-60 bp, 60-70 bp, 70-80 bp, 80-90 bp, 90-100 bp, 100-110 bp, 110-120 bp, 120-130 bp, 130-140 bp, 140-150 bp, 150-160 bp, 160-170 bp, 170- 180 bp. 180-190 bp, 190-200 bp, 200-210 bp, 210-220 bp, 220-230 bp, 230-240 bp, 240-250 bp, 250-260 bp, 260-270 bp. 270-280 bp, 280-290 bp. 290-300 bp, 300-310 bp, 310-320 bp, 320-330 bp, 330-340 bp, 340-350 bp, 350-360 bp, 360-370 bp, 370-380 bp, 380-390 bp, 390- 400 bp, or any intermediate length.

[0171] In some embodiments, the set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of inverted terminal repeat (ITR) targets. In some embodiments, the set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of inverted terminal repeat (ITR) targets. In some embodiments, the set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six inverted terminal repeat (ITR) targets. In some embodiments, the set of vector targets comprises one inverted terminal repeat (ITR) target. In some embodiments, the set of vector targets comprises two inverted terminal repeat (ITR) targets. In some embodiments, the set of vector targets comprises three inverted terminal repeat (ITR) targets. In some embodiments, the set of vector targets comprises four inverted terminal repeat (ITR) targets. In some embodiments, the set of vector targets does not comprise an inverted terminal repeat (ITR) target.

[0172] In some embodiments, the set of vector targets comprises a long terminal repeat (LTR) target. In some embodiments, the long terminal repeat (LTR) target comprises a long terminal repeat (LTR) from a wild-type retrovirus or a variant thereof. In some embodiments, the long terminal repeat (LTR) target comprises a long terminal repeat (LTR) from a wildtype retrovirus. In some embodiments, the long terminal repeat (LTR) target comprises a long terminal repeat (LTR) from a variant of a wild-type retrovirus. In some embodiments, the long terminal repeat (LTR) target comprises a long terminal repeat (LTR) from wild-type lentivirus or a variant thereof. In some embodiments, the long terminal repeat (LTR) targetAtty. DocketNo.: 43161-65181 / WO (004WO) comprises a long terminal repeat (LTR) from wild-type lentivirus. In some embodiments, the long terminal repeat (LTR) target comprises a long terminal repeat (LTR) from a variant of wild-type lentivirus.

[0173] In some embodiments, the long terminal repeat (LTR) target has a length of up to 100 bp, up to 150 bp, up to 200 bp, up to 250 bp, up to 300 bp, up to 350 bp, up to 400 bp, up to 450 bp, up to 500 bp, up to 550 bp, up to 600 bp, up to 650 bp, up to 700 bp, up to 750 bp, up to 800 bp, up to 850 bp, up to 900 bp, up to 950 bp, up to 1000 bp, or greater.

[0174] In some embodiments, the long terminal repeat (LTR) target has a length of at least100 bp, at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least400 bp. at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least700 bp. at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, at least1000 bp, or greater.

[0175] In some embodiments, the long terminal repeat (LTR) target has a length of about 100-200 bp, 200-300 bp, 300-400 bp, 400-500 bp, 500-600 bp, 600-700 bp, 700-800 bp, 800- 900 bp. 900-1000 bp, or any intermediate length.

[0176] In some embodiments, the set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of long terminal repeat (LTR) targets. In some embodiments, the set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of long terminal repeat (LTR) targets. In some embodiments, the set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six long terminal repeat (LTR) targets. In some embodiments, the set of vector targets comprises one long terminal repeat (LTR) target. In some embodiments, the set of vector targets comprises two long terminal repeat (LTR) targets. In some embodiments, the set of vector targets comprises three long terminal repeat (LTR) targets. In some embodiments, the set of vector targets comprises four long terminal repeat (LTR) targets. In some embodiments, the set of vector targets does not comprise a long terminal repeat (LTR) target.

[0177] In some embodiments, the set of vector targets comprises a Rep target. In some embodiments, the Rep target comprises a rep gene encoding a Rep protein. In some embodiments, the Rep protein is selected from a Rep 78 protein, a Rep 68 protein, a Rep 52 protein, and a Rep 40 protein. In some embodiments, the rep gene encodes at least one Rep protein selected from a Rep 78 protein, a Rep 68 protein, a Rep 52 protein, and a Rep 40 protein. In some embodiments, the rep gene encodes at least two Rep protein selected from a Rep 78 protein, a Rep 68 protein, a Rep 52 protein, and a Rep 40 protein. In someAtty. DocketNo.: 43161-65181 / WO (004WO) embodiments, the rep gene encodes at least three Rep protein selected from a Rep 78 protein, a Rep 68 protein, a Rep 52 protein, and a Rep 40 protein. In some embodiments, the rep gene encodes at least four Rep protein selected from a Rep 78 protein, a Rep 68 protein, a Rep 52 protein, and a Rep 40 protein. In some embodiments, the Rep protein is from an AAV seroty pe selected from AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV 11, AAV 12, AAV 13, and a variant thereof. In some embodiments, the Rep target comprises at least one promoter.

[0178] In some embodiments, the set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of Rep targets. In some embodiments, the set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of Rep targets. In some embodiments, the set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six Rep targets. In some embodiments, the set of vector targets comprises one Rep target. In some embodiments, the set of vector targets comprises two Rep targets. In some embodiments, the set of vector targets comprises three Rep targets. In some embodiments, the set of vector targets comprises four Rep targets. In some embodiments, the set of vector targets does not comprise a Rep target.

[0179] In some embodiments, the set of vector targets comprises a helper target. In some embodiments, the helper target comprises a helper virus gene. In some embodiments, the helper virus gene is selected from one or more of: Adenovirus 5, Adenovirus 2, E1A / E1B. E2A, E4ORF6, and VA RNA. In some embodiments, the helper virus gene is selected from one or more of: Adenovirus 5 or Adenovirus 2. In some embodiments, the helper virus gene comprises E1A / E1B, E2A, E4 ORF6, and VA RNA.

[0180] In some embodiments, the set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of helper targets. In some embodiments, the set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of helper targets. In some embodiments, the set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six helper targets. In some embodiments, the set of vector targets comprises one helper target. In some embodiments, the set of vector targets comprises two helper targets. In some embodiments, the set of vector targets comprises three helper targets. In some embodiments, the set of vector targets comprises four helper targets. In some embodiments, the set of vector targets does not comprise a helper target.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0181] In some embodiments, vector targets of the set of vector targets are linked (i.e., a linked set of vector targets). In some embodiments, the linked set of vector targets comprises at least a cargo target. In some embodiments, the linked set of vector targets comprises at least a promoter target. In some embodiments, the linked set of vector targets comprises at least a transcription terminator target. In some embodiments, the linked set of vector targets comprises at least an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises at least a long terminal repeat (LTR) target. In some embodiments, the linked set of vector targets comprises at least a Rep target. In some embodiments, the linked set of vector targets comprises at least a helper target. In some embodiments, the linked set of vector targets comprises at least a cargo target and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises at least a cargo target, a promoter target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises at least a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises flanking inverted terminal repeat (ITR) targets.

[0182] In some embodiments, the linked set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of cargo targets. In some embodiments, the linked set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six. or a greater number of cargo targets. In some embodiments, the linked set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six cargo targets. In some embodiments, the linked set of vector targets comprises one cargo target. In some embodiments, the linked set of vector targets comprises two cargo targets. In some embodiments, the linked set of vector targets comprises three cargo targets. In some embodiments, the linked set of vector targets comprises four cargo targets. In some embodiments, the linked set of vector targets does not comprise a cargo target.

[0183] In some embodiments, the linked set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of promoter targets. In some embodiments, the linked set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of promoter targets. In some embodiments, the linked set of vector targets comprises less than one, less than two. less than three, less than four, less than five, or less than six promoter targets. In some embodiments, the linked set of vector targets comprises one promoter target. In someAtty. DocketNo.: 43161-65181 / WO (004WO) embodiments, the linked set of vector targets comprises two promoter targets. In some embodiments, the linked set of vector targets comprises three promoter targets. In some embodiments, the linked set of vector targets comprises four promoter targets. In some embodiments, the linked set of vector targets does not comprise a promoter target.

[0184] In some embodiments, the linked set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of transcription terminator targets. In some embodiments, the linked set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of transcription terminator targets. In some embodiments, the linked set of vector targets comprises less than one. less than two, less than three, less than four, less than five, or less than six transcription terminator targets. In some embodiments, the linked set of vector targets comprises one transcription terminator target. In some embodiments, the linked set of vector targets comprises two transcription terminator targets. In some embodiments, the linked set of vector targets comprises three transcription terminator targets. In some embodiments, the linked set of vector targets comprises four transcription terminator targets. In some embodiments, the linked set of vector targets does not comprise a transcription terminator target.

[0185] In some embodiments, the linked set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of inverted terminal repeat (ITR) targets. In some embodiments, the linked set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of inverted terminal repeat (ITR) targets. In some embodiments, the linked set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six inverted terminal repeat (ITR) targets. In some embodiments, the linked set of vector targets comprises one inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises two inverted terminal repeat (ITR) targets. In some embodiments, the linked set of vector targets comprises three inverted terminal repeat (ITR) targets. In some embodiments, the linked set of vector targets comprises four inverted terminal repeat (ITR) targets. In some embodiments, the linked set of vector targets does not comprise an inverted terminal repeat (ITR) target.

[0186] In some embodiments, the linked set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of long terminal repeat (LTR) targets. In some embodiments, the linked set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or aAtty. DocketNo.: 43161-65181 / WO (004WO) greater number of long terminal repeat (LTR) targets. In some embodiments, the linked set of vector targets comprises less than one. less than two, less than three, less than four, less than five, or less than six long terminal repeat (LTR) targets. In some embodiments, the linked set of vector targets comprises one long terminal repeat (LTR) target. In some embodiments, the linked set of vector targets comprises two long terminal repeat (LTR) targets. In some embodiments, the linked set of vector targets comprises three long terminal repeat (LTR) targets. In some embodiments, the linked set of vector targets comprises four long terminal repeat (LTR) targets. In some embodiments, the linked set of vector targets does not comprise a long terminal repeat (LTR) target.

[0187] In some embodiments, the linked set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of Rep targets. In some embodiments, the linked set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six, or a greater number of Rep targets. In some embodiments, the linked set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six Rep targets. In some embodiments, the linked set of vector targets comprises one Rep target. In some embodiments, the linked set of vector targets comprises two Rep targets. In some embodiments, the linked set of vector targets comprises three Rep targets. In some embodiments, the linked set of vector targets comprises four Rep targets. In some embodiments, the linked set of vector targets does not comprise a Rep target.

[0188] In some embodiments, the linked set of vector targets comprises one or more, two or more, three or more, four or more, five or more, six or more, or a greater number of helper targets. In some embodiments, the linked set of vector targets comprises at least one, at least two, at least three, at least four, at least five, at least six. or a greater number of helper targets. In some embodiments, the linked set of vector targets comprises less than one, less than two, less than three, less than four, less than five, or less than six helper targets. In some embodiments, the linked set of vector targets comprises one helper target. In some embodiments, the linked set of vector targets comprises two helper targets. In some embodiments, the linked set of vector targets comprises three helper targets. In some embodiments, the linked set of vector targets comprises four helper targets. In some embodiments, the linked set of vector targets does not comprise a helper target.

[0189] In some embodiments, the linked set of vector targets comprises one or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR)Atty. DocketNo.: 43161-65181 / WO (004WO) target, a Rep target, and a helper target. In some embodiments, the linked set of vector targets comprises two or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR) target, a Rep target, and a helper target. In some embodiments, the linked set of vector targets comprises three or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR) target, a Rep target, and a helper target. In some embodiments, the linked set of vector targets comprises four or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR) target, a Rep target, and a helper target. In some embodiments, the linked set of vector targets comprises five or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR) target, a Rep target, and a helper target. In some embodiments, the linked set of vector targets comprises six or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR) target, a Rep target, and a helper target. In some embodiments, the linked set of vector targets comprises seven or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR) target, a Rep target, and a helper target. In some embodiments, the linked set of vector targets comprises eight or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a long terminal repeat (LTR) target, a Rep target, and a helper target. In some embodiments, the linked set of vector targets comprises flanking inverted terminal repeat (ITR) targets. In some embodiments, the linked set of vector targets comprises flanking long terminal repeat (LTR) targets.

[0190] In some embodiments, the linked set of vector targets comprises one or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises two or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises three or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In someAtty. DocketNo.: 43161-65181 / WO (004WO) embodiments, the linked set of vector targets comprises four or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises five or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises six or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises seven or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises eight or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises flanking inverted terminal repeat (ITR) targets.

[0191] In some embodiments, the sample comprises a viral vector. In some embodiments, the set of vector targets comprises a viral vector. In some embodiments, the viral vector is selected from an adeno-associated virus (AAV) viral vector, an adenovirus (AdV) viral vector, a lentivirus viral vector, a retrovirus viral vector, and any other suitable viral vector. In some embodiments, the viral vector comprises an adeno-associated virus (AAV) viral vector. In some embodiments, the viral vector comprises an adenovirus (AdV) viral vector. In some embodiments, the viral vector comprises a lentivirus viral vector. In some embodiments, the viral vector comprises a retrovirus viral vector. In some embodiments, the viral vector comprises one or more vector targets of the set of vector targets. In some embodiments, the viral vector comprises a vector genome. In some embodiments, the vector genome comprises one or more vector targets of the set of vector targets. In some embodiments, the vector genome is a full-length vector genome. In some embodiments, the full-length vector genome comprises a linked set of vector targets of the set of vector targets. In some embodiments, the linked set of vector targets is characteristic of the vector genome of the viral vector of the sample and / or set of vector targets. In some embodiments, the linked set of vector targets comprises at least a cargo target. In some embodiments, the linked set of vector targets comprises at least a promoter target. In some embodiments, the linked set of vector targets comprises at least a transcription terminator target. In some embodiments, the linked set of vector targets comprises at least an inverted terminal repeat (ITR) target. InAtty. DocketNo.: 43161-65181 / WO (004WO) some embodiments, the linked set of vector targets comprises at least a cargo target and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises at least a cargo target, a promoter target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises at least a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the adeno-associated virus (AAV) viral vector comprises a vector genome, wherein the full-length vector genome comprises a linked set of vector targets of the set of vector targets, the linked set of vector targets comprising at least a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target. In some embodiments, the linked set of vector targets comprises flanking inverted terminal repeat (ITR) targets.

[0192] In some embodiments, the full-length vector genome has a length of up to 100 bp, up to 150 bp, up to 200 bp, up to 250 bp, up to 300 bp, up to 350 bp, up to 400 bp, up to 450 bp, up to 500 bp, up to 550 bp, up to 600 bp, up to 650 bp, up to 700 bp, up to 750 bp, up to 800 bp, up to 850 bp. up to 900 bp, up to 950 bp, up to 1 (kilo base pairs) kb, up to 1.1 kb, up to 1.2 kb, up to 1.3 kb, up to 1.4 kb, up to 1.5 kb, up to 1.6 kb, up to 1.7 kb, up to 1.8 kb, up to 1.9 kb, up to 2 kb, up to 2. 1 kb, up to 2.2 kb, up to 2.3 kb, up to 2.4 kb, up to 2.5 kb, up to 2.6 kb, up to 2.7 kb, up to 2.8 kb, up to 2.9 kb, up to 3 kb, up to 3. 1 kb, up to 3.2 kb, up to 3.3 kb, up to 3.4 kb, up to 3.5 kb, up to 3.6 kb, up to 3.7 kb, up to 3.8 kb, up to 3.9 kb, up to 4 kb, up to 4. 1 kb. up to 4.2 kb. up to 4.3 kb, up to 4.4 kb, up to 4.5 kb, up to 4.6 kb, up to 4.7 kb. up to 4.8 kb, up to 4.9 kb, up to 5 kb, up to 5. 1 kb, up to 5.2 kb, up to 5.3 kb, up to 5.4 kb, up to 5.5 kb, up to 5.6 kb, up to 5.7 kb, up to 5.8 kb, up to 5.9 kb, up to 6 kb, up to 6.5 kb, up to 7 kb, up to 7.5 kb, up to 8 kb, up to 8.5 kb, up to 9 kb, up to 9.5 kb, up to 10 kb, or greater. In some embodiments, the full-length vector genome has a length of up to 1 kb. In some embodiments, the full-length vector genome has a length of up to 2 kb. In some embodiments, the full-length vector genome has a length of up to 3 kb. In some embodiments, the full-length vector genome has a length of up to 4 kb. In some embodiments, the full-length vector genome has a length of up to 5 kb. In some embodiments, the full-length vector genome has a length of up to 6 kb. In some embodiments, the full-length vector genome has a length of up to 7 kb. In some embodiments, the full-length vector genome has a length of up to 8 kb. In some embodiments, the full-length vector genome has a length of up to 9 kb. In some embodiments, the full-length vector genome has a length of up to 10 kb.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0193] In some embodiments, the full-length vector genome has a length of at least 100 bp, at least 150 bp. at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, at least 1 (kilo base pairs) kb, at least 1.1 kb, at least 1.2 kb, at least 1.3 kb, at least 1.4 kb, at least 1.5 kb, at least 1.6 kb, at least 1.7 kb, at least 1.8 kb. at least 1.9 kb, at least 2 kb, at least 2.1 kb, at least2.2 kb, at least 2.3 kb, at least 2.4 kb, at least 2.5 kb, at least 2.6 kb, at least 2.7 kb, at least 2.8 kb, at least 2.9 kb, at least 3 kb, at least 3.1 kb, at least 3.2 kb, at least 3.3 kb, at least 3.4 kb, at least 3.5 kb, at least 3.6 kb, at least 3.7 kb, at least 3.8 kb, at least 3.9 kb, at least 4 kb, at least 4. 1 kb, at least 4.2 kb, at least 4.3 kb. at least 4.4 kb, at least 4.5 kb, at least 4.6 kb, at least 4.7 kb, at least 4.8 kb, at least 4.9 kb. at least 5 kb. at least 5. 1 kb, at least 5.2 kb, at least5.3 kb, at least 5.4 kb, at least 5.5 kb, at least 5.6 kb, at least 5.7 kb, at least 5.8 kb, at least 5.9 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, at least 10 kb, or greater.

[0194] In some embodiments, the full-length vector genome has a length of about 100-500 bp, about 500 bp-1 kb, about 1-1.5 kb, about 1.5-2 kb, about 2-2.5 kb, about 2.5-3 kb, about 3-3.5 kb, about 3.5-4 kb, about 4-4.5 kb, about 4.5-5 kb, about 5-5.5 kb, about 5.5-6 kb, about 6-6.5 kb, about 6.5-7 kb, about 7-7.5 kb, about 7.5-8 kb, about 8-8.5 kb, about 8.5-9 kb, about 9-9.5 kb, about 9.5-10 kb, or any intermediate length.8.3.1.2. Assay Materials and Compositions for Competitive Target- Specific Assays

[0195] Step S 130 recites: combining the sample with a set of processing materials, which functions to tag and amplify multiple targets of the sample in parallel. Step 210 recites: generating a plurality of partitions (e.g., at least 500,000 partitions) within a single closed container, wherein the plurality of partitions comprises: a set of targets of a sample, and a set of processing materials. In variations of the Step 210, the set of targets comprises and / or is: a set of target proteins (e.g., Step 210b), a set of vector targets (e.g., Step 210c), or a set of undesired DNA targets (Step 210d). The set of processing materials disclosed herein can include fewer components (e.g., requiring just forward and reverse primers, requiring single primers with tandem adapters, using shared probes / quencher oligonucleotides for primers targeting different targets, etc.) to provide detection of multiple targets in parallel. For instance, for a set of colors / wavelengths used for detection and digital quantitation of multiple targets based on color combinatorics, the set of processing materials can includeAtty. DocketNo.: 43161-65181 / WO (004WO) only one probe for each of the set of colors / wavelengths, rather than one probe per target of interest. As such, probes can be designed against a common PCR adapter tagged to forward and / or reverse primers of the set of processing materials, where the number of probes used has a number corresponding to the number of channels for detection, rather than the number of targets, thereby significantly reducing assay cost. As such, the set of processing materials can implement chemistry’ for differential discrimination of partition contents based on color combinatorics, where color combinatorics of a set of color combinatorics are paired with targets of a set of targets of interest, and where the set of targets has a total number greater than the number of color channels used to detect colors corresponding to the set of color combinatorics.

[0196] In embodiments, as shown in FIG. 2, the set of processing materials can include: a) for each of the set of targets, a set of target-specific (e.g., allele-specific) forward primers corresponding to different alleles of a respective target of the set of targets, and a common reverse primer for the set of target-specific (e.g., allele-specific) forward primers, and b) a master mixture including amplification reagent as well as: for each of the set of targets, a set of target-specific (e.g., allele-specific) flanking sequences corresponding to different targets of the set of targets.

[0197] For each target (e.g., SNP, CNV, loci of interest, insertion, deletion, other target) with allelic variations, the set of target-specific forward primers can include an allele-specific forward primer for each allele. As such, the set of target-specific forward primers can include two allele-specific forward primers, or greater than two allele-specific forward primers (e g., 3 allele-specific forward primers, 4 allele-specific forward primers, 5 allele-specific forward primers, etc.) for multiallele variations. The allele-specific forward primers include sequence portions complementary to alleles of targets, such that the primer groups encode the target (e.g., SNP, other target) being evaluated, and colors / fluorophores detected provide indication of which allele of a target is present.

[0198] The allele-specific forward primers for each target are configured to be competing, and include unique tail sequences for each allele. In variations, the tail sequences include oligonucleotides with a label corresponding to a dye / fluorophore that can be detected after sample processing. The label can be positioned at the 5’ end of the forward primer or the 3’ end of the forward primer, or can otherwise be positioned (e.g., at a position intermediate the 3’ and 5’ ends). In variations, each forward primer can include multiple labels. The targetspecific forward primers can incorporate mismatches at or near penultimate sites (e.g., depending upon destabilization effects of allele combinations associated with targets beingAtty. DocketNo.: 43161-65181 / WO (004WO) evaluated). In variations, the common reverse primer can additionally or alternatively include one or more labels, and / or the set of processing materials can include multiple reverse primers.

[0199] Concentrations of forward primers can range from 50 nM to 300 nM in solution, or alternatively, less than 50 nM or greater than 300 nM in solution. Concentrations of reverse primers can range from 100 nM to 600 nM in solution, or alternatively, less than 100 nM or greater than 600 nM in solution. Concentrations of reporter oligonucleotides (e.g., fluorescent reporter oligonucleotides) can range from 30 nM to 200 nM in solution, or alternatively, less than 30 nM or greater than 200 nM in solution. Concentrations of quencher oligonucleotides can range from 100 nM to 600 nM in solution, or alternatively, less than 100 nM or greater than 600 nM in solution.

[0200] Primers (e.g., forward primers, reverse primers) can have lengths of 20 base pairs, 21 base pairs, 22 base pairs, 23 base pairs, 24 base pairs, 25 base pairs, 26 base pairs, 27 base pairs, 28 base pairs, 29 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, an intermediate number of base pairs, or a greater number of base pairs. In variations, primers can incorporate sequence regions corresponding to probes and target sequences (e.g., a 20 base pair target sequence, a target sequence having another suitable length, etc.), and be designed for various levels of plexy (e.g., 1-plex conditions, 2-plex conditions, 3-plex conditions, 4-plex conditions, 5-plex conditions, 6-plex conditions, 7-plex conditions, etc.) as disclosed herein. In variations, forward primers can be longer than reverse primers, and in specific examples, used of forward primers having lengths 5-10 base pairs longer (e.g., than reverse primers, than another reference length) produced higher counts (e.g., 8-10% higher counts) and higher SNR values (e g., 12-17% higher SNR values) in relation to shorter primer lengths, when detecting of targets from partitions, thereby providing higher detection performance.

[0201] Primers (e.g., forward primers, reverse primers) can have annealing temperatures from 480to 65 °C or another suitable annealing temperature range based upon reactions performed according to various assays. Primers (e.g., forward primers, reverse primers) can have melting temperatures from 65 °C to 70 °C (e.g., from 67 °C to 68.8 °C) or another suitable melting temperature range based upon reactions performed according to various assays.

[0202] Characteristics of forward and reverse primers disclosed herein can be reversed (e.g., the set of processing materials can include a forward primer and a set of target-specific reverse primers). Still alternatively, both forward and reverse primers can be target-specific.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0203] As noted herein and shown in FIG. 2, in embodiments of the methods, systems, and compositions disclosed herein, the master mixture can include amplification reagents and, for each of the set of targets, a set of target-specific flanking sequences corresponding to different targets of the set of targets, in order to support multiplexed processing, detection, and digital quantitation. As such, in one variation, the set of processing materials can include, for a target of the set of targets: a primer set comprising: a common primer and a set of target-specific primers configured to interact with a target region of the target, the set of target-specific primers comprising a first target-specific primer comprising a first flanking sequence, and a first fluorophore-labeled oligonucleotide corresponding to the flanking sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified.

[0204] In some embodiments, the set of processing materials comprises, for a first target of the set of targets, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the first target, the set of target-specific primers comprising: a target-specific primer comprising a first flanking sequence, and a first fluorophore-labeled oligonucleotide corresponding to the first flanking sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second target of the set of targets, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the second target, the set of target-specific primers comprising: a target-specific primer comprising a second flanking sequence, and a second fluorophore-labeled oligonucleotide corresponding to the second flanking sequence, the second fluorophore- labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, the set of processing materials comprises, for a first target of the set of targets, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the first target, the set of target-specific primers comprising: a target-specific primer comprising a first adapter sequence, and a first fluorophore-labeled oligonucleotide corresponding to the first adapter sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second target of the set of targets, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the second target, the set of target-Atty. DocketNo.: 43161-65181 / WO (004WO) specific primers comprising: a target-specific primer comprising a second adapter sequence, and a second fluorophore-labeled oligonucleotide corresponding to the second adapter sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, first adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, second adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, the set of processing materials comprises, for a first target of the set of targets: a forward primer corresponding to a target region and comprising a first adapter sequence; a reverse primer; and a first fluorophore- labeled oligonucleotide corresponding to the first adapter sequence, wherein the first fluorophore-labeled oligonucleotide comprises a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second target of the set of targets: a forward primer corresponding to a target region and comprising a second adapter sequence; a reverse primer; and a second fluorophore-labeled oligonucleotide corresponding to the second adapter sequence, wherein the second fluorophore-labeled oligonucleotide comprises a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, first adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, second adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, the set of processing materials comprises, for a first target and a second target of the set of targets, a primer set comprising: at least one primer configured to tag the first target with a first probe having a first fluorophore, and at least one primer configured to tag the second target with a second probe having a second fluorophore. In some embodiments, the set of processing materials comprises a set of non-hydrolysis probes, the method further comprising tagging the set of targets with a set of permutations of the set of non-hydrolysis probes, wherein detecting signals indicative of the set of targets from the subset of the plurality of partitions comprises detecting signals corresponding to the set of permutations for differential detection of the set of targets. In some embodiments, the set of processing materials comprises a set of hydrolysis probes, the method further comprising tagging the set of targets with a set of combinations of the set of hydrolysis probes, wherein detecting signals indicative of the set of targets from the subset of the plurality' of partitions comprises detecting signals corresponding to the set of combinations for differential detection of the set of targets. In some embodiments, the set of processing materials comprises a set of probes configured to: associate with targets of the setAtty. DocketNo.: 43161-65181 / WO (004WO) of targets upon reacting the set of processing materials with the set of targets, and emit fluorescent signals upon associating with targets of the set of targets. In some embodiments, the set of processing materials comprises a set of probes configured to: associate with amplicons of the set of targets upon reacting the set of processing materials with the set of targets, and emit fluorescent signals upon associating with amplicons of the set of targets. In some embodiments, the set of processing materials comprises a primer and / or a probe comprising a nucleotide sequence selected from SEQ ID NOs: 13-20. In some embodiments, the set of processing materials further comprises a probe additive reagent configured to reduce background noise.

[0205] In some embodiments, the set of processing materials comprises, for a first target protein of the set of target proteins, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the first target protein, the set of target-specific primers comprising: a target-specific primer comprising a first flanking sequence, and a first fluorophore-labeled oligonucleotide corresponding to the first flanking sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second target protein of the set of target proteins, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the second target protein, the set of target-specific primers comprising: a target-specific primer comprising a second flanking sequence, and a second fluorophore-labeled oligonucleotide corresponding to the second flanking sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, the set of processing materials comprises, for a first target protein of the set of target proteins, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the first target protein, the set of target-specific primers comprising: a target-specific primer comprising a first adapter sequence, and a first fluorophore-labeled oligonucleotide corresponding to the first adapter sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second target protein of the set of target proteins, a primer set comprising: a common primer, and a set of targetspecific primers configured to interact with a target region of the second target protein, the set of target-specific primers comprising: a target-specific primer comprising a second adapterAtty. DocketNo.: 43161-65181 / WO (004WO) sequence, and a second fluorophore-labeled oligonucleotide corresponding to the second adapter sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, first adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, second adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, the set of processing materials comprises, for a first target protein of the set of target proteins: a forward primer corresponding to a target region and comprising a first adapter sequence; a reverse primer; and a first fluorophore-labeled oligonucleotide corresponding to the first adapter sequence, wherein the first fluorophore-labeled oligonucleotide comprises a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second target protein of the set of target proteins: a forward primer corresponding to a target region and comprising a second adapter sequence; a reverse primer; and a second fluorophore-labeled oligonucleotide corresponding to the second adapter sequence, wherein the second fluorophore-labeled oligonucleotide comprises a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, first adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, second adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, the set of processing materials comprises, for a first target protein and a second target protein of the set of target proteins, a primer set comprising: at least one primer configured to tag the first target protein with a first probe having a first fluorophore, and at least one primer configured to tag the second target protein with a second probe having a second fluorophore. In some embodiments, the set of processing materials comprises a set of non-hydrolysis probes, the method further comprising tagging the set of target proteins with a set of permutations of the set of non-hydrolysis probes, wherein detecting signals indicative of the set of target proteins from the subset of the plurality' of partitions comprises detecting signals corresponding to the set of permutations for differential detection of the set of target proteins. In some embodiments, the set of processing materials compnses a set of hydrolysis probes, the method further comprising tagging the set of target proteins with a set of combinations of the set of hydrolysis probes, yvherein detecting signals indicative of the set of target proteins from the subset of the plurality of partitions comprises detecting signals corresponding to the set of combinations for differential detection of the set of targets. In some embodiments, the set of processing materials comprises a set of probes configured to:Atty. DocketNo.: 43161-65181 / WO (004WO) associate with target proteins of the set of target proteins upon reacting the set of processing materials with the set of target proteins, and emit fluorescent signals upon associating with target proteins of the set of target proteins. In some embodiments, the set of processing materials comprises a set of probes configured to: associate with amplicons of the set of target proteins upon reacting the set of processing materials with the set of target proteins, and emit fluorescent signals upon associating with amplicons of the set of target proteins. In some embodiments, the set of processing materials comprises a pnmer and / or a probe comprising a nucleotide sequence selected from SEQ ID NOs: 13-20. In some embodiments, the set of processing materials further comprises a probe additive reagent configured to reduce background noise.

[0206] In some embodiments, the set of processing materials comprises, for a first vector target of the set of vector targets, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the first vector target, the set of target-specific primers comprising: a target-specific primer comprising a first flanking sequence, and a first fluorophore-labeled oligonucleotide corresponding to the first flanking sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second vector target of the set of vector targets, a primer set comprising: a common primer, and a set of targetspecific primers configured to interact with a target region of the second vector target, the set of target-specific primers comprising: a target-specific primer comprising a second flanking sequence, and a second fluorophore-labeled oligonucleotide corresponding to the second flanking sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, the set of processing materials comprises, for a first vector target of the set of vector targets, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the first vector target, the set of targetspecific primers comprising: a target-specific primer comprising a first adapter sequence, and a first fluorophore-labeled oligonucleotide corresponding to the first adapter sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second vector target of the set of vector targets, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the second vector target, the set of target-specific primersAtty. DocketNo.: 43161-65181 / WO (004WO) comprising: a target-specific primer comprising a second adapter sequence, and a second fluorophore-labeled oligonucleotide corresponding to the second adapter sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, first adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, second adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, the set of processing materials comprises, for a first vector target of the set of vector targets: a forward primer corresponding to a target region and comprising a first adapter sequence; a reverse primer; and a first fluorophore-labeled oligonucleotide corresponding to the first adapter sequence, wherein the first fluorophore- labeled oligonucleotide comprises a first fluorophore configured to transmit a first target signal if the target region is amplified. In some embodiments, the set of processing materials further comprises, for a second vector target of the set of vector targets: a forward primer corresponding to a target region and comprising a second adapter sequence; a reverse primer; and a second fluorophore-labeled oligonucleotide corresponding to the second adapter sequence, wherein the second fluorophore-labeled oligonucleotide comprises a second fluorophore configured to transmit a second target signal if the target region is amplified. In some embodiments, first adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, second adapter sequence comprises a nucleotide sequence selected from SEQ ID NOs: 1-4. In some embodiments, the set of processing materials comprises, for a first vector target and a second vector target of the set of vector targets, a primer set comprising: at least one primer configured to tag the first vector target with a first probe having a first fluorophore, and at least one primer configured to tag the second vector target with a second probe having a second fluorophore. In some embodiments, the set of processing materials comprises a set of non-hydrolysis probes, the method further comprising tagging the set of vector targets with a set of permutations of the set of nonhydrolysis probes, wherein detecting signals indicative of the set of vector targets from the subset of the plurality of partitions comprises detecting signals corresponding to the set of permutations for differential detection of the set of vector targets. In some embodiments, the set of processing materials comprises a set of hydrolysis probes, the method further comprising tagging the set of vector targets with a set of combinations of the set of hydrolysis probes, wherein detecting signals indicative of the set of vector targets from the subset of the plurality of partitions comprises detecting signals corresponding to the set of combinations for differential detection of the set of targets. In some embodiments, the set of processingAtty. DocketNo.: 43161-65181 / WO (004WO) materials comprises a set of probes configured to: associate with vector targets of the set of vector targets upon reacting the set of processing materials with the set of vector targets, and emit fluorescent signals upon associating with vector targets of the set of vector targets. In some embodiments, the set of processing materials comprises a set of probes configured to: associate with amplicons of the set of vector targets upon reacting the set of processing materials with the set of vector targets, and emit fluorescent signals upon associating with amplicons of the set of vector targets. In some embodiments, the set of processing materials comprises a primer and / or a probe comprising a nucleotide sequence selected from SEQ ID NOs: 13-20. In some embodiments, the set of processing materials further comprises a probe additive reagent configured to reduce background noise.

[0207] For tagging a target with probes configured to emit multiple colors (where tandem probes are disclosed in more detail herein), the set of target-specific primers can further include a second target-specific primer comprising a second flanking sequence, and the set of processing materials further comprises a second fluorophore-labeled oligonucleotide corresponding to the second flanking sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified, such that the target can be positively detected based upon the first target signal and the second target signal. Alternatively, a single primer can be used to tag the target, along with tandem adapters corresponding to the probes used to tag the targets. As such, the set of processing materials can include at least one primer configured to tag the target w ith a first probe having a first fluorophore and a second probe having a second fluorophore (and / or additional probes with additional fluorophores), wherein the first fluorophore and the second fluorophore (and optional additional fluorophores) correspond to two (or more) color channels of the number of color channels.

[0208] The master mixture can include a probe including a dye / fluorophore with complementary quencher for each target, a polymerase (e.g., Taq polymerase), dNPTs, and buffer components.

[0209] With respect to tagging implemented using the forward primers, and corresponding dyes / fluorophore families of probes, dyes / fluorophores can be associated with chemical families including: acridine derivatives, arylmethine derivatives, fluorescein derivatives, anthracene derivatives, tetrapyrrole derivatives, xanthene derivatives, oxazine derivatives, dipyrromethene derivatives, cyanine derivatives, squaraine derivates, squaraine rotaxane derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, pyreneAtty. DocketNo.: 43161-65181 / WO (004WO) derivatives, and / or other chemicals. Such fluorophores can further be attached to other functional groups as needed for tagging of targets in a detectable manner.

[0210] In examples, dyes (e.g., for tagging of RNAs, DNAs, oligonucleotides, etc.) can include one or more of: FAM, (e.g., 6-FAM), Cy3TM, Cy5TM, Cy5.5TM, TAMRATM (e.g., 5-TAMRA, 6-TAMRA, etc ), MAX, JOE, TETTM, ROX, TYETM (e.g., TYE 563, TYE 665, TYE 705, etc.), Yakima Yellow ®, HEX, TEX (e.g.. TEX 615), SUN. ATTOTM (e.g., ATTO 488, ATTO 490LS. ATTO 532, ATTO 550. ATTO 565, ATTO RholOl, ATTO 590. ATTO 633, ATTO 647, ATTO 647N, etc.), Alexa Fluor ® (e.g., Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 750, etc.), IRDyes® (e.g., 5’IRDye 700, 5’IRDye 800, 5’IRDye 800CW, etc.), Rhodamine (e.g.. Rhodamine Green. Rhodamine Red. Texas Red ®, Lightcycler ®, Dy-482XL, Dy- 508XL, Dy-526XL, Dy 750, Hoechst dyes, DAPI dyes, SYTOX dyes, chromomycin dyes, mithramycin dyes, YOYO dyes, ethidium bromide dyes, acridine orange dyes, TOTO dytes, thiazole dyzes, CyTRAK dyes, propidium iodide dyes, LDS dyes, BODIPY dyes, and / or other dyes. In some embodiments of the systems, methods, devices, and compositions disclosed herein, the dyes used are selected from: Alexa Fluor 488, Alexa Fluor 594, ATTO 490LS, ATTO 532, ATTO 647N, Cy5TM, FAM, Dy 482XL, Dy 508XL, Dy 526XL, and combinations thereof.

[0211] In examples, cell function dyes for tagging of target material and detection can include one or more of: DCFH, DHR, SNARF, indo-1, Fluo-3, Fluo-4, and / or other dyes.

[0212] In examples, fluorescent proteins for tagging of target material and detection can include one or more of: cerulean, mCFP, mTurquoise, T-Sapphire, CyPet, ECFP, CFP, EBFP, Azurite, and / or other fluorescent proteins.

[0213] In an embodiment, dyes used for 10-plex or greater multiplexing of targets using the matrix 410 or other sample formats can include: Alexa Fluor™ 488, Atto™ 532, Alexa Fluor™ 594, Atto™ 647N, Cy5, FAM, Dy-526XL, Dy-508XL, Dy-482XL, and Atto™ 490LS.

[0214] Dyes / fluorophores implemented can correspond to wavelength ranges in the visible spectrum and / or non-visible spectrum of electromagnetic radiation. Furthermore, dyes / fluorophores implemented can be configured to prevent overlapping wavelengths (e.g., of emission) and / or signal bleed through with respect to multiplexed detection and achieving high SNR values involving detection of signals from packed partitions. In variations, the set of processing materials can include components for 7 wavelength ranges for multiplexed detection of targets; however, the set of processing materials can include components for lessAtty. DocketNo.: 43161-65181 / WO (004WO) than 7 wavelength ranges (e.g., one wavelength, two wavelengths, three wavelengths, four wavelengths, five wavelengths) or more than 7 wavelength ranges.

[0215] Quencher oligonucleotides implemented can include a quencher molecule configured such that, when the quencher oligonucleotide anneals with a primer having a fluorophore, the quencher molecule is in proximity to (e.g., directly opposite) the fluorophore in order to quench the fluorophore. Additionally or alternatively, quenchers can include one or more of: black hole quenchers, static quenchers, self-quenchers (e.g., fluorophores that self-quench under certain conditions by producing secondary structures or other structures), and / or other suitable quenchers. Variations of positions of quenchers (e.g., when tandem probes are involved) are disclosed in more detail herein.

[0216] The set of processing materials of Step SI 30. Step 200, and variations thereof can additionally or alternatively include implementation of components configured to improve signal-to-noise ratio (SNR) characteristics in the context of multiplexed detection, by increasing signal characteristics and / or reducing background (e.g., noise other artifacts). The components can include one additive for each wavelength range / color for detection (as opposed to one additive for each target / SNP being evaluated). Additionally or alternatively, the additives can have from 5-20 bases or another suitable number of bases. Additionally or alternatively, modified nucleic acids (e.g., such as locked nucleic acids (LNA) or other modified nucleic acids) can be incorporated into forward and / or reverse primers of the set of processing materials to improve SNR. In variations. LNA content can occupy a percentage (e.g., 10-60% LNA content) of the respective primer to improve SNR, where LNA content can be biased toward the 3’ end, the 5’ end, or intermediate the 3’ and 5’ ends.

[0217] However, the set of processing materials can additionally or alternatively include other suitable components and / or be configured in another suitable manner.

[0218] Furthermore, with respect to different wavelength ranges, different targets can be tagged with dye / fluorophore colors in a manner that promotes discrimination of results (e.g., without overlap) upon detection of signals from processed sample material. Furthermore, different targets can be matched with different combinations of colors / associated wavelengths in order to provide distinction upon detection of signals from processed sample materials. Variations and examples of multiplexing based upon color combinatorics and other features are provided herein.Atty. DocketNo.: 43161-65181 / WO (004WO)8.3.1.3. Multiplexing Based Upon Color Combinatorics

[0219] In relation to Step S130, Step 210, and variations thereof (e.g., Steps 230, 230b, 230c, 230d, etc.), the methods can include: detecting signals from the set or plurality of partitions of a partition matrix, wherein the signals correspond to a set of color combinatorics paired with targets of a set of targets potentially represented in the sample and contained within partitions of the set or plurality of partitions, and wherein the set of targets has a total number greater than the number of color channels used to detect colors corresponding to the set of color combinatorics S 132. In some embodiments, the methods disclosed herein can include detecting signals indicative of targets of the set of targets from at least a subset of the plurality of partitions. In variations, the set of color combinatorics involves combinations of up to 3 colors detectable from each of the set or plurality of partitions, up to 4 colors detectable from each of the set or plurality of partitions, up to 5 colors detectable from each of the set or plurality of partitions, up to 6 colors detectable from each of the set or plurality of partitions, up to 7 colors detectable from each of the set or plurality of partitions, up to 8 colors detectable from each of the set or plurality of partitions, or another suitable number of colors detectable from each of the set or plurality of partitions.

[0220] Multiplexing with color combinatorics is practically achievable using embodiments of the systems, methods, and processing materials disclosed herein, especially due to the large number of partitions available (as disclosed herein), and in some instances, low occupancy of partitions by targets (as disclosed herein). For instance, as shown in FIG. 3A (left), low-partition systems are subject to greater prevalence of multiple targets within a single partition (e.g., as in doublets, as in triplets), thereby contributing to a lower degree of multiplexed assay sensitivity. However, high-partition systems, also shown in FIG. 3A (right) are subject to lower prevalence of multiple targets within a single partition (e.g., as in doublets, as in triplets), thereby contributing to a higher degree of multiplexed assay sensitivity when color combinatorics are used. As such, a higher percentage of different targets that are tagged with combinations of colors can be accurately discriminated with the high partition platform disclosed herein. Table 1 below provides exemplar}' scenarios indicating doublet rates observable for systems with 20,000 partitions and 30,000,000 partitions (as in system embodiments disclosed herein), respectively, where doublet rates are provided for various inputs / counts per color channel used for scanning (and a significantly lower percentage of doubles are observed with a high number of partitions).Atty. DocketNo.: 43161-65181 / WO (004WO)

[0221] Furthermore, when implementing four or more colors for tagging a set of targets for multiplexed detection, the methods 100 and / or 200 can include tagging targets with combinations of three or more colors, in order to reduce or otherwise eliminate error due to presence of doublets (i. e. , two targets within a partition), where doublet targets, each tagged with single colors, would result in dual color partitions. Similarly, the methods 100 and / or 200 can include tagging targets with combinations of four or more colors, in order to reduce or otherwise eliminate error due to presence of triplets (i.e., three targets within a partition), where triplet targets, each tagged with single colors, would result in tricolor partitions.

[0222] In relation to the set of processing materials disclosed herein, labeling a target with multiple colors can be performed using multiple primers per target (e.g., gene), where each of a set of primers (e.g., forward primers) used to tag a respective target can tag the respective target with one of a set of dyes / fluorophores (2 primers / tandem probes are shown in representative FIG. 3B, but the methods can be adapted to more than 2 primers / tandem probes). Alternatively, labeling a target with multiple colors can be performed using a single primer (e.g., forw ard primer) for the target, along with tandem adapters (show n in FIG. 3B), where tandem adapters and probes are disclosed in more detail herein. Different tagging strategies (e.g., multiple primers per target vs. single primer per target) can be implemented. For instance, a set of primers (e.g., forw ard primers) can be used for tagging a first target with multiple colors, and a single primer (e.g., forward primer) can be used for tagging a second target with one or more colors. When using multiple primers, in silico primer designAtty. DocketNo.: 43161-65181 / WO (004WO) can be used to prevent undesired primer-primer, primer-probe, primer-amplicon (non-target) interactions and / or self-primer interactions (e.g., undesired hairpin structures, primer-dimer interactions, etc.) that could produce increased background during scanning.

[0223] In one exemplary case, in silico primer design can include generating multiple pairs of primers for individual targets of interest, with check steps to remove candidates with potential for amplicon / primer / probe interactions, to remove primers with multiple continuous matches (e.g., 11 or more continuous matches) to reduce cross-channel interactions in signal positive partitions and primer-probe interactions or background noise before amplification. Primer sets can be selected from the best primer pairs for each target, with secondary' check steps for undesired primer-primer interactions and primer-amplicon interactions, removal of primers with multiple base pair (bp) continuous matches or 9 bp continuous matches in a region (e.g., last 10 bases). Such design steps can be used to create primer sets for which background is reduced for every' partition (during use), and for which cross-channel interactions in positive partitions are reduced. A resulting output of the in silico primer design operation produces a panel of compatible primers (e.g., with one primer pair per target of interest) for a specific multiplexing assay.

[0224] In relation to multiplexing with color combinatorics, labeling a target with multiple colors can be performed with combinations of colors, where the order of the colors used to tag a target is unaccounted for. Alternatively, labeling a target with multiple colors can be performed with permutations of colors, where the order of the colors used to tag a target is accounted for in relation to discrimination of a target. Permutation-based multiplexing is achievable using tandem probes used to tag targets, where tandem probes are disclosed in more detail herein. Implementation of fluorophores having transitionable fluorophore states (e.g., in response to a stimulus, in relation to exhibition of Foerster resonance energy transfer behavior, etc.) can also be used to achieve higher degrees of multiplexing, as disclosed in more detail herein.

[0225] An example of targets (e.g., SNPs), corresponding alleles, and corresponding tagged- color combinations for detection and differentiation is shown in FIG. 3C. which enables encoding of 26 targets (e.g., 13 SNP loci) with a 5-color system (such that each partition can exhibit a color combinatoric of up to 5 colors). An example of targets (e.g., SNPs) and corresponding tagged-color combinations for detection and differentiation is shown in FIG. 3D, which enables encoding of 15 targets with a 4-color system (such that each partition can exhibit a color combinatoric of up to 4 colors). The example shown in FIGs. 3C and 3D can be adapted for systems greater than 5 colors or less than 4 colors. As such, with the ultra-highAtty. DocketNo.: 43161-65181 / WO (004WO) partition setting disclosed herein involving limited dilution, each target of interest can be confidently labeled with a unique set of colors, for subsequent detection and digital quantitation without requiring multiplexing based upon signal amplitudes.

[0226] Assays can be designed such that targets are differentially tagged with combinations of colors in a manner that improves ability to discriminate color signals upon scanning and / or to use probes in a conservative and efficient manner. For instance, targets that are anticipated to be most prevalent can be tagged with fewer colors, and targets that are anticipated to be least prevalent can be tagged with more colors. Additionally or alternatively, non-similar targets (e.g., a first target and a second target that is non-similar to the first target) can be tagged with opposite color combinations, in order to improve the ability- to discriminate non- similar targets upon scanning. For instance, in the context of microbiome analyses, bacteria- derived targets can be tagged with a first combination of colors and fungal targets can be tagged with a second combination of colors (e.g., colors that are complementary to the first combination of colors, colors that are near complementary- to the first combination of colors, etc.) that allows for improved discrimination of bacterial targets and fungal targets upon scanning.

[0227] However, assay designs involving multiplexing with color combinatorics can be applied in another suitable manner.8.3.1.4. Multiplexing with Tandem Probes, Conjugated Polymers, and Foerster Resonance Energy Transfer (FRET)

[0228] As noted herein, higher degrees of multiplexing for partition-based systems can be achieved using tandem probes (e.g., a set of probes configured to tag the same target, for instance, with a single primer and tandem adapters for the target), such that each target being analyzed can be tagged with one or more of a set of probes (i.e., different probes configured to produce different color combinations of detectable signals). An example of a tandem probe design in shown in FIG. 3E, where individual probe sequences can be conjugated with one of a set of fluorophore / quencher combinations, and tagging a target with a subset of probes produces a signals that that can be detected with color channels appropriate to the subset of probes. As such, a target can be positively detected if signals corresponding to the subset of probes are detected from a partition upon scanning the set or plurality of partitions with color channels corresponding to the subset of probes.

[0229] In variations, the fluorophores of the set of probes can all be positioned near a first end (e.g., 3' end, 5' end) of the respective probe, and the quenchers of the set of probes can allAtty. DocketNo.: 43161-65181 / WO (004WO) be positioned near a second end (e.g., 5' end, 3' end) of the respective probe, such that, as shown in FIG. 3F (top), the quencher of a first probe is positioned near the fluorophore of a second probe when the first probe and the second probe have tagged a target in tandem. Alternatively, as shown in FIG. 3F (middle), a first probe and a second probe to be used for tagging of a target in tandem can be configured such that the quencher of the first probe and the quencher of the second probe are positioned near each other when the first probe and the second probe have tagged a target in tandem. Alternatively, as shown in FIG. 3F (bottom), a first probe and a second probe to be used for tagging of a target in tandem can be configured such that the quencher of the first probe and the quencher of the second probe are positioned far from each other when the first probe and the second probe have tagged a target in tandem. However, in variations, the fluorophores and / or quenchers of probes can be positioned away from ends (e.g., 3' ends, 5' ends) of respective probes. Fluorophore and quencher positions along the lengths of respective probes can thus be configured to improve signal detection (e.g., in relation to detection of fluorescent signals from partitions of a partition matrix, with desired quenching performance (e.g., with respect to quencher interference or quencher enhancement within a partition) and with desired background reduction performance).

[0230] While only tw o tandem primers / tandem probes are shown in FIGs, methods can be adapted and expanded to incorporate more than tw o primers / probes for target tagging. In particular, the methods disclosed herein can involve up to 2 tandem probes for target tagging, up to 3 tandem probes for target tagging, up to 4 tandem probes for target tagging, up to 5 tandem probes for target tagging, up to 6 tandem probes for target tagging, up to 7 tandem probes for target tagging, up to 8 tandem probes for target tagging, up to 9 tandem probes for target tagging, up to 10 tandem probes for target tagging, or greater numbers of tandem probes for target tagging.

[0231] Additionally or alternatively, when the fluorophores of the set of probes can are positioned near a first end (e.g., 3' end, 5' end) of the respective probe, and the quenchers of the set of probes can all be positioned near a second end (e.g., 5' end, 3' end) of the respective probe, ordered positioning of a first probe with a first fluorophore and a first quencher and a second probe with a second fluorophore and a second quencher can be used to create different permutations of ordered probes that result in amplitude differentiation of signals for target detection. In particular, the effect of positioning quenchers and fluorophores in tandem and resulting signal intensity changes based upon positioning is due to FRET behavior, where, as a quencher of one probe is placed next to a fluorophore of another probe, the intensity of the fluorophore is reduced. Thus, by modulating relative positioning of quenchersAtty. DocketNo.: 43161-65181 / WO (004WO) and fluorophores of different probes used to tag a target, differential signal amplitudes for respective probes involved can be achieved and detected.

[0232] In the examples shown in FIG. 3G, when two fluorophore colors (i.e., a first color and a second color) are available, tagging a target in tandem with a first probe having a first color and a second probe having the first color (FIG. 3G, top), enables detection of the target with a high amplitude signal corresponding to the first color. Tagging a target in tandem with a first probe having a first color and a second probe having a second color (FIG. 3G, second from top), enables detection of the target with a medium-high amplitude signal corresponding to the first color and a medium-low amplitude signal corresponding to the second color. Tagging a target in tandem with a first probe having a second color and a second probe having a first color (FIG. 3G, third from top), enables detection of the target with a medium- high amplitude signal corresponding to the second color and a medium-low amplitude signal corresponding to the first color. Tagging a target in tandem with a first probe having a second color and a second probe having a second color (FIG. 3G, bottom), enables detection of the target with a high amplitude signal corresponding to the second color. As such, when two colors are available, 4 permutations of ordered pairs of tandem probes are available for differential target tagging (with detectable signals based upon signal amplitude in different color channels corresponding to the colors available). When four colors are available, 16 permutations of ordered pairs of tandem probes are available (e.g., 4 permutations for each pair of colors), for differential target tagging, if only two probes are used per template. When four colors are available, 256 permutations are available (i.e., 4 probes with 4 colors x 4 probes with 4 colors). Examples of signal amplitudes, for a scenario w here four colors are available and pairs of tandem probes are used to tag targets, are shown in FIG. 3G. Variations of the examples shown in FIG. 3G may not have the fluorophores and quenchers positioned at opposite ends of their respective probe, to provide differentiation of signals from targets using such tandem probes.

[0233] Additionally or alternatively, a first quencher can be added to a first probe used to tag a target, and based on the position of the first quencher of the first probe, the amplitude of a signal produced by a second fluorophore of a second probe used to tag a target in tandem with the first probe is reduced during detection. The amplitude reduction of the second fluorophore of the second probe can then enable discernment of the order of w hich the first probe (with the first fluorophore) and the second probe (with the second fluorophore) are placed.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0234] Additionally or alternatively, tandem probes can be configured to have a spacer region positioned between different probes used to tag the same target. The spacer region can reduce quenching effects provided by a quencher of a first probe and a fluorophore of a second probe positioned next to the first probe, where the length of the spacer modulates the quenching effect. As such, a longer spacer increases the resultant signal amplitude of a second probe spaced from the first probe by the spacer. Furthermore, resulting amplitudes associated with probes used can be modulated by tuning spacer length, in order to achieve additional granularity of amplitude levels of each dye used to tag one or more targets (and thus more permutations for multiplexing of targets). In variations, the spacer can have a length from 1 to 25 base pairs, and have a specific sequence or a random sequence, in relation to primer aspects disclosed herein.

[0235] In variations, as shown in FIG. 3H (left), a target (e.g., target gene) can be tagged using a target-specific primer (e.g., forward primer) with one or more detectable probes (e.g., Probe C and / or Probe D shown in FIG. 3H). As shown in FIG. 3H (right), multiple targets (e.g., target genes) can be tagged using primers (e.g., forward primers) with single probes and / or combinations of tandem probes, with signal detection for target identification from partitions performed as disclosed herein. As such, tandem primer / probes and non-tandem primer / probes can be combined within the set of processing materials for tagging of different targets.

[0236] Alternatively, tandem probes where fluorophores are positioned near each other (as shown in FIG. 31) and capable of producing and / or responding a Foerster resonance energy transfer (FRET) effect (e.g., with one or more emitting fluorophores and one or more reporting fluorophores) can be used to enable signal detection based on FRET behavior. For instance, a first probe of a tandem probe can be excited by a first wavelength of light that matches the excitation spectrum of the first fluorophore, and FRET transfer to a reporter dye of a second probe can excite the second probe for detection of a target (e.g., using emission filter specific for the emission spectrum of the second fluorophore), thereby enabling differential detection of targets from different partitions given that the excitation wavelength profiles and the emission wavelength profiles for the set or plurality of partitions is identifiable with scanning. As such, in relation to a first fluorophore and a second fluorophore included in processing materials as disclosed herein, embodiments of the method can further include causing Foerster resonance energy transfer (FRET) from the first fluorophore to the second fluorophore upon exciting the first fluorophore with a first wavelength profile of light, such that detecting signals from the set or plurality of partitions with the number of colorAtty. DocketNo.: 43161-65181 / WO (004WO) channels can include detecting the target from a partition upon scanning the set or plurality of partitions with a second wavelength profile of light corresponding to the second fluorophore. As shown in FIG. 31, for an exemplar}' set of four fluorophores that exhibit FRET behavior in tandem with excitation of a cooler color first probe and FRET transfer and detection of a warmer color second probe, differential tagging and detection can be achieved for 7 targets.

[0237] As shown in FIG. 3J, the set of processing materials can additionally or alternatively include conjugated polymer probes for fluorescence enhancement, where such conjugated polymers operate as optical harvesters that exhibit long-range FRET capability. In more detail, as shown in FIG. 3 J, a variation of a conjugated polymer probe includes a set of optical segments that harvest incident light with a large absorption cross-section. The structure of the conjugated polymer and set of optical segments determines the excitation w avelength. The set of optical segments operates as a molecular antenna and drive energy migration to a reporter dye of the probe, thereby achieving FRET transfer to the reporter dye.

[0238] Additionally or alternatively, conjugated polymer probes can include cationic polymers with complex structures (e.g., kinked structures, twisted structures, coiled structures, zigzagging structures, etc.) capable of FRET transfer from such complex structures to a reporter dye for amplification of signals produced by the reporter dye, with or without an emission spectrum shift by the reporter dye. As such, a limited number of excitation spectra (e.g., associated with a limited number of color channels) can be used to achieve color detection across more colors than the limited number of color channels. Exemplary dyes that are included with or otherwise compatible with conjugated polymer probes include, for example, BD HorizonTM BB515, Alexa FluorTM 488, FITC, BD HorizonTM BB630-P2, BD HorizonTM BB660-P2, PerCP-Cy5.5**, PerC**, BD HorizonTM BB700, BD HorizonTM BB755-P, BD HorizonTM BB790-P, and other BD HorizonTM components. Implementation of such conjugated polymer probes can, in exemplary embodiments, be used to achieve detection of targets within partitions, with 12- color multiplexing, using only 2 colors of light sources.

[0239] Additionally or alternatively, conjugated polymer probes can form complexes (e.g., electrostatic complexes) with quantum dots or other structures in a manner that can produce a cascading FRET effect, whereby energy is transferred to the quantum dots or other structures, from the conjugated polymer structure(s), upon being subject to excitation wavelenghts of light, and energy is transferred from the quantum dots or other structures to the dye of the probe, in order to produce higher sensitivity.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0240] Reporter dyes can include dyes discussed herein and / or other dyes with distinct emission behavior, where FRET-based amplification of dyes can result in an orders of magnitude (e.g., one order of magnitude, two orders of magnitude, three orders of magnitude, etc.) or greater amplification effect in comparison to dyes without a conjugated polymer component.8.3.1.5. Multiplexing Based Upon Stimulus-Responsive Dyes and Fluorophores

[0241] Additionally or alternatively, in the context of partitions that are fixed in position or otherwise addressable (e g., with barcoding, with identification of relative positions of partitions) across a set of scanning runs with the same color channel or different color channels, the methods 100 and / or 200 can further include implementing stimulus-responsive dyes and / or fluorophores for tagging of targets, where scanning the set or plurality of partitions before and after applying a stimulus to the stimulus-responsive dyes and / or fluorophores enables additional levels of multiplexing to be achieved when using a limited number of color channels. As such, in relation to a base level of multiplexing achievable for a set of available color channels, the level of multiplexing can be expanded beyond the base level by a factor corresponding to the number of stimulus-responsive states available for each dye / fluorophore. For instance, multiplexing ability can be doubled (e.g., expanded by a factor of 2) for a color channel and use of a first fluorophore that is photobleaching resistant when irradiated using the color channel, and a second fluorophore that is photobleachable when irradiated using the color channel. Additional examples are provided herein.

[0242] In variations, the dye(s) / fluorophore(s) can transition between states based upon application of a stimulus or stimuli, where the stimuli can involve one or more of: a stimulus involving irradiation, a stimulus involving a change in pH, a stimulus involving a change in temperature, a stimulus involving a change in pressure, a stimulus involving a change in electric field, a stimulus involving a change in magnetic field, or another suitable stimulus. Transition states of the dye(s) / fluorophore(s) can be binary (e.g., a first state and a second state) in response to an applied stimulus. Alternatively, a fluorophore can undergo transitions between a set of states (e.g., to different degrees), depending upon a method of application of the applied stimulus or stimuli, and / or number of same stimulus-response fluorophores attached to a target. For irradiation-responsive materials, stimulus application parameters can include intensity7, wavelength, exposure duration, and other factors, and the amount of exposure to the stimulus can achieve different levels of photobleaching (which can have aAtty. DocketNo.: 43161-65181 / WO (004WO) more differentiable effect when multiple fluorophores are used to tag the same target). For pH-responsive materials, stimulus application parameters can include pH value, temperature, duration of exposure, and other factors and the amount of exposure to the stimulus can achieve different levels of photobleaching (which can have a more differentiable effect when multiple fluorophores are used to tag the same target). For temperature-responsive materials, stimulus application parameters can include temperature, duration of exposure, and other factors and the amount of exposure to the stimulus can achieve different levels of photobleaching (which can have a more differentiable effect when multiple fluorophores are used to tag the same target).

[0243] For multiplexing, the set of processing materials can include materials for tagging groups of dyes / fluorophores including one or more dyes / fluorophores that are resistant to the stimulus (or are not responsive to application of the stimuli) for a respective color channel, and one or more dyes / fluorophores that are sensitive to the stimulus.

[0244] Examples of photostimulus-responsive dyes / fluorophores include: Atto 495 (Blue color channel). FAM (Blue color channel), Bodipy-TMR (Green color channel), ROX (Yellow color channel), Cyanine 5 (Red color channel), Dy636 (Red color channel), Atto680(Crimson color channel), and Cyanine 5.5 (Crimson color channel). Examples of photostimulus-resistant dyes / fluorophores include: Dy490 (Blue color channel), Atto488 (Blue color channel), Alexa488 (Blue color channel), Cyanine 3 (Green color channel), AttoRho 6G (Green color channel), HEX (Green color channel), VIC (Green color channel). SUN (Green color channel), Rhodamin 6G (Green color channel), Atto532 (Green color channel), Cyanin 3.5 (Yellow color channel), TEX615 (Orange color channel), Texas Red (Orange color channel), CAL Fluor 610 (Orange color channel), Bodipy-TR-X (Orange color channel). Atto590 (Orange color channel), Atto647N (Red color channel), and Dy682 (Crimson color channel).

[0245] Examples of pH sensitive fluorophores include pHrodoTM Green AM, pHrodoTM Red AM, fluorescein, LysoSensor Yellow, LysoSensor Blue, LysoSensor Green, Oregon Green 514, Oregon Green 488, Dichlorofluorescein derivatives, ACMA, HPTS, FAM, pHluorin, and pHluorin2.

[0246] Examples of temperature sensitive fluorophores include rhodamine B, Rhodamine 6G, Rhodamine C, Benzothiadiazoles, aza-BODIPY, phthalocyanines, perylene bisimide, and others.

[0247] Fluorophores or other colorimetric indicators can be differentially-responsive to the stimuli discussed, such that an applied stimulus produces differential responses in theAtty. DocketNo.: 43161-65181 / WO (004WO) fluorophores / colorimetric indicators. Alternatively, fluorophores or other colorimetric indicators can be equally-responsive or near-equally-responsive to an applied stimulus.

[0248] As such, implementation of the methods disclosed herein can involve exposing tagged target analytes with stimulus-responsive tagging components, which can be differentially detectable before and / or after application of a stimulus, thereby increasing the level of plexy achievable with a limited number of color channels.

[0249] In relation to Step S130, Step 210, and variations thereof (e.g.. Steps 230, 230b, 230c, 230d, etc.), the methods 100 and / or 200 can additionally or alternatively include (for one or more color channels of a set of color channels involved in detection, and for a first fluorophore of the set of processing materials) use of a first fluorophore for achieving higher degrees of multiplexing, wherein the first fluorophore is a stimulus-responsive (e.g., photo- bleachable, photo-responsive, pH responsive, temperature-sensitive, etc.) fluorophore, and wherein detecting signals from the set or plurality of partitions comprises scanning the set or plurality7of partitions prior to and post applying a stimulus to the first fluorophore, thereby- transitioning the first fluorophore between a first state and a second state SI 33 (an example of which is shown in FIG. 3K). In a specific example, the applied stimulus is a light stimulus, and scanning can be performed with a first wavelength range of light to detect signals from the set or plurality7of partitions prior to photobleaching, and after a second wavelength range of light configured to bleach the first fluorophore is applied. As such, the methods 100 and / or 200 can include detecting signals from the set or plurality of partitions in a first phase of analysis upon scanning the set or plurality of partitions with the first wavelength range of light SI 34, and detecting signals from the set or plurality of partitions in a second phase of analysis upon scanning the set or plurality of partitions and bleaching the first fluorophore with the second wavelength range of light SI 35, as shown in FIG. 3K. As such, the set of processing materials disclosed herein can include at least one primer configured to tag a first target with a first probe having a first fluorophore and a second target with a second probe having a second fluorophore, wherein the first fluorophore is a photo-bleachable fluorophore, thereby enabling differential detection of the first target and the second target. Alternatively, for Taqman™ chemistry, the set of processing materials can include, for a first target and a second target of the set of targets: a primer set comprising: a first primer configured to tag the first target w ith a first probe having a first fluorophore and a second primer configured to tag the second target w ith a second probe having a second fluorophore, w herein the first fluorophore is a photo-bleachable fluorophore, and wherein detecting signals from the set or plurality of partitions includes scanning the set or plurality of partitions with a firstAtty. DocketNo.: 43161-65181 / WO (004WO) wavelength range of light and a second wavelength range of light configured to bleach the first fluorophore. thereby enabling differential detection of the first target and the second target.

[0250] Wavelengths of light used for scanning can be in the visible or non-visible spectrum. Light sources implemented can include laser light, light emitting diodes (LEDs), and / or other light sources. Laser powers implemented can include laser powers of 10 mW through 80 mW (or alternatively less than 10 mW or greater than 80 mW laser powers). However, other low power lasers can be implemented.

[0251] In a specific example, where the first fluorophore is FAM, scanning the set or plurality of partitions can include: scanning a set of planes of partitions within a collecting container, with a laser having a power of 50 mW and focused with optics to a 20 micron- thick light sheet (where other thicknesses less than 20 microns or greater than 20 microns can be implemented), where the duration of scanning for the set of planes (e.g., 500 planes) is at most 3 minutes, and wherein scanning bleaches the first fluorophore. Bleaching can be performed across a single scan of the set of planes, or across multiple scans of the set of planes. However, variations of the specific example can use other light sources (e.g., LEDs) that are lower power, with longer exposure times to achieve bleaching or other states of signal emission characteristics. Detecting signals from the set or plurality' of partitions can be performed prior to bleaching in a first characterization of the set or plurality of partitions, and post bleaching the first fluorophore for a second characterization of the set or plurality of partitions, in order to achieve higher degrees of multiplexing for differential detection of targets based upon the first characterization and the second characterization. Given that the locations of the partitions do not change (e.g., are fixed in position within a stable emulsion), the first characterization can describe a first local signal amplitude for each partition, and the second characterization can describe a second local signal amplitude for each partition after the light stimulus is applied, where local amplitude characterizations can be achieved for partitions arranged in bulk with embodiments, variations, and examples of the platforms disclosed herein.

[0252] Furthermore, scanning can be performed using different light wavelengths, powers, exposure times, and other parameters, for other transitionable fluorophores implemented in addition to the first fluorophore.

[0253] An example of signal discrimination pre and post photobleaching is show n in FIG. 3K.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0254] Alternatively, for primarily non-photo responsive fluorophores, scanning the set or plurality of partitions can include: scanning a set of planes of partitions within a collecting container prior to application of a stimulus (e.g., temperature change, electric field, pH shift, mechanical stimulus, etc.); detecting signals from the set of planes of partitions for a first characterization of the set or plurality7of partitions; scanning the set of planes of partitions within the collecting container post application of the stimulus (e g., temperature change, electric field, pH shift, mechanical stimulus, etc.); detecting signals from the set of planes of partitions for a second characterization of the set or plurality of partitions; and characterizing targets of the sample in a multiplexed manner based upon the first characterization and the second characterization.

[0255] As such, in relation to other stimuli, the method 100 and / or 200 can include scanning the set or plurality of partitions with other wavelength(s) of light prior to and / or postapplication of a stimulus (e.g., temperature change, electric field, pH shift, mechanical stimulus, etc.), where the stimulus causes changes in signal emission from the set or plurality7of partitions appropriate to the wavelength(s) of light used for scanning.8.3.1.6. Multiplexing with Different Levels of Plexy at Different Regions of a Density Gradient

[0256] In variations where the set or plurality of partitions comprise partition of a partition matrix, the partition matrix can include subregions at different depths within the collecting container (e.g., vertical depths, radial depths, etc.), each subregion associated with a different level of plexy. For instance, a first subregion of the partition matrix can include a first sample being assessed for targets of a first panel, and a second subregion of the partition matrix can include a second sample being assessed for targets of a second panel, where the degree of multiplexing required to characterize the first panel of targets of the first sample is different from the degree of multiplexing required to characterize the second panel of targets of the second sample.

[0257] In variations where the partition matrix is generated upon applying a force (e.g., centrifugal force, pressure, etc.) to the sample(s) through a porous membrane or plate with openings, the first sample can have a first density and the second sample can have a second density, such that the first sample and the second sample self-arrange at different depths of the collecting container and have processing materials for different levels of plexy, at each depth of the collecting container. However, a gradient of regions can be formed in anotherAtty. DocketNo.: 43161-65181 / WO (004WO) suitable manner, in order to have different levels of assay plexy at different subregions of the collecting container, for different sets of targets of different samples.8.3.1.7. Combined Multiplexing Options to Further Expand Levels of Plexy

[0258] In relation to the variations of multiplexing disclosed herein (e.g., with color combinatorics, with stimulus-responsive materials, with density gradients involving layers of a container each having different degrees of plexy, etc.), multiplexing can be performed in a combinatorial manner, by implementing a number of different strategies, including color combinatorics, signal amplitude-based multiplexing (e g., where discrimination of various targets is based upon signal amplitude with varied concentrations of primers, and when levels of background noise allow for accurate characterizations of signal amplitude corresponding to each target), stimulus-responsive fluoro phores / dyes, different amplification and tagging chemistries (e.g., TaqMan-based chemistries disclose in more detail herein, KASP-based chemistries, etc.) where a first level of plexy can be achieved with a first chemistry / assay design and a second level of plexy can be achieved with a second chemistry / assay design, chemistries involving Foerster resonance energy’ transfer (FRET) to produce a cascade of emission for different partitions, chemistries of the set of processing materials with nonhydrolysis probes, and other multiplexing technologies. For instance, chemistries with nonhydrolysis probes that are capable of FRET behavior can be used to tag the set of targets with a set of permutations of the set of non-hydrolysis probes, where detecting signals from the set or plurality of partitions comprises detecting signals (e.g.. based upon FRET from a first fluorophore of a first non-hydrolysis probe to a second fluorophore of a second nonhydrolysis probe) corresponding to the set of permutations, for differential detection of the set of targets. Additionally or alternatively, the set of processing materials can include a set of hydrolysis probes, such that the method includes tagging the set of targets with a set of combinations of the set of hydrolysis probes, and wherein detecting signals from the set or plurality of partitions includes detecting signals corresponding to the set of combinations for differential detection of the set of targets.

[0259] Exemplary’ FIG. 4A shows the number of different targets that can be differentially tagged with four colors, without combinations or permutations of colors, with combinations of colors, and with permutations of colors. Without combinations or permutations of colors, four different targets can be differentially tagged and detected using four colors. With combinations four colors (with position-agnostic arrangements / orders of the colors used toAtty. DocketNo.: 43161-65181 / WO (004WO) tag the respective targets), 15 different targets can be differentially tagged and detected using four colors, and 10 different targets can be differentially tagged and detected using four colors if only up to two colors are selected from the set of four colors. With permutations of four colors (with position-sensitive arrangements / orders of the colors used to tag the respective targets), 21 different targets can be differentially tagged and detected using four colors (with two colors of tandem probes), and 16 different targets can be differentially tagged and detected using four colors if only up to two colors are selected from the set of four colors.

[0260] As disclosed herein, the levels of multiplexing achieved can be enhanced (e.g., in an additive manner) with co-implementation of multiple multiplexing strategies and mechanisms. As shown in FIG. 4B, when combinations of colors are used, the number of targets that can be differentially tagged can be represented by expression [1] below, where n represents the number of available colors, and r represents the number of selected colors from the number of available colors. When permutations of colors are used, the number of targets that can be differentially tagged can be represented by expression [2], , where n represents the number of available colors, and r represents the number of selected colors from the number of available colors.

[0261] nCr = n! / [r! (n-r)!] [1]

[0262] nPr = n ! / [ (n-r) ! ] + n [2]

[0263] Thus, with four available colors and up to three colors used in combination to tag targets, the number of targets that can be differentially tagged is 14. With four available colors and only 2 colors used in permutation to tag targets, the number of targets that can be differentially tagged is 12.

[0264] When involving probes capable of FRET behavior, with four available base colors and 3 additional FRET-discriminable colors for a total of 7 signal types used in combination to tag targets, the number of targets that can be differentially tagged is 63 if up to 3 signal ty pes are selected and 28 if up to two signal ty pes are selected.

[0265] When additionally involving photobleachable probes, with four available base colors, 3 additional FRET-discriminable colors, and 2 photobleachable probes for a total of 9 signal types used in combination to tag targets, the number of targets that can be differentially tagged is 127 if up to 3 signal types are selected and 45 if up to two signal types are selected.

[0266] As such, the methods, systems, and compositions disclosed herein can achieve high levels of multiplexing even when a 3D imaging technique (e.g.. light sheet imaging, 3D confocal microscopy, etc.) is involved for target detection, where background noise is muchAtty. DocketNo.: 43161-65181 / WO (004WO) higher than for systems (e.g., microwell systems, etc.) where ID and 2D imaging techniques are sufficient for target detection.8.3.1.8. Assay Materials and Compositions Involving Allele-Targeting Probes

[0267] In alternative variations, the set of processing materials can utilize, for each target, a set of fluorophore-conjugated versions of the same probe sequence, where the set of fluorophore-conjugated versions produce a detectable combination of signals (e.g.. color signals) that enable positive identification of the respective target. As such, the set of fluorophore-conjugated versions for each respective probe sequences can enable multiplexing based on color combinatorics to provide a high degree of multiplexing. In more detail, the set of processing materials can include: a) for a respective target region of the set of targets, a forward primer, a reverse primer, and a set of fluorophore-conjugated probes, each of the set of fluorophore-conjugated probes targeting an allele of the respective target region and b) a master mixture including amplification reagents. The set of processing materials can additionally or alternatively include a probe additive for each of the set of fluorophore- conjugated probes, where the probe additive functions to quench background fluorescence resulting from the probes (i. e. , is configured to reduce background noise), thereby improving signal-to-noise ratio (SNR). The set of fluorophore-conjugated probes can thus be configured to tag different alleles within a partition with different combinations of colors (corresponding to different fluorophores used), in order to provide discrimination of partition contents upon detection of signals from contents of each partition. A probe additive reagent can further include one or more quenchers configured to interact with at least one of the 3' region and the 5' region of a fluorophore-labeled oligonucleotide.

[0268] FIG. 5A depicts an example of fluorophore-conjugated probes that provide target detection and digital quantitation of different targets based on color combinatorics.

[0269] The probe additive can have a melting temperature (Tm) of 40 °C - 48 °C to be nonreacting during thermocycling (above 55 °C in general). However, in variations, the Tm of the probe additive can be greater than 48 °C or greater than 55 °C. In examples the Tm of the probe additive can be from 47 °C - 79 °C. greater than or equal to 79 °C, or an intermediate value. In particular, with implementation of multiple and different probe additives, as primer diversity increases, primers can effectively compete with the probe additives to bind to respective probes, and some of the undesired primer-probe interactions cannot be eliminated via in silico design alone.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0270] The set of probes can include TaqmanTM probes and / or other dual-labeled probes to differentiate alleles of a target region. Probes can include dyes / fluorophores associated with chemical families including: acridine derivatives, arylmethine derivatives, anthracene derivatives, tetrapyrrole derivatives, xanthene derivatives, oxazine derivatives, dipyrromethene derivatives, cyanine derivatives, squaraine derivates, squaraine rotaxane derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, pyrene derivatives, and / or other chemicals. Such fluorophores can further be attached to other functional groups as needed for tagging of targets in a detectable manner. Dyes / fluorophores can additionally or alternatively include compositions disclosed herein.

[0271] Primer concentration (e.g., forward primer concentration), probe concentration, and probe additive concentrations can be configured to improve detection performance (e.g.. in relation to number of positive counts corresponding to positive targets, in relation to SNR values, etc.). In variations, the primer concentration (e.g., forward primer concentration) : probe concentration : probe additive concentration can have a ratio of: 10: 10:30; 10:20:60; 10:40: 120; 10:80:240; 20: 10:30; 20:20:30; 20:20:60; 20:40: 120; 20:80:240; 40: 10:30;40:20:60; 40:40: 120; 40:80:240; 80: 10:30; 80:20:60; 80:40: 120; 80:80:240; ratio values intermediate to those disclosed; or other ratio values. Concentrations can be provided in terms of molarity7or another suitable unit.

[0272] The example of targets (e.g., SNPs) and corresponding tagged-color combinations for detection and differentiation shown in FIGs. 3A and 3B can be similarly detected using the set of processing materials disclosed herein. Similarly, the example shown in FIGs. 3C and 3D can be adapted for systems greater than 5 colors or less than 4 colors.

[0273] Quenchers of Taqman™ and / or other dual-label probes can be configured to quench signal of the fluorophore if the quencher is in proximity to the fluorophore below a threshold distance). Additionally or alternatively, quenchers can include one or more of: black hole quenchers, static quenchers, self-quenchers (e.g., fluorophores that self-quench under certain conditions by producing secondary' structures or other structures), and / or other suitable quenchers. Quenchers can be used to suppress background signals (e.g., for 3D imaging applications, for other detection applications).

[0274] However, the set of processing materials can additionally or alternatively include other suitable components and / or be configured in another suitable manner.Atty. DocketNo.: 43161-65181 / WO (004WO)8.3.2. Method - Partitioning of Sample with Processing Materials

[0275] Distributing the sample combined with the set of processing materials, across a set or plurality of partitions in step S140, and / or generating a plurality of partitions (e.g., at least 500,000 partitions) within a single closed container, wherein the plurality of partitions comprises: a set of targets of a sample, and a set of processing materials in step S210 (and variations thereof, including steps S210b, S210c, S210d, etc.) can include receiving a sample (variations and examples of which are disclosed herein) at a vessel passively or actively (e.g., with applied force, such as with gravitational force, with centrifugal force, with pressurization, etc.). The sample and processing materials can be delivered manually (e.g., with a fluid aspiration and delivery device, such as a pipettor). The sample and processing materials can additionally or alternatively be delivered with automation (e.g., using liquid handling apparatus or other sample handling apparatus).

[0276] In variations, vessel formats can include: tubes (e.g., PCR tubes) containing partitions of the sample (e.g., in partition matrix format, in emulsion format, in another format), wells (e.g., microwells, nanowells, etc.), channels, chambers, and / or other suitable containers. Additionally or alternatively, alternative variations of step S140 and / or Step S210 (and variations thereof, including steps S210b, S210c, S210d, etc.) can include receiving the sample at other suitable substrates (e.g., slides, plates, etc.) functionalized with material components configured to interact with target material of the sample. For instance, sample material can be spotted onto substrates with material components configured to interact with target material of the sample and in a detectable manner.

[0277] Embodiments, variations, and examples of the methods disclosed herein can be implemented by or by way of embodiments, variations, and examples of components of system 200 shown in FIG. 6, with a first substrate 210 defining a set of reservoirs 214 (for carrying sample / mixtures for partition generation), each having a reservoir inlet 215 and a reservoir outlet 216; one or more membranes (or alternatively, partition-generating substrates) 220 positioned adjacent to reservoir outlets of the set of reservoirs 214, each of the one or more membranes 220 including a distribution of holes 225; and optionally, a sealing body 230 positioned adjacent to the one or more membranes 120 and including a set of openings 235 aligned with the set of reservoirs 214; and optionally, one or more fasteners (including fastener 240) configured to retain the first substrate 210, the one or more membranes 220, and optional sealing body 230 in position relative to a set of collecting containers 250. In variations, the system 200 can additionally include a second substrate 260,Atty. DocketNo.: 43161-65181 / WO (004WO) wherein the one or more membranes 220 and optionally, the sealing body 230, are retained in position between the first substrate 210 and the second substrate 260 by the one or more fasteners. In using embodiments, variations, and examples of the system 200, material derived from each sample is retained in its own tube and does not require batching and pooling, allowing for scalable batch size.

[0278] In variations, the distribution of holes 225 can be generated in bulk material with specified hole diameter(s), hole depth(s) (e.g., in relation to membrane thickness), aspect ratio(s), hole density, and hole orientation, where, in combination with fluid parameters, the structure of the membrane can achieve desired flow rate characteristics, with reduced or eliminated poly dispersity and merging, suitable stresses (e.g., shear stresses) that do not compromise the single cells but allow for partitioning of the single cells, and steady formation of partitions (e.g., without jetting of fluid from holes of the membrane).

[0279] In variations, the hole diameter can range from 0.02 micrometers to 30 micrometers, and in examples, the holes can have an average hole diameter of 0.02 micrometers, 0.04 micrometers. 0.06 micrometers, 0.08 micrometers, 0.1 micrometers, 0.5 micrometers, 1 micrometers, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, any intermediate value, or greater than 30 micrometers (e.g., with use of membrane having a thickness greater than or otherwise contributing to a hole depth greater than 100 micrometers).

[0280] In variations, the hole depth can range from 1 micrometer to 200 micrometers (e.g., in relation to thickness of the membrane layer) or greater, and in examples the hole depth (e.g., as governed by membrane thickness) can be 1 micrometers, 5 micrometers, 10 micrometers. 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, 125 micrometers, 150 micrometers, 175 micrometers, 200 micrometers, or any intermediate value.

[0281] In variations, the hole aspect ratio can range from 5: 1 to 200: 1, and in examples, the hole aspect ratio can be 5: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1. 100: 1, 125: 1, 150: 1, 175:1, 200: 1, or any intermediate value.

[0282] In variations, the hole-to-hole spacing can range from 5 micrometers to 200 micrometers or greater, and in examples, the hole-to-hole spacing is 5 micrometers, 10 micrometers. 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, 125Atty. DocketNo.: 43161-65181 / WO (004WO) micrometers, 150 micrometers, 175 micrometers, 200 micrometers, or greater. In a specific example, the hole-to-hole spacing is greater than 10 micrometers.

[0283] In examples, the hole orientation can be substantially vertical (e.g., during use in relation to a predominant gravitational force), otherwise aligned with a direction of applied force through the distribution of holes, or at another suitable angle relative to a reference plane of the membrane or other partition generating substrate 120.

[0284] Additionally or alternatively, embodiments, variations, and examples of the methods disclosed herein can be implemented by or by way of embodiments, variations, and examples of components described in U.S. Application No. 17 / 687,080 filed 04-MAR-2022, U.S. Patent No. 11,242,558 granted 08-FEB-2022, U.S. Application No. 16 / 309,093 filed 25- MAY-2017, and PCT Application PCT / CN2019 / 093241 filed 27-JUN-2019. each of which is herein incorporated in its entirety by this reference. However, methods disclosed herein can additionally or alternatively implement other system elements for sample reception and processing.8.3.3. Method - Target-Specific Tagging and Amplification

[0285] Step S150 recites: performing target-specific (e.g., allele-specific) tagging and amplification (and / or providing suitable environments for supporting such operations), with the set of processing materials, for target regions associated with the set of targets across a set of stages. Step 220 recites: reacting the set of processing materials with the set of target proteins within the plurality of partitions. In variations of the Step 220, the set of targets comprises and / or is: a set of target proteins (e.g., Step 220b), a set of vector targets (e.g.. Step 220c), or a set of undesired DNA targets (Step 220d). Steps SI 50, S220, and associated Steps S 151, SI 52, and SI 53 (disclosed in more detail herein and shown in the schematic of exemplary FIG. 7 and FIG. 8) preferably occur within partitions generated from the sample.

[0286] In one embodiment, the sample can be combined with an embodiment, variation, or example of the set of processing materials disclosed herein, and then partitioned such that template molecules of the sample occupy individual partitions with minimal overlap between different template molecules. Partitioning can be performed by passing the sample combined with the set of processing materials through a partitioning device (e.g., to generate partitions, to generate a partition matrix with partitions provided in a continuous phase, etc.).Partitioning can alternatively be performed by distribution of the sample combined with the set of processing materials across a set of containers (e.g., microwells, nanowells, etc.).Atty. DocketNo.: 43161-65181 / WO (004WO)Partitioning can still alternatively be performed by distributing the sample combined with the set of processing materials across a substrate (e.g., as spots) and / or in another suitable manner.

[0287] In embodiments of the methods, systems, and compositions disclosed herein, reacting the set of processing materials with the set of targets comprises binding the set of targets with probes of a set of probes. In some embodiments, reacting the set of processing materials with the set of target proteins comprises binding the set of target proteins with probes of a set of probes. In some embodiments, reacting the set of processing materials with the set of vector targets comprises binding the set of vector targets with probes of a set of probes.

[0288] As shown in FIG. 7, Steps S150 and / or S220 function to denature the template material (e.g., DNA template) of the sample, anneal components to the target region(s) being evaluated, and amplify the target region(s) in a first stage, with generation of complements of allele-specific tail sequences in a second stage. Then, in subsequent stages of amplification, amounts of allele-specific tagged sequences increase in a manner that can be detected (e.g., through an optical-based detection method).

[0289] As show n in FIG. 7, the first stage of sample processing S151 can include denaturing of sample material (e.g., sample DNA) and processing the denatured sample material with primers (i.e., allele-specific forward primers grouped with corresponding reverse primers for each target). In the first stage, one of the allele-specific forward primers of the set of sample processing materials matches the target (e g., target SNP) and, with the common reverse primer, amplifies the target region. As such, targets (e.g., target SNPs) present in the sample are amplified upon interacting with respective allele-specific forw ard primers.

[0290] As shown in FIG. 7, the second stage of sample processing S152 can include generation of allele-specific sequences (e.g., tail sequences), where the common reverse primer binds to, elongates, and produces a complementary copy of a labeled tail sequence corresponding to the target allele.

[0291] As shown in FIG. 7, the third stage of sample processing SI 53 and subsequent stages can include one or more rounds of amplification / PCR to produce a detectable signal, whereby levels of tagged allele-specific sequences increase until a detection threshold is reached and / or surpassed. In the third stage SI 53 and subsequent stages of sample processing, labeled oligonucleotides bind to new' complementary' sequences (e.g., tail sequences), releasing fluorophores from corresponding quenchers to produce detectable signals for each target (e.g., target SNP, other target) present in the sample. How ever, fluorophores corresponding toAtty. DocketNo.: 43161-65181 / WO (004WO) targets that are not present are not released and thus continue to be quenched during rounds of amplification. In particular, with regard to parameters associated with threshold cycles at which or beyond which amplified targets become detectable (e.g., Ct, Cp, Cq, etc.), step S 153 can include detecting and / or returning results indicative of target presence prior to the endpoint of the process and / or at the end-point of the process (e.g., as in end-point PCR). Additionally or alternatively, real-time measurement of signals can be performed contemporaneously with each cycle of amplification.

[0292] In relation to the one or more stages of sample processing, activation-associated steps can be performed at a temperature or temperature profile (e.g., 90 °C, 92 °C, 94 °C, another suitable temperature), for a duration of time (e.g., 10 minutes, 12 minutes, 15 minutes, another suitable duration of time), and / or for a number of cycles (e.g.. 1 cycle, 2 cycles, another suitable number of cycles). In relation to the one or more stages of sample processing, denaturation-associated steps can be performed at a temperature (e.g., 90°C, 92 °C, 94 °C, another suitable temperature) or temperature profile, for a duration of time (e.g., 10 seconds, 15 seconds, 20 seconds, 25 seconds,, another suitable duration of time), and / or for a number of cycles (e.g., 1 cycle. 5 cycles, 10 cycles, 20 cycles, another suitable number of cycles). In relation to the one or more stages of sample processing, anneal ing / elongati on- associated steps can be performed at a temperature or temperature profile (e.g., 52 °C - 70 °C with a ramp down rate, another suitable temperature profile), for a duration of time (e.g., 20 seconds, 30 seconds. 60 seconds, 90 seconds, another suitable duration of time), and / or for a number of cycles (e.g., 1 cycle, 5 cycles, 10 cycles, 20 cycles, 25 cycles, 30 cycles, another suitable number of cycles).

[0293] In a specific example, activation-associated steps in a first stage of sample processing can be performed at a temperature of 94 °C, for 15 minutes, with 1 cycle. In the specific example, denaturation-associated steps in a second stage of processing can be performed at 94 °C for 20 seconds, with annealing / elongation performed from 61 °C - 55 °C (with a drop of 0.6 °C / cycle), for 60 seconds and for 10 cycles. In the specific example, denaturation-associated steps in a third stage of processing can be performed at 94 °C for 20 seconds, with annealing / elongation performed at 55 °C for 60 seconds and for 26 cycles. Additional denaturation-associated steps can be performed at 94 °C for 20 seconds, with annealing / elongation performed at 57 °C for a suitable number of cycles (e.g., 3 cycles).

[0294] In another specific example, activation-associated steps in a first stage of sample processing can be performed at a temperature of 94 °C, for 15 minutes, with 1 cycle. In the specific example, denaturation-associated steps in a second stage of processing can beAtty. DocketNo.: 43161-65181 / WO (004WO) performed at 94 °C for 20 seconds, with annealing / elongation performed from 65 °C - 57 °C (with a drop of 0.8 °C / cycle), for 60 seconds and for 10 cycles. In the specific example, denaturation-associated steps in a third stage of processing can be performed at 94 °C for 20 seconds, with annealing / elongation performed at 57 °C for 60 seconds and for 30 cycles. Additional denaturation-associated steps can be performed at 94 °C for 20 seconds, with annealing / elongation performed at 57 °C for a suitable number of cycles (e.g., 3 cycles).

[0295] Stages of sample processing in Blocks S I 50 and / or S220 can further include implementation of additives (as disclosed herein) to improve signal-to-noise ratio (SNR) characteristics in the context of multiplexed detection, by increasing signal characteristics and / or reducing background (e.g., noise other artifacts). Additionally or alternatively, stages of sample processing in Steps S150 and / or S220 can implement other components (e.g., density gradient mediums) to improve SNR.

[0296] In particular, in the context of emulsion digital PCR with the numbers of partitions disclosed herein, such multiplexed assay design aspects disclosed can produce significantly improved signal-to-noise (SNR) values with reduced background, in relation to detection techniques disclosed herein (e.g., based on lightsheet imaging, etc.). In examples, target signals can be at least 102greater than background noise signals, 103greater than background noise signals, 104greater than background noise signals, IO5greater than background noise signals, 106greater than background noise signals, 107greater than background noise signals, or better. Background noise can be attributed to fluorescence from adjacent partitions and adjacent planes of the set of planes of partitions in the context of emulsion digital PCR, or attributed to other sources with closely-positioned partitions. Determining the SNR can include scanning a set of planes of the set or plurality of partitions, determining a target signal value and a noise signal value for the set of planes, and determining the SNR from the target signal value and the noise signal value SI 80, where a variation of determining the target signal value and the noise signal value is disclosed herein.

[0297] In examples associated with reaction materials disclosed herein and used for partition-based digital PCR, determining the target signal value and a noise signal value can include: for each plane of a set of planes of partitions under interrogation (e.g., by lightsheet detection, fluorescent microscopy, confocal microscopy, detection by photodiodes, by another method of detection, etc.): determining a categorization (of a set of categorizations for the respective plane) based upon a profde of signal-positive partitions represented in a respective plane S181. determining a target signal distribution and a noise signal distribution specific to the profile SI 82. Here, a target signal value can be determined from the targetAtty. DocketNo.: 43161-65181 / WO (004WO) signal distribution SI 83, and can be an average value (or other representative value) of the target signal intensities determined from the set of planes. Similarly, a noise signal value can be determined from the noise signal distribution S 184, and the background noise signal value can be an average value (or other representative value) of the noise signal intensities determined from the set of planes. A schematic is show n in FIG. 5B.

[0298] How ever, materials used for the amplification and / or detection reactions can be otherwise configured to improve SNR.8.3.4. Method - Signal Detection and Returned Outputs

[0299] As shown in FIG. 1A, the method 100 can include Step SI 10, which recites detecting signals indicative of a profile of a set of targets, from a processed sample (e.g., within a single vessel, within a set of vessels) SI 10. As shown in FIG. IF, the method 200 can include Step S230. which recites detecting signals indicative of targets of the set of targets from at least a subset of the plurality of partitions (e.g., within a single vessel, within a set of vessels) S230. In variations of the Step 230, the set of targets comprises and / or is: a set of target proteins (e.g., Step 230b), a set of vector targets (e.g., Step 230c), or a set of undesired DNA targets (Step 230d). Steps SI 10, S230, and variations thereof can function to enable detection of signals from dyes / fluorophores that are released upon processing the sample with the set of processing materials, thereby providing indications of presence of targets (e.g., SNP targets, other targets) within the sample. In particular, with regard to parameters associated with threshold cycles at which or beyond which amplified targets become detectable (e.g., Ct, Cp, Cq. etc.), steps SI 10 and / or S230 can include detecting and / or returning results indicative of target presence prior to the end-point of the process and / or at the end-point of the process (e.g., as in end-point PCR). Additionally or alternatively, real-time measurement of signals can be perform contemporaneously with each cycle of amplification.

[0300] Processed sample material can include samples processed according to methods disclosed herein, with respect to multiplexed tagging of alleles of targets of interest.

[0301] In embodiments of the methods, systems, and compositions disclosed herein, detecting signals indicative of targets of the set of targets comprises detecting signals emitted from probes of a set of probes associated with targets of the set of targets. In some embodiments, detecting signals indicative of target proteins of the set of target proteins comprises detecting signals emitted from probes of a set of probes associated w ith targetAtty. DocketNo.: 43161-65181 / WO (004WO) proteins of the set of target proteins. In some embodiments, detecting signals indicative of vector targets of the set of vector targets comprises detecting signals emitted from probes of a set of probes associated with vector targets of the set of vector targets.

[0302] In variations, detection of signals can include irradiating processed sample material with suitable excitation wavelengths of light, and / or receiving emitted wavelengths of light corresponding to released dyes / fluorophores. As such detection of signals can be implemented by an optical signal detection system (e.g., imaging system), including embodiments, variations, and examples of systems disclosed herein. In particular, detection systems can be configured for detection of signals from partitions (e.g., by light sheet imaging, by fluorescence microscopy, by confocal microscopy, by another suitable optical detection system, etc.) using combinations of filters and / or color channels, where signals from individual partitions are detected in a high-partition number but low-occupancy regime. As such, detection can be performed for partitions arranged in 3D (e.g., as in partitions of a partition matrix within a container), in 2D (e.g., for a monolayer or bi-layer of partitions at a substrate), and / or in another suitable format.

[0303] In variations, the sample can be processed with the set of processing materials in coordination with distribution of the sample across a set or plurality of partitions, where the set or plurality7of partitions can include partitions of a partition matrix or other partitions (e.g., partitions of an emulsion, partitions provided in a sheathing fluid, gel partitions, other forms of partitions), microchambers, microwells, spotted samples on a substrate, and / or other partitions. As such, the partitions can be provided within one or more of: a container configured for centrifugation (e.g., a centrifuge tube, a microcentrifuge tube, etc.), a process container for PCR (e.g., a PCR tube), a strip tube, a plate having wells (e.g., a microtiter plate, a multi-well plate, a microwell plate, a nanowell plate, etc.), or another suitable collecting container. Additionally or alternatively, partitions can include regions of sample provided in another manner upon a substrate (e.g., spotted onto a substrate / slide).

[0304] With respect to sample processing using the set of processing materials, reactions within individual partitions can thus produce signals that are detected by systems that can detect signals from multiple partitions or all partitions simultaneously in a distinguishable manner (e.g., with a 3D scanning technique, as disclosed herein). Alternatively, reactions within individual partitions can produce signals that are detected by systems that can detect signals from individual partitions in a sequential manner.

[0305] Characterizing a set of targets of a sample (e.g., in relation to presence / absence, in relation to counts of targets), upon scanning the set or plurality of partitions, can include:Atty. DocketNo.: 43161-65181 / WO (004WO) generating a multivariable vector of emission values (e.g., emitted intensity values across the set of available color channels), for each detected emitted signal from a respective partition, paired with the excitation parameters used to excite the set or plurality of partitions (e.g., in the context of probes that exhibit FRET and / or probes that can be photobleached); performing a clustering operation with the multivariable vectors of emission values generated from the set or plurality of partitions; sorting partitions of the set or plurality of partitions into a set of categories corresponding to targets of the set of targets, based upon the clustering operation and a known set of probes used to tag the set of targets; and generating a count of each of the set of targets represented in the set or plurality of partitions, based upon said sorting. In variations, the clustering operation can include performance of a co-localization operation, whereby scanning deviations are corrected for in order to further delineate / provide stronger discrimination between different clusters of partitions. Additionally or alternatively, clustering algorithms can further include one or more of: principal component analysis (PC A), k-means clustering, t-stochastic neighbor embedding (t-SNE), UMAP clustering, and / or other algorithms. In variations, characterizing partitions can further include identifying partitions that are signal positive in more than one channel, in relation to color combinatorics disclosed herein.

[0306] Step S120 recites: returning a characterization of the processed sample based upon the profile. Step S240 recites: returning a characterization of detected targets of the set of targets of the sample. In variations of the Step 240, the set of targets comprises and / or is: a set of target proteins (e g., Step 240b), a set of vector targets (e.g., Step 240c), or a set of undesired DNA targets (Step 240d). Steps S120, S240, and variations thereof can function to provide information pertaining to presence or absence of the set of targets associated with the sample being evaluated, and / or presence or absence of variants of the set of targets. The characterization can then be used to provide diagnostics and / or to support diagnostics of the organism(s) from which the processed sample was sourced, and / or to provide quality' for conclusiveness of diagnostic results. Additionally or alternatively, the characterization can be used to guide provision of therapeutics (e.g., personalized therapeutics) corresponding to determined states of the organism(s) from which the processed sample was sourced, in order to improve or maintain health statuses of the organism(s).

[0307] In specific applications, the characterization can be used to inform diagnostics, provide other characterizations (e.g., of disease resistance, of disease predisposition, of genetic relationships, etc.) and / or guide generation of therapeutics associated with non- invasive prenatal testing (disclosed herein). More broadly, outputs of steps SI 20, S240, andAtty. DocketNo.: 43161-65181 / WO (004WO) variations thereof can be used to characterize (e.g., based on relative abundance measurements) self genetic material (e.g., genetic material of an organism) and non-self genetic material (e.g., genetic material not of the organism, genetic material of a different organism) from a sample.

[0308] Additionally or alternatively, in specific applications, the characterization can be used to inform characterizations of a subject from which the sample is sourced, in relation to one or more of: cancers, integumentary system conditions, skeletal system conditions, muscular system conditions, lymphatic system conditions, respiratory system conditions, digestive system conditions, nervous system conditions, endocrine system conditions, cardiovascular system conditions, urinary system conditions, reproductive system conditions, and / or other conditions.

[0309] Outputs can additionally or alternatively support at least one of: pathogen detection, non-invasive prenatal testing, organ transplantation analysis, forensics, and oncology7, based upon the quantitative analysis.

[0310] In other specific applications, the characterization can be used to inform diagnostics, provide other characterizations (e.g., of disease resistance, of disease predisposition, of genetic relationships, etc.) and / or guide generation of therapeutics in the context of other multicellular organisms, plants, fungi, unicellular organisms, viruses, and / or other subjects.

[0311] In embodiments of the methods, systems, and compositions disclosed herein, returning the characterization comprises a linkage analysis of a full-length vector genome comprising a linked set of vector targets characteristic of the full-length vector genome, the linked set of vector targets characteristic of the full-length vector genome comprising two or more vector targets of the set of vector targets. In some embodiments, the linkage analysis provides a metric characterizing integrity of the full-length vector genome or other genome. In some embodiments, the metric characterizing the integrity of the full-length vector genome or other genome is determined from counts of the linked set of targets characteristic of the full-length genome, counts of fragments of the linked set of targets characteristic of the full- length genome, counts of unlinked vector targets of the set of targets, or a combination thereof. In some embodiments, the linked set of targets characteristic of the full-length genome, the fragments of the linked set of targets characteristic of the full-length genome, the unlinked targets of the set of targets, and amplicons thereof, comprise a length of up tolOO bp, up to 150 bp, up to 200 bp, up to 250 bp, up to 300 bp, up to 350 bp, up to 400 bp, up to 450 bp. up to 500 bp, up to 550 bp, up to 600 bp, up to 650 bp, up to 700 bp, up to 750 bp. up to 800 bp, up to 850 bp, up to 900 bp, up to 950 bp, up to 1000 bp, 1100 bp, up to 1150 bp.Atty. DocketNo.: 43161-65181 / WO (004WO) up to 1200 bp, up to 1250 bp, up to 1300 bp, up to 1350 bp, up to 1400 bp, up to 1450 bp, up to 1500 bp. up to 1550 bp, up to 1600 bp, up to 1650 bp, up to 1700 bp, up to 1750 bp. up to 1800 bp, up to 1850 bp, up to 1900 bp, up to 1950 bp, up to 2000 bp, or greater. In some embodiments, the linked set of targets characteristic of the full-length genome, the fragments of the linked set of targets characteristic of the full-length genome, the unlinked targets of the set of targets, and amplicons thereof, comprise a length of up to 2000 base pairs.

[0312] In some embodiments, returning the characterization comprises identification of a fraction of virions (e g., AAV virions) of the sample comprising a full-length vector genome. In some embodiments, returning the characterization provides a metric characterizing full capsid values associated with virions of the sample, a metric characterizing partially filled capsid values associated with virions of the sample, a metric characterizing empty capsid values associated with virions of the sample, a metric characterizing full versus empty capsid ratios associated with virions of the sample, a metric characterizing full versus partial filled capsid ratios associated with virions of the sample, or a combination thereof. In some embodiments, the ratio of full versus empty capsids is at least 1:1, 1:2, 1:4, 1:6, 1:8, 1:10,1:12. 1:12. 1:14. 1:16, 1:18, 1:20, 1:22, 1:24, 1:26, 1:28, 1:30, 1:32, 1:34, 1:36. 1:38. 1:40.1:42, 1:44, 1:46, 1:48, 1:50, 1:52, 1:54, 1:56, 1:58, 1:60, 1:62, 1:64, 1:66, 1:68, 1:70, 1:72,1:74, 1:76, 1:78, 1:80, 1:82, 1:84, 1:86, 1:88, 1:90, 1:92, 1:94, 1:96, 1:98, 1:100, 1:102,1:104, 1:106. 1:108, 1:110, 1:112, 1:114, 1:116, 1:118, 1:120, 1:122, 1:124, 1:126, 1:128, 1:130, 1:132. 1:134, 1:136, 1:138. 1:140, 1:142. 1:144, 1:146, 1:148, 1:150, or another suitable ratio. In some embodiments, the ratio of full versus partially filled capsids is at least 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:12, 1:14, 1:16, 1:18, 1:20, 1:22, 1:24, 1:26, 1:28, 1:30, 1:32, 1:34, 1:36, 1:38, 1:40, 1:42, 1:44, 1:46, 1:48, 1:50, 1:52, 1:54, 1:56, 1:58, 1:60, 1:62, 1:64, 1:66, 1:68, 1:70, 1:72, 1:74, 1:76, 1:78, 1:80, 1:82, 1:84, 1:86, 1:88, 1:90. 1:92. 1:94. 1:96, 1:98, 1:100, 1:102, 1:104, 1:106, 1:108, 1:110, 1:112, 1:114, 1:116, 1:118, 1:120, 1:122, 1:124, 1:126, 1:128, 1:130, 1:132, 1:134, 1:136, 1:138, 1:140, 1:142, 1:144, 1:146, 1 : 148, 1 : 150, or another suitable ratio.

[0313] In some embodiments, returning the characterization comprises identification of a concentration of a full-length vector genome of the sample. In some embodiments, returning the characterization provides a count of the full-length vector genomes of the sample.

[0314] In some embodiments, returning the characterization comprises two or more of: a linkage analysis of a full-length vector genome comprising a linked set of vector targets characteristic of the full-length vector genome; identification of a fraction of virions of theAtty. DocketNo.: 43161-65181 / WO (004WO) sample comprising a full-length vector genome; and identification of a concentration of a full-length vector genome of the sample.

[0315] In some embodiments, the methods, systems, and compositions disclosed herein provide at least a 1-log, at least a 2-log, at least a 3-log, at least a 4-log, at least a 5-log, at least a 6-log, at least a 7-log, or at least an 8-log dynamic range that enables the simultaneous characterization of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 1-log dynamic range that enables the simultaneous characterization of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 2-log dynamic range that enables the simultaneous characterization of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 3-log dynamic range that enables the simultaneous characterization of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 4- log dynamic range that enables the simultaneous characterization of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 5-log dynamic range that enables the simultaneous characterization of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 6-log dynamic range that enables the simultaneous characterization of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 7-log dynamic range that enables the simultaneous characterization of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least an 8-log dynamic range that enables the simultaneous characterization of one or more targets of the set of targets.

[0316] In some embodiments, the methods, systems, and compositions disclosed herein provide at least a 1-log, at least a 2-log, at least a 3-log, at least a 4-log, at least a 5-log, at least a 6-log, at least a 7-log, or at least an 8-log dynamic range that enables the simultaneous quantification of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 1-log dynamic range that enables the simultaneous quantification of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 2-log dynamic range that enables the simultaneous quantification of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 3-log dynamic range that enables the simultaneous quantification of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 4-logAtty. DocketNo.: 43161-65181 / WO (004WO) dynamic range that enables the simultaneous quantification of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 5-log dynamic range that enables the simultaneous quantification of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 6-log dynamic range that enables the simultaneous quantification of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least a 7-log dynamic range that enables the simultaneous quantification of one or more targets of the set of targets. In some embodiments, the methods, systems, and compositions provide at least an 8-log dynamic range that enables the simultaneous quantification of one or more targets of the set of targets.

[0317] In some embodiments, the methods, systems, and compositions disclosed herein provide at least a 1-log, at least a 2-log, at least a 3-log, at least a 4-log, at least a 5-log, at least a 6-log, at least a 7-log, or at least an 8-log dynamic range that enables the simultaneous characterization of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a l-log dynamic range that enables the simultaneous characterization of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 2-log dynamic range that enables the simultaneous characterization of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 3-log dynamic range that enables the simultaneous characterization of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 4-log dynamic range that enables the simultaneous characterization of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 5-log dynamic range that enables the simultaneous characterization of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 6-log dynamic range that enables the simultaneous characterization of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 7-log dynamic range that enables the simultaneous characterization of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least an 8-log dynamic range that enables the simultaneous characterization of one or more target proteins of the set of target proteins.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0318] In some embodiments, the methods, systems, and compositions disclosed herein provide at least a 1-log, at least a 2-log, at least a 3-log, at least a 4-log. at least a 5-log, at least a 6-log, at least a 7-log, or at least an 8-log dynamic range that enables the simultaneous quantification of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 1-log dynamic range that enables the simultaneous quantification of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 2- log dynamic range that enables the simultaneous quantification of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 3-log dynamic range that enables the simultaneous quantification of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 4-log dynamic range that enables the simultaneous quantification of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 5-log dynamic range that enables the simultaneous quantification of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 6-log dynamic range that enables the simultaneous quantification of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least a 7-log dynamic range that enables the simultaneous quantification of one or more target proteins of the set of target proteins. In some embodiments, the methods, systems, and compositions provide at least an 8-log dynamic range that enables the simultaneous quantification of one or more target proteins of the set of target proteins.

[0319] In some embodiments, the methods, systems, and compositions disclosed herein provide at least a 1-log, at least a 2-log, at least a 3-log, at least a 4-log, at least a 5-log, at least a 6-log, at least a 7-log, or at least an 8-log dynamic range that enables the simultaneous characterization of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 1-log dynamic range that enables the simultaneous characterization of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 2-log dynamic range that enables the simultaneous characterization of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 3-log dynamic range that enables the simultaneous characterization of one or more vector targets of the set of vector targets. In someAtty. DocketNo.: 43161-65181 / WO (004WO) embodiments, the methods, systems, and compositions provide at least a 4-log dynamic range that enables the simultaneous characterization of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 5-log dynamic range that enables the simultaneous characterization of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 6-log dynamic range that enables the simultaneous characterization of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 7-log dynamic range that enables the simultaneous characterization of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least an 8-log dynamic range that enables the simultaneous characterization of one or more vector targets of the set of vector targets.

[0320] In some embodiments, the methods, systems, and compositions disclosed herein provide at least a 1-log, at least a 2-log, at least a 3-log, at least a 4-log, at least a 5-log, at least a 6-log, at least a 7-log. or at least an 8-log dynamic range that enables the simultaneous quantification of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 1-log dynamic range that enables the simultaneous quantification of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 2- log dynamic range that enables the simultaneous quantification of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 3-log dynamic range that enables the simultaneous quantification of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 4-log dynamic range that enables the simultaneous quantification of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 5-log dynamic range that enables the simultaneous quantification of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 6- log dynamic range that enables the simultaneous quantification of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least a 7-log dynamic range that enables the simultaneous quantification of one or more vector targets of the set of vector targets. In some embodiments, the methods, systems, and compositions provide at least an 8-log dynamic range that enables the simultaneous quantification of one or more vector targets of the set of vector targets.Atty. DocketNo.: 43161-65181 / WO (004WO)8.3.5. Method - Signal Detection and Returned Outputs for Fetal Fraction and Non-Invasive Prenatal Testing Applications

[0321] As show n in FIG. 1C, in a variation, a method 100b for determination of fetal fraction (FF) can include: Step SI 10c, which recites: detecting signals indicative of a profile of a set of single nucleotide polymorphisms from a sample. Step S 110c functions to enable characterizations of presence or absence of alleles of a set of SNPs from a sample (e.g., a maternal sample, other sample), which can be used to determine FF and inform conclusiveness of results in NIPT applications. In particular, with regard to parameters associated with threshold cycles at which or beyond which amplified targets become detectable (e.g., Ct, Cp, Cq, etc.), step SI 10c can include detecting and / or returning results indicative of SNP allele presence prior to the end-point of the process and / or at the end-point of the process (e.g., as in end-point PCR).

[0322] Processed sample material can include samples processed according to methods disclosed herein, with respect to multiplexed tagging of alleles of SNPs of interest.

[0323] In variations, detection of signals can include irradiating processed sample material with suitable excitation wavelengths of light, and / or receiving emitted wavelengths of light corresponding to released dyes / fluorophores. As such detection of signals can be implemented by an optical signal detection system (e.g., imaging system), including embodiments, variations, and examples of systems disclosed herein. In particular, detection systems can be configured for detection of signals from partitions (e.g., by light sheet imaging, by another suitable optical detection system, etc.) using combinations of filters and / or color channels, where signals from partitions are detected in a high-partition number but low-occupancy regime.

[0324] In variations, the sample can be processed with the set of processing materials in coordination with distribution of the sample across a set or plurality of partitions. Additionally or alternatively, the partitions can be provided within one or more of: a container configured for centrifugation (e.g., a centrifuge tube, a microcentrifuge tube, etc.), a process container for PCR (e.g., a PCR tube), a strip tube, a plate having wells (e.g., a microtiter plate, a multi-well plate, a microwell plate, a nanowell plate, etc.), or another suitable collecting container. Additionally or alternatively, partitions can include regions of sample provided in another manner upon a substrate (e.g., spotted onto a substrate / slide).Atty. DocketNo.: 43161-65181 / WO (004WO)

[0325] Step SI 20c recites: returning a characterization of relative abundance of alleles of each SNP in the set of SNPs to generate an estimate of fetal DNA fraction in the sample, which functions to enable determinations of conclusiveness of NIPT results.

[0326] In variations, SNP alleles processed and evaluated in a massively parallel manner to determine FF in step S120c can include SNPs associated with chromosomes 1, 13, 18, 21, X, and / or Y, at various loci (e.g., from 10 to 20,000 polymorphic loci); however, SNPs characterized to determine FF can additionally or alternatively be associated with other chromosomes and / or loci. SNPs evaluated can be biallelic or multiallelic, with more than two alleles per SNP. SNPs evaluated can further be characterized by a high minor allele fraction (MAF), with an MAF above a suitable threshold (e.g., MAF >0.2, MAF >0.3, MAF >0.4, etc.); however. SNPs evaluated can be characterized with other MAF values. SNPs evaluated can be for coding regions (e.g., synonymous, non-synonymous, missense, nonsense) and / or non-coding regions.

[0327] With respect to determination of FF in Step SI 20c, target panels undergoing evaluation can be designed such that FF associated with fetus of any gender can be determined, without requiring detection of chromosome Y markers. As such, for a male fetus. FF can be estimated by the amount of chromosome Y fragments present in the sample (e.g., maternal sample) relative to the amount of other non-sex chromosomes. For determination of FF for a female fetus, the set of SNPs evaluated are selected such that for each fetus-mother pair, there would be at least a few SNPs in the common SNP panel that are homozygous in mother and heterozygous in fetus. The count of the alternate allele from the fetus, when compared to the count of the homozygous allele (from mother, and also half from fetus), would yield FF for a female fetus (or non-male fetus, such as in intersex conditions).

[0328] In a specific application, the method can implement counting requirements per reference chromosome to provide indications of confidence in NIPT assay results with respect to threshold FF values. In a specific example, for a counting requirement of 400,000 counts per reference chromosome, the lowest FF (e.g., DNA FF) in which an anueploidy assay would be confident in calling a true negative is ~4%; thus, the FF assay estimates <4% DNA FF, then the results from the aneuploidy assay would be inconclusive. However, if the FF assay estimates > 4% DNA FF, then the results from the aneuoploidy assay would be more conclusive with increasing FF.

[0329] However, in other specific examples, the counting requirement per reference chromosome can be set at another value (e.g., less than 400,000 counts, greater than 400,000Atty. DocketNo.: 43161-65181 / WO (004WO) counts, etc.) in relation to other FF threshold values (e.g., 3%, 5%, 6%, other percentages, etc.).

[0330] Expansions of the methods can be applied to detection of sex aneuplodies (e.g., Klinefelter syndrome, Turner syndrome, etc.), trisomies (e.g., Downs syndrome, Edwards syndrome, Palau syndrome, etc.), and / or other genetic conditions.8.3.6. Method - Signal Detection and Returned Outputs for Transplant Rejection Applications

[0331] In another example, materials and methods disclosed herein can be adapted for characterization and / or early detection of transplant rejection in a subject. Methods disclosed herein can be used to detect and digitally quantify donor-specific genetic material (e.g., dd- cfDNA, ds-DNA, GcfDNA) in a sample from a subject who has received a transplant, such that the sample potentially contains a quantifiable amount of self genetic material and donor genetic material. Furthermore, longitudinal characterization of the amount of donor genetic material in samples acquired from the subject at different time points can be used to assess onset of transplant rejection, where increases in donor genetic material over time can serve as a proxy for transplant rejection.

[0332] The biological rationale behind the utility’ of donor genetic material as a biomarker for transplant rejection is that the immune system of the subject receiving a transplant is activated upon recognition of the transplanted material (e.g., organ, cells, etc.) and produces antibodies in response. The antibodies attack the transplanted material, which leads to apoptosis or cell necrosis. The ruptured or dead cells then release their contents into the subject’s blood plasma and thereafter, the subject carries the genetic material of the donor, in a detectable manner.

[0333] The methods disclosed (e.g., 100. 100b, 100c, lOOd, 200, 200b, 200c, 200d) can, however, include other suitable steps and / or enable other downstream applications.

[0334] For instance, in another specific use case, the methods can be adapted for evaluation of minimal residual disease (MRD) based upon detection of numbers of cancer cell targets present in a sample from a subject after one or more phases of cancer treatment (e.g.. treatment of leukemia, treatment of lymphoma, treatment of multiple myeloma, etc.).

[0335] In another specific use case, the methods can be adapted for single nucleotide polymorphism genotyping (SNPtyping) to measure genetic variations of SNPs between members (e.g., members of a species). Additionally, the invention(s) can be used for single nucleotide variant genotyping (SNV typing) for germline DNA samples.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0336] The invention(s) can also be used for applications involving disease prediction generation and monitoring with multiplexed detection of markers of a gene expression marker panel (e.g., for pregnancy-associated complications, for other applications).

[0337] In another specific use case, the methods can be adapted for ribosomal 16S and / or ITS characterization, where current sequencing technologies are fraught with high false positive rates and / or high PCR error. In relation to the specific use case, systems, methods, and compositions disclosed can be used to disperse a sample of I6S and / or ITS ribosomal RNA (rRNA) across a plurality of partitions (as disclosed in more detail herein), where processing materials disclosed enable detection of regions / sequences of interest (e.g., V3 region, V4 region, V5, region, other hypervariable regions, etc.), and subsequently, for operational taxonomic unit (OTU) or amplicon sequence variant (ASV) categorizations. For instance, detection of V3, V4, and / or V5 regions can be used for bacterial microbiome analyses, fungal microbiome analyses, other microbiome analyses, rare species detection, and / or other applications. Additionally or alternatively, such rRNA characterizations can be used for antimicrobial susceptibility testing (e.g., with a sample having one or more antibiotics being assessed, combined with bacteria and materials that can be used to indicate bacteria responses to the antibiotic(s)). Additionally or alternatively, such rRNA characterizations can be used for detection of a set of pathogens (e.g., up to 30 pathogens, up to 40 pathogens, up to 50 pathogens, up to 60 pathogens, up to 70 pathogens, etc.) and quantification (e.g., in relation to detection of presence or absence of various pathogens, in relation to characterization of infectious agents and potential prognoses). Additionally or alternatively, for microbial pathogen detection / quantification, any part of microbial genomics of a sample (e.g., non-rRNA targets) can be targeted, and subsequent detection can involve detection of sample composition (e.g.. microbial composition, microbiome composition, etc.) without performance of next generation sequencing (NGS). In a related use case, detection / quantification of targets of a sample in a multiplexed manner can be used to differentiate betw een viral, fungal, and / or microbial infections (e.g., for a respiratory illness panel).

[0338] Embodiments of the methods disclosed herein can be further adapted for other applications of use.Atty. DocketNo.: 43161-65181 / WO (004WO)8.3.7. Method - Universal Multiplexing

[0339] In some embodiments, the systems, methods, compositions, and kits disclosed herein employ a Universal Multiplexing approach.

[0340] Universal Multiplexing (UM) is a versatile, cost-effective solution for assaying multiple targets (e.g., with Countable PCR). Universal Multiplexing offers a solution to develop multiplex assays for up to four targets at approximately 1 / 10 the cost of conventional hydrolysis probe (HP) multiplexed assays. Using standard unmodified primers, UM delivers the same performance and specificity as multiplexed hydrolysis probe assays. Combining UM with Countable PCR delivers a versatile, straightforward solution for developing multiplex PCR assays with single-molecule precision.

[0341] Multiplex PCR is a powerful technique that enables the simultaneous detection of multiple targets in a single reaction. Rather than detecting individual targets separately and comparing results, multiplexing reduces reaction size and sample consumption and improves quantification accuracy by minimizing pipetting errors. However, developing multiplex PCR assays presents challenges that have limited their widespread adoption.

[0342] Countable PCR overcomes multiplexing challenges through true single-molecule amplification, as disclosed herein. In conventional qPCR or dPCR, balancing amplification kinetics between amplicons is difficult because target molecules compete for resources in the same reaction vessel or partition. This varying amplification efficiency can lead to amplification bias between targets, resulting in inaccurate quantification and potential target dropouts. In contrast, Countable PCR isolates each molecule (e g., DNA molecule) in its compartment within a gel-like matrix for independent amplification. Multiplexing becomes achievable with minimal optimization, without competition between targets within compartments.

[0343] To address challenges associated with traditional multiplexed assays, UM was developed. A UM assay and its comparative performance with HP -based assays on the Countable PCR platform are disclosed herein.

[0344] In one aspect, the present disclosure provides a UM assay. In some embodiments, the UM assay comprises a 1-plex UM assay. In some embodiments, the UM assay comprises a 2- plex UM assay. In some embodiments, the UM assay comprises a 3-plex UM assay. In some embodiments, the UM assay comprises a 4-plex UM assay. In some embodiments, the UM assay comprises a level of plexy of 4 or more.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0345] FIG. 31 illustrates the assay principle of UM chemistry . UM uses generic prefixed probe sequences with target-specific primers. In UM, one primer is appended with a UM adapter sequence. During initial PCR cycles, the primer with the UM adapter binds to the template and extends. In subsequent cycles, the non-UM primer (typically R primer) binds to the forward template (now with UM adapter) and extends to create a UM probe complementary sequence. Detection occurs when the UM probe hybridizes to the probe binding site within the amplicon. The reaction can involve a 1 :5 ratio (or another suitable ratio) of primer with UM adapter to the non-UM primer to drive the generation of antisense templates with the probe binding site. In some embodiments, the reaction can involve a 1 : 1, 1:2, 1 :3, 1 :4, 1 :5. 1:6, 1:7, 1 :8, 1:9, 1 :10, 1 : 11, 1: 12. 1: 13, 1: 14, 1: 15, 1: 16, 1: 17, 1:18, 1: 19, 1:20, 1:21, 1:22, 1:23, 1 :24, 1 :25, 1 :26, 1 :27, 1:28, 1:29, 1 :30, or another suitable ratio of primer with UM adapter to the non-UM primer.

[0346] To convert an existing probe-based or DNA intercalating dye-based assay, a UM adapter sequence can be added to the 5’ end of either the forward or reverse primers to create a UM primer. The other primer can remain unchanged. In some embodiments, the UM adapter sequence is added to the 5’ end of the forward primer to create the UM primer. In some embodiments, the UM adapter sequence is added to the 5’ end of the reverse primer to create the UM primer.

[0347] A Universal Multiplexing kit can be used to develop multiplex assays for multiple targets per reactions. In some embodiments, the Universal Multiplexing kit comprises a UM probe. In some embodiments, the Universal Multiplexing kit comprises one UM probe. In some embodiments, the Universal Multiplexing kit comprises two UM probes. In some embodiments, the Universal Multiplexing kit comprises three UM probes. In some embodiments, the Universal Multiplexing kit comprises four UM probes. In some embodiments, the Universal Multiplexing kit comprises four or more UM probes. In some embodiments, the Universal Multiplexing kit comprises a UM probe comprising an adapter sequence selected from SEQ ID NOs: 1-4. In some embodiments, the Universal Multiplexing kit comprises a UM probe comprising an adapter sequence of SEQ ID NO: 1. In some embodiments, the Universal Multiplexing kit comprises a UM probe comprising an adapter sequence of SEQ ID NO: 2. In some embodiments, the Universal Multiplexing kit comprises a UM probe comprising an adapter sequence of SEQ ID NO: 3. In some embodiments, the Universal Multiplexing kit comprises a UM probe comprising an adapter sequence of SEQ ID NO: 4.Atty. DocketNo.: 43161-65181 / WO (004WO)

[0348] In some embodiments, the Universal Multiplex kit can be used to develop multiplex assays for one target per reaction. In some embodiments, the Universal Multiplex kit can be used to develop multiplex assays for up to two targets per reaction. In some embodiments, the Universal Multiplex kit can be used to develop multiplex assays for up to three targets per reaction. In some embodiments, the Universal Multiplex kit can be used to develop multiplex assays for up to four targets per reaction. In some embodiments, the Universal Multiplex kit can be used to develop multiplex assays for four or more targets per reaction.

[0349] In some embodiments, the target comprises a target as disclosed herein (e.g., target component, rare target component, etc.).

[0350] In a specific example, a Universal Multiplex kit comprising UM probes was used to develop multiplex assays for up to four targets per reaction. Table 4 lists the UM adapter sequences for the UM-1, UM-2, UM-3, and UM-4 probes within the exemplary Universal Multiplex kit.Table 2. Four UM adapter sequences in the exemplary Universal Multiplex kit.

[0351] Table 3 shows the sequences of both UM primers and non-UM primers designed for each of the four targets in the UM assays of the specific example. Table 3 also shows how these primers were derived from forward and reverse primers from a previously designed HPbased assay.Table 3. Primer sequences used for the exemplary’ UM assays. A UM adapter ...

Claims

Atty. DocketNo.: 43161-65181 / WO (004WO)CLAIMSWhat is claimed is:

1. A method for high sensitivity detection of target proteins of a sample, the method comprising: generating a plurality of partitions comprising at least 500,000 partitions within a single closed container, wherein the plurality of partitions comprises: a set of target proteins of the sample, and a set of processing materials. wherein each partition of the plurality of partitions contains at most one target protein of the set of target proteins; reacting the set of processing materials with the set of target proteins within the plurality of partitions; detecting signals indicative of target proteins of the set of target proteins from at least a subset of the plurality of partitions upon scanning the single closed container with a detection system; and returning a characterization of detected target proteins of the set of target proteins of the sample from the detected signals.

2. The method of claim 1, wherein the set of target proteins comprises a capsid protein.

3. The method of claim 2, wherein the capsid protein is a VP 1, VP2, or VP3 capsid protein.

4. The method of claim 2, wherein the capsid protein is selected from a wild-type or modified capsid protein selected from: AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-B; hu.45-B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh, 19-B; rh.49-B; rh.52-B; rh, 13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu. 9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.l-C; hu, 18-C; hu.3-C; hu.25-C; hu, 15-C; hu,16-C; hu.l l-C; hu.lO-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E: hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40- E; hu.67-E; hu, 17-E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81;Atty. DocketNo.: 43161-65181 / WO (004WO)Anc82; Anc83; Anc84; Anc94; And 13; Ancl26; Ancl27; Anc80L27; Anc80L59; Anc80L60: Anc80L62; Anc80L65; Anc80L33; Anc80L36; Anc80L44; Anc80Ll; And 10; and Anc80DI.

5. A method for high sensitivity detection of vector targets of a sample, the method comprising: generating a plurality' of partitions comprising at least 500,000 partitions within a single closed container, wherein the plurality of partitions comprises: a set of vector targets of the sample, and a set of processing materials, wherein each partition of the plurality of partitions contains at most one vector target of the set of vector targets or one linked set of vector targets of the set of vector targets; reacting the set of processing materials with the set of vector targets within the plurality of partitions; detecting signals indicative of vector targets of the set of vector targets from at least a subset of the plurality' of partitions upon scanning the single closed container with a detection system; and returning a characterization of detected vector targets of the set of vector targets of the sample from the detected signals.

6. The method of claim 5, wherein the set of vector targets comprises an adeno- associated virus (AAV) viral vector, an adenovirus (AdV) viral vector, a lentivirus viral vector, or a retrovirus viral vector.

7. The method of claim 6, wherein the adeno-associated virus (AAV) viral vector comprises a vector genome, wherein the full-length vector genome comprises a linked set of vector targets of the set of vector targets, the linked set of vector targets comprising at least a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target.

8. The method of claim 7, wherein the full-length vector genome has a length of up to 5 kb.

9. The method of claim 5, wherein the plurality of partitions comprises at least 1 million partitions.Atty. DocketNo.: 43161-65181 / WO (004WO)10. The method of claim 5, wherein the plurality' of partitions is characterized by less than 15% occupancy of partitions by the set of vector targets.

11. The method of claim 5, wherein the plurality’ of partitions is stabilized in position as a partition matrix in a close-packed format.

12. The method of claim 5, wherein the set of vector targets comprises a cargo target, a promoter target, a transcription terminator target, an inverted terminal repeat (ITR) target, a Rep target, and a helper target.

13. The method of claim 5, wherein the linked set of vector targets comprises two or more vector targets independently selected from: a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target.

14. The method of claim 13, wherein the linked set of vector targets comprises at least a cargo target, a promoter target, a transcription terminator target, and an inverted terminal repeat (ITR) target.

15. The method of claim 12, wherein the cargo target is a gene therapy cargo target.

16. The method of claim 12, wherein the promoter target is selected from CMV, SV40, EFla, CAG, PGK, Ubc, human beta actin, CBh, CaMKIIa, TEF1, Hl, p5, p40, and p41.

17. The method of claim 12, wherein the transcription terminator target is selected from SV40, hGH, BGH, and rbGlob.

18. The method of claim 12, wherein the transcription terminator target further comprises a polyA signal, wherein the polyA signal comprises a AAUAAA nucleotide sequence motif.

19. The method of claim 12, wherein the inverted terminal repeat (ITR) target comprises an inverted terminal repeat (ITR) from wild-type adeno-associated virus (AAV) or a variant thereof.

20. The method of claim 5, wherein the set of vector targets comprises one or more Rep targets.Atty. DocketNo.: 43161-65181 / WO (004WO)21. The method of claim 20, wherein the one or more Rep targets comprise a rep gene encoding a Rep protein.

22. The method of claim 21, wherein the Rep protein is selected from a Rep 78 protein, a Rep 68 protein, a Rep 52 protein, and a Rep 40 protein.

23. The method of claim 22. wherein the Rep protein is from an AAV serotype selected from AAV 1 , AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV1 1 , AAV12, and AAV13.

24. The method of claim 20, wherein the one or more Rep targets comprise at least one promoter.

25. The method of claim 5, wherein the linked set of vector targets comprises two or more Rep targets.

26. The method of claim 5, wherein the set of vector targets comprises a helper target.

27. The method of claim 5, wherein the linked set of vector targets comprises two or more helper targets.

28. The method of claim 27. wherein the helper target comprises a helper virus gene.

29. The method of claim 28, wherein the helper virus gene is selected from one or more of Adenovirus 5 or Adenovirus 2.

30. The method of claim 5, wherein the set of processing materials comprises, for a first vector target of the set of vector targets, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the first vector target, the set of target-specific primers comprising: a target-specific primer comprising a first adapter sequence, and a first liuorophore-labeled oligonucleotide corresponding to the first adapter sequence, the first fluorophore-labeled oligonucleotide comprising a first fluorophore configured to transmit a first target signal if the target region is amplified.Atty. DocketNo.: 43161-65181 / WO (004WO)31. The method of claim 30, wherein the set of processing materials further comprises, for a second vector target of the set of vector targets, a primer set comprising: a common primer, and a set of target-specific primers configured to interact with a target region of the second vector target, the set of target-specific primers comprising: a target-specific primer comprising a second adapter sequence, and a second fluorophore-labeled oligonucleotide corresponding to the second adapter sequence, the second fluorophore-labeled oligonucleotide comprising a second fluorophore configured to transmit a second target signal if the target region is amplified.

32. The method of claim 5, wherein the set of processing materials comprises, for a first vector target and a second vector target of the set of vector targets, a primer set comprising: at least one primer configured to tag the first vector target with a first probe having a first fluorophore, and at least one primer configured to tag the second vector target with a second probe having a second fluorophore.

33. The method of claim 5, wherein the set of processing materials comprises a set of probes configured to: associate with vector targets of the set of vector targets upon reacting the set of processing materials with the set of vector targets, and emit fluorescent signals upon associating with vector targets of the set of vector targets.

34. The method of claim 5, wherein reacting the set of processing materials with the set of vector targets comprises binding the set of vector targets with probes of a set of probes.

35. The method of claim 5, wherein detecting signals indicative of vector targets of the set of vector targets comprises detecting signals emitted from probes of a set of probes associated with vector targets of the set of vector targets.

36. The method of claim 5, wherein returning the characterization comprises a linkage analysis of a full-length vector genome comprising a linked set of vector targets characteristicAtty. DocketNo.: 43161-65181 / WO (004WO) of the full-length vector genome, the linked set of vector targets characteristic of the full- length vector genome comprising two or more vector targets of the set of vector targets.

37. The method of claim 36, wherein the linkage analysis provides a metric characterizing integrity of the full-length vector genome.

38. The method of claim 37. wherein the metric characterizing the integrity of the full-length vector genome is determined from counts of the linked set of vector targets characteristic of the full-length vector genome, counts of fragments of the linked set of vector targets characteristic of the full-length vector genome, counts of unlinked vector targets of the set of vector targets, or a combination thereof.

39. The method of claim 38. wherein the linked set of vector targets characteristic of the full- length vector genome, the fragments of the linked set of vector targets characteristic of the full-length vector genome, the unlinked vector targets of the set of vector targets, and amplicons thereof, comprise a length of up to 2000 base pairs.

40. The method of claim 5, wherein returning the characterization comprises identification of a fraction of virions of the sample comprising a full-length vector genome.

41. The method of claim 40, wherein returning the characterization provides a metric characterizing full capsid values associated with virions of the sample, a metric characterizing partially filled capsid values associated with virions of the sample, a metric characterizing empty capsid values associated with virions of the sample, a metric characterizing full versus empty capsid ratios associated with virions of the sample, a metric characterizing full versus partially filled capsid ratios associated with virions of the sample, or a combination thereof.

42. The method of claim 5, wherein returning the characterization comprises identification of a concentration of a full-length vector genome of the sample.

43. The method of claim 42, wherein returning the characterization provides a count of the full-length vector genomes of the sample.

44. The method of claim 5, wherein returning the characterization comprises two or more of:Atty. DocketNo.: 43161-65181 / WO (004WO) a linkage analysis of a full-length vector genome comprising a linked set of vector targets characteristic of the full-length vector genome; identification of a fraction of virions of the sample comprising a full-length vector genome; and identification of a concentration of a full-length vector genome of the sample.

45. The method of claim 5, further comprising performing a proximity ligation assay (PL A) with contents of the plurality of partitions.

46. The method of claim 5, wherein the method provides at least a 6-log dynamic range that enables simultaneous quantification of one or more vector targets of the set of vector targets.

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