Systems and methods of sequencing polynucleotides with four labeled nucleotides

Abstract

The application relates to DNA sequencing systems and methods. Systems and methods for determining the nucleotide sequence of a polynucleotide may include introducing a fourth labeled nucleotide in a two-channel sequencing by synthesis system that allows for encoding space for a detecting a fifth labeled nucleotide or empty well detection.

Description

ILLINC.764WO / IP-2536-PCT PATENT SYSTEMS AND METHODS OF SEQUENCING POLYNUCLEOTIDES WITH FOUR LABELED NUCLEOTIDES INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 511,364 filed June 30, 2023, the content of which is incorporated by reference in its entirety. BACKGROUND Field

[0002] The present disclosure relates to DNA sequencing systems and methods. In particular, this disclosure relates to improved detection methods for detecting four or more nucleotides by labeling four nucleotides. Background

[0003] Current sequencing technologies involve determining DNA or RNA sequences by deciphering four natural bases in the genome: A, T (U), G, and C. However, many of these DNA or RNA sequences have modified nucleotide bases. These modified bases play essential roles in biological processes such as epigenetic studies, epi-transcriptomics, human diseases, and cancer. A common form of DNA modification is Methylated C (5-methylcytosine or 5-MeC) found in CpG dinucleotides. RNA modifications can also arise from noncoding RNA, such as ribosomal RNA and transfer RNA. The current standard for DNA methylation analysis typically uses genome sequencing of bisulfite-converted DNA. Since uracil, read as thymine, will bind to complementary adenosine, 5-MeC can be partially inferred with only four base detection. However, as the number and complexity of chemical base modifications continue to grow, it may be advantageous to be able to detect more than the four unmodified bases during a DNA sequencing process.

[0004] Current base calling schemes for four bases generally include two and four- channel base calling. Some sequencing systems, such as those from Illumina, Inc. (San Diego, CA) use onboard real-time analysis (RTA) to turn raw image data into base calls. This process can be massively parallelized in order to occur in real time on the instrument. The number of images fedinto the RTA base calling software could be either four images (referred to as four-channel base calling or four-dye base calling) or two images (referred to as two-channel base calling).

[0005] Normally in such systems, clusters of the polynucleotide inserts to be sequenced are formed within wells on a flowcell. One cluster is positioned into each well so that a single well should cluster with many identical copies of the insert to be sequenced using SBS methods. In current systems, three of the nucleotides are labeled, and the third nucleotide (typically G) remains unlabeled. This is referred to as G being in an “off state.” However, during SBS sequencing runs on the flowcell, the wells in the flowcell that are not occupied by a DNA cluster might be erroneously called as a G nucleotide because the empty well will not fluoresce, similar to if there was an unlabeled G nucleotide present. This issue can also occur in random flowcells without wells due to spurious spots on the flowcell that are assigned as clusters. During a sequencing run, clusters which are dim, empty, or otherwise not fluorescing within enough intensity to be detected by a sequencing system may produce sequencing errors. The sequencing system may assume that the cluster contains an unlabeled G nucleotide, whereas it may actually contain a different nucleotide which is only dimly fluorescent, for example. If these errors are not caught by the sequencing system, then an incorrect sequence may be attributed to the insert bound to the cluster. Without a fluorophore attached to a G nucleotide, these types of errors may occur more since any error in fluorescence may be called as a G nucleotide in the sequencing process. SUMMARY

[0006] An aspect of the disclosure is directed to a method of sequencing polynucleotides bound to a flowcell, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a second intensity that is different from the first intensity; and determining the sequence of the polynucleotides based on the detected fluorescent emissions and intensities.

[0007] In some embodiments, the flowcell may comprise wells configured to bind polynucleotides. The incorporation of one of the at least four labeled nucleotide may conjugate into a well may be detected from at least one signal state. In some embodiments, the presence ofan empty well may be determined from a dark state. In some embodiments, the first intensity may be approximately double the second intensity.

[0008] In some embodiments, the fourth labeled nucleotide may be guanine. In some embodiments, the fluorescent emissions from the first, second, third and fourth labeled nucleotides may be plotted onto a cloud plot of intensity and wavelength. In some embodiments, the modified nucleotide may be a 5-methylcytosine, a N6-methyladenine, or an inosine. In some embodiments, the fourth labeled nucleotide may be a modified nucleotide.

[0009] In some embodiments, the first wavelength may be different from the second wavelength. Detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths. Detecting fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a second intensity that may be different from the first intensity. Determining the sequence of the polynucleotides based on the detected fluorescent emission and intensity.

[0010] In some embodiments, the flowcell may include wells configured to bind polynucleotides. Further. The incorporation of the at least four labeled nucleotides may conjugate into a well may be detected from at least one signal state. In some embodiments, the presence of an empty well may be determined from a dark state. In some embodiments, the first intensity may be approximately double the second intensity.

[0011] In some embodiments, the fourth labeled nucleotide may be guanine. In some embodiments, the fluorescent emissions from the first, second, third and fourth labeled nucleotides may be plotted onto a cloud plot of intensity and wavelength. In some embodiments, the fourth labeled nucleotide may be a modified nucleotide.

[0012] An aspect of the disclosure relates to mitigating potential issues with assigning bases (or empty wells) that have a low probability of occurring, due to, for example, low base diversity. Clusters that are not incorporating or are empty wells on a patterned flowcell may erroneously generate G basecalls because the G nucleotide is not labeled. Furthermore, when clusters sequence the whole insert and are no longer incorporating, poly G will be called for the remainder of the read. By partially labeling the G, a fifth cloud may be present in a two- dimensional plot of intensities in 2-channel SBS. An aspect of the disclosure is directed to addressing the fifth cloud.

[0013] An aspect of the disclosure is directed to effective solutions where, in some embodiments, the disclosed methods provide a sufficient signal-to-noise ratio to distinguish emptycells and partially labeled G bases. If there is an overlap between empty clouds and G clouds, then a new error mode will be introduced of miscalling G as empty and vice versa. In some embodiments, the G cloud may be moved far enough away from the empty cloud so they do not overlap. In some embodiments, the cloud for a different base than G may be replaced for G, and the same sequencing may be run where G is no longer in the "empty" location, and thereby G may be moved far enough away from the empty cloud, so they do not overlap. In some embodiments, the disclosure provides methods to address an issue where the effectiveness of procedures depends on the occupancy and the population of the empty cloud. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear. While the disclosure has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The disclosure is not limited to the disclosed embodiments. Variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed disclosure, from a study of the drawings, the disclosure and the appended claims.

[0015] FIG. 1A schematically illustrates an example sequencing system that can perform embodiments of the disclosed sequencing technology.

[0016] FIG. 1B schematically illustrates an example imaging system to be used in embodiments of the disclosed sequencing technology.

[0017] FIG.2 is a flowchart illustrating a process of sequencing polynucleotides bound to a flow cell with two detection channels.

[0018] FIG. 3 shows an illustration of one embodiment of a two channel chemistry method employing a labeled G nucleotide.

[0019] FIG. 4 shows an illustration of one embodiment of a two channel chemistry method employing a labeled G nucleotide and an unlabeled fifth base.

[0020] FIG.5A shows a two-dimensional scatterplot of an experiment to demonstrate labeled bases with two channel chemistry.

[0021] FIG. 5B shows a two-dimensional scatterplot of an experiment to demonstrate labeled bases with two channel chemistry with empty wells identified.

[0022] FIG. 6 depicts an embodiment of a system including a memory comprising a sequencing module.

[0023] Fig.7 shows an X-Y scatterplot of one embodiment of a two channel chemistry method. DETAILED DESCRIPTION

[0024] All patents, applications, published applications and other publications referred to herein are incorporated herein by reference to the referenced material and in their entireties. If a term or phrase is used herein in a way that is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the use herein prevails over the definition that is incorporated herein by reference.

[0025] The disclosure provides for systems and methods for determining the sequence of a polynucleotide by labeling at least four nucleotides in a sequencing by synthesis (SBS) system. In some embodiments, the polynucleotides have four unmodified bases, and each of the A,T,C and G nucleotides are labeled. In this embodiment, a two-dimensional scatterplot showing the measured wavelengths and intensities from each fluorescent label will have at least four distinct cloud formations, one for each fluorescent label. For example, a first base A may be labeled with a first fluorophore bound to all of the A bases which are to be incorporated into the insert during SBS sequencing reactions. A second base may be labeled with a second fluorophore bound to all of the C bases which are to be incorporated into the insert during the SBS sequencing reactions. A third base T may be labeled with both the first and the second fluorophore on each of the T bases to be incorporated into the insert during SBS sequencing reactions. And finally, the fourth base G may be labeled with the first fluorophore, but with only a percentage of the labeled G bases being incorporated into the insert during SBS sequencing reactions. For example, only 60% of the G bases may be labeled with the first fluorophore. In this way, the intensity of the G nucleotides can be distinguished from the intensity of the A nucleotides even though they are both labeled with the same fluorophore. In this embodiment, some wells will be empty and not contain any clusters ascluster formation does not always occur in every well. In these empty wells, a dark state cloud may be formed on the two-dimensional scatterplot from the lack of fluorescent signals corresponding to the empty wells. Thus, in this embodiment there may be five clouds formed on a two- dimensional scatterplot.

[0026] It should also be realized that some inserts may be smaller than others, and so each insert may complete its SBS cycles at different times. For example, an insert which is 100 bases long will complete its sequencing run well before an insert that is 300 bases long. For the relatively short inserts, the nucleotide bases which occur after the insert has been fully sequenced, but prior to completion of the sequence run of the system will form a dark state fifth cloud that can be differentiated from any of the four labeled nucleotides. This disclosure provides for systems and methods that accurately handle empty wells. Empty wells or erroneous dark reads may arise from a variety of scenarios.

[0027] As one non-limiting example, an erroneous dark read may arise in a paired-end sequencing run if a second read does not properly initialize. Generally, in a paired-end sequencing run a DNA fragment is first fragmented into smaller pieces. Then, adapters are ligated to each end of the fragments. A reversal of the DNA fragment is accomplished through the use of adapters ligated to both ends of the fragment during library preparation. These adapters may include specific sequences that serve as priming sites for the amplification and sequencing steps and may be specific to the sequencing platform being used.

[0028] The DNA fragments with adapters attached are then amplified and immobilized on a solid surface and subjected to cycles of DNA synthesis. In each cycle, the sequencing instrument may read the sequential nucleotides incorporated into the DNA strand, allowing for the determination of the DNA sequence. During library preparation, the DNA fragment is initially ligated to the forward adapter, which enables sequencing from one end of the fragment (Read 1). Once the clusters are formed and Read 1 has concluded, the fragment may be reversed by denaturing the DNA strands and removing the forward adapter. The reverse adapter, which may be ligated to the opposite end of the DNA fragment, may be then used as a priming site for the sequencing of the complementary strand from the other end (Read 2). With paired-end sequencing, a system may read the sequence from one end of the fragment (Read 1) and then reverses the fragment to read the sequence from the other end (Read 2). This process will usually generate two separate sequence reads for each DNA fragment, potentially with a gap in between the sequencereads. However, after a paired-end turn, some DNA fragments may fail to turn which consequently leads to empty wells during the second read.

[0029] Another embodiment is a system and method for determining the sequence of a polynucleotide which has modified bases, wherein the modified bases are labeled with a fluorophore at a distinguishable intensity from the other bases. Thus, instead of labeling a G nucleotide with the first fluorophore and a different intensity, as mentioned above, the G nucleotide is left unlabeled and the modified base is labeled with the first fluorophore at a different intensity. For example, methylated C nucleotides in the polynucleotide insert to be sequenced may be labeled with the first fluorophore, but only 60% of the methylated C nucleotides in the cluster within a well are labeled. In this way, a cloud on the two-dimensional scatterplot of wavelengths and intensities from each cluster will have five clouds. One for each of A, C, T, and the modified base at a lower intensity, with G being a dark state cloud. This will be explained more fully with reference to the description below. However, it should be realized that the intensity of fluorophores used in embodiments of the invention can be any detectable percentage of a full-intensity fluorophore by mixing labeled and unlabeled nucleotides together prior to contact with the flowcell. For example, a mixture of 50% labeled C nucleotides and 50% unlabeled nucleotides will result in the sequencing system detecting an intensity of C nucleotides that is 50% of the expected full fluorescence of the fluorophore as compared to a mixture of all labeled C nucleotides. Of course, any percentage mixture of labeled and unlabeled nucleotides may be used so that a desired percentage intensity may be provided during sequencing runs. For example, 30, 40, 50, 60, 70, or 80 percent of labeled nucleotides may be used, along with unlabeled nucleotides, to result in an intensity that is 30, 40, 50, 60, 70, or 80 percent, respectively, of a full intensity signal for that particular flurophore.

[0030] One aspect of the disclosure is a method of sequencing polynucleotides bound in clusters to a flowcell. This method can include detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity. For example, wherein a first reversibly terminated nucleotide C is labeled with a blue dye which emits light at a first wavelength. The method then includes detecting fluorescent emissions from a second reversibly terminated nucleotide T which is labeled with a green dye which emits light at a second wavelength, and wherein the first wavelength is different from the second wavelength. The method then detectsfluorescent emissions from a third reversibly terminated nucleotide A which is labeled with both the blue and green dyes and which emits light at the first and second wavelengths. Finally, the method includes detecting fluorescent emissions from a fourth reversibly terminated nucleotide G which emits fluorescent emissions at the first wavelength from the blue dye, but at an intensity that is different from the first intensity. For example, the percentage of G nucleotides which are labeled within a cluster may be 60%, so the cluster would emit blue light at 60% of the intensity of the blue light emitted by a cluster labeled with a C nucleotide. The method then determines the sequence of the polynucleotides based on the detected fluorescent emissions and intensities. In this method described above, the embodiment used a two-dye sequencing system within a method of labeling a G nucleotide at a lower intensity than the C nucleotide. Of course, it should be realized that this method would work by using different dyes linked to different nucleotides without departing from the spirit of the invention.

[0031] Moreover, in some aspects, the disclosure herein relates to a system for performing the above method of sequencing polynucleotides bound to a flowcell. The method would include using a memory linked to one or more processors which are configured to execute machine-readable instructions, which, when executed by the one or more processors, cause the system to perform above method steps.

[0032] Another embodiment is related to the above method, but instead of using a reduced intensity dye to label a particular nucleotide, such as G, the method uses a reduced intensity dye to label a modified base. For example, the method may include labeling a first labeled nucleotide at a first wavelength and a first intensity. For example, wherein a first reversibly terminated nucleotide C is labeled with a blue dye which emits light at a first wavelength. The method then includes detecting fluorescent emissions from a second reversibly terminated nucleotide T which is labeled with a green dye which emits light at a second wavelength, and wherein the first wavelength is different from the second wavelength. The method then detects fluorescent emissions from a third reversibly terminated nucleotide A which is labeled with both the blue and green dyes and which emits light at the first and second wavelengths. The method then includes detecting the absence of fluorescent emissions from an unlabeled fourth reversibly terminated nucleotide G. Moreover, during the library preparation process, particularly modified nucleotides, such as met-C, may have a specific percentage of the modified nucleotides labeled such that a detectable reduction in the light being emitted at the first wavelength can be detected.Any clusters having the reduced intensity of light being emitted at the first wavelength may be determined to have a met-C (or any other chosen modified base) during that cycle of the SBS sequencing reaction. Definitions

[0033] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0034] It is noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless expressly and unequivocally limited to one referent. It will be apparent to those skilled in the art that various modifications and variations can be made to various embodiments described herein without departing from the spirit or scope of the present teachings. Thus, it is intended that the various embodiments described herein cover other modifications and variations within the scope of the appended claims and their equivalents.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have”, “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0036] All references cited herein are incorporated herein by reference in their entirety. 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.

[0037] As used herein, common organic abbreviations are defined as follows: °C Temperature in degrees CentigradedATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate ddNTP Dideoxynucleotide triphosphate ffA Fully functionalized A nucleotide ffC Fully functionalized C nucleotide ffG Fully functionalized G nucleotide ffN Fully functionalized nucleotide ffT Fully functionalized T nucleotide h Hour(s) RT Room temperature SBS Sequencing by Synthesis A Adenosine (may refer to nucleotide base calls) C Cytosine G Guanine T Thymine

[0038] As used herein, a “peptide” refers to two or more amino acids joined together by an amide bond (that is, a “peptide bond”). Peptides comprise up to or include 50 amino acids. Peptides may be linear or cyclic. Peptides may be Į, ȕ, Ȗ, į, or higher, or mixed. Peptides may comprise any mixture of amino acids as defined herein, such as comprising any combination of D, L, Į, ȕ, Ȗ, į, or higher amino acids.

[0039] As used herein, a “protein” refers to an amino acid sequence having 51 or more amino acids.

[0040] As used herein, “nucleobase” is a heterocyclic base such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclic derivative, analog, or tautomer thereof. A nucleobase can be naturally occurring or synthetic. Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purines substituted at the 8 position with methyl or bromine, 9-oxo-N6-methyladenine, 2- aminoadenine, 7-deazaxanthine, 7-deazaguanine, 7-deaza-adenine, N4-ethanocytosine, 2,6- diaminopurine, N6-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)- alkynylcytosine, 5- fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4- triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine, 5,6- dihydrouracil, 4-methyl-indole, ethenoadenine and the non-naturally occurring nucleobases described in U.S. Pat. Nos. 5,432,272 and 6,150,510 and PCT applications WO 92 / 002258, WO 93 / 10820, WO 94 / 22892, and WO 94 / 24144, and Fasman ("Practical Handbook of Biochemistryand Molecular Biology", pp.385-394, 1989, CRC Press, Boca Raton, LO), all herein incorporated by reference in their entireties.

[0041] As used herein, the term “nucleotide” is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some examples also includes a nucleobase. A nucleotide that lacks a nucleobase may be referred to as “abasic.” In some embodiments, a “nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are monomeric units of a nucleic acid sequence. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides (RNA), the sugar is a ribose, and in deoxyribonucleotides (DNA), the sugar is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present at the 2' position in ribose. The nitrogen containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine. The phosphate groups may be in the mono-, di-, or tri-phosphate form. These nucleotides are natural nucleotides, but it is to be further understood that non-natural nucleotides, modified nucleotides or analogs of the aforementioned nucleotides can also be used.

[0042] Examples of nucleotides may include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate(dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0043] Examples of nucleotides may also be intended to encompass any nucleotide analogue which is a type of nucleotide that includes a modified nucleobase, sugar, backbone, and / or phosphate moiety compared to naturally occurring nucleotides. Nucleotide analogues also may be referred to as “modified nucleic acids.” Example modified nucleobases include inosine, xathanine, hypoxathanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5- hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5- propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4- thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8- thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7- deazaadenine, 3-deazaguanine, 3-deazaadenine or the like. As is known in the art, certain nucleotide analogues cannot become incorporated into a polynucleotide, for example, nucleotide analogues such as adenosine 5'-phosphosulfate. Nucleotides may include any suitable number of phosphates, e.g., three, four, five, six, or more than six phosphates. Nucleotide analogues also include locked nucleic acids (LNA), peptide nucleic acids (PNA), and 5-hydroxylbutynl-2'- deoxyuridine (“super T”).

[0044] In some embodiments, the term “modification” as used herein is intended to refer not only to a chemical modification of a nucleic acids, but also to a variation in nucleic acid conformation or composition, interaction of an agent with a nucleic acid (e.g., bound to the nucleic acid), and other perturbations associated with the nucleic acid. As such, a location or position of a modification is a locus (e.g., a single nucleotide or multiple contiguous or noncontiguous nucleotides) at which such modification occurs within the nucleic acid. For a double-stranded template, such a modification may occur in the strand complementary to a nascent strand synthesized by a polymerase processing the template or may occur in the displaced strand. For example, modified nucleotides may include 5-methylcytosine, N6-methyladenosine, N3- methyladenosine, N7-methylguanosine, 5-hydroxymethylcytosine, pseudouridine, thiouridine, isoguanosine, isocytosine, dihydrouridine, queuosine, wyosine, inosine, triazole, diaminopurine, ȕ-D-glucopyranosyloxymethyluracil (a.k.a., ȕ-D-glucosyl-HOMedU, ȕ-glucosyl-hydroxymethyluracil, “dJ,” or “base J”), 8-oxoguanosine, and 2ƍ-O-methyl derivatives of adenosine, cytidine, guanosine, and uridine. Modified DNA and RNA bases are further described, for example, in Narayan P, et al. (1987) Mol Cell Biol 7(4):1572-5; Horowitz S, et al. (1984) Proc Natl Acad Sci U.S.A. 81(18):5667-71; “RNA's Outfits: The nucleic acid has dozens of chemical costumes,” (2009) C&EN; 87(36):65-68; Kriaucionis, et al. (2009) Science 324 (5929): 929-30; and Tahiliani, et al. (2009) Science 324 (5929): 930-35; Matray, et al. (1999) Nature 399(6737):704-8; Ooi, et al. (2008) Cell 133: 1145-8; Petersson, et al. (2005) J Am Chem Soc. 127(5):1424-30; Johnson, et al. (2004) 32(6):1937-41; Kimoto, et al. (2007) Nucleic Acids Res. 35(16):5360-9; Ahle, et al. (2005) Nucleic Acids Res 33(10):3176; Krueger, et al., Curr Opinions in Chem Biology 2007, 11(6):588); Krueger, et al. (2009) Chemistry & Biology 16(3):242; McCullough, et al. (1999) Annual Rev of Biochem 68:255; Liu, et al. (2003) Science 302(5646):868-71; Limbach, et al. (1994) Nucl. Acids Res.22(12):2183-2196; Wyatt, et al. (1953) Biochem. J.55:774-782; Josse, et al. (1962) J. Biol. Chem.237:1968-1976; Lariviere, et al. (2004) J. Biol. Chem. 279:34715-34720; and in International Application Publication No. WO / 2009 / 037473, the disclosures of which are incorporated herein by reference in their entireties.

[0045] Modifications may further include the presence of non-natural base pairs in the nucleic acid, including but not limited to hydroxypyridone and pyridopurine homo- and hetero- base pairs, pyridine-2,6-dicarboxylate and pyridine metallo-base pairs, pyridine-2,6- dicarboxamide and a pyridine metallo-base pairs, metal-mediated pyrimidine base pairs T-Hg(II)- T and C-Ag(I)-C, and metallo-homo-basepairs of 2,6-bis(ethylthiomethyl)pyridine nucleobases Spy, and alkyne-, enamine-, alcohol-, imidazole-, guanidine-, and pyridyl-substitutions to the purine or pyridimine base (Wettig, et al. (2003) J Inorg Biochem 94:94-99; Clever, et al. (2005) Angew Chem Int Ed 117:7370-7374; Schlegel, et al. (2009) Org Biomol Chem 7(3):476-82; Zimmerman, et al. (2004) Bioorg Chem 32(1):13-25; Yanagida, et al. (2007) Nucleic Acids Symp Ser (Oxf) 51:179-80; Zimmerman (2002) J Am Chem Soc 124(46):13684-5; Buncel, et al. (1985) Inorg Biochem 25:61-73; Ono, et al. (2004) Angew Chem 43:4300-4302; Lee, et al. (1993) Biochem Cell Biol 71:162-168; Loakes, et al. (2009), Chem Commun 4619-4631; and Seo, et al. (2009) J Am Chem Soc 131:3246-3252, the disclosures of which are incorporated herein by reference in their entireties). Other types of modifications include, e.g, a nick, a missing base (e.g., apurinic or apyridinic sites), a ribonucleoside (or modified ribonucleoside) within a deoxyribonucleoside-based nucleic acid, a deoxyribonucleoside (or modifieddeoxyribonucleoside) within a ribonucleoside-based nucleic acid, a pyrimidine dimer (e.g., thymine dimer or cyclobutane pyrimidine dimer), a cis-platin crosslinking, oxidation damage, hydrolysis damage, other methylated bases, bulky DNA or RNA base adducts, photochemistry reaction products, interstrand crosslinking products, mismatched bases, and other types of “damage” to the nucleic acid. Modified nucleotides can be caused by exposure of the DNA to radiation (e.g., UV), carcinogenic chemicals, crosslinking agents (e.g., formaldehyde), certain enzymes (e.g., nickases, glycosylases, exonucleases, methylases, other nucleases, glucosyltransferases, etc.), viruses, toxins and other chemicals, thermal disruptions, and the like.

[0046] As used herein, the term “polynucleotide” refers to a molecule that includes a sequence of nucleotides that are bonded to one another. A polynucleotide is one nonlimiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogues thereof such as locked nucleic acids (LNA) and peptide nucleic acids (PNA). A polynucleotide may be a single stranded sequence of nucleotides, such as RNA or single stranded DNA, a double stranded sequence of nucleotides, such as double stranded DNA, or may include a mixture of a single stranded and double stranded sequences of nucleotides. Double stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single stranded DNA (ssDNA) can be converted to dsDNA and vice-versa. Polynucleotides may include non-naturally occurring DNA, such as enantiomeric DNA, LNA, or PNA. The precise sequence of nucleotides in a polynucleotide may be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, expressed sequence tag (EST) or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer or amplified copy of any of the foregoing.

[0047] The terms “oligonucleotide” and “polynucleotide” may be used interchangeably herein. The different terms are not intended to denote any particular difference in size, sequence, or other property unless specifically indicated otherwise. For clarity of description, the terms may be used to distinguish one species of polynucleotide from another when describing a particular method or composition that includes several polynucleotide species.

[0048] The term “nucleic acid” and “polynucleotide” may be used interchangeably to refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides, such as peptide nucleic acids (PNAs) and phosphorothioate DNA. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof. Nucleotides include, but are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, dITP, 2- amino-adenosine-TP, 2-amino-deoxyadenosine-TP, 2-thiothymidine triphosphate, pyrrolo- pyrimidine triphosphate, and 2-thiocytidine, as well as the alphathiotriphosphates for all of the above, and 2ƍ-O-methyl-ribonucleotide triphosphates for all the above bases. Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP.

[0049] As used herein, a “nucleoside” is structurally similar to a nucleotide, but is missing the phosphate moieties. An example of a nucleoside analogue would be one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule. The term “nucleoside” is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside comprising a ribose moiety and a deoxyribonucleoside comprising a deoxyribose moiety. A modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced with a carbon and / or a carbon has been replaced with a sulfur or an oxygen atom. A “nucleoside” is a monomer that can have a substituted base and / or sugar moiety. Additionally, a nucleoside can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0050] The term “purine base” is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term “pyrimidine base” is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. A non-limiting list of optionally substituted purine-bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g. 7-methylguanine), theobromine, caffeine, uric acid and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (e.g., 5-methylcytosine).

[0051] The term “nucleobase” as used herein, is a purine base or a pyrimidine base. Non-limiting examples of purine nucleobases include adenine (A), guanine (G), and derivatives oranalogs thereof. Non-limiting examples of pyrimidine nucleobases include cytosine (C), thymine (T), uracil (U), and derivatives or analogs thereof.

[0052] As used herein, when an oligonucleotide or polynucleotide is described as “comprising” a nucleoside or nucleotide described herein, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. Similarly, when a nucleoside or nucleotide is described as part of an oligonucleotide or polynucleotide, such as “incorporated into” an oligonucleotide or polynucleotide, it means that the nucleoside or nucleotide described herein forms a covalent bond with the oligonucleotide or polynucleotide. In some such embodiments, the covalent bond is formed between a 3^ hydroxy group of the oligonucleotide or polynucleotide with the 5^ phosphate group of a nucleotide described herein as a phosphodiester bond between the 3^ carbon atom of the oligonucleotide or polynucleotide and the 5^ carbon atom of the nucleotide.

[0053] As used herein, the term “array” refers to a population of different probe molecules that are attached to one or more substrates such that the different probe molecules can be differentiated from each other according to relative location. An array can include different probe molecules that are each located at a different addressable location on a substrate. Alternatively, or additionally, an array can include separate substrates each bearing a different probe molecule, wherein the different probe molecules can be identified according to the locations of the substrates on a surface to which the substrates are attached or according to the locations of the substrates in a liquid. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those including beads in wells as described, for example, in U.S. Patent No. 6,1055,331 B1, US 2002 / 0102578 and PCT Publication No. WO 00 / 63437. Exemplary formats that can be used in the invention to distinguish beads in a liquid array, for example, using a microfluidic device, such as a fluorescent activated cell sorter (FACS), are described, for example, in US Pat. No. 6,524,793. Further examples of arrays that can be used in the invention include, without limitation, those described in U.S. Pat Nos. 5,329,807; 5,336,1027; 5,561,071; 5,583,911; 5,658,734; 5,837,858; 5,874,919; 5,919,523; 6,836,969; 6,987,768; 6,987,776; 6,988,920; 6,997,006; 6,991,893; 6,1046,313; 6,316,949; 6,382,591; 6,514,751 and 6,610,382; and WO 93 / 17126; WO 95 / 11995; WO 95 / 35505; EP 742987; and EP 799897.

[0054] A nucleotide analog may be attached to or associated with one or more photo- detectable labels to provide a detectable signal. In some embodiments, a photo-detectable labelmay be a fluorescent compound, such as a small molecule fluorescent label. Fluorescent molecules (fluorophores) suitable as a fluorescent label include, but are not limited to: 1,5 IAEDANS; 1,8- ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); fluorescein amidite (FAM); 5-carboxynapthofluorescein; tetrachloro-6-carboxyfluorescein (TET); hexachloro-6-carboxyfluorescein (HEX); 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE); VIC®; NED™; tetramethylrhodamine (TMR); 5-carboxytetramethylrhodamine (5- TAMRA); 5-HAT (Hydroxy Tryptamine); 5-hydroxy tryptamine (HAT); 5-ROX (carboxy-X- rhodamine); 6-carboxyrhodamine 6G; 6-JOE; Light Cycler® red 610; Light Cycler® red 640; Light Cycler® red 670; Light Cycler® red 705; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine; 6-methoxy-N- (4-aminoalkyl)quinolinium bromide hydrochloride (ABQ); Acid Fuchsin; ACMA (9-amino-6- chloro-2-methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; AFPs-AutoFluorescent Protein-(Quantum Biotechnologies); Texas Red; Texas Red-X conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC (TetramethylRodamine-lsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; WW 781; X-Rhodamine; X-Rhodamine-5-(and-6)-Isothiocyanate (5(6)-XRITC); Xylene Orange; Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; interchelating dyes such as YOYO-3, Sybr Green, Thiazole orange; members of the Alexa Fluor® dye series (from Molecular Probes / Invitrogen) which cover a broad spectrum and match the principal output wavelengths of common excitation sources such as Alexa Fluor 350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750; members of the Cy Dye fluorophore series (GE Healthcare), also covering a wide spectrum such as Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7; members of the Oyster® dye fluorophores (Denovo Biolabels) such as Oyster- 500, -550, -556, 645, 650, 656; members of the DY-Labels series (Dyomics), for example, with maxima of absorption that range from 418 nm (DY-415) to 844 nm (DY-831) such as DY-415, - 495, -505, -547, -548, -549, -550, -554, -555, -556, -560, -590, -610, -615, -630, -631, -632, -633, -634, -635, -636, -647, -648, -649, -650, -651, -652, -675, -676, -677, -680, -681, -682, -700, -701, -730, -731, -732, -734, -750, -751, -752, -776, -780, -781, -782, -831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL; members of the ATTO series of fluorescent labels (ATTO-TECGmbH) such as ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594, 610, 611X, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740; members of the CAL Fluor® series or Quasar® series of dyes (Biosearch Technologies) such as CAL Fluor® Gold 540, CAL Fluor® Orange 560, Quasar® 570, CAL Fluor® Red 590, CAL Fluor® Red 610, CAL Fluor® Red 635, Quasar® 570, and Quasar® 670. In some embodiments, a first photo-detectable label interacts with a second photo-detectable moiety to modify the detectable signal, e.g., via fluorescence resonance energy transfer (“FRET”; also known as Förster resonance energy transfer).

[0055] The fluorescent labels utilized by the systems and methods disclosed herein can have different peak absorption wavelengths, for example, ranging from 400 nm to 800 nm. In some embodiments, the peak absorption wavelengths of the fluorescent labels can be, or be about, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or a range between any two of these values. In some embodiments the peak absorption wavelengths of the fluorescent labels can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm.

[0056] The fluorescent labels can have different peak emission wavelength, for example, ranging from 400 nm to 800 nm. In some embodiments, the peak emission wavelengths of the fluorescent labels can be, or be about, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or a range between any two of these values. In some embodiments the peak emission wavelengths of the fluorescent labels can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm.

[0057] The fluorescent labels can have different Stokes shift, for example, ranging from 10 nm to 200 nm. In some embodiments, the stoke shift can be, or be about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or a number or a range between any two of these values. In some embodiments, the stoke shift can be at least, or atmost, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm.

[0058] In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can vary, for example, ranging from 10 nm to 200 nm. In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can be, or be about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or a number or a range between any two of these values. In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can be at least, or at most, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm.

[0059] A “light source” may be any device capable of emitting energy along the electromagnetic spectrum. A light source may be a source of visible light (VIS), ultraviolet light (UV) and / or infrared light (IR). “Visible light” (VIS) generally refers to the band of electro- magnetic radiation with a wavelength from about 400 nm to about 750 nm. “Ultraviolet (UV) light” generally refers to electromagnetic radiation with a wavelength shorter than that of visible light, or from about 10 nm to about 400 nm range. “Infrared light” or infrared radiation (IR) generally refers to electromagnetic radiation with a wavelength greater than the VIS range, or from about 750 nm to about 50,000 nm. A light source may also provide full spectrum light. Light sources may output light from a selected wavelength or a range of wavelengths. In some embodiments of the invention, the light source may be configured to provide light above or below a predetermined wavelength, or may provide light within a predetermined range. A light source may be used in combination with a filter, to selectively transmit or block light of a selected wavelength from the light source. A light source may be connected to an intensity source by one or more electrical connectors; an array of light sources may be connected to an intensity source in series or in parallel. An intensity source may be a battery, or a vehicle electrical system or a building electrical system. The light source may be connected to an intensity source via control electronics (control circuit); control electronics may comprise one or more switches. The one or more switches may be automated, or controlled by a sensor, timer or other input, or may be controlled by a user, or a combination thereof. For example, a user may operate a switch to turn on a UV light source; the light source may be applied on a constant basis until it is turned off, or it may be pulsed (repeated on / off cycles) until it is turned off. In some embodiments, the lightsource may be switched from a continuously-on state to a pulsed state, or vice versa. In some embodiments, the light source may be configured to be brightening or darkening over time.

[0060] For operation, the light source may be connected to a intensity source capable of providing sufficient intensity to illuminate the sample. Control electronics may be used to switch the intensity on or off based on input from a user or some other input, and can also be used to modulate the intensity to a suitable level (e.g. to control brightness of the output light). Control electronics may be configured to turn the light source on and off as desired. Control electronics may include a switch for manual, automatic, or semi-automatic operation of the light sources. The one or more switches may be, for example, a transistor, a relay or an electromechanical switch. In some embodiments, the control circuit may further comprise an AC-DC and / or a DC-DC converter for converting the voltage from the voltage source to an appropriate voltage for the light source. The control circuit may comprise a DC-DC regulator for regulation of the voltage. The control circuit may further comprise a timer and / or other circuitry elements for applying electric voltage to the optical filter for a fixed period of time following the receipt of input. A switch may be activated manually or automatically in response to predetermined conditions, or with a timer. For example, control electronics may process information such as user input, stored instructions, or the like.

[0061] One or more of a plurality of light sources may be provided. In some embodiments, each of the plurality of light sources may be the same. Alternatively, one or more of the light sources may vary. The light characteristics of the light emitted by the light sources may be the same or may vary. A plurality of light sources may or may not be independently controllable. One or more characteristic of the light source may or may not be controlled, including but not limited to whether the light source is on or off, brightness of light source, wavelength of light, intensity of light, angle of illumination, position of light source, or any combination thereof.

[0062] In some embodiments, light output from a light source may be from about 350 to about 750 nm, or any amount or range therebetween, for example from about 350 nm to about 360, 370, 380, 390, 400, 410, 420, 430 or about 450 nm, or any amount or range therebetween. In other embodiments, light from a light source may be from about 550 to about 700 nm, or any amount or range therebetween, for example from about 550 to about 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690 or about 700 nm, or any amount or range therebetween. In some embodiments, the wavelength of the light generated by the light source can vary, forexample, ranging from 400 nm to 800 nm. In some embodiments, the wavelength of the light generated by the light source can be, or be about, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or a range between any two of these values. In some embodiments, the wavelength of the light generated by the light source can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm. The light source may be capable of emitting electromagnetic waves in any spectrum. In some embodiments, the light source may have a wavelength falling between 10 nm and 100 ^m. In some embodiments, the wavelength of light may fall between 100 nm to 5000 nm, 300 nm to 1000 nm, or 400 nm to 800 nm. In some embodiments, the wavelength of light may be less than, and / or equal to 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1500 nm, 1750 nm, 2000 nm, 2500 nm, 3000 nm, 4000 nm, or 5000 nm.

[0063] In one example, a light source may be a light-emitting diode (LED) (e.g., gallium arsenide (GaAs) LED, aluminum gallium arsenide (AlGaAs) LED, gallium arsenide phosphide (GaAsP) LED, aluminum gallium indium phosphide (AlGaInP) LED, gallium(III) phosphide (GaP) LED, indium gallium nitride (InGaN) / gallium(III) nitride (GaN) LED, or aluminum gallium phosphide (AlGaP) LED). In another example, a light source can be a laser, for example a vertical cavity surface emitting laser (VCSEL) or other suitable light emitter such as an Indium-Gallium-Aluminum-Phosphide (InGaAIP) laser, a Gallium-Arsenic Phosphide / Gallium Phosphide (GaAsP / GaP) laser, or a Gallium-Aluminum-Arsenide / Gallium-Aluminum-Arsenide (GaAIAs / GaAs) laser. Other examples of light sources may include but are not limited to electron stimulated light sources (e.g., Cathodoluminescence, Electron Stimulated Luminescence (ESL light bulbs), Cathode ray tube (CRT monitor), Nixie tube), incandescent light sources (e.g., Carbon button lamp, Conventional incandescent light bulbs, Halogen lamps, Globar, Nernst lamp), electroluminescent (EL) light sources (e.g., Light-emitting diodes—Organic light-emitting diodes, Polymer light-emitting diodes, Solid-state lighting, LED lamp, Electroluminescent sheets Electroluminescent wires), gas discharge light sources (e.g., Fluorescent lamps, Inductive lighting, Hollow cathode lamp, Neon and argon lamps, Plasma lamps, Xenon flash lamps), or high-intensity discharge light sources (e.g., Carbon arc lamps, Ceramic discharge metal halide lamps,Hydrargyrum medium-arc iodide lamps, Mercury-vapor lamps, Metal halide lamps, Sodium vapor lamps, Xenon arc lamps). Alternatively, a light source may be a bioluminescent, chemiluminescent, phosphorescent, or fluorescent light source.

[0064] Optical filters may be tuned in terms of clarity or haze, translucency, transparency or opacity, light transmittance (LT), switching speed, durability, photostability, contrast ratio, state of light transmittance (e.g. dark state or light state). “Light transmittance” (LT) refers to the quantity of light that is transmitted or passes through an optical filter, or device or apparatus comprising same. LT may be expressed with reference to a change in light transmission and / or a particular type of light or wavelength of light (e.g. from about 10% visible light transmission (LT) to about 90% LT, or the like). LT may alternately be expressed as absorbance, and may optionally include reference to one or more wavelengths that are absorbed. According to some embodiments, an optical filter may be selected, or configured to have in one state, a LT of less than 80%, or less than 70%, or less than 60%, or less than 50%, or less than 40%, or less than 30%, or less than 20% or less than 10%, or any amount or range therebetween. According to some embodiments, an optical filter may be selected, or configured to have in another state, a LT of greater than 80%, or greater than 70%, or greater than 60%, or greater than 50%, or greater than 40%, or greater than 30%, or greater than 20% or greater than 10%, or any amount or range therebetween.

[0065] A filter can be a bandpass filter and can have peak transmittance of varying wavelength, ranging from 400 nm to 800 nm. In some embodiments, the peak transmittance can be, or be about, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a number or a range between any two of these values. In some embodiments, the peak transmittance can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm. The width of the transmission window of a filter can vary, for example, ranging from 1 nm to 50 nm. In some embodiments, the width of the filter can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nm, or a number or a range between any two of these values. In some embodiments, the width of the filter can be at least, or at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nm. A shortpass filter may be considered a special bandpass filter having the lower limit of the transmissionwindow close to 0 nm. A longpass filter may be considered a special bandpass filter having the upper limit of the transmission window close to infinity. A bandstop filter may be defined as complementary to some bandpass filter.

[0066] As used herein, an “optical channel” is a predefined profile of optical frequencies (or equivalently, wavelengths). For example, a first optical channel may have wavelengths of 500 nm–600 nm. To take an image in the first optical channel, one may use a detector which is only responsive to 500 nm–600 nm light, or use a bandpass filter having a transmission window of 500 nm–600 nm to filter the incoming light onto a detector responsive to 300 nm–800 nm light. A second optical channel may have wavelengths of 300 nm–450 nm and 850 nm–900 nm. To take an image in the second optical channel, one may use a detector responsive to 300 nm–450 nm light and another detector responsive to 850 nm–900 nm light and then combine the detected signals of the two detectors. Alternatively, to take an image in the second optical channel, one may use a bandstop filter which rejects 451 nm–849 nm light in front of a detector responsive to 300 nm–900 nm light. Example Sequencer

[0067] In FIG. 1A, an example sequencing system 100 which can perform the disclosed sequencing technology is illustrated. The sequencing system 100 can be configured to utilize disclosed sequencing methods based on a single optical excitation and a single detection channel. Non-limiting examples of the sequencing reactions utilized can include variations of sequencing-by-synthesis processes, such as those used in Illumina® dye sequencing or HeliScope® single molecule sequencing.

[0068] The sequencing system 100 can include an optics system 102 configured to generate raw sequencing data using sequencing reagents supplied by a fluidics system 104 that is part of the sequencing system 100. The raw sequencing data can include fluorescent images captured by the optics system 102. The sequencing system 100 can further include a computer system 106 that can be configured to control the optics system 102 and the fluidics system 104 via communication channels 108a and 108b. For example, a computer interface 110 of the optics system 102 can be configured to communicate with the computer system 106 through the communication channel 108a.

[0069] During sequencing reactions, the fluidics system 104 can direct the flow of reagents through one or more reagent tubes 112 to and from a flowcell 114 positioned on a mounting stage 116. The reagents can include, for example, fluorescently labeled nucleotides, buffers, enzymes, and cleavage reagents. The flowcell 114 can include at least one fluidic channel. The flowcell 114 can be a patterned array flowcell or a random array flowcell. The flowcell 114 can include multiple clusters of single-stranded polynucleotides to be sequenced in the at least one fluidic channel. The lengths of the polynucleotides can vary ranging, for example, from about 50 bases, 100 bases, 150 bases, 200 bases, 300 bases, 500 bases, to about 1000 bases. The polynucleotides can be attached to one or more fluidic channels of the flowcell 114. In some embodiments, the flowcell 114 can include a plurality of wells, wherein each well can include a cluster comprising multiple identical copies of a target polynucleotide to be sequenced. The mounting stage 116 can be configured to allow proper alignment and movement of the flowcell 114 in relation to the other components of the optics system 102. In one embodiment, the mounting stage 116 can be used to align the flowcell 114 with a lens 118.

[0070] The optics system 102 can include a two light sources 120, such as lasers or a LEDs, with each light source configured to generate light having wavelengths distributed at around a predetermined wavelength. However, embodiments are not limited to any particular wavelength of light. The light source only needs to be configured to generate the correct wavelength of light which excites the fluorescent labels attached to the nucleotides on the flowcell.

[0071] The light generated by the light source 120 can pass through fiber optic cables 122 to excite fluorescent labels in the flowcell 114. The lens 118, mounted on a focuser 124, can move along the z-axis. The focused fluorescent emissions can be detected by detectors 126, for example charge-coupled device (CCD) sensors or a complementary metal oxide semiconductor (CMOS) sensors. In some embodiments, nucleotide incorporations can be detected with zero mode waveguides as described, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett.33, 1026-1028 (2008); and Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties.

[0072] A filter assembly 128 of the optics system 102 can be configured to filter the fluorescent emissions from the fluorescent labels in the flowcell 114. The filter assembly 128 can include a plurality of optical filters, where a correct filter can be selected depending on the particular fluorophores used in a sequencing reaction. In one alternate embodiment, the computersystem 106 may automatically determine which optical filter should be used for a sequencing reaction, e.g., by scanning labels and / or barcodes attached to a sample vial and determining the particular fluorophores to be used in a sequencing reaction based on the labels and / or barcodes, or by retrieving information stored in the memory relating to previous sequencing reactions, and then control the filter assembly 128 to select and use the desired optical filter. The selected filter can be a longpass filter, a shortpass filter, a bandstop filter, or a bandpass filter, depending on the types of fluorescent molecules being used in the system. For example, the selected filter can be a bandpass filter selected to match the peak of the emission spectrum of a particular fluorescent label.

[0073] In some embodiments, the detectors 126 include one or more sub-detector while the filters of the filter assembly 128 may be mechanically switched or rotated in front of the sub- detector, such that differently filtered images can be taken by the sub-detector sequentially. In some embodiments, the detectors 126 include one sub-detector and the filter assembly 128 may include at least one layer of switchable material which has a light transmittance that is variable upon application of a stimulus, where the stimulus may be light, electricity, temperature, or any combination thereof. As a result, the filter assembly 128 can provide a plurality of optical filters such that differently filtered images can be taken by the sub-detector sequentially. In some embodiments, the detectors 126 may each include one sub-detector and the filter assembly 128 may include one or more switchable filters base on the micro-electromechanical system technology, such that differently filtered images can be taken by the sub-detector sequentially.

[0074] In some embodiments, the detectors 126 can include two or more sub-detectors to be selected depending on the set of fluorophores used, for example a first detector coupled with a first filter and a second detector coupled with a second filter. In some embodiments, the optics system 102 may include two or more dichroic mirrors / beamsplitters configured to split the fluorescent emissions, such that after splitting the fluorescent emissions with the dichroic mirrors, the detectors 126 can take two differently filtered images simultaneously (or close in time) using the two sub-detectors coupled with two different filters. In some embodiments, the detectors 126 can include two or more sub-detectors stacked along the incoming direction of the fluorescent emissions. Different wavelengths of the fluorescent emissions may differentially decay or be differentially absorbed along the incoming direction, such that sub-detectors at different positionsalong the incoming direction can be selected depending on the set of fluorophores used, or be configured to take differently filtered images simultaneously (or close in time).

[0075] In use, a sample having a polynucleotide to be sequenced may be loaded into the flowcell 114 and placed in the mounting stage 116. The computer system 106 may then activate the fluidics system 104 to begin a sequencing cycle. During sequencing reactions, the computer system 106 may instruct the fluidics system 104, through the communication interface 108b, to supply reagents, for example labeled nucleotide analogs, to the flowcell 114. Through the communication interface 108a and the computer interface 110, the computer system 106 may control the light source 120 of the optics system 102 to generate light at around a predetermined wavelength and excite nucleotide analogs incorporated into growing primers hybridized to the polynucleotide being sequenced, for example. The computer system 106 may control the detector 126 of the optics system 102 to capture images of the diffraction-limited spots of DNA clusters having the fluorescently labeled nucleotide analogs. The computer system 106 can receive the fluorescent images from the detector 126 and process the fluorescent images received to determine the nucleotide sequence of the polynucleotide being sequenced.

[0076] In FIG.1B, an example of an imaging system 10000 to be used in the disclosed sequencing technology is illustrated. For example, the imaging system 10000 may be used in the example sequencing system 100 illustrated in FIG. 1A. The imaging system 10000 may include a light source 11000 that can provide light at one or more wavelengths to excite fluorophores at targeted points on a sample. The light source 11000 can include one or more lasers, light-emitting diodes, or other optical sources, such that the light source 11000 can provide a variety of wavelengths of light. In some embodiments, the light source 11000 can be configured to selectively provide light with a predetermined range of wavelengths that are tuned to the set of fluorophores being used. In some embodiments, the light source 11000 can be configured to output light at an optical frequency corresponding to a wavelength in a predefined range of wavelengths of light. In some embodiments, a user of the disclosed sequencing systems may choose a specific optical frequency to be output from the light source 11000, depending on the particular fluorophores used in a sequencing reaction. In one alternate embodiment, the computer system 106 may automatically determine which optical frequency should be output from the light source 11000, e.g., by scanning labels and / or barcodes attached to a sample vial and determining the particular fluorophores to be used in a sequencing reaction based on the labels and / or barcodes, orby retrieving information stored in the memory relating to previous sequencing reactions, and then control the light source 11000 to select and output the desired optical frequency.

[0077] The imaging system 10000 may include an optical path 12000 from the light source 11000 to the sample 13000, e.g., a microfluidic device including one or more flow chambers where one or more sequencing reactions occur. In some embodiments, the optical path 12000 can include a combination of one or more of mirrors, lenses, prisms, quarter wave plates, half wave plates, polarizers, filters, dichroic mirrors, beam splitters, beam combiners, objective lenses, wide field optics configured to spread light from a light source over a relatively large region of a sample, etc. The optical path 12000 can be configured to direct light from the light source 11000 to the sample 13000. In addition, the optical path 12000 may include optical components which can be configured to direct light emitted from the sample 13000 to an integration detection system 15000. In some embodiments, a portion of the optical elements that are used to direct light from the light source 11000 to the sample 13000 are also used to direct light from the sample 13000 to the integration detection system 15000. Further examples of optical paths and optical systems may be found in U.S. Pat. No. 7,589,315, U.S. Pat. No. 8,951,781, or U.S. Pat. No. 9,193,996, each of which is incorporated by reference herein in its entirety.

[0078] The imaging system 10000 may include a scanning system 14000 to effectively move light relative to the sample 13000 to scan the sample to generate an image. In some embodiments, the scanning system 14000 can be implemented within the optical path 12000. For example, the scanning system 14000 can include one or more scanning mirrors that move relative to one another within the optical path 12000 to effectively move the light from the light source 11000 across the sample. In some embodiments, the scanning system 14000 can be implemented as a mechanical system that physically moves the sample 13000 so that the sample moves relative to the light from the light source 11000. In some embodiment, the scanning system 14000 can be a combination of optical components in the optical path 12000 and a mechanical system for physically moving the sample 13000 so that the light from the light source 11000 and the sample 13000 move relative to one another.

[0079] The imaging system 10000 may include an integration detection system 15000 that includes one or more light detectors as well as associated electronic circuitry, processors, data storage, memory, and the like to acquire and process image data of the sample 13000. In some embodiments, the integration detection system 15000 can include photomultiplier tubes, avalanchephotodiodes, image sensors (e.g., CCDs, CMOS sensors, etc.), and the like. In some embodiments, the light detectors of the integration detection system 15000 can include components to amplify light signals and may be sensitive to single photons. In some embodiments, the light detectors of the integration detection system 15000 can have a plurality of channels or pixels. The integration detection system 15000 can acquire one or more images based on the light detected from the sample 13000.

[0080] In some embodiments, the optical path 12000 may include an array generator 12100 that can generate a plurality of light exposure regions on the sample 13000. In some embodiments, the array generator 12100 can generate a certain light exposure pattern on the sample 13000. These light exposure regions can be scanned over the sample 13000 using the scanning system 14000 to selectively illuminate areas of the sample 13000 for imaging. The integration detection system 15000 can integrate signals corresponding to particular points on the sample 13000 as the plurality of light exposure regions are scanned over the sample 13000. For example, for an individual point on the sample 13000, the integration detection system 1500 can selectively aggregate detected signals corresponding to the individual point where the individual point is illuminated at different times by different light exposure regions. In some embodiments, the combination of the array generator 12100 and the integration detection system 15000 can detect light simultaneously, or near-simultaneously, from a plurality of points on the sample 13000. In some embodiments, the combination of the array generator 12100 and the integration detection system 15000 can integrate the detected light from a plurality of points on the sample over time.

[0081] In some embodiments, a plurality of sequencing reactions may be run parallelly in a plurality of flow chambers of the sample 13000. For example, a plurality of sequencing reactions may be performed for a plurality of biological specimen. In some embodiments, the plurality of sequencing reactions may use different sets of fluorophores. In some embodiments, the light source 11000, the array generator 12100, and the scanning system 14000 can be configured to selectively illuminate different areas of the sample 13000 with different optical frequencies of light, depending on the different sets of fluorophores used for the sequencing reactions occurring in different areas of the sample 13000.Four Labeled Nucleotides in Two Channels

[0082] As mentioned above, one aspect of the disclosure is directed to systems and methods that utilize two fluorescent dyes to perform SBS sequencing with four labeled nucleotides. In some embodiments, systems and methods may utilize two dye sequencing with four labeled nucleotides in a 2 channel 2 excitation (2ch-2ex) system. Using four nucleotides labeled with fluorescent dyes allows for additional an additional encoding space (corresponding to a dark state), as compared to traditional 2 channel 2 excitation systems, which may use three nucleotides labeled with dyes and one dark nucleotide. The additional encoding space corresponding to a dark state may be used for a fifth nucleotide, such as a modified nucleotide, or an empty well.

[0083] In some embodiments, by labeling G nucleotides using one of the fluorescent dyes, but at a different intensity, any unoccupied (empty) wells may form a fifth cloud in a 2- dimensional scatter plot. Similarly, in some embodiments, by labeling G nucleotides, any base calls which follow after a short insert completes its sequencing run may also be found in a fifth cloud that is differentiated from any of the four labeled nucleotides. In some embodiments, unlabeled wells may be identified as a fifth cloud in a 2-dimensional scatter plot. In other embodiments, a fifth base, such as a modified base, may be identified as a fifth cloud in a 2- dimensional scatter plot.

[0084] Figure 2 shows a flowchart of a method 300 of sequencing clusters of labeled polynucleotides bound to a flowcell according to one embodiment. The method may begin at a start step 302, by, for example, gathering DNA samples, and putting the sample into a sequencing system, such as described above. Sequencing systems according to the disclosure may include flow cell sequencers such as those produced by ILLUMINA®, INC. (San Diego, CA). For example, when the method 300 begins at step 302, a flowcell may have been prepared by embedding the flowcell with fragmented polynucleotides (e.g., fragmented single- or double- stranded polynucleotide fragments). Fragmented polynucleotides may be generated from a deoxyribonucleic acid (DNA) sample. DNA samples may be from various sources, for example, a biological sample, a cell sample, an environmental sample, or any combination thereof. The lengths of fragmented polynucleotide fragments may range from, for example, 100 bases to 1000 bases, or more.

[0085] Polynucleotide fragments may be bridge-amplified into clusters of polynucleotide fragments attached to the inside surface of one or more channels of a flowcell. An inside surface of the one or more flowcell channels may include two types of primers, for example a first primer type (P1) and a second primer type (P2) and the DNA fragments may be amplified by well-known methods to generate clusters. In some embodiments the flowcells are patterned with wells and each well contains a single cluster.

[0086] After generating clusters within the flowcell, the method 300 may begin a Sequencing by Synthesis process. During each sequencing cycle, four or more types of nucleotide analogs are added and incorporated onto the growing primer-polynucleotides. The four or more types of nucleotide analogs may have different modifications. For example, a first type of nucleotide may be an analog of deoxyguanosine triphosphate (dGTP), which is partially conjugated via a linker with a first type of fluorescent label that, after an excitation, can emit light at a first emission wavelength. In some embodiments, only a fraction of the dGTP nucleotides are labeled so that each cluster which has a dGTP as the next incorporarted nucleotide will fluoresce with a reduced intensity as compared to a cluster where every incorporated nucleotide was labeled. For example, only 10, 20, 30, 40, 50, 60, or 70 percent of the nucleotides in a dGTP mixture that is used during SBS may be labled. Thus, the intensity of the fluorescence coming from a cluster that is labeled with dGTP would be detectibly lower than clusters having every growing strand labeled with a fluorescent dye. This allows the same dye to be used on dGTP nucleotides as on another nucleotide, but still have the dGTP clusters be detectibly different from the other clusters.

[0087] A second type of nucleotide may be an analog of deoxythymidine triphosphate (dTTP), which may be labeled with the same fluorescent label as dGTP, but with every nucleotide being labeled. Thus, as compared to dGTP, each cluster which is labeled with dTTP would have a higher intensity than clusters labeled with dGTP. A third type of nucleotide may be an analog of deoxycytidine triphosphate (dCTP), which is conjugated via a linker with the second type fluorescent label that, after an excitation, can emit at a second emission wavelength. A fourth type of nucleotide may be an analog of deoxyadenosine triphosphate (dATP), which is conjugated via a linker with both the first and second types of fluorescent labels, and can after a corresponding excitation, can emit at both first and second emission wavelengths. A fifth type of nucleotide may be an analog of 5-methylcytosine, which may not be conjugated with any fluorescent label, but still may be conjugated with a linker.

[0088] In a separate embodiment, the dGTP may be unlabeled and the modified base, such as 5-methylcytosine may be labeled with the first fluorescent label, but at a reduced intensity. Thus, in this embodiment, detecting a well with a dark state would indicate the presence of the dGTP nucleotide and detection of the reduced intensity of the first fluorescent label would indicate the presence of a modified nucleotide.

[0089] The linkers may include one or more cleavage groups. Prior to the subsequent sequencing cycle, the fluorescent labels may be removed from the nucleotide analogs. For example, a linker attaching a fluorescent label to a nucleotide analog may include an azide and / or an alkoxy group, for example on the same carbon, such that the linker may be cleaved after each incorporation cycle by a phosphine reagent, thereby releasing the fluorescent label from subsequent sequencing cycles.

[0090] Once the clusters are created on the flowcell, the method 300 moves to a step 310 of detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity. For example, step 310 may include exciting all of the clusters of labeled polynucleotides on a flowcell at a first excitation wavelength. A single light source such as a laser or an LED source may excite a fluorescent label at the predetermined excitation wavelength. In some embodiments, the single laser or the LED source may be non-tunable. Step 310 may also include detecting any fluorescent emissions from the clusters at a first detection wavelength to detect the presence of a first labeled nucleotide. In general, the first detection wavelength will be red-shifted to a longer wavelength relative to the first excitation wavelength.

[0091] Image processing yields base calling, described in further detail below, where the complementary nucleotides added to the molecules in a cluster during a cycle are identified. In some embodiments, the fluorescent images may be stored for later processing offline. In some embodiments, the fluorescent images may be processed to determine the sequence of the growing primer-polynucleotides in each cluster in real time. After detecting the fluorescence emissions from the clusters at step 310, the method 300 moves to a step 320, where the method detects fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength.

[0092] When the method detects fluorescent emissions from a first wavelength in step 310 different than a second wavelength in step 320, the method may be described as employing at least two channel base calling. A “channel” may be used to describe an emission / detection processfor a labeled or unlabeled base that is specific for a particular excitation wavelength and detection wavelength pair. Thus, a channel may include a particular first range of wavelengths of light used to excite a fluorescent dye and a second range of wavelengths of light used to detect the fluorescent emissions from the excited dyes. The disclosure provides for, inter alia, two-channel base calling for four or more bases.

[0093] After detecting the fluorescence emissions from the clusters at step 310, the method 300 moves to a step 330, where the method detects fluorescent emissions from a third labeled nucleotide at the first and second wavelengths. For example, at step 330 fluorescent emissions from a third labeled nucleotide may be detected at the second wavelength at some intensity and at the first wavelength may be detected at the first intensity. In some embodiments, fluorescent emissions from a third labeled nucleotide at the first and second wavelength may be detected at some non-zero intensity other than the first intensity.

[0094] After detecting the fluorescence emissions from the clusters at step 310, the method 300 moves to a step 340, where the method detects fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a second intensity that is different from the first intensity. For example, at step 340, the method may detect fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a second intensity that is greater than the first intensity. In some embodiments the second intensity is less than the first intensity. The intensity of the detected emissions at the first wavelength may be quantified as an absolute measurement in terms of photon flux or counts per second. As described herein, the first and second intensities may also be quantified as relative measurement for a cluster labeled with a single nucleotide as compared to a cluster labeled with two different nucleotides. In some embodiments, at step 340, the method may detect fluorescent emissions from the fourth labeled nucleotide at the first wavelength and also at a second and / or third wavelength. In some embodiments, at step 340, the method may detect fluorescent emissions from the fourth labeled nucleotide only at the first wavelength.

[0095] The method 300 then moves to a decision step 345 to determine if the method 300 should repeat for an additional SBS cycle of reading sequence data. A determination may be made at decision step 345 whether to detect more nucleotides based on, for example, the quality of the signal or after a predetermined number of bases. If more nucleotides are to be detected, then the method 300 may loop back to the step 310 to start a next sequencing cycle.

[0096] The method 300 may then move to a step 350 wherein the system may determine the nucleotide sequence of the polynucleotide based on the detected fluorescent emissions and intensities. For example, at the step 350 the method may identify clusters that have added nucleotides with emissions at wavelengths and intensities corresponding to the first, second, third and / or fourth labeled nucleotides. In some embodiments, at the step 350 the method may identify clusters which had no fluorescent emissions following excitation at the first and / or second excitation wavelength and determine that the cluster corresponds to an empty well or to a short insert that has completed sequencing.

[0097] As mentioned above, a cluster may be identified by the lack of any fluorescence after excitation at either the first or second wavelengths. Because the lack of fluorescent emissions is dark, undetectable, or otherwise “missing” on an image, the system may track the position of each cluster on a flow cell. Here, the term “undetectable” refers to fluorescent emissions from a nucleotide that are intentionally or unintentionally reduced to an intensity that is not effectively distinguishable from noise by a detection scheme. Once a cluster has been identified at a particular position, the system may then note in subsequence sequencing rounds whether there is a fluorescent emission at the position of the cluster. If no fluorescent emissions are noted at a known position of a cluster, the system may determine that the nucleotide added in the latest sequencing round was the unlabeled nucleotide. For example, in some cases the unlabeled nucleotide may be a “G”. In another example, the unlabeled nucleotide may correspond to no nucleotide being sequenced—from either an empty well or a finished cluster. However, it should be realized that any nucleotide could be chosen as the one which is unlabeled and still be within the scope of the invention.

[0098] In some embodiments, prior to the next sequencing cycle, the fluorescent labels attached to each nucleotide may be removed from the incorporated nucleotide analogs, and the reversible 3ƍ blocks may be removed so that another nucleotide analog may be added onto each extending primer-polynucleotide. If a determination is made at the decision step 345 that there are no more additional rounds of sequencing necessary, the method 300 then moves to an end step 360 and terminates the method 300. If the system is employing offline fluorescent imaging processing, when there is no additional nucleotide to be detected at decision block 345, the fluorescent images comprising the fluorescent signals detected may be processed after step 360, and the bases of the nucleotides incorporated into the fragmented polynucleotide may be determined remotely. Foreach nucleotide base determined, a quality score may be determined. After all the fluorescent images are processed, the method 300 may terminate at the step 360.

[0099] In some embodiments, the disclosure provides for systems and methods for DNA sequencing using detection schemes other than fluorescence. For example, some DNA sequencing systems and methods employ voltage detectors instead of light detectors to detect specific nucleotides within a polynucleotide. In some embodiments, these techniques may or may not require amplification of the DNA or RNA sample into a cluster, for example, to be analyzed. In some embodiments, these techniques may or may not require the labelling of the DNA or RNA sample in order to be analyzed. The methods disclosed herein may be applied to voltage-based systems via an analogous process to the fluorescent detection systems. In some embodiments, instead of detecting fluorescent emissions from a labelled nucleotide and determining the nucleotide based on the emission wavelength and intensity, a voltage detection system may detect various parameters of a voltage signal, such as amplitude, frequency, or waveform characteristics, to extract relevant information to determine the presence of a particular nucleotide. In some embodiments, various parameters of a voltage signal, such as amplitude, frequency, or waveform characteristics may be characterized as a channel, with “on” and “off” states. For example, a voltage signal corresponding to a “T” nucleotide may be detected as a voltage signal with a characteristic frequency and at a first amplitude. In some embodiments, a voltage signal corresponding to a “C” nucleotide may be detected at the same characteristic frequency but at a second amplitude.

[0100] An aspect of the disclosure is directed to providing a sequencing system with fluorescent emissions with different relative intensities as compared to prior systems which may have only utilized an “on” versus “off” intensity. An increase or reduction in brightness may be quantified as an absolute measurement in terms of photon flux or counts per second. The increase in brightness may also be quantified as relative measurement for a cluster labeled with a single nucleotide as compared to a cluster labeled with two different nucleotides. For SNR optimization reasons, some sequencing systems employ a square constraint on a scatterplot of a two-channel detection, wherein a cluster labeled with a single nucleotide is constrained to have a similar intensity as a cluster labeled with two nucleotides (e.g. base C in a corner cloud). Such constraints in intensity / brightness of a cluster may be controlled by diluting labeled nucleotides with a non- fluorescing tag, such that all clusters are emitting at approximately half of the potential brightness.Relaxing this constraint according to this disclosure may improve increase signal to noise ratio (SNR).

[0101] It should be realized that on any flowcell, the different clusters may have varying brightness. For example, some clusters can be bright, and some clusters can be dim in comparison to each other. In embodiments, the intensity values vary between base calling cycles and thus the classification of bright and dim may also change between cycles. Some examples of intensity value ratios of emissions between bright and dim clusters include 0.55:0.45, 0.60:0.40, 0.65:0.35, 0.70:0.30, 0.75:0.25, 0.80:0.20, 0.85:0.15, 0.90:0.10, and 0.95:0.05. During each sampling event (e.g., each illumination stage or each image acquisition stage), a detector may image clusters with different intensities or clusters generating different types of signals.

[0102] In some embodiments, a method according to this disclosure may allow for each cluster to be labeled with a single dye, and that dye, or faction of that dye, may be chosen to increase the brightness of the cluster. In some embodiments, methods and systems may not require clusters to be labeled with two nucleotides. Some embodiments may result in an increase in the brightness of a cluster relative to an intensity of fluorescent emissions from a labeled nucleotide labeled with two fluorescent dyes, wherein the two fluorescent dyes are detected in two different channels. Some embodiments may result in an increase in the brightness of a cluster relative to an intensity of fluorescent emissions from a nucleotide forming a corner cloud in a two-excitation, two-channel detection method.

[0103] Some embodiments may include a first labeled nucleotide that is labeled with one fluorescent dye at a first intensity, wherein the first intensity is at least 10% higher than an intensity of fluorescent emissions from, for example, a fourth labeled nucleotide at the second intensity. In some embodiments, the first intensity may be at least 20% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 30% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 40% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 50% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 100% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 110% higher relative to a fourth labeled nucleotide. In some embodiments, the first intensity may be at least 120% higher relative to a fourth labeled nucleotide.

[0104] The brightness of any cluster may also be affected by fragment length distribution of the sample. The varying brightness of the cluster population can have the effect of elongating the ‘on’ populations in the base calling scatterplot. By way of example, without normalizing the level of amplification before trying to increase the brightness of all the clusters, some over-amplified AT rich sequences may be much brighter than similar GC rich sequences, and become even more ‘over amplified’ than the GC rich clusters. In some embodiments, it may be advantageous to normalize each cluster’s intensity by its mean intensity in the first 10 cycles to reduce population intensity variation. For example, in the first ten cycles, for every non- guanine(G) base call, two radii can be calculated: the distance of the population intensity from the origin, and the distance of the corresponding Gaussian mean from the origin. Cluster scaling can include normalizing to the mean of the ratio of these two radii averaged over, for example, the first 10 cycles. All cluster intensities can be normalized by this scaling factor before phase correction and base calling are performed. Cluster scaling can advantageously increase throughput and decrease error rates, for example, for samples with large fragment length distributions.

[0105] It is also possible to improve the brightness of all clusters, for example, by carrying out a higher number of amplification cycles, or by changing the chemistry / fraction of the dyes, or by changing the detection scheme (two to three-channel detection as described herein). In some embodiments, the methods and systems may be employed in a two-channel system, however, one of skill in the art will understand that the labeling in one channel with different / same dyes for two nucleotides may be applied to, for example, a four-channel system.

[0106] In some aspects, the techniques described herein relate to a method of sequencing polynucleotides bound to a flowcell, using a two channel, two excitation system. In some embodiments, a method may consist essentially of: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a second intensity that is different from the first intensity; and determining the sequence of the polynucleotides based on the detected fluorescent emissions and intensities.

[0107] In some embodiments, the techniques described herein relate to a method, wherein the flowcell includes wells configured to bind polynucleotides; further wherein the incorporation of each of the at least four labeled nucleotide conjugates into a well is detected from at least one signal state. In some embodiments, the techniques described herein relate to a method, wherein the presence of an empty well is determined from a dark state.

[0108] In some embodiments, the techniques described herein relate to a method, wherein the first intensity is approximately double the second intensity. The disclosure is believed to be the first reporting of using dual labeling in one channel with different dyes for two nucleotides, because previous systems relied on an off state, or additional channel to differentiate between nucleotides with emission in the same channel. Usually the unlabeled nucleotide was “G.” In some embodiments of this disclosure, the techniques described herein relate to a method, wherein the fourth labeled nucleotide is guanine. In some embodiments, the techniques described herein relate to a method, wherein the modified nucleotide is a 5-methylcytosine, a N6- methyladenine, or an inosine.

[0109] An aspect of the disclosure is directed to 2 channel dyes, methods and systems that utilize four labeled nucleotides. In some embodiments, methods and systems of the disclosure may employ dyes regularly used for a typical 2 channel 2 excitation system, but with four nucleotides labeled with dyes. In some embodiments, methods and systems utilizing four labeled nucleotides allow for an additional encoding space for empty wells.

[0110] Fig. 3 shows an illustration of one embodiment of a two-channel chemistry method employing four labeled nucleotides, including a labeled G nucleotide. A graphic for each labeled nucleotide or empty well is shown above each summary of the nucleotides. The column on the left includes labels for the remaining five columns, where the emission detections for wavelength 1, wavelength 2, and resulting base calls are shown below each graphic of labeled nucleotides.

[0111] In Fig. 3, the column corresponding to empty wells is designated with an “X,” and is shown as not detecting either wavelength 1 or wavelength 2 (depicted with greyed out boxes). The bottom box in the column shows the resulting determination of an empty well. One of skill in the art will understand that the results of an empty column may also be applied to short inserts that have already completed sequencing. Accordingly, where a short sequence has completed sequencing the lack of signal may be ascribed to the lack of additional nucleotides,instead of ascribed to a dark base (typically “G”) that would create an artefact of a long sequence of repeated “GGG.”

[0112] The nucleotide A is represented in the next column of Fig. 3, and the graphic above shows a nucleotide labeled with two fluorescent dyes. A green half-circle represents a fluorescent dye detected at wavelength 1 in green and a red half-circle represents a fluorescent dye detected at wavelength 2. Note that the half-circle for each fluorescent dye does not necessarily represent that there are equal proportions of the two dyes. Similarly, the half circles do not necessarily represent that the corresponding dye is present at half of the usual intensity. The box below the top graphic shows a green circle indicating that that the fluorescent dye associated with wavelength 1 is detected for the fluorescently labeled nucleotide “A.” Similarly, the next box below shows a red circle indicating that that the fluorescent dye associated with wavelength 2 is detected for the fluorescently labeled nucleotide A. The bottom box in the column shows that the nucleotide “A” is given the correct base call of “A.”

[0113] The nucleotides “G” and “T” are represented in the next two columns of Fig.3. The labeled nucleotide corresponding to “G” is depicted as a checkered green circle, while the labeled nucleotide corresponding to “T” is depicted as a solid green circle. The checkered green circle for nucleotide “G” indicates that the intensity of the fluorescent emissions is lower than that for the labeled nucleotide corresponding to “T.” Note that the relative intensities of the nucleotides “G” and “T” may be switched and may correspond to any nonzero intensity ranging from about 10% to about 200% of a typical intensity of fluorescent emissions, such as the intensity of emissions at wavelength 1 for “A” nucleotides or the intensity of emissions at wavelength 2 for “C” nucleotides. The lower boxes show that wavelength 2 is not detected for “G” and “T.” The bottom boxes in the columns show that the nucleotides are given the correct base calls of “G” and “T,” respectively.

[0114] The nucleotide “C” is represented in the last column of Fig. 3, and the graphic shows a nucleotide labeled with a single dye. The greyed out box for wavelength 1 indicates that no emissions are detected for nucleotide “C” at wavelength 1. The solid red circle represents a single dye with emissions detected at wavelength 2, and not wavelength 1. The bottom box in the column shows the resulting determination of nucleotide “C.”

[0115] The five columns of Fig.3 represent the five possible options for base calls for one embodiment of a two channel chemistry method employing a labeled G nucleotide. Asdescribed above, a typical 2-channel sequencing by synthesis system will present an issue where wells not occupied by a DNA cluster are called G, because the G nucleotide in those typical systems is not labeled. This issue is solved as summarized above by labeling all four nucleotides. By labeling the “G” nucleotide here, the unoccupied wells will form a fifth cloud in a 2- dimensional scatter plot of intensities. Additionally, experiments seeking to sequence an insert shorter than the sequencing read length can continue sequencing after the whole insert is sequenced, but any calls after beyond the sequence length will be incorrectly called as “G.” Fig.3 shows a solution to this issue because any calls after the insert has been sequenced will also be in a fifth cloud that is differentiated from any of the four labeled nucleotides.

[0116] An aspect of the disclosure is directed to methods and systems capable of sequencing five types of nucleotides by using four labeled nucleotides and an additional dark base. In some embodiments, methods and systems of the disclosure may employ dyes regularly used for a typical 2 channel 2 excitation system, but with an additional nucleotide that is unlabeled or labeled with a dark dye. In some embodiments, methods and systems utilizing four labeled nucleotides allows for an additional encoding space for empty wells.

[0117] Fig. 4 shows an illustration of one embodiment of a two channel chemistry method employing a labeled G nucleotide and a fifth nucleotide that is unlabeled. The column on the left includes labels for the remaining five columns, where the emission detections for wavelength 1 and wavelength 2, and resulting base calls are shown below each individual graphic of labeled nucleotides. A graphic for each unlabeled or labeled nucleotide is shown above each summary of the nucleotides.

[0118] In Fig.4, the column corresponding to a fifth base, here Methyl-C, is designated with an “X,” and is shown as not detecting either wavelength 1 or wavelength 2 (depicted with greyed out boxes). The bottom box in the column shows the resulting determination of a fifth base.

[0119] As in Fig.3, the next column of Fig.4 represents nucleotide A, and the graphic above shows a nucleotide labeled with two fluorescent dyes. A green half-circle represents a fluorescent dye detected at wavelength 1 in green and a red half-circle represents a fluorescent dye detected at wavelength 2. The box below the top graphic shows a green circle indicating that that the fluorescent dye associated with wavelength 1 is detected for the fluorescently labeled nucleotide “A.” Similarly, the next box below shows a red circle indicating that that the fluorescentdye associated with wavelength 2 is detected for the fluorescently labeled nucleotide A. The bottom box in the column shows that the nucleotide “A” is given the correct base call of “A.”

[0120] The nucleotides “G” and “T” are represented in the next two columns of Fig.4. The labeled nucleotide corresponding to “G” is depicted with half of a checkered green circle and the remaining half with a checkered red circle. , The labeled nucleotide corresponding to “T” is depicted as a solid green circle. As in Fig.3, the checkered green circle for nucleotide “G” indicates that the intensity of the fluorescent emissions is lower than that for the labeled nucleotide corresponding to “T,” and may be lower than the green component of the fluorescent emissions corresponding to the labeled nucleotide “A.” In some embodiments, nucleotide “G” may be partially labeled with one dye as shown in Fig. 3 or two dyes as shown in Fig. 4. For example, in some embodiments, the nucleotide “G” may be labeled with 10 percent green and 10 percent red, with the remainder unlabeled. The lower boxes show that wavelength 2 is partially detected for “G” and not detected for “T.” The bottom boxes in the columns show that the nucleotides are given the correct base calls of “G” and “T,” respectively.

[0121] The nucleotide “C” is represented in the last column of Fig. 4, and the graphic shows a nucleotide labeled with a single dye. The greyed-out box for wavelength 1 indicates that no emissions are detected for nucleotide “C” at wavelength 1. The solid red circle represents a single dye with emissions detected at wavelength 2, and not wavelength 1. The bottom box in the column shows the resulting determination of nucleotide “C.”

[0122] An aspect of the disclosure is directed to increasing the encoding space for base calls from two channels to three-channels. Fig. 5A shows a two-dimensional scatterplot of intensities of a traditional two excitation two channel sequencing system. Fig. 5B shows a two- dimensional scatterplot of a two excitation two channel sequencing system 2 channel SBS system with four labeled nucleotides, and a fifth cloud corresponding to empty wells or base calls past the read length of an insert.

[0123] The Y axes 401, and X axes 402 in Fig.5A and 5B correspond to intensities of fluorescent signals detected in a detection channel. In some embodiments, as shown here, the Y axis 401 may correspond to intensities of fluorescence detected from a channel detecting green wavelengths and the X axis 402 may correspond to intensities of fluorescence detected from a channel detecting red wavelengths. Note that the intensities of each axis may be normalized andthe total non-normalized intensities may be increased by using a dye with a higher quantum yield of fluorescence, or a larger percentage of a dye with a high quantum yield.

[0124] Nucleotide “T” 410 is shown as a cloud in the top left corner of both Fig. 5A and 5B, and corresponds to detections of fluorescence in the green channel, and no detections (or minimal detections) in the red channel. Nucleotide “A” 420 is shown as a cloud in the top right corner of both Fig.5A and 5B, and corresponds to a detection of fluorescence in the green channel and in the red channel. The separation between nucleotide “A” 420 and Nucleotide “T” 410 is reduced in Fig.5B as compared to 5A. The separation between some of the clouds may reduce due to cross-talk between the some of the fluorescent labels, and an aspect of the disclosure is related to minimizing this effect and increasing the signal to noise of the sequencing system. In some embodiments, the signal to noise may be improved by increasing the brightness of the dye associated with either the green channel or the red channel. Nucleotide “C” 430 is shown as a cloud in the bottom right corner of both Fig. 5A and 5B, and corresponds to a detection of fluorescence in the red channel and no detections (or minimal detections) in the green channel.

[0125] Nucleotide “G” 440 is shown as a cloud in the bottom left corner of Fig. 4A, and corresponds to typical dark detection of nucleotide “G”—no detections (or minimal detections) in the green and red channels.

[0126] Fig. 5B shows two different clouds in bottom left corner, 441 and 450. Empty wells or base calls continuing beyond the sequencing read length are shown in the dashed circle 450, and corresponds to typical dark detection, but the lack of detections may properly be associated with an empty well. Nucleotide “G” 441 in Fig. 5B is shown above the dashed circle 450, and corresponds to a partial detection of fluorescence in the green channel and no detections (or minimal detections) in the red channel. Fig.5B shows the results from an experiment performed during the first cycle of a typical 2-channel system, and so the cloud at 450 would correspond to empty wells and not short inserts, though the technique is applicable to both scenarios.

[0127] In some embodiments, the techniques described herein relate to a method, wherein the fluorescent emissions from the first, second, third and fourth labeled nucleotides are plotted onto a cloud plot of intensity and wavelength, as shown in Fig. 5A and 5B. In some embodiments, the techniques described herein relate to a method, wherein detecting the fluorescent emissions from the second labeled nucleotide at a second wavelength includes detecting the second labeled nucleotide fluorescent emissions at a third intensity, and the method further includes:detecting fluorescent emissions from a fifth labeled nucleotide at the second wavelength and at fourth intensity which is different from the third intensity.

[0128] In some embodiments, the techniques described herein relate to a method, wherein the fourth labeled nucleotide is a modified nucleotide. Examples of modified nucleotides are includes throughout the disclosure, including the definition section. However, one skilled in the art will understand that new modified nucleotides beyond those currently discovered may be used as one of the modified bases.

[0129] An aspect of the disclosure relates to mitigating potential issues with assigning bases (or empty wells) that have a low probability of occurring, due to, for example, low base diversity. Clusters that are not incorporating or are empty wells on a patterned flowcell generate may erroneously generate G basecalls because the G nucleotide is not labeled. In some embodiments, software methods may remove these by filtering on any cluster with 9 or 10 Gs in the first 10 cycles. However, some embodiments with filters may also remove any cluster whose true sequence was 9 or 10 Gs in the first 10 cycles, so there may be some false detection to the extent that sequence context is present in the samples and it starts at cycle 1. Furthermore, when clusters sequence the whole insert and are no longer incorporating, poly G will be called for the remainder of the read. This may be trimmed in adapter trimming by specifying poly G as an adapter sequence during fastq generation. By partially labeling the G, a fifth cloud may be present in a two-dimensional plot of intensities in 2-channel SBS.

[0130] An aspect of the disclosure is directed to addressing the fifth cloud. Some requirements of such an embodiment include an underlying assumption of “base call diversity” baked into the base calling model – meaning at any given cycle for any given population of clusters, the base representation is roughly equal in ratio. While this is true for naturally occurring DNA bases, additional bases representing specific features in the genome (such as methylated bases) do not necessarily satisfy the equal diversity assumption. In addition, amplitude multiplexing may have relatively tighter requirements on signal to noise ratio of the raw signal. For sequencing systems dealing with phasing, intensity decay and general noise, utilizing more than two levels in a channel could lead to poorer performance of the overall system.

[0131] Fig. 7 shows an X-Y scatterplot showing five clouds corresponding to the fluorescence emissions of dyes bound to each nucleotide and a shaded and dashed cloud corresponding to empty wells. Here, the Y axis corresponds to intensity of fluorescent emissionsin a green channel, and the X axis corresponds to the intensity of fluorescent emissions in a blue channel. Each of the four nucleotides are labeled, and the four solid circles in Fig.7 correspond to the fluorescence emissions of nucleotide “T” 710, nucleotide “C” 720, nucleotide “A” 730, and nucleotide “G” 740. The fifth cloud corresponds to empty clusters 743. The “G” nucleotide 740 is shown as a circle with three-quarters shaded black and the remaining quarter shaded green, and illustrates that “G” nucleotide 740 may be partially labeled with a dye that fluoresces in the same channel as the “T” nucleotide 710, but at a lower intensity than the “T” nucleotide 710. The partial labeling of the “G” nucleotide 740 is highlighted by the green arrow underneath the cloud of G” nucleotide 740.

[0132] In some embodiments, the “G” nucleotide 740 may be labeled with the same fluorescent dye as the “T” nucleotide 710, but a fraction of the “G” nucleotides 740 may be unlabeled such that the intensity in the green channel is lower than for a cluster with a base call corresponding to the “T” nucleotide 710. In some embodiments, the intensity of emissions for “G” nucleotide 740 may be quantified as a percentage of the intensity of emissions for “T” nucleotide 710, wherein the percentage may be one of 90%, 80%, 70%, 60%, 50%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, and 5% of the intensity of emissions of “T” nucleotide 710 in the green channel. In some embodiments, the “G” nucleotide 740 may be partially labeled with the same fluorescent dye as the “T” nucleotide 710 and also partially labeled with the same fluorescent dye as the “A” nucleotide 730.

[0133] The intensity of the fluorescent emissions of the “T” nucleotide 710 in the green channel is shown as approximately the same as the intensity of the fluorescent emissions of the “C” nucleotide 720 in the green channel. The “C” nucleotide 720 is shown as a circle with halves shaded in green and blue, corresponding to the labeling of the fluorescent dyes that emit in the green and blue channels. The “C” nucleotide 720 is shown in the top right corner of the scatterplot and illustrates that a nucleotide may be labeled in two channels. The “A” nucleotide 730 is shown as a circle with one half shaded black and the remaining half shaded blue, and illustrates that “A” nucleotide 730 may be partially labeled with a dye that fluoresces in the same channel and intensity as the “C” nucleotide 720.

[0134] By labeling nucleotide “G” with different levels of labeling and / or brighter dyes may be used to optimize the separation of clouds in a scatterplot. In some embodiments, the G cloud may be sufficiently separated in the X-Y plane from the empty cloud such that there is nooverlap between the clouds. In some embodiments, a separation of the G nucleotide cloud may remove an error mode related to miscalling G nucleotides as empty wells and vice versa.

[0135] Fig. 7 shows the fifth cloud that corresponds to empty clusters 743 as smaller than the clouds corresponding to the other nucleotides. In some embodiments, the total size of the clouds does not refer to the signal to noise ratio, the total prevalence of the signal, or any other indication aside from the general location of the cloud in the X-Y plane. In other embodiments, the smaller size of fifth cloud may be indicative of the population of the empty cloud. In some embodiments, the smaller size of fifth cloud may be indicative of one or more statistic of the empty cloud that differentiates the empty cloud from the other nucleotides. In some embodiments, the systems and methods provide for effective handling of a fifth cloud across a large range of loading concentrations that will yield may differences in the population of the fifth cloud.

[0126] In some embodiments, traditional 2-dimensional data could still be modeled using 4 clouds and the empty wells would likely be called G. The detection of poly G could still be done with 9 or 10 Gs in the first 10 cycles. In addition, or in the alternative, in some embodiments, disclosed methods may take the population of the clusters called G and segment them, using binary segmentation such as Otsu’s method, in the y dimension into two populations, empty and occupied G. This method may be performed for one or more cycles to build confidence in the wells being assigned as empty. In some embodiments, the disclosed methods may modify the model for the data to include five populations, the four populations for A, C, G, and T and one more for the empties at the origin and then perform expectation maximization to learn the maximum likelihood parameters of the 5-cloud model. In some embodiments, other segmentation algorithms such as k-means could also be used in place of expectation maximization. The disclosure provides for several permutations on this method. For example, one method may only use the 5-cloud model for a single cycle, a few early cycles, or every cycle in the sequencing run. In some embodiments, the fifth cloud may be assigned as “N” until the cluster is filtered out after some number of early cycles. In some embodiments, this method may be present in the model for every cycle. In some embodiments, disclosed methods may be employed for detecting that an insert had been completely sequenced because it returned an “empty” well. The following non-limiting examples demonstrate methods for mitigating potential issues with assigning bases that have low base diversity. One mitigation may include introducing a known sequence that includes multiples of a less abundant fifth base and allow the RTA to train on that data set. In other examples, such asbiotechnological applications like a tumor selection, methods may first artificially increase the diversity of base pairs by adding in unnatural base pairs and then sequencing with more than four base pairs.

[0136] An aspect of the disclosure is directed to effective solutions where, in some embodiments, the disclosed methods provide a sufficient signal-to-noise ratio to distinguish empty cells and partially labeled G bases. If there is an overlap between empty clouds and G clouds, then a new error mode will be introduced of miscalling G as empty and vice versa. In some embodiments, the G cloud may be moved far enough away from the empty cloud so they do not overlap. In some embodiments, the cloud for a different base than G may be replaced for G, and the same sequencing may be run where G is no longer in the "empty" location, and thereby G may be moved far enough away from the empty cloud, so they do not overlap. In some embodiments, the disclosure provides methods to address an issue where the effectiveness of procedures depends on the occupancy and the population of the empty cloud.

[0137] Disclosed solutions may be robust across an extensive range of loading concentrations and may yield differences in the population of the fifth cloud. Some embodiments may adjust dye concentrations, ratios, or wavelengths such that the clouds for each base are not aligned with an axis. Some embodiments may control the degree of labeling to maximize the distance between bases. Some embodiments may locate the G cloud by, for example, adjusting ratios, dyes, and wavelengths to minimize the quenching of other adjacent clouds or subsequent bases in the sequences.

[0138] Embodiments of the present disclosure also include a system for analyzing and assembling sequences of polynucleotides. Fig. 6 is a block diagram of an exemplary computing system 500 that may be used in connection with an illustrative sequencing system. The computing system 500 may be configured to determine a DNA sequence by using the sequencing and assembly methods disclosed herein. The general architecture of the computing system 500 depicted in Fig. 6 includes an arrangement of computer hardware and software components. The computing system 500 may include many more (or fewer) elements than those shown in Fig.6. It is not necessary, however, that all of these generally conventional elements be shown in order to provide an enabling disclosure.

[0139] As illustrated, the computing system 500 includes a processing unit 510, a network interface 520, a computer-readable medium drive 530, an input / output device interface540, a display 550, and an input device 560, all of which may communicate with one another by way of a communication bus. The network interface 570 may provide connectivity to one or more networks or computing systems. The processing unit 510 may thus receive information and instructions from other computing systems or services via a network. The processing unit 510 may also communicate to and from memory 570 and further provide output information for an optional display 550 via the input / output device interface 540. The input / output device interface 540 may also accept input from the optional input device 560, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0140] The memory 570 may contain computer program instructions (grouped as modules or components in some embodiments) that the processing unit 510 executes in order to implement one or more embodiments. The memory 570 generally includes RAM, ROM and / or other persistent, auxiliary or non-transitory computer-readable media. The memory 570 may store an operating system 572 that provides computer program instructions for use by the processing unit 510 in the general administration and operation of the computing device 500. The memory 570 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0141] For example, in one embodiment, the memory 570 includes a two-channel sequencing module 574 for analyzing and assembling sequences of polynucleotides. The two- channel sequencing module 574 can perform the methods disclosed herein, including the method described with respect to the flow diagrams of Fig. 1. In addition, memory 570 may include or communicate with the data store 590 and / or one or more other data stores that store one or more inputs, one or more outputs, and / or one or more results (including intermediate results) of determining a DNA sequence and providing an assembly process according to the present disclosure.

[0142] Particular embodiments of the method of sequencing may utilize a one- excitation, two-channel detection system (also known as 1Ex-2Ch) or a two-excitation, two- channel detection system (also known as 2Ex-2Ch). Detailed disclosures are provided in WO 2018 / 165099 and U.S. Ser. No. 17 / 338590, each of which is incorporated by reference in its entirety. However, 1Ex-2Ch and a two-excitation, two-channel detection system (2Ex-2Ch)configurations are not considered mutually exclusive and can be used in various combinations. For example, some dyes used in 1Ex-2Ch may be used in 2Ex-2Ch configurations.

[0143] In some embodiments, methods according to the disclosure may be performed on an automated sequencing instrument, and wherein the automatic sequencing instrument may comprise two light sources operating at different wavelengths (e.g., at 350-360 nm (blue), 520- 530 nm (green), 630 nm-670 nm (red)). The incorporation of the first type of the nucleotide conjugates is determined by a signal state in the first imaging event and a dark state in the second imaging event. The incorporation of the second type of the nucleotide conjugates is determined by a dark state in the first imaging event and a signal state in the second imaging event. The incorporation of the third type of the nucleotide conjugates is determined by a signal state in both the first imaging event and the second imaging event. The incorporation of the fourth type of the nucleotide conjugates is determined by a dark state in the first imaging event and a partial signal state in the second imaging event. The incorporation of the fifth type of the nucleotide conjugates is determined by a dark state in both the first imaging event and the second imaging event.

[0144] In some embodiments, the automatic sequencing instrument may comprise a single light source operating with a blue laser at about 350 nm to about 360 nm. The incorporation of the first type of the nucleotide may determined by detection in the one of the blue or green channel / region (e.g., at a blue region with a wavelength ranging from about 372 to about 520 nm, or at a green region with a wavelength ranging from about 540 nm to about 640nm). The incorporation of the second type of nucleotide is determined by detection in the other one of the blue or green detection channel / region. The incorporation of the third type of nucleotide is determined by detection in both the blue and green channels / regions. The incorporation of the fourth type of nucleotide is determined by a partial detection in the blue channel / region but no detection green channel / region. The incorporation of the fifth type of nucleotide is determined by no detection in either the blue or detection green channels / regions.

[0145] In some embodiments, the disclosed systems and methods may involve approaches for shifting or distributing certain sequence data analysis features and sequence data storage to a cloud computing environment or cloud-based network. User interaction with sequencing data, genome data, or other types of biological data may be mediated via a central hub that stores and controls access to various interactions with the data. In some embodiments, the cloud computing environment may also provide sharing of protocols, analysis methods, libraries,sequence data as well as distributed processing for sequencing, analysis, and reporting. In some embodiments, the cloud computing environment facilitates modification or annotation of sequence data by users. In some embodiments, the systems and methods may be implemented in a computer browser, on-demand or on-line.

[0146] In some embodiments, software written to perform the methods as described herein is stored in some form of computer readable medium, such as memory, CD-ROM, DVD- ROM, memory stick, flash drive, hard drive, SSD hard drive, server, mainframe storage system and the like. Samples

[0147] In some embodiments, the sample comprises or consists of a purified or isolated polynucleotide derived from a tissue sample, a biological fluid sample, a cell sample, and the like. Suitable biological fluid samples include, but are not limited to blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, lymph, saliva, cerebrospinal fluid, ravages, bone marrow suspension, vaginal flow, trans-cervical lavage, brain fluid, ascites, milk, secretions of the respiratory, intestinal and genitourinary tracts, amniotic fluid, milk, and leukophoresis samples. In some embodiments, the sample is a sample that is easily obtainable by non-invasive procedures, e.g., blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow, saliva or feces. In certain embodiments the sample is a peripheral blood sample, or the plasma and / or serum fractions of a peripheral blood sample. In other embodiments, the biological sample is a swab or smear, a biopsy specimen, or a cell culture. In another embodiment, the sample is a mixture of two or more biological samples, e.g., a biological sample can comprise two or more of a biological fluid sample, a tissue sample, and a cell culture sample. As used herein, the terms “blood,” “plasma” and “serum” expressly encompass fractions or processed portions thereof. Similarly, where a sample is taken from a biopsy, swab, smear, etc., the “sample” expressly encompasses a processed fraction or portion derived from the biopsy, swab, smear, etc.

[0148] In certain embodiments, samples can be obtained from sources, including, but not limited to, samples from different individuals, samples from different developmental stages of the same or different individuals, samples from different diseased individuals (e.g., individuals with cancer or suspected of having a genetic disorder), normal individuals, samples obtained at different stages of a disease in an individual, samples obtained from an individual subjected todifferent treatments for a disease, samples from individuals subjected to different environmental factors, samples from individuals with predisposition to a pathology, samples individuals with exposure to an infectious disease agent, and the like.

[0149] In one illustrative, but non-limiting embodiment, the sample is a maternal sample that is obtained from a pregnant female, for example a pregnant woman. The maternal sample can be a tissue sample, a biological fluid sample, or a cell sample. In another illustrative, but non-limiting embodiment, the maternal sample is a mixture of two or more biological samples, e.g., the biological sample can comprise two or more of a biological fluid sample, a tissue sample, and a cell culture sample.

[0150] In certain embodiments samples can also be obtained from in vitro cultured tissues, cells, or other polynucleotide-containing sources. The cultured samples can be taken from sources including, but not limited to, cultures (e.g., tissue or cells) maintained in different media and conditions (e.g., pH, pressure, or temperature), cultures (e.g., tissue or cells) maintained for different periods of length, cultures (e.g., tissue or cells) treated with different factors or reagents (e.g., a drug candidate, or a modulator), or cultures of different types of tissue and / or cells.

[0151] In some embodiments, the use of the disclosed sequencing technology does not involve the preparation of sequencing libraries. In other embodiments, the sequencing technology contemplated herein involve the preparation of sequencing libraries. In one illustrative approach, sequencing library preparation involves the production of a random collection of adapter-modified DNA fragments (e.g., polynucleotides) that are ready to be sequenced.

[0152] Sequencing libraries of polynucleotides can be prepared from DNA or RNA, including equivalents, analogs of either DNA or cDNA, for example, DNA or cDNA that is complementary or copy DNA produced from an RNA template, by the action of reverse transcriptase. The polynucleotides may originate in double-stranded form (e.g., dsDNA such as genomic DNA fragments, cDNA, PCR amplification products, and the like) or, in certain embodiments, the polynucleotides may originated in single-stranded form (e.g., ssDNA, RNA, etc.) and have been converted to dsDNA form. By way of illustration, in certain embodiments, single stranded mRNA molecules may be copied into double-stranded cDNAs suitable for use in preparing a sequencing library. The precise sequence of the primary polynucleotide molecules is generally not material to the method of library preparation, and may be known or unknown. In one embodiment, the polynucleotide molecules are DNA molecules. More particularly, in certainembodiments, the polynucleotide molecules represent the entire genetic complement of an organism or substantially the entire genetic complement of an organism, and are genomic DNA molecules (e.g., cellular DNA, cell free DNA (cfDNA), etc.), that typically include both intron sequence and exon sequence (coding sequence), as well as non-coding regulatory sequences such as promoter and enhancer sequences. In certain embodiments, the primary polynucleotide molecules comprise human genomic DNA molecules, e.g., cfDNA molecules present in peripheral blood of a pregnant subject.

[0153] Methods of isolating nucleic acids from biological sources may differ depending upon the nature of the source. One of skill in the art can readily isolate nucleic acids from a source as needed for the method described herein. In some instances, it can be advantageous to fragment large nucleic acid molecules (e.g. cellular genomic DNA) in the nucleic acid sample to obtain polynucleotides in the desired size range. Fragmentation can be random, or it can be specific, as achieved, for example, using restriction endonuclease digestion. Methods for random fragmentation may include, for example, limited DNase digestion, alkali treatment and physical shearing. Fragmentation can also be achieved by any of a number of methods known to those of skill in the art. For example, fragmentation can be achieved by mechanical means including, but not limited to nebulization, sonication and hydroshear.

[0154] In some embodiments, sample nucleic acids are obtained from as cfDNA, which is not subjected to fragmentation. For example, cfDNA, typically exists as fragments of less than about 300 base pairs and consequently, fragmentation is not typically necessary for generating a sequencing library using cfDNA samples.

[0155] Typically, whether polynucleotides are forcibly fragmented (e.g., fragmented in vitro), or naturally exist as fragments, they are converted to blunt-ended DNA having 5’- phosphates and 3’-hydroxyl. Standard protocols, e.g., protocols for sequencing using, for example, the Illumina platform, instruct users to end-repair sample DNA, to purify the end-repaired products prior to dA-tailing, and to purify the dA-tailing products prior to the adaptor-ligating steps of the library preparation.

[0156] In various embodiments, verification of the integrity of the samples and sample tracking can be accomplished by sequencing mixtures of sample genomic nucleic acids, e.g., cfDNA, and accompanying marker nucleic acids that have been introduced into the samples, e.g., prior to processing.Sequencing Techniques

[0157] The disclosed sequencing systems and methods may be compatible with any sequencing techniques based on optical detection, for example, next-generation sequencing (NGS), fluorescent in situ sequencing (FISSEQ), and Massively Parallel Signature Sequencing (MPSS). In one embodiment, the disclosed systems and methods may be compatible with NGS technologies that allow multiple samples to be sequenced individually as genomic molecules (i.e., singleplex sequencing) or as pooled samples comprising indexed genomic molecules (e.g., multiplex sequencing) on a single sequencing run. These methods can generate up to several hundred million reads of DNA sequences.

[0158] The disclosed technology may implement sequencing reactions such as those incorporating sequencing-by-synthesis methods described in U.S. Patent Application Publication Numbers 2007 / 0166705, 2006 / 0188901, 2006 / 0240439, 2006 / 0281109, 2005 / 0100900, U.S. Patent Number 7,057,026, PCT Application Publication Numbers WO 2005 / 065814, WO 2006 / 064199, and WO 2007 / 010251, the disclosures of which are incorporated herein by reference in their entireties. In some embodiments, the sequencers may implement sequencing-by-synthesis methods similar to those used in the HiSeq, MiSeq, or HiScanSQ systems from Illumina (San Diego, Calif.).

[0159] Alternatively, sequencing by ligation techniques may be used in the disclosed technology, such as described in U.S. Patent Numbers 6,969,488, 6,172,218, and 6,306,597, the disclosures of which are incorporated herein by reference in their entireties. Sequencing by ligation techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides.

[0160] The disclosed technology may be implemented in some sequencing techniques which are available commercially, such as the sequencing-by-hybridization platform from Affymetrix Inc. (Sunnyvale, CA) and the sequencing-by-synthesis platforms from 454 Life Sciences (Bradford, CT) and Helicos Biosciences (Cambridge, MA), the sequencing-by-ligation platform from Applied Biosystems (Foster City, CA), or the SMRT technology of Pacific Biosciences.

[0161] In one illustrative, but non-limiting, embodiment, the methods described herein comprise obtaining sequence information for the nucleic acids in a sample using Illumina’ssequencing-by-synthesis and reversible terminator-based sequencing chemistry (e.g. as described in Bentley et al., Nature 6:53-59

[2009] ). Illumina’s sequencing technology may include the attachment of fragmented genomic DNA to a planar, optically transparent surface on which oligonucleotide anchors are bound. For example, template DNA is end-repaired to generate 5’- phosphorylated blunt ends, and the polymerase activity of Klenow fragment is used to add a single A base to the 3’ end of the blunt phosphorylated DNA fragments. This addition prepares the DNA fragments for ligation to oligonucleotide adapters, which have an overhang of a single T base at their 3’ end to increase ligation efficiency. The adapter oligonucleotides are complementary to the flowcell anchor oligos. Under limiting-dilution conditions, adapter-modified, single-stranded template DNA is added to the flowcell and immobilized by hybridization to the anchor oligos. Attached DNA fragments are extended and bridge amplified to create an ultra-high density sequencing flowcell with hundreds of millions of clusters, each containing about 1,000 copies of the same template. In one embodiment, the randomly fragmented genomic DNA is amplified using PCR before it is subjected to cluster amplification. Alternatively, an amplification-free (e.g., PCR free) genomic library preparation is used, and the randomly fragmented genomic DNA is enriched using the cluster amplification alone (Kozarewa et al., Nature Methods 6:291-295

[2009] ). The sequencing-by-synthesis reaction may employ reversible terminators with removable fluorescent dyes. Short sequence reads of about tens to a few hundred base pairs are aligned against a reference genome and unique mapping of the short sequence reads to the reference genome are identified. After completion of the first read, the templates can be regenerated in situ to enable a second read from the opposite end of the fragments. Thus, either single-end or paired end sequencing of the DNA fragments can be used. Detailed information about paired end sequencing can be found in US Patent No. 7601499 and US Patent Publication No. 2012 / 0,053,063, which are incorporated by reference.

[0162] In some embodiments, the sequencing by synthesis platform by Illumina involves clustering fragments. Clustering is a process in which each fragment molecule is isothermally amplified. In some embodiments, the fragment has two different adaptors attached to the two ends of the fragment, the adaptors allowing the fragment to hybridize with the two different oligos on the surface of a flowcell lane. The fragment further includes or is connected to two index sequences at two ends of the fragment, where index sequences provide labels to identify different samples in multiplex sequencing.

[0163] In some implementation, a flowcell for clustering in the Illumina platform is a glass slide with lanes. Each lane is a glass channel coated with a lawn of two types of oligos. Hybridization is enabled by the first of the two types of oligos on the surface. This oligo is complementary to a first adapter on one end of the fragment. A polymerase creates a compliment strand of the hybridized fragment. The double-stranded molecule is denatured, and the original template strand is washed away. The remaining strand, in parallel with many other remaining strands, is clonally amplified through bridge application.

[0164] In bridge amplification, a strand folds over, and a second adapter region on a second end of the strand hybridizes with the second type of oligos on the flowcell surface. A polymerase generates a complimentary strand, forming a double-stranded bridge molecule. This double-stranded molecule is denatured resulting in two single-stranded molecules tethered to the flowcell through two different oligos. The process is then repeated over and over, and occurs simultaneously for millions of clusters resulting in clonal amplification of all the fragments. After bridge amplification, the reverse strands are cleaved and washed off, leaving only the forward strands. The 3’ ends are blocked to prevent unwanted priming.

[0165] After clustering, sequencing starts with extending a first sequencing primer to generate the first read. With each cycle, fluorescently tagged nucleotides compete for addition to the growing chain. Only one is incorporated based on the sequence of the template. After the addition of each nucleotide, the cluster is excited by a light source, and a characteristic fluorescent signal is emitted. The number of cycles determines the length of the read. The emission wavelength and the signal intensity determine the base call. For a given cluster all identical strands are read simultaneously. Hundreds of millions of clusters, or thousands to tens of thousands of millions of clusters, are sequenced in a massively parallel manner. At the completion of the first read, the read product is washed away.

[0166] In processes involving two index primers, an index 1 primer is introduced and hybridized to an index 1 region on the template. Index regions provide identification of fragments, which is useful for de-multiplexing samples in a multiplex sequencing process. The index 1 read is generated similar to the first read. After completion of the index 1 read, the read product is washed away and the 3’ end of the strand is de-protected. The template strand then folds over and binds to a second oligo on the flowcell. An index 2 sequence is read in the same manner as index 1. Then an index 2 read product is washed off at the completion of the step.

[0167] After reading two indices, read 2 initiates by using polymerases to extend the second flowcell oligos, forming a double-stranded bridge. This double-stranded DNA is denatured, and the 3’ end is blocked. The original forward strand is cleaved off and washed away, leaving the reverse strand. Read 2 begins with the introduction of a read 2 sequencing primer. As with read 1, the sequencing steps are repeated until the desired length is achieved. The read 2 product is washed away. This entire process generates millions of reads, representing all the fragments. Sequences from pooled sample libraries are separated based on the unique indices introduced during sample preparation. For each sample, reads of similar stretches of base calls are locally clustered. Forward and reversed reads are paired creating contiguous sequences. These contiguous sequences are aligned to the reference genome for variant identification. Systems and Instruments

[0168] In some embodiments, the methods may be written in any of various suitable programming languages, for example compiled languages such as C, C#, C++, Fortran, and Java. Other programming languages could be script languages, such as Perl, MatLab, SAS, SPSS, Python, Ruby, Pascal, Delphi, R and PHP. In some embodiments, the methods are written in C, C#, C++, Fortran, Java, Perl, R, Java or Python. In some embodiments, the method may be an independent application with data input and data display modules. Alternatively, the method may be a computer software product and may include classes wherein distributed objects comprise applications including computational methods as described herein.

[0169] In some embodiments, the methods may be incorporated into pre-existing data analysis software, such as that found on sequencing instruments. Software comprising computer implemented methods as described herein are installed either onto a computer system directly, or are indirectly held on a computer readable medium and loaded as needed onto a computer system. Further, the methods may be located on computers that are remote to where the data is being produced, such as software found on servers and the like that are maintained in another location relative to where the data is being produced, such as that provided by a third party service provider.

[0170] An assay instrument, desktop computer, laptop computer, or server which may contain a processor in operational communication with accessible memory comprising instructions for implementation of systems and methods. In some embodiments, a desktop computer or a laptop computer is in operational communication with one or more computer readable storage media ordevices and / or outputting devices. An assay instrument, desktop computer and a laptop computer may operate under a number of different computer based operational languages, such as those utilized by Apple based computer systems or PC based computer systems. An assay instrument, desktop and / or laptop computers and / or server system may further provide a computer interface for creating or modifying experimental definitions and / or conditions, viewing data results and monitoring experimental progress. In some embodiments, an outputting device may be a graphic user interface such as a computer monitor or a computer screen, a printer, a hand-held device such as a personal digital assistant (i.e., PDA, Blackberry, iPhone), a tablet computer (for example, iPAD), a hard drive, a server, a memory stick, a flash drive and the like.

[0171] A computer readable storage device or medium may be any device such as a server, a mainframe, a supercomputer, a magnetic tape system and the like. In some embodiments, a storage device may be located onsite in a location proximate to the assay instrument, for example adjacent to or in close proximity to, an assay instrument. For example, a storage device may be located in the same room, in the same building, in an adjacent building, on the same floor in a building, on different floors in a building, etc. in relation to the assay instrument. In some embodiments, a storage device may be located off-site, or distal, to the assay instrument. For example, a storage device may be located in a different part of a city, in a different city, in a different state, in a different country, etc. relative to the assay instrument. In embodiments where a storage device is located distal to the assay instrument, communication between the assay instrument and one or more of a desktop, laptop, or server is typically via Internet connection, either wireless or by a network cable through an access point. In some embodiments, a storage device may be maintained and managed by the individual or entity directly associated with an assay instrument, whereas in other embodiments a storage device may be maintained and managed by a third party, typically at a distal location to the individual or entity associated with an assay instrument. In embodiments as described herein, an outputting device may be any device for visualizing data.

[0172] An assay instrument, desktop, laptop and / or server system may be used itself to store and / or retrieve computer implemented software programs incorporating computer code for performing and implementing computational methods as described herein, data for use in the implementation of the computational methods, and the like. One or more of an assay instrument, desktop, laptop and / or server may comprise one or more computer readable storage media forstoring and / or retrieving software programs incorporating computer code for performing and implementing computational methods as described herein, data for use in the implementation of the computational methods, and the like. Computer readable storage media may include, but is not limited to, one or more of a hard drive, a SSD hard drive, a CD-ROM drive, a DVD-ROM drive, a floppy disk, a tape, a flash memory stick or card, and the like. Further, a network including the Internet may be the computer readable storage media. In some embodiments, computer readable storage media refers to computational resource storage accessible by a computer network via the Internet or a company network offered by a service provider rather than, for example, from a local desktop or laptop computer at a distal location to the assay instrument.

[0173] In some embodiments, computer readable storage media for storing and / or retrieving computer implemented software programs incorporating computer code for performing and implementing computational methods as described herein, data for use in the implementation of the computational methods, and the like, is operated and maintained by a service provider in operational communication with an assay instrument, desktop, laptop and / or server system via an Internet connection or network connection.

[0174] In some embodiments, a hardware platform for providing a computational environment comprises a processor (i.e., CPU) wherein processor time and memory layout such as random access memory (i.e., RAM) are systems considerations. For example, smaller computer systems offer inexpensive, fast processors and large memory and storage capabilities. In some embodiments, graphics processing units (GPUs) can be used. In some embodiments, hardware platforms for performing computational methods as described herein comprise one or more computer systems with one or more processors. In some embodiments, smaller computer are clustered together to yield a supercomputer network.

[0175] In some embodiments, computational methods as described herein are carried out on a collection of inter- or intra-connected computer systems (i.e., grid technology) which may run a variety of operating systems in a coordinated manner. For example, the CONDOR framework (University of Wisconsin-Madison) and systems available through United Devices are exemplary of the coordination of multiple stand-alone computer systems for the purpose dealing with large amounts of data. These systems may offer Perl interfaces to submit, monitor and manage large sequence analysis jobs on a cluster in serial or parallel configurations. One aspect of the disclosure is directed to a workflow module that may be integrated into existing workflows. Insome embodiments, a workflow module may be a two-channel sequencing module and may be integrated into a NGS sequence analysis platform, for example the DRAGEN™ Bio-ID platform from Illumina.

Claims

1. A method of sequencing polynucleotides bound to a flowcell, comprising: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a second intensity that is different from the first intensity; and determining the sequence of the polynucleotides based on the detected fluorescent emissions and intensities.

2. The method of claim 1, wherein the flowcell comprises wells configured to bind polynucleotides; further wherein the incorporation of each of the at least four labeled nucleotide conjugates into a well is detected from at least one signal state.

3. The method of claim 2, wherein the presence of an empty well is determined from a dark state.

4. The method of claim 1, wherein the first intensity is approximately double the second intensity.

5. The method of claim 1, wherein the fourth labeled nucleotide is guanine.

6. The method of claim 1, wherein the fluorescent emissions from the first, second, third and fourth labeled nucleotides are plotted onto a cloud plot of intensity and wavelength.

7. The method of claim 1, wherein the fourth labeled nucleotide is a modified nucleotide.

8. The method of claim 7, wherein detecting the fluorescent emissions from the second labeled nucleotide at a second wavelength comprises detecting the second labeled nucleotide fluorescent emissions at a third intensity, and the method further comprises: detecting fluorescent emissions from a fifth labeled nucleotide at the second wavelength and at fourth intensity which is different from the third intensity.

9. The method of claim 6, wherein the modified nucleotide is a 5-methylcytosine, a N6- methyladenine, or an inosine.

10. A system for sequencing polynucleotides bound to a flowcell, comprising:a machine-readable memory; and a processor configured to execute machine-readable instructions, which, when executed by the processor, cause the system to perform steps including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detecting fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a second intensity that is different from the first intensity; and determining the sequence of the polynucleotides based on the detected fluorescent emission and intensity.

11. The system of claim 10, wherein the flowcell comprises wells configured to bind polynucleotides; further wherein the incorporation of the at least four labeled nucleotide conjugates into a well is detected from at least one signal state.

12. The system of claim 11, wherein the presence of an empty well is determined from a dark state.

13. The system of claim 10, wherein the first intensity is approximately double the second intensity.

14. The system of claim 10, wherein the fourth labeled nucleotide is guanine.

15. The system of claim 10, wherein the fluorescent emissions from the first, second, third and fourth labeled nucleotides are plotted onto a cloud plot of intensity and wavelength.

16. The system of claim 10, wherein the fourth labeled nucleotide is a modified nucleotide.

17. The system of claim 16, wherein detecting the fluorescent emissions from the second labeled nucleotide at a second wavelength comprises detecting the second labeled nucleotide fluorescent emissions at a third intensity, and the method further comprises: detecting fluorescent emissions from a fifth labeled nucleotide at the second wavelength and at fourth intensity which is different from the third intensity.

18. The system of claim 17, wherein the modified nucleotide is a 5-methylcytosine, a N6- methyladenine, or an inosine.

19. A non-transitory computer-readable medium storing a polynucleotide sequencing program including instructions that, when executed by a processor, causes a polynucleotide sequencing apparatus, to: detect fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detect fluorescent emissions from a second labeled nucleotide at a second wavelength, wherein the first wavelength is different from the second wavelength; detect fluorescent emissions from a third labeled nucleotide at the first and second wavelengths; detect fluorescent emissions from the fourth labeled nucleotide at the first wavelength and a second intensity that is different from the first intensity; and determine the sequence of the polynucleotides based on the detected fluorescent emission and intensity.

20. The non-transitory computer-readable medium of claim 19, wherein the fourth labeled nucleotide is a modified nucleotide.

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