Polyphenolic Additives in Sequencing by Synthesis

Abstract

The invention relates to methods, compositions, devices, systems and kits as described including, without limitation, reagents and mixtures for determining the identity of nucleic acids in nucleotide sequences using, for example, sequencing by synthesis methods. In particular, the present invention contemplates the use of polyphenolic compounds, known as antioxidant additives, to improve the efficiency of Sequencing-By-Synthesis reactions. For example, gallic acid (GA) is shown herein to be one of many exemplary SBS polyphenolic additives.

Claims

1. A method of incorporating labeled nucleotides, comprising: a) providing i) a plurality of nucleic acid primers and template molecules, ii) a polymerase, iii) a cleave reagent comprising a reducing agent and a polyphenolic compound, and iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base; b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers; c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated labeled nucleotide analogue; d) detecting said incorporated labeled nucleotide analogue; and e) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.

2. The method of claim 1, wherein said polyphenolic compound is selected from the group consisting of gallic acid, gentisic acid, pryocatechol, pyrogallol, hydroquinone and resorcinol.

3. The method of claim 1, wherein said reducing agent of said cleave reagent comprises TCEP (tris(2-carboxyethyl)phosphine).

4. The method of claim 1, wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′-OH group.

5. The method of claim 3, wherein the cleaving of step e) removes the removable chemical moiety capping the 3′-OH group.

6. The method of claim 5, wherein the method further comprises: f) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.

7. The method of claim 1, wherein said label is fluorescent.

8. A cleave reagent comprising i) a reducing agent, and ii) a polyphenolic compound.

9. The cleave reagent of claim 8, wherein said polyphenolic compound is selected from the group consisting of gallic acid, gentisic acid, pryocatechol, pyrogallol, hydroquinone, and/or resorcinol.

10. The cleave reagent of claim 8, wherein said reducing agent is TCEP Tris(2-carboxyethyl)phosphine).

11. A kit, comprising i) the cleave reagent of claim 8 and ii) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base.

12. A system comprising primers hybridized to template in solution, said solution comprising the cleave reagent of claim 8.

13. The system of Clam 11, wherein said hybridized primers and template are immobilized.

14. The system of claim 12, wherein said hybridized primers and template are in a flow cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 presents exemplary data showing a comparison of sequencing performance for runs containing gallic acid (addG) in Cleave solution vs Baseline runs. Sequencing parameters include average error rate, average percentage of perfect (error free) reads, phasing parameters (lead and lag).

[0035] FIG. 2 presents exemplary data showing a comparison of sequencing performance for runs containing gallic acid (addG) in Cleave solution vs Baseline runs. Sequencing included assessment of called variants (true positives —TP, false positives —FP).

[0036] FIG. 3 presents exemplary data showing a comparison of sequencing performance for nuns containing gallic acid (addG) in Cleave solution vs Baseline runs. Sequencing included assessment of average error rate per cycle.

[0037] FIG. 4 presents exemplary data showing a comparison of sequencing performance for runs containing gallic acid (addG) in Cleave solution vs Baseline runs. Sequencing included assessment of the following performance indicators: average error rate per cycle, average percentage of perfect reads, percent of signal retention, false positive rate and lead/lag. Values higher than 0.95 on the third bar indicate statistical significance, first bar corresponds to baseline, second to experiment with relative improvement factor.

[0038] FIG. 5 presents exemplary data showing an LC-MS analysis of a cleaved spacer arm terminating with free SH group and exposure to Cleave without additives. Formation of alkene moiety detected as a result of side reactions during cleavage step.

[0039] FIG. 6 presents exemplary data showing a comparison of sequencing performance for runs containing gallic acid (addG) in Cleave solution vs solutions with gallic acid and Tween detergent. Sequencing included assessment of the following performance indicators: average error rate per cycle, average percentage of perfect reads, percent of signal retention, false positive rate and lead/lag. Values higher than 0.95 on the third bar indicate statistical significance, first bar corresponds to baseline, second to experiment with relative improvement factor

[0040] FIG. 7 presents exemplary data showing a comparison of sequencing performance for runs containing addG alternatives in Cleave solution (pyrogallol, pyrocatechol, gentisic acid) vs baseline runs without any additive. Results are provided for two sample types (Clones and Gene Panel). Improvements are noted for sequencing KPIs such as error rate, percent perfect and lead/lag when addG alternatives such as pyrogallol, pyrocatechol, gentisic acid are added to Cleave.

[0041] FIG. 8 presents representative electropherograms of sequencing products after sequencing under baseline conditions (A) and with gallic acid additive in Cleave (B). Higher yield of full length product observed in case with additive (B).

[0042] FIG. 9 presents exemplary RP-HPLC cleavage studies of labeled dCTP nucleotide in the absence (baseline) and presence of additive (examples of gallic and gentisic acid). Byproducts are clearly visible in baseline and absent in chromatograms with additives.

[0043] FIG. 10 presents exemplary data showing a comparison of sequencing performance for runs containing baseline imaging buffer vs imaging buffer containing additives: GR 5.10: Replace baseline Imaging buffer with a single HEPES pH 7.5 buffer with Gentisic acid at final concentration of 25 mM; GR 5.4: Add Gentisic acid (25 mM) to Image B; GR 5.5: Image B buffer with Trolox removed and Gentisic acid added (25 mM).

[0044] FIG. 11 presents exemplary data showing the relative bead loss in flow cells during runs comparing indole-3-propionic acid (IPA: AddC_15FC) and gallic acid (GA: AddG_7FC).

[0045] FIG. 12 presents exemplary data comparing the raw error rate for ascorbic acid (Add AA), additive C (Add C), additive G (light) along with an additive-free control (No add) where the results were generated in a sequencing run using the NA12878/101X gene panel as template. The raw error rate for ascorbic acid is significantly better than i) the no additive run, and ii) the run with additive C (while performing comparably to additive G).

DETAILED DESCRIPTION OF THE INVENTION

[0046] The invention relates to methods, compositions, devices, systems and kits as described including, without limitation, reagents and mixtures for determining the identity of nucleic acids in nucleotide sequences using, for example, sequencing by synthesis methods. In particular, the present invention contemplates the use of polyphenolic compounds, known as antioxidant additives, to improve the efficiency of Sequencing-By-Synthesis reactions. For example, gallic acid (GA) is shown herein to be one of many exemplary SBS polyphenolic additives.

I. Sequencing-By-Synthesis (SBS)

[0047] One step in the sequencing-by-synthesis workflow is the removal of the fluorescent label which is covalently attached via a cleavable linker molecule to the ring-position of the heterocyclic base of the nucleotide (reversible terminator) involved in the incorporation step. The efficacy of the cleave step is reflected not only in the efficiency of the fluorescent label cleavage but also in the mitigation of reaction by-products that could accumulate in the flow cell and interfere with subsequent base incorporation step. Examples of such compounds are radical by-products that may form due to radical pathways involved in the omolytic scission of the linker molecule to release the fluorescent label and excess cleave reagent (i. e., tris(2-carboxyethyl)phosphine or TCEP). These may build up in the flow cell and carry over into the subsequent base extension step thus causing premature de-protection of the 3 ′-OH moiety and causing more than one base to incorporate. An effective cleave step is important for single nucleotide incorporation throughout the sequencing reaction, as well as a prerequisite for low error rate and long read length. To improve the efficacy of the cleave step, molecules that quench radical pathways and oxidize excess TCEP are contemplated, such as ascorbic acid, so as to enhance the efficacy of this reactive step.

[0048] In one embodiment, the present invention contemplates a series of method steps performed by an automated sequencing by synthesis instrument. See U.S. Pat. No. 9,145,589, hereby incorporated by reference. In one embodiment, the instrument is comprised of numerous reagent reservoirs. Each reagent reservoir has a specific reactivity reagent dispensed within the reservoir to support the SBS process, for example:

[0049] One reactive step in a method for sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators comprises cleaving a fluorescent label from a nucleotide analogue molecule. It is not intended that the present invention be limited by the nature of the cleaving agent.

[0050] In one embodiment, the SBS method comprises doing different steps at different stations. By way of example, each station is associated with a particular step. While not limited to particular formulations, some examples for these steps and the associated reagents are shown below: [0051] 1) Extend A Reagent: Comprises reversibly terminated labeled nucleotides and polymerase. The composition of Extend A is as follows:

TABLE-US-00001 Component Conc PNSE (% wt/vol) 0.005% Tris × HCl (pH 8.8), mM 50 NaCl (mM) 50 EDTA (mM) 1 MgSO4 (mM) 10 Cystamine (mM) 1 Glycerol (% wt/vol)  0.01% Therminator IX* (U/ml) 10 N3-dCTP (μM) 3.83 N3-dTTP (μM) 3.61 N3-dATP (μM) 4.03 N3-dGTP (μM) 0.4 Alexa488-dCTP (nM) 550 R6G-dUTP (nM) 35 ROX-dATP (nM) 221 Cy5-dGTP (nM) 66 *with Alkylated free Cysteine [0052] 2) Extend B Reagent: Comprises reversibly terminated unlabeled nucleotides and polymerase, but lacks labeled nucleotide analogues. The composition of Extend B is as follows:

TABLE-US-00002 Component Conc PNSE (% wt/vol) 0.005% Tris × HCl (pH 8.8), mM 50 NaCl (mM) 50 EDTA (mM) 1 MgSO4 (mM) 10 Glycerol (% wt/vol)  0.01% Therminator IX* (U/ml) 10 N3-dCTP (μM) 21 N3-dTTP (μM) 17 N3-dATP (μM) 21 N3-dGTP (μM) 2 *Alkylated free Cysteine [0053] 3) Wash solution 1 with a detergent (e.g., polysorbate 20) citrate buffer (e.g., saline) [0054] 4) Cleave Reagent: A cleaving solution composition is as follows:

TABLE-US-00003 Component Conc NaOH (mM) 237.5 TrisHCl (pH 8.0) (mM) 237.5 TCEP (mM) 50 [0055] 5) Wash solution 2 with a detergent (e.g., polysorbate 20) a tris(hydroxymethyl)-aminomethane (Tris) buffer.

II. Polyphenolic Sequencing Additives

[0056] In one embodiment, the present invention contemplates compositions and compounds that arc polyphenolic compounds as antioxidant additives which improve methods of sequencing by synthesis. In one embodiment, the polyphenolic compound includes, but is not limited to, gallic acid, gentisic acid, pryocatechol, pyrogallol, hydroquinone, and/or resorcinol.

[0057] One embodiment of the invention includes addition of polyphenolic additives in sequencing reactions of Cleavage solution to improve lifetime of solution, to reduce undesirable free radical driven side reactions, allow premixing, and as a result improve sequencing performance. Another embodiment includes addition of polyphenolic compounds to Imaging solution to improve lifetime of solution and to reduce undesirable free radical driven side reactions.

[0058] Yet another embodiment of the invention is addition of polyphenolic compounds to Extend solution to improve lifetime of solution and to reduce undesirable free radical driven side reactions. In one embodiment of the invention the polyphenolic compounds are antioxidants. In yet another embodiment the polyphenolic compounds have free radical scavenging properties.

[0059] Current sequencing processes includes a Cleave solution with buffered phosphine to deprotect 3′-OH groups and disulfide dye linker. This solution has limited activity window due to oxygen absorption from the air and open Cleave container on GeneReader instrument. Literature reports indicate that phosphines such as TCEP (Tris(carboxyethyl)phosphine) can lead to by-products with thiol-based compounds. One example is conversion of cysteine to dehydroalanine residues in peptides. The process is thought to involve a free radical path. Zhouxi et al., Rapid Commun Mass Spectrom. (2010) 24(3):267-275. Analysis of SBS nucleotides cleavage reactions in solution by means of LC-MS indicates formation of additional species in addition to expected products. Analysis of sequencing products by means of denaturing capillary electrophoresis indicates presence of non-full length products.

[0060] One Imaging solution currently used on GeneReader uses an active oxygen scavenging system and radical/triplet state scavenger. Extend A/B solutions do not contain reducing agents due to compatibility with sequencing chemistry (disufide bridges). In another embodiment, polyphenolic anti-oxidant compounds are identified that actively scavenge dissolved oxygen out of a Cleave solution and prolong useful life time of a Cleave solution and increase its efficiency. In another embodiment, improved performance of a Cleave step and reduction of side reactions is disclosed.

[0061] Preliminary data included tests with polyphenolic additives to a Cleave reagent to assess improvements SBS performance. The results suggested that several polyphenolic compounds had a high antioxidant potential. For example, two promising polyphenolic compounds were chosen for further studies: gallic acid and gentisic acid. In addition to reducing available oxygen and having positive impact on the lifetime of a Cleave solution (reducing agent) these two polyphenolic compounds had additional positive impact on sequencing performance possibly due to reducing side reactions.

[0062] Sequencing SBS chemistry performance was assessed using standard baseline SBS conditions (50 mM TCEP at pH=8.5) versus runs with additives at the same pH. To this effect, the following conclusions were made based on experimental data and described further in detail: [0063] 1. Analysis of cleavage reactions at nucleotide level by means of analytical HPLC and LC-MS (labeled and terminating nucleotide) in the absence and presence of additives was performed. These analyses revealed that cleavage reactions containing antioxidant compounds (50 mM) including, but not limited to, gallic acid, gentisic acid, pyrocatechol or pyrogallol revealed fewer side products. [0064] 2. Sequencing runs containing Cleave solution with phosphine only or containing gallic acid, gentisic acid, pyrocatechol, pyrogallol were conducted at varying concentrations. Analysis of sequencing KPIs indicates better performance as indicated by lower error rate, higher signal margin and lower lead values as well as lower false positive rate for variants effectively extending usable read length by 25-50%. Analysis by CE reveals higher yield of full length sequencing products. Flowcell data homegeneity was also improved indicating possibly beneficial impact on Cleave solution clearance from the flowcell. [0065] 3. Sequencing runs containing gallic acid, gentisic acid, pyrocatechol, or pyrogallol in Imaging solution indicated better performance as shown by lower error rate, higher signal margin and lower lead values as well as lower false positive rate for variants Analysis by CE reveals higher yield of full length sequencing products. [0066] 4. Identification of additional compounds with similar properties were identified. Additional compunds evalauted as Cleave additives showed similar benefits as demonstrated in 1-3 above. These compounds contain poly-phenolic groups or have antioxidant properties. Compounds tested include pyrogallol, pyrocatechol, hydroquinone, resorcinol, but it is not intended that the invention is limited to this set of compounds.

[0067] A. Gallic Acid

[0068] Gallic acid (GA) has been shown to improve sequencing performance and allow the system to provide a filtered trimmed sequence output of 1 Gb.

[0069] Gallic acid is found in a number of land plants, such as the parasitic plant, Cynomorium coccineum, the aquatic plant, Myriophyllum spicatum, and the blue-green alga, Microcystis aeruginosa. Zucca et al., “Evaluation of Antioxidant Potential of “Maltese Mushroom” (Cynomorium coccineum) by Means of Multiple Chemical and Biological Assays” Nutrients 5(1):149-161; and Nakai, S (2000). “Myriophyllum spicatum-released allelopathic polyphenols inhibiting growth of blue-green algae Microcystis aeruginosa” Water Research 34(11):3026-3032. Gallic acid is a trihydroxybenzoic acid, a type of phenolic acid, a type of organic acid, also known as 3,4,5-trihydroxybenzoic acid, found in gallnuts, sumac, witch hazel, tea leaves, oak bark, and other plants. The chemical fonnula is C.sub.6H.sub.2(OH).sub.3COOH, having the following structure:

##STR00001##

[0070] Gallic acid is found both free and as part of hydrolyzable tannins The gallic acid groups are usually bonded to form dimers such as ellagic acid. Hydrolysable tannins break down on hydrolysis to give gallic acid and glucose or ellagic acid and glucose, known as gallotannins and ellagitannins respectively. Gallic acid may also form intermolecular esters (depsides) such as digallic and trigallic acid, and cyclic ether-esters (depsidones) and is commonly used in the pharmaceutical industry. Fiuza et al., “Phenolic acid derivatives with potential anticancer properties—a structure-activity relationship study. Part 1: Methyl, propyl and octyl esters of caffeic and gallic acids”. Bioorganic & Medicinal Chemistry (Elsevier) 12 (13): 3581-3589. Gallic acid is easily freed from gallotannins by acidic or alkaline hydrolysis. When gallic acid is heated with concentrated sulfuric acid, rufigallol is produced by condensation. Oxidation with arsenic acid, permanganate, persulfate, or iodine yields ellagic acid, as does reaction of methyl gallate with iron(III) chloride.

[0071] Gallic acid is formed from 3-dehydroshikimate by the action of the enzyme shikimate dehydrogenase to produce 3,5-didehydroshikimate. This latter compound tautomerizes to form the redox equivalent gallic acid, where the equilibrium lies essentially entirely toward gallic acid because of the coincidentally occurring aromatization. Dewick et al., (1969) “Phenol biosynthesis in higher plants. Gallic acid”. Biochemical Journal 113 (3): 537-542. Gallate dioxygenase and gallate decarboxylase are enzymes responsible for the degradation of gallic acid.

[0072] The data presented herein demonstrate that SBS runs with gallic acid showed no bead loss as shown by its comparison to SBS runs using indole-3-propionic acid (IPA). See, FIG. 11. These data show that gallic acid, like IPA, does not undergo any bead loss during SBS, contradicting previous reports. Although it is not necessary to understand the mechanism of an invention, it is believed that when a certain pH value is reached, Gallic acid undergoes an irreversible transition to a new chemical entity. It is believed that this chemical transition generates an active gallic acid derivative that is responsible for previously observed bead loss. In one embodiment, the present invention contemplates an SBS reagent (e.g., for example, a Cleave 1 buffer, where gallic acid is not mixed in the absence of TCEP. When gallic acid and TCEP are both present in the SBS reagent, no bead loss is observed.

[0073] This lack of bead loss is reflected in data showing improved error rates when SBS runs were compared between the presence of gallic acid, IPA and ascorbic acid. All three additives improved SBS error rates when compared to no additive. See, FIG. 12. The data show that gallic acid (AddG) results in a raw error rate that is significantly better than no additive and IPA (AddC).

[0074] B. Gentisic Acid

[0075] Gentisic acid is a dihydroxybenzoic acid. It is a derivative of benzoic acid and a minor (1%) product of the metabolic break down of aspirin. It is also found in the African tree Alchornea cordifolia and in wine.

[0076] Gentisic acid may be produced by carboxylation of hydroquinone:

C.sub.6H.sub.4(OH).sub.2+CO.sub.2.fwdarw.C.sub.6H.sub.3(CO.sub.2H)(OH).sub.2

This conversion is an example of a Kolbe-Schmitt reaction and results in the following structure:

##STR00002##

Alternatively the compound can be synthesized from Salicylic acid via Elbs persulfate oxidation (50% yield). Schock Jr. et al., (1951) “The Persulfate Oxidation of Salicylic Acid. 2,3,5-Trihydroxybenzoic Acid” The Journal of Organic Chemistry 16(11):1772-1775. As a hydroquinone, gentisic acid is readily oxidized and is used as an antioxidant excipient in some pharmaceutical preparations. In the laboratory, it is used as a sample matrix in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, and has been shown to conveniently detect peptides incorporating the boronic acid moiety by MALDI. Strupat et al., (1991) “2,5-Dihidroxybenzoic acid: a new matrix for laser desorption-ionization mass spectrometry” Int. J. Mass Spectrom. Ion Processes 72(111):89-102; and Crumpton et al., (2011) “Facile Analysis and Sequencing of Linear and Branched Peptide Boronic Acids by MALDI Mass Spectrometry” Analytical Chemistry 83(9):3548-3554.

[0077] C. Pryocatechol

[0078] Pyrocatechol, also known as catechol or 1,2-dihydroxybenzene, is an organic compound with the molecular foiiiiula C.sub.6H.sub.4(OH)2. It is the ortho isomer of the three isomeric benzenediols. This colorless compound occurs naturally in trace amounts. It was first discovered by destructive distillation of the plant extract catechin. About 20 million kg are now synthetically produced annually as a commodity organic chemical, mainly as a precursor to pesticides, flavors, and fragrances.

Catechol is produced industrially by the hydroxylation of phenol using hydrogen peroxide:

C.sub.6H.sub.5OH+H.sub.2O.sub.2.fwdarw.C.sub.6H.sub.4(OH).sub.2+H.sub.2O

and results in the following structure:

##STR00003##

Previously, it was produced by hydrolysis of 2-substituted phenols, especially 2-chlorophenol, with hot aqueous solutions containing alkali metal hydroxides. Its methyl ether derivative, guaiacol, converts to catechol via hydrolysis of the CH3-O bond as promoted by hydriodic acid.

[0079] D. Pyrogallol

[0080] Pyrogallol is an organic compound with the formula C.sub.6H.sub.3(OH).sub.3 having the following chemical structure:

##STR00004##

It is a white solid although because of its sensitivity toward oxygen, samples are typically brownish. It is one of three isomeric benzenetriols. It is produced by heating gallic acid that results in decarboxylation. An alternate preparation involves treating para- chlorophenoldisulphonic acid with potassium hydroxide.

[0081] E. Hydroquinone

[0082] Hydroquinone, also benzene-1,4-diol or quinol, is an aromatic organic compound that is a type of phenol, a derivative of benzene, having the chemical formula C.sub.6H.sub.4(OH).sub.2, having the following structure:

##STR00005##

Its chemical structure features two hydroxyl groups bonded to a benzene ring in a para position. It is a white granular solid. Substituted derivatives of this parent compound are also referred to as hydroquinones.

[0083] The reactivity of hydroquinone's O—H groups resembles other phenols, being weakly acidic. The resulting conjugate base undergoes easy O-alkylation to give mono- and diethers. Similarly, hydroquinone is highly susceptible to ring substitution by Friedel-Crafts reactions such as alkylation. This reaction is exploited en route to popular antioxidants such as 2-tert-butyl-4-methoxyphenol (“BHA”). The useful dye quinizarin is produced by diacylation of hydroquinone with phthalic anhydride. Hydroquinone undergoes oxidation under mild conditions to give benzoquinone. This process can be reversed. Some naturally occurring hydroquinone derivatives exhibit this sort of reactivity, one example being coenzyme Q. Industrially this reaction is exploited both with hydroquinone itself but more often with its derivatives where one OH has been replaced by an amine.

[0084] There are various other uses associated with its reducing power. As a polymerization inhibitor, hydroquinone prevents polymerization of acrylic acid, methyl methacrylate, cyanoacrylate, and other monomers that are susceptible to radical-initiated polymerization. This application exploits the antioxidant properties of hydroquinone.

[0085] Hydroquinone can undergo mild oxidation to convert to the compound parabenzoquinone, C6H4O2, often called p-quinone or simply quinone. Reduction of quinone reverses this reaction back to hydroquinone. Some biochemical compounds in nature have this sort of hydroquinone or quinone section in their structures, such as Coenzyme Q, and can undergo similar redox interconversions.

[0086] Hydroquinone can lose an H+from both to form a diphenolate ion. The disodium diphenolate salt of hydroquinone is used as an alternating comonomer unit in the production of the polymer PEEK.

[0087] F. Resorcinol

[0088] Resorcinal is the 1,3-isomer (or meta-isomer) of benzenediol with the formula C.sub.6H.sub.4(OH).sub.2, having the following structure:

##STR00006##

Resorcinol crystallizes from benzene as colorless needles that are readily soluble in water, alcohol, and ether, but insoluble in chloroform and carbon disulfide. Sodium amalgam reduces it to dihydroresorcin, which when heated to 150 to 160° C. with concentrated barium hydroxide solution gives γ-acetylbutyric acid and when fused with potassium hydroxide, resorcinol yields phloroglucin, pyrocatechol, and diresorcin.

EXPERIMENTAL

Example 1

[0089] In one embodiment, the present invention contemplates a SBS method comprising the steps shown in Table 1. See Olejink et al., “Methods And Compositions For Inhibiting Undesired Cleaving Of Labels” U.S. Pat. Number 8,623,598 (herein incorporated by reference in its entirety).

TABLE-US-00004 TABLE 1 An Exemplary SBS Workflow Fluid Movements Volume Speed Station Temp Time Step Reagent mL mL/s Number ° C. [s]  1. Dispense Reagent 1 100 67 3 65 7 Reagent  2. Incubate Reagent 1 n/a n/a 3 65 210 Reagent  3. Dispense Reagent 2 100 67 4 65 7 Reagent  4. Incubate Reagent 2 n/a n/a 4 65 210 Reagent  5. Dispense Reagent 3 330 27 5 Ambient 12 Reagent  6. Dispense Reagent 4 + 5 200 27 5 Ambient 15 Reagent  7. Image n/a n/a n/a 11 Ambient 210  8. Dispense Reagent 3 330 27 20 65 12 Reagent  9. Dispense Reagent 6 100 67 1 65 7 Reagent 10. Incubate Reagent 6 n/a n/a 1 65 210 Reagent 11. Incubate Reagent 6 n/a n/a 2 65 210 Reagent 12. Dispense Reagent 7 990 27 2 65 37 Reagent 13. Go to Step 1 Reagent 1 = Extend A; Reagent 2 = Extend B; Reagent 3 = Wash; Reagent 4 =Image A; Reagent 5 = Image B; Reagent 6 = Cleave; and Reagent 7 = Wash 11

[0090] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art and in fields related thereto are intended to be within the scope of the following claims.