Scavenger Compounds for Improved Sequencing-by-Synthesis
20170233725 · 2017-08-17
Inventors
Cpc classification
C12N15/1068
CHEMISTRY; METALLURGY
C12Q2527/125
CHEMISTRY; METALLURGY
C12Q2527/125
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention discloses methods of applications of indole-3-propionic acid, L-carnitine and O-acetyl-L-carnitine in one or more different reactive steps of a sequencing-by-synthesis workflow. The reactive steps employing these compounds include, but are not limited to, cleaving, imaging, incorporating bases and washing. The use of these new compounds provides improved sequencing performance including, but not limited to, lower error rates, higher sequence outputs and/or longer read lengths.
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 scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based 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 nucleotide analogue; and d) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.
2. The method of claim 1, wherein said scavenger is indole-3-propionic acid.
3. The method of claim 1, wherein said scavenger is L-carnitine.
4. The method of claim 1, wherein said scavenger is O-acetyl-L-carnitine.
5. The method of claim 1, wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′-OH group.
6. The method of claim 5, wherein the cleaving of step d) removes the removable chemical moiety capping the 3′-OH group.
7. The method of claim 6, wherein the method further comprises: d) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.
8. The method of claim 7, wherein said incorporating of a second nucleotide analogue is performed in the presence of a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound
9. A method of incorporating nucleotides, comprising: a) providing i) a plurality of nucleic acid primers and template molecules, and ii) an extend reagent, said extend reagent comprising polymerase, a plurality of nucleotide analogues, and a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound; b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers; and c) exposing said hybridized primers to said extend reagent under conditions such that a first nucleotide analogue is incorporated into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue.
10. The method of claim 9, wherein said incorporated nucleotide analogue comprises a label attached through a cleavable disulfide linker to the base.
11. The method of claim 10, wherein said label is fluorescent.
12. The method of claim 9, wherein said extend reagent further comprises cystamine.
13. The method of claim 10, further comprising: d) cleaving the cleavable linker of said incorporated nucleotide analogue with a cleave reagent comprising a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound.
14. The method of claim 9, wherein said scavenger is indole-3-propionic acid.
15. The method of claim 9, wherein said scavenger is L-carnitine.
16. The method of claim 9, wherein said scavenger is O-acetyl-L-carnitine.
17. The method of claim 13, wherein said incorporated nucleotide analogues prior to step d) further comprise a removable chemical moiety capping the 3′-OH group.
18. The method of claim 17, wherein the cleaving of step d) removes the removable chemical moiety capping the 3′-OH group.
19. A method of incorporating nucleotides, comprising: a) providing i) a plurality of nucleic acid primers and template molecules, ii) an extend reagent comprising polymerase and a plurality of nucleotide analogues, and iii) a wash reagent comprising a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound; 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) exposing said hybridized primers to said extend reagent under conditions such that a first nucleotide analogue is incorporated into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue; d) washing said extended primers with said wash reagent.
20. The method of claim 19, wherein said incorporated nucleotide analogue comprises a label attached through a cleavable disulfide linker to the base.
21. The method of claim 20, wherein said label is fluorescent.
22. The method of claim 20, wherein said extend reagent further comprises cystamine.
23. The method of claim 20, further comprising: e) detecting said label of a first labeled nucleotide analogue.
24. The method of claim 23, wherein said detecting of step e) is performed in the presence of a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound.
25. The method of claim 19, wherein said scavenger is indole-3-propionic acid.
26. The method of claim 19, wherein said scavenger is L-carnitine.
27. The method of claim 19, wherein said scavenger is O-acetyl-L-carnitine
28. A method of incorporating labeled nucleotides, comprising: a) providing i) a plurality of nucleic acid primers and template molecules, ii) an extend reagent comprising polymerase and a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled, and iii) an image reagent comprising a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound; 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) exposing said hybridized primers to said extend reagent under conditions such that a first labeled nucleotide analogue is incorporated into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue; and d) detecting said label of said first labeled nucleotide analogue with said image reagent.
29. The method of claim 28, wherein said label is attached through a cleavable disulfide linker to the base.
30. The method of claim 29, wherein said label is fluorescent.
31. The method of claim 28, wherein said extend reagent further comprises cystamine.
32. The method of claim 29, further comprising: e) cleaving the cleavable linker of said incorporated nucleotide analogue with a cleave reagent comprising a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound.
33. The method of claim 28, wherein said scavenger is indole-3-propionic acid.
34. The method of claim 28, wherein said scavenger is L-carnitine.
35. The method of claim 28, wherein said scavenger is O-acetyl-L-carnitine.
36. The method of claim 32, wherein said incorporated labeled nucleotide analogue of step d) further comprises a removable chemical moiety capping the 3′-OH group.
37. The method of claim 36, wherein the cleaving of step e) removes the removable chemical moiety capping the 3′-OH group.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0044] 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, data obtained from sequencing by synthesis methods.
[0045] In one embodiment, the present invention contemplates a method comprising indole-3-propionic acid (IPA), a potent radical scavenger and a singlet oxygen quencher, as an additive to a cleave reagent utilized in sequencing by synthesis (SBS). Although it is not necessary to understand the mechanism of an invention, it is believed that the presently disclosed method provides a significant improvement in the efficacy of a cleave reaction thus allowing diminished sequencing error rate and enhancement of filtered sequence read output. It is further believed that IPA can also be used during the steps of imaging, base incorporation (e.g., extension) and washing to achieve longer sequencing reads.
[0046] In one embodiment, the present invention also contemplates the use of additional compounds for use as either cocktail components with IPA or standalone additives to further improve cleave chemistry and sequencing performance. These additional compounds include, but are not limited to, L-carnitine and/or O-acetyl-L-carnitine. In one embodiment, the present invention also contemplates the use of radical scavenger including, but not limited to, indole-3-propionic acid, L-carnitine and/or O-acetyl-L-carnitine.
I. Sequencing-By Synthesis (SBS)
[0047] The invention relates to methods and compositions for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. In sequencing by synthesis, nucleotides conjugated with fluorescent markers that incorporate into a growing double-stranded nucleic acid from the single strand are detected. For example, one may immobilize template DNA on a solid surface by its 5′ end. One may accomplish this by annealing a sequencing primer to a consensus sequence and introducing DNA polymerase and fluorescent nucleotide conjugates (alternatively, a self-priming hairpin can be introduced by PCR or ligation to the template). One detects nucleotide incorporation using a laser microarray scanner or fluorescent microscope by correlating a particular fluorescent marker to a specific nucleotide. After each nucleotide is incorporated and the fluorescent signal is detected, one bleaches or removes the fluorescent moiety from the nucleotide conjugate so as to prevent the accumulation of a background signal.
[0048] In one embodiment, the present invention contemplates DNA sequencing by synthesis using an automated instrument, as well as methods and compositions useful for sequencing using such an instrument. In one embodiment, the instrument comprises a flow cell with at least two fluidics ports, a substrate with sequenceable nucleic acid molecules attached to the substrate, reagent and waste reservoirs and fluidic system connecting the reservoirs to the flow cell. The flow cell is interfaced with a detection system to monitor the incorporation of the nucleotides.
[0049] In one embodiment, the sequencing by synthesis is carried out using reversibly terminating nucleotides and polymerase. The nucleotides comprise a protective group at their 3′-OH which prevents multiple incorporations and allows for accurate decoding of the sequence. Once incorporated, the protective groups can be cleaved with high efficiency and specificity to allow subsequent nucleotide incorporations. The nucleotides may also comprise a detectable label which can be cleaved after the detection.
[0050] In one embodiment, the present invention contemplates a SBS method comprising the steps shown in Table 1. Olejink et al., “Methods And Compositions For Inhibiting Undesired Cleaving Of Labels” U.S. Pat. No. 8,623,598 (herein incorporated by reference in its entirety).
TABLE-US-00001 TABLE 1 An Exemplary SBS Workflow Fluid Movements Vol- ume 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 + 200 27 5 Ambient 15 Reagent 5 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
[0051] Washing solution compositions may include, but are not limited to:
TABLE-US-00002 Component Conc Wash (9, 10) Tween 0.05% TrisHCl (pH 8.8) (mM) 50 NaCl (mM) 50 EDTA (mM) 1 Methylenediphosphonic acid (PcPi) (mM) 1 Wash (11) Tween 0.05% TrisHCl (pH 8.8) (mM) 50 NaCl (mM) 50 EDTA (mM) 1 Cystamine (mM) 10 Wash (12) Tween 0.05% TrisHCl (pH 8.8) (mM) 50 NaCl (mM) 50 EDTA (mM) 1
[0052] 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:
[0053] A. SBS Cleavage Step
[0054] A 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. In one embodiment, the fluorescent label may be covalently attached via a linker molecule to the heterocyclic base of an incorporated nucleotide analogue molecule (See U.S. Pat. No. 6,664,079, hereby incorporated by reference). Conceivably, the efficacy of the cleaving step may be reflected not only in the efficiency of the fluorescent label cleavage but also in the mitigation of by-product formation due to radical pathways involved in the omolytic scission of the linker molecule to release the fluorescent label. Although it is not necessary to understand the mechanism of an invention, it is believed that an effective cleave step plays a role in single nucleotide incorporation throughout the sequencing reaction which may control the accuracy of high throughput sequencing.
[0055] Gallic acid (GA) has been shown to improve sequencing performance and allow the system to provide a filtered trimmed sequence output of 1 Gb. Gallic acid is used herein for performance benchmarking of indole-3-propionic acid.
[0056] B. Extension Step
[0057] 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 (see, Table 1). Each reagent reservoir has a specific reactivity reagent dispensed within the reservoir to support the SBS process, for example: [0058] 1) Extend A Reagent: Comprises reversibly terminated labeled nucleotides and polymerase. The composition of Extend A is as follows:
TABLE-US-00003 Component Conc PNSE (% wt/vol) 0.005% Tris x 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 [0059] 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-00004 Component Conc PNSE (% wt/vol) 0.005% Tris x 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 [0060] 3) Wash solution 1 [0061] a detergent (e.g., polysorbate 20) [0062] citrate buffer (e.g., saline) [0063] 4) Cleave Reagent: A cleaving solution composition is as follows:
TABLE-US-00005 Component Conc NaOH (mM) 237.5 TrisHCl (pH 8.0) (mM) 237.5 TCEP (mM) 50 [0064] 5) Wash solution 2 [0065] a detergent (e.g., polysorbate 20) [0066] a tris(hydroxymethyl)aminomethane (Tris) buffer.
Of course, the present invention is not limited to any particular concentrations of reagents in these solutions and other buffers and detergents can be employed. Nonetheless, in order to achieve high throughput rates, the incorporation reactions and the cleavage reactions are desired to be fast. In one embodiment, high reaction rates are achieved by increasing the concentration of reagents, agitation, pH or temperature (or the combination of all these factors). The incorporation rate in addition is dependent on the specific activity and processivity of the polymerase used. In one particular embodiment (which is provided by way of a non-limiting example), the reagent solutions may have the following compositions and concentration ranges: [0067] 1) Extend A Reagent—reversibly terminated labeled (1 nM to 1 μM) and non-labeled nucleotides (1 μM to 100 μM) and a first polymerase (1-500 μg/ml)); [0068] 2) Extend B Reagent—reversibly terminated non-labeled nucleotides (1 μM to 100 μM) and a second polymerase (1-500 μg/ml)); [0069] 3) Wash 1 Reagent: (3×SSC, 0.02% Tween 20); [0070] 4) Cleave Reagent: (50-100 mM TCEP); [0071] 5) Wash 2 Reagent: (100 mM Tris-HCl, 0.02% Tween 20, 10 mM KCl, 20 mM (NH.sub.2).sub.2SO.sub.4.
[0072] In one embodiment, a first polymerase incorporates labeled nucleotides better than a second polymerase, which incorporates unlabeled nucleotides more efficiently. Examples of commercially available polymerases that can be used include, but are not limited to, Therminator I-III. These polymerases are derived from Thermococcus sp. and carry mutations allowing for incorporation of modified nucleotides.
[0073] In one embodiment, a sequenceable DNA molecule (i.e., for example, a DNA molecule that is preferably loaded on the chip or slide) is subjected to SBS reagents and compositions compatible with an SBS instrument, and the sequencing is performed using an automated protocol (see, Table 1). Again, it is not intended that the present invention be limited to any precise protocol or series of method steps. The order and number of steps can vary, as well as the time taken for each step. By way of a non-limiting example, in one embodiment, the instrument protocol comprises (and is configured) as follows: [0074] 1. Extend A Reagent: 0.5-5 minutes (delivery+agitation) [0075] 2. Extend B Reagent: 1-20 minutes (delivery+agitation) [0076] 3. Wash 2 Reagent: 5-10 minutes (10-20× delivery and agitation followed by flow cell evacuation) [0077] 4. Image Reagent. The imaging reagent solution (either solution A or B) is as follows:
TABLE-US-00006 Component Conc Image A Hepes-Na, pH 7.5, mM 100 NaI (mM) 1 Glucose oxidase (U/ml) 5 EGTA (μM) 25 Glycerol (% wt/vol) 0.25 Image B Hepes-Na, pH 7.5, mM 100 NaCl (mM) 300 Glucose (mM) 100 Trolox (mM) 2.4 [0078] 5. Cleave Reagent (e.g., Cleave A and B): 1-5 minutes (delivery+agitation) [0079] 6. Wash 1 Reagent: 5-10 minutes (10-20x delivery and agitation followed by flow cell evacuation) [0080] 7. Wash 2 Reagent: 5-10 minutes (10-20x delivery and agitation followed by flow cell evacuation) [0081] 8. Go to step 1
This series of steps may be repeated as a cycle as many times as desired and images may be taken and subsequently analyzed to decode the DNA sequence of the template DNA molecule present at each location. As noted above, in one embodiment, one or more of these steps is associated with an instrument “station” wherein each station has the requisite reagent and/or dispensing elements to perform the step. Flow cells are moved from one station to another station in order to carry out each step of the sequencing protocol. Any one of these steps can be done at two stations if desired, i.e. a step taking a longer time can be completed over the course of two stations, each station doing a part (e.g. half of the step).
[0082] In one embodiment, a cycle may comprise incubating with eight nucleotide analogues including, but not limited to, four nucleotide analogues (e.g., A, T, C, G) that are cleavably labeled and reversibly terminated and/or four nucleotide analogues (e.g., A, T, G, C) that are unlabeled and reversibly terminated.
[0083] In one embodiment, the concentration of the labeled nucleotide analogues are at a relatively low concentration. Although it is not necessary to understand the mechanism of an invention, it is believed that the labeled nucleotide analogue concentration is just low enough to be incorporated into a substantial portion of the plurality of primers such that the label is visible and can be detected. Detection may be observed whether the primers are detached or self-priming hairpins hybridized to a template DNA.
[0084] In one embodiment, the concentration of the unlabeled analogues are at a relatively high concentration. Although it is not necessary to understand the mechanism of an invention, it is believed that the unlabeled analogue high concentration is capable of driving extensions to completion, and avoid the use of primers, whether they be detached primers or self-priming hairpins, that lack incorporation of a first nucleotide analogue. It has been found empirically that the use of unlabeled nucleotides improves read lengths, and reduces lead and lag.
III. Reactive Oxygen Species Scavengers
[0085] In one embodiment, the present invention contemplates an SBS method comprising a radical oxygen species scavenger compound including, but not limited to, indole-3-propionic acid, L-carnitine and/or O-acetyl-L-carnitine. Any one of these compounds, or any combination of these compounds, are contemplated as radical scavengers in any SBS reactive step, as well as in multiple steps. See, for example, Table 1 and the series of steps (provided by way of example). In some embodiments, one or more of these compounds may be included in the cleaving step, an imaging step, a base incorporation step (extension) and/or a wash step (in each step or combination of steps, or even in all of these steps). Although it is not necessary to understand the mechanism of an invention, it is believed that oxygen radical species may form due to an interaction between organic dyes and radiation during SBS and may be responsible for DNA photodamage and read length scission. It is further believed that quenching of radical oxygen species can lead to longer read length and a more efficient SBS method. For example, radical oxygen species that form during a cleaving step may carry over into subsequent SBS steps of the workflow that can be responsible for less efficient base incorporation. Therefore, the presence of a scavenger (e.g. radical oxygen scavenger) in the cleaving step (e.g., Cleave Reagent additive), base incorporation step (e.g., Extend A or Extend B Reagent additive), imaging step (e.g., Image Reagent additive) and wash steps (e.g., Wash Reagent additive) can lead to improved overall efficiency of the sequencing-by-synthesis reaction thereby resulting in longer read lengths.
[0086] A. Indole-3-propionic Acid (IPA)
[0087] Indole-3-propionic acid (IPA), a close relative molecule of melatonin, is an endogenous substance and may be found in the plasma and cerebrospinal fluid of humans. It is believed to be a potent radical scavenger and singlet oxygen quencher. Below a model mechanism of action for scavenging free radical is outlined. Upon initial reaction with a hydroxyl radical IPA is oxidized to a kynuric acid. Such mechanism can be extended to free radicals other than hydroxyl.
##STR00001##
[0088] IPA has been demonstrated to prevent formation of beta-amyloid fibrils, leading to neuroprotective properties against Alzheimer disease. J. Biol. Chem. 274:21937 (1999); and J. Biol. Chem. 262:7213 (1987). As IPA is believed devoid of polyphenolic OH groups that are present in the reference reagent gallic acid, these groups are thought to be responsible for gallic acid's SiO.sub.2 attack. Consequently, no reactivity of IPA with SiO.sub.2 is anticipated based on its chemical architecture.
[0089] Due to its efficacy at quenching radical pathways, coupled with its mild chemical nature, IPA was evaluated for enhancement of Cleave Reagent performance in the sequencing workflow. The cleaving reactive step involves omolytic cleavage of a di-sulfide bond in the linker arm off of the heterocyclic base with concomitant release of a fluorescent label from the incorporating nucleotide. Although it is not necessary to understand the mechanism of an invention, it is believed that during this cleaving reaction radical oxygen species may form and their build up within the flow cell may impair efficiency of the next base incorporation cycle.
[0090] In one embodiment, the present invention contemplates a cleaving reactive step comprising an effective radical scavenger that shuts down radical pathways and prevents formation of radical species. In one embodiment, the reduced concentration of oxidative radicals improves the efficiency of the subsequent nucleotide base extension steps. Although it is not necessary to understand the mechanism of an invention, it is believed that this improved base incorporation efficiency beneficially impacts lead values, error rates, filtered sequence outputs and false positive rates.
[0091] IPA testing was performed using four different reading instruments and a gene panel pool across all sequencing runs. See, Example III. IPA and the reference cleave reagent (e.g., gallic acid) were used to sequence clonally amplified gene panel beads (e.g., for example, an NA12878 barcoded library and/or a 101× gene panel) on each instrument. Finally, sequencing metrics were analyzed to provide both system and application KPI's, i. e., error rate/output (Gb) and false positive rate. See,
[0092] IPA was initially tested for solubility and stability in Cleave Reagent formulations. It was found to be highly soluble over a range of concentrations and stable against discoloration and precipitation even upon prolonged storage at room temperature. IPA was then tested by sequencing in a head-to-head comparison with the reference cleaving reagent, gallic acid, to provide a performance benchmark. IPA was implemented into a sequencing workflow as a standalone powder component to the cleave reagent. See, Example IV.
[0093] Subsequently, IPA was evaluated in a pre-system verification testing paradigm to generate a larger volume of sequencing statistics to determine both performance KPI's and instrument reliability KPPs. See,
[0094] The system performance and reliability was also evaluated. It was found that the performance of gallic acid and IPA was comparable and the higher throughput observed for gallic acid can be explained by the presence of more mapped reads. See,
[0095] B. Carnitines
[0096] In some embodiments, the SBS method comprises a reactive oxygen species scavenger comprising a carnitine-based compound. In one embodiment, the carnitine-based compound is L-carnitine. In one embodiment, the carnitine-based compound is O-acetyl-carnitine. The data presented herein provides a preliminary early feasibility study to determine whether carnitine-based compounds can improve efficacy of a cleaving reagent during SBS. In particular, the structures of L-carnitine and O-acetyl-carnitine are shown below.
##STR00002##
[0097] Carnitine and O-acetyl carnitine are similar to IPA with respect to radical scavenging behavior and anti-Alzheimer activity. Life Sci. 78:803 (2006); and Neurology 11:1726 (2006). In one embodiment, the present invention contemplates an SBS method comprising carnitine. In one embodiment, the present invention contemplates an SBS method comprising O-acetyl carnitine. In one embodiment, the present invention contemplates an SBS method comprising carnitine and O-acetyl carnitine. In one embodiment, the present invention contemplates an SBS method comprising carnitine and IPA. In one embodiment, the present invention contemplates an SBS method comprising O-acetyl carnitine and IPA. In one embodiment, the present invention contemplates an SBS method comprising carnitine, O-acetyl carnitine and IPA.
[0098] L-carnitine and O-acetyl-carnitine were tested for solubility and stability in cleave reagent formulations similarly to IPA. They were found to be highly soluble over a range of concentrations and stable against discoloration and precipitation upon storage at room temperature. See, Example V.
IV. An SBS Automated Instrument
[0099] In one embodiment, the present invention contemplates using an optical system, for exciting and measuring fluorescence on or in samples comprising fluorescent materials (e.g., fluorescent labels, dyes or pigments). In a further embodiment, a device is used to detect fluorescent labels on nucleic acid. In another embodiment, the device comprises a fluorescent detection system and a flow cell for processing biomolecules (e.g., nucleic acid samples) arrayed on a “chip” or other surface (e.g., microscope slide, etc.). The flow cell permits the user to perform biological reactions, including but not limited to, hybridization and sequencing of nucleic acids.
[0100] It is not intended that the present invention be limited to particular light sources. By way of example only, the system can employ ultra-bright LEDs (such as those available from Philips Lumileds Lighting Co., San Jose, Calif.) of different colors to excite dyes attached to the arrayed nucleic acids. These LEDs are more cost effective and have a longer life than conventionally used gas or solid state lasers. Other non-lasing sources of lights such as incandescent or fluorescent lamps may also be used.
[0101] It is not intended that the present invention be limited to particular light collection devices. By way of example only, the system may employ a high sensitivity CCD camera (such as those available from Roper Scientific, Inc., Photometric division, Tucson Ariz. or those available from Apogee Instruments, Roseville, Calif.) to image the fluorescent dyes and make measurements of their intensity. The CCD cameras may also be cooled to increase their sensitivity to low noise level signals. These may also be CMOS, vidicon or other types of electronic camera systems.
[0102] In one embodiment, the chip containing the array of nucleic acid spots is processed in a transparent flow cell incorporated within the instrument, which flows reagent past the spots and produces the signals required for sequencing. In a particular embodiment, the chip remains in the flow cell while it is imaged by the LED detector. The flow cell and associated reagents adds the nucleic acids, enzymes, buffers, etc. that are required to produce the fluorescent signals for each sequencing step, then the flow cell delivers the required reagents to remove the fluorescent signals in preparation for the next cycle. Measurement by the detector occurs between these two steps. In order for reactions to take place, the flow channels are configured to be of sufficient dimensions. For example, the flow-cell fluid channel formed by the array and the flat surface of the flow cell are at least 0.1 mm in depth (more particularly 0.5 mm in depth) and the volume formed by the chip, the block and the seal is at least 100 microliters in volume (more particularly, between 100 and 700 microliters, and still more particularly, between 150 and 300 microliters, e.g. 200 microliters, in volume).
[0103] In one embodiment, the flow cell is motionless (i.e., not moved during reactions or imaging). On the other hand, the flow cell can readily be mounted on a rotary or one or more linear stages, permitting movement. For example, in a two flow cell embodiment, the two flow cells may move up and down (or side to side) across the imaging system. Movement may be desired where additional processes are desired (e.g., where exposure to UV light is desired for photochemical reactions within the flow cell, such as removal of photocleavable fluorescent labels), when multiple flow cells share a single camera, or when the field of view of the detection system is smaller than the desired area to be measured on the flow cell. The detector system may also be moved instead of or in addition to the flow cell.
[0104] In a further embodiment, the flow cell is in fluid communication with a fluidics system. In one embodiment, each bottle is pressurized with a small positive gas pressure. Opening the appropriate valve allows reagent to flow from the source bottle through the flow cell to the appropriate collection vessel(s). In one embodiment, the nucleotides and polymerase solutions are recovered in a separate collection bottle for re-use in a subsequent cycle. In one embodiment, hazardous waste is recovered in a separate collection bottle. The bottle and valve configuration allow the wash fluid to flush the entire valve train for the system as well as the flow cell. In one embodiment, the process steps comprise: 1) flushing the system with wash reagent, 2) introducing nucleotides (e.g. flowing a nucleotide cocktail) and polymerase, 3) flushing the system with wash reagent, 4) introducing de-blocking reagent (enzyme or compounds capable of removing protective groups in order to permit nucleic acid extension by a polymerase), 5) imaging, 6) introducing label removing reagent (enzyme or compounds capable of removing fluorescent labels), and 7) flushing the system with wash reagent.
IV. Nucleotides
[0105] The invention's compositions and methods contemplate using nucleotide sequences that contain nucleotides. The term “nucleotide” refers to a constituent (or building block) of nucleic acids (DNA and RNA) that contain a purine base, such as adenine (A) and guanine (G), or a pyrimidine base, such as cytosine (C), uracil (U), or thymine (T)), covalently linked to a sugar, such as D-ribose (in RNA) or D-2-deoxyribose (in DNA), with the addition of from one to three phosphate groups that are linked in series to each other and linked to the sugar. The term “nucleotide” includes native nucleotides and modified nucleotides.
[0106] “Native nucleotide” refers to a nucleotide occurring in nature, such as in the DNA and RNA of cells. In contrast, “modified nucleotide” refers to a nucleotide that has been modified by man, such as using chemical and/or molecular biological techniques compared to the native nucleotide. The terms also include nucleotide analogs attached to one or more probes to facilitate the determination of the incorporation of the corresponding nucleotide into the nucleotide sequence. In one embodiment, nucleotide analogues are synthesized by linking a unique label through a cleavable linker to the nucleotide base or an analogue of the nucleotide base, such as to the 5-position of the pyrimidines (T, C and U) and to the 7-position of the purines (deaza-G and deaza-A), to use a small cleavable chemical moiety to cap the 3′-OH group of the deoxyribose or ribose, and to incorporate the nucleotide analogues into the growing nucleotide sequence strand as terminators (e.g. reversible terminators). In one embodiment, detection of the label will yield the sequence identity of the nucleotide. Upon removing the label (by cleaving the linker) and the 3′-OH capping group, the polymerase reaction will proceed to incorporate the next nucleotide analogue and detect the next base. Exemplary fluorescent moieties re described in Ju et al., U.S. Pat. No. 6,664,079, hereby incorporated by reference.
[0107] Other nucleotide analogs that contain markers, particularly cleavable markers, are also contemplated, such as those configured using allyl groups, azido groups, and the like, and which are further described below. The nucleotide compositions of the invention are particularly useful in massively parallel DNA Sequencing By Synthesis (SBS) approaches utilizing fluorophores as markers.
Experimental
EXAMPLE 1
Verification And Testing Cleave Reagent
[0108] Materials
TABLE-US-00007 Component Supplier Item # Cleave solution 1, unadjusted In-house (OPS-style) n.a. (Legacy buffer) Cleave solution 2 In-house (OPS-style) n.a. (Legacy TCEP) Add C Sigma Aldrich 57410 (New component: IPA) Notes: 1. When assembling this reagent, be sure to pipet accurately. To aliquot 1.74 mL of solution, use P1000 pipet. When aliquoting 49.5 mL, use 50 mL pipet and a P1000 to get accurate measurements.
[0109] Procedure for Preparing ˜36 mL of 50mM IPA in Cleave Solution for 2 or 3FC Run: [0110] 1. Add 328 mg of IPA to conical tune. [0111] 2. Add 33 mL of Cleave 1, unadjusted, to IPA. Invert several times to dissolve IPA. Add 1.74 mL of Cleave 2. [0112] 3. Mix the final solution by inverting the tube up and down at least ten times. [0113] 4. Required: Check pH and record in a Set up sheet.
[0114] Procedure for Preparing ˜53 mL of 50 mM IPA in Cleave Solution for 4 FC Run: [0115] 1. Add 491 mg of IPA to conical tube. [0116] 2. Add 49.5 mL of Cleave 1 to IPA. Invert several times to dissolve IPA. Add 2.61 mL of Cleave 2. [0117] 3. Mix the final solution by inverting the tube up and down at least ten times. [0118] 4. Required: Check pH and record.
[0119] After the run: Record the pH of the cleave solution.
EXAMPLE II
Optimized Cleave Reagent
[0120] The scheme (below) shows which reagents are contained in each of the Sequencing Q Buffers (Box 1) and Sequencing Q Add-Ons (Box 2) packages, and how they are combined for 1 flow cell.
Cleave reagent components are contained in Box 1 and in the new kit configuration have been re-labeled as follows: [0121] (a) Cleave Solution (Legacy buffer) [0122] (b) Cleave Additive 1 (New: IPA) [0123] (c) Cleave Additive 2 (Legacy: TCEP)
See tables in following pages for preparation of new Cleave Reagent containing IPA.
TABLE-US-00008 TABLE 3 Reagent preparation for Sequencing Q Kit (1) for running 1 flow cell. Sequencing Q Buffers (Box 1) Fill Number component volume of tubes Add-On to be added by user Extend Premix A1 17.35 ml 1 177 μl Extend A2 and 177 μl of Pol Extend from Box 2 Extend Premix B1 17.35 ml 1 177 μl Extend B2 and 177 μl of Pol Extend from Box 2 Cleavage Solution 16.5 ml 1 164 mg/l tube of Cleave Additive 1 and 870 μl/1 tube of Cleave Additive 2 from Box 1 Image Premix A1 17.35 ml 1 354 μl Image A2 from Box 2 Image Premix B1 17.64 ml 1 360 μl Image B2 from Box 2
[0124] Preparation of IPA Cleave Reagent for 1 Flow Cell:
[0125] Add 164 mg of Cleave Additive 1 to Cleavage Solution. Mix the contents in the tube by inverting the tube at least 10 times, until Cleave Additive 1 is completely dissolved. If any residual Cleave Additive 1 remains in its original tube, pipette 500 ul of the Cleavage Solution into Cleave Additive 1 tube, vortex to mix. Transfer all liquid back into Cleavage Solution tube. Then, Add 870 ul of Cleave Additive 2 to the combined Cleavage Solution. Mix the contents in the tube by inverting the tube at least 10 times.
TABLE-US-00009 TABLE 4 Reagent preparation for Sequencing Q Kit (4) for running 2 flow cells. Sequencing Q Buffers (Box 1) Fill Number component volume of tubes Add-On to be added by user Extend Premix A1 2 tubes of 2 2 tubes of Extend A2 (177 μl 17.35 ml from each tube) and 1 tube of Pol Extend (total of 354 μl) from Box 2 Extend Premix B1 2 tubes of 2 2 tubes of Extend B2 (177 μl 17.35 ml from each tube) and 1 tube of Pol Extend (total of 354 μl) from Box 2 Cleavage Solution 2 tubes of 2 2 tubes of Cleave Additive 1 16.5 ml (164 mg in each tube); 2 tubes of Cleave Additive 2 (870 μl in each tube) from Box 1 Image Premix A1 2 tubes of 2 2 tubes of Image A2 (354 μl 17.35 ml in each tube) from Box 2 Image Premix B1 2 tubes of 2 2 tubes of Image B2 (360 μl 17.64 ml in each tube) from Box 2
[0126] Preparation of IPA Cleave Reagent for 2 Flow Cells:
[0127] Add 164 mg of Cleave Additive 1 to each of the Cleave Solution tubes. After addition, mix the contents in the tube by inverting the tubes at least 10 times, until Cleave Additive 1 is completely dissolved. If any residual Cleave Additive 1 remains in its original tube, pipette 500 ul of the Cleavage Solution into each of the Cleave Additive 1 tubes, vortex to mix. Transfer liquid back into corresponding Cleavage Solution tubes. Then, add 870 ul of Cleave Additive 2 to each of the two single Cleavage Solution tubes. After addition, mix the contents in the tube by inverting the tubes at least 10 times and finally pool in to one single tube.
TABLE-US-00010 TABLE 5 Reagent preparation for Sequencing Q Kit (4) for running 3 or 4 flow cells. Sequencing Q Buffers (Box 1) Fill Number component volume of tubes Add-On to be added by user Extend Premix A1 3 tubes of 3 3 tubes of Extend A2 (177 μl 17.35 ml from each tube) and 1.5 tubes of Pol Extend (total of 531 μl) from Box 2 Extend Premix B1 3 tubes of 3 3 tubes of Extend B2 (177 μl 17.35 ml from each tube) and 1.5 tubes of Pol Extend (total of 531 μl) from Box 2 Cleavage Solution 3 tubes of 3 3 tube of Cleave Additive 1 16.5 ml (164 mg in each tube); 3 tubes of Cleave Additive 2 (870 μl from each tube) from Box 1 Image Premix A1 3 tubes of 3 3 tubes of Image A2 (354 μl 17.35 ml in each tube) from Box 2 Image Premix B1 3 tubes of 3 3 tubes of Image B2 (360 μl 17.64 ml in each tube) from Box 2
[0128] Preparation of IPA Cleave Reagent for 3-4 Flow Cells:
[0129] Add 164 mg of Cleave Additive 1 to each of the three single Cleave Solution tubes. After addition, mix the contents in the tube by inverting the tubes at least 10 times, until Cleave Additive 1 is completely dissolved. If any residual Cleave Additive 1 remains in its original tube, pipette 500 ul of the Cleavage Solution into each of the Cleave Additive 1 tubes, vortex to mix. Transfer liquid back into corresponding Cleavage Solution tubes. Then, add 870 ul of Cleave Additive 2 to each of the three single Cleavage Solution tubes. After addition, mix the contents in the tube by inverting the tubes at least 10 times and finally pool in to one single tube.
EXAMPLE III
GDP4 Testing: IPA Versus Gallic Acid
[0130] This example tests IPA and gallic acid in a head-to-head comparison to evaluate performance equivalency using back-to-back assays on four (4) reading instruments. A total of eight runs were performed where four (4) runs used IPA and four (4) runs used gallic acid. The IPA runs evaluated fifteen (15) flow cells and the gallic acid runs evaluated seven (7) flow cells. See,
[0131] In this initial study, IPA and gallic acid appear to be performance equivalent based on System KPI's and FP rate. One (1) out of seven (7) flow cells showed an approximate 30% bead loss for gallic acid whereas no flow cells showed any bead loss when using IPA. See,
[0132] Testing on 8-series with new baseline configuration (88 tile and 101× panel) has been performed to verify initial observations from this feasibility study.
EXAMPLE IV
IPA Solubility
[0133] This example tests alternative formulations and chemistry for a cleave mix including TCEP as a standard reducing agent and IPA as a radical oxygen scavenger. The goal is to test both solubility and the ease of implementation into a kit configuration.
[0134] Formation of solid components directly within a Cleave 1 solution using an IPA concentration curve (10mM, 25 mM and 50mM) was assessed by measuring solubility, color, precipitate observation and pH level.
[0135] The results of these observations are presented in Table 6.
TABLE-US-00011 TABLE 6 IPA Solubility Concentration Curve Time IPA Conc/ Point Temperature (hours) Solubility Color Precipitate pH 10 mM/RT 0 Excellent Transparent None 12.88 No color 10 mM/2-8 C.° 0 Excellent Transparent None 12.81 No color 25 mM RT 0 Excellent Transparent None 12.78 Pale yellow 25 mM/2-8 C.° 0 Excellent Transparent None 12.75 Pale yellow 50 mM/RT 0 Excellent Transparent None 12.61 Pale yellow 50 mM/2-8 C.° 0 Excellent Transparent None 12.67 Pale yellow 10 mM/RT 8 Excellent Transparent None 12.83 No color 10 mM/2-8 C.° 8 Excellent Transparent None 12.80 No color 25 mM/RT 8 Excellent Transparent None 12.67 Pale yellow 25 mM/2-8 C.° 8 Excellent Transparent None 12.71 Pale yellow 50 mM/RT 8 Excellent Transparent None 12.69 Pale yellow 50 mM/2-8 C.° 8 Excellent Transparent None 12.61 Pale yellow 10 mM/RT 48 Excellent Transparent None 12.79 No color 10 mM/2-8 C.° 48 Excellent Transparent None 12.83 No color 25 mM/RT 48 Excellent Transparent None 12.71 Pale yellow 25 mM/2-8 C.° 48 Excellent Transparent None 12.76 Pale yellow 50 mM/RT 48 Excellent Transparent None 12.72 Pale yellow 50 mM/2-8 C.° 48 Excellent Transparent None 12.64 Pale yellow
These results show that IPA is highly soluble in aqueous solution at a pH ˜12 and demonstrates relative stability as shown by lack of discoloration and/or formation of precipitate.
EXAMPLE V
Carnitine Solubility
[0136] This example tests carnitine-based compounds for solubility into Cleave reagent. Various concentrations (10 mM, 25 mM and 50 mM) of carnitine and acetylcarnitine were tested for solubility and stability. The data (not shown) demonstrates that both compounds are very soluble across the tested range of concentrations. The solutions also appeared stable at room temperature (RT) in that no discoloration or precipitate were observed after a few hours of storage.