Photoprotective mixtures as imaging reagents 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 photoprotective mixture of compounds as imaging reagents to improve stability and storage of fluorescent compounds, including but not limited to, nucleotides with fluorescent labels.

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 first imaging reagent comprising a mixture of at least one fluorescence quenching inhibitor, at least one antioxidant, and at least one radical scavenger 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 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; and d) imaging said incorporated labeled nucleotide analogue in the presence of said first imaging reagent.

2. The method of claim 1, wherein said at least one fluorescence quenching inhibitor is 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

3. The method of claim 1, wherein said at least one antioxidant is selected from the group consisting of gentisic acid, protocatechuic acid and protocatechuate ethyl ester.

4. The method of claim 1, wherein said at least one radical scavenger compound is carnitine.

5. The method of claim 1, further comprising step (e) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.

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

7. An imaging reagent comprising: i) 2-Amino-2-hydroxymethyl-propane-1,3-diol (TRIS) HCl buffer; ii) carnitine ranging in concentration between approximately 5-50 mM; iii) 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid ranging in concentration between approximately 5-15 mM; iv) 2,5 dihydroxybenzoic acid ranging in concentration between approximately 10-50 mM; and v) 3,4, dihydroxybenzoic acid ethyl ester ranging in concentration between approximately 10-20 mM.

8. The method of claim 1, wherein said at least one antioxidant comprises gentisic acid, said at least one radical scavenger compound comprises carnitine, and said at least one fluorescence quenching inhibitor comprises 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

9. The method of claim 1, wherein said at least one antioxidant comprises protocatechuic acid ethyl ester, said at least one radical scavenger compound comprises carnitine, and said at least one fluorescence quenching inhibitor comprises 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

10. The method of claim 1, wherein said imaging delivers an average read length that is substantially similar to a read length obtained when said first imaging reagent is replaced by a second imaging reagent comprising 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, glucose oxidase, glucose, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A-B presents a comparative workflow between a conventional imaging buffer configuration and photoprotective imaging buffer configuration.

(2) FIG. 1A: An illustrative workflow for a conventional imaging buffer kit configuration.

(3) FIG. 1B: A photoprotective imaging buffer kit configuration comprising new and improved Cleave and Imaging Buffer Consumables,

(4) FIG. 2 presents exemplary data showing that SBS metrics are comparable between a conventional imaging buffer and a Photoprotective imaging buffer 7B.

(5) FIG. 3A-D presents exemplary data showing comparative read length distributions between a conventional imaging buffer and a Photoprotective imaging buffer 7B subsequent to each SBS run.

(6) FIG. 3A: Run 8.3

(7) FIG. 3B; Run 8.5

(8) FIG. 3C; Run 8.6

(9) FIG. 3D: Run 8.41 (retest)

(10) FIG. 4A-D presents exemplary data showing raw error plots between a conventional imaging buffer and a Photoprotective imaging buffer 7B subsequent to each SBS run.

(11) FIG. 4A: Run 8.3

(12) FIG. 4B; Run 8.5

(13) FIG. 4C; Run 8.6

(14) FIG. 4D: Run 8.41 (retest)

(15) FIG. 5 presents exemplary data showing a comparison of average read length data between a conventional imaging buffer (IB_Baseline) and three (3) versions of a Photoprotective imaging buffer SC-P version 9.

(16) FIG. 6A-C presents exemplary data showing a comparison of raw error plot data between a conventional imaging buffer (IB_Baseline) and three (3) versions of a Photoprotective imaging buffer SC-P version 9.

(17) FIG. 6A: Photoprotective imaging buffer SC-P 9.

(18) FIG. 6B: Photoprotective imaging buffer SC-P 9B.

(19) FIG. 6C: Photoprotective imaging buffer SC-P 9C.

(20) FIG. 7 presents exemplary data showing the effect of lower ionic and aromatic compound concentrations in an imaging buffer on nucleotide signal retention.

(21) FIG. 8 presents exemplary data showing raw error rates in sequencing runs using SC-P9C imaging reagent versus a baseline IB.

(22) FIG. 9A-B presents exemplary data showing raw error rates in sequencing runs using SC-P9C imaging reagent formulated with either a HEPES buffer or a TRIS buffer versus a baseline IB.

(23) FIG. 9A Results using a HEPES buffer.

(24) FIG. 9B: Results using a TRI S buffer.

(25) FIG. 10 presents exemplary data showing the effects of carnitine concentration on MFST data output in both 101x gene panels (blue) and BRCA gene panels (red).

(26) FIG. 11 presents exemplary data showing the effects of carnitine concentration on percent perfect parameters in both 101x gene panels (blue) and BRCA gene panels (red).

(27) FIG. 12 presents exemplary data showing the effects of carnitine concentration on average read length in both 101x gene panels (blue) and BRCA gene panels (red).

(28) FIG. 13 presents exemplary data showing the effects of carnitine concentration on percent error rate in both 101x gene panels (blue) and BRCA gene panels (red).

(29) FIG. 14A-B presents exemplary data showing the effects of carnitine concentration on raw error rate in both 101x gene panels (blue) and BRCA gene panels (red).

(30) FIG. 14A: SC-9 IB and SC9B IB versus reference IB.

(31) FIG. 14B: SC-9D IB versus reference IB.

(32) FIG. 15A-B presents exemplary data showing the effects of carnitine concentration on read length distribution in both 101x gene panels (blue) and BRCA gene panels (red).

(33) FIG. 15A: SC-9 IB and SC9B IB versus reference IB.

(34) FIG. 15B: SC-9D IB versus reference IB.

(35) FIG. 16 presents exemplary data showing the effects of carnitine concentration on nucleotide signal retention in both 101x gene panels (blue) and BRCA gene panels (red).

DETAILED DESCRIPTION OF THE INVENTION

(36) 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 nucleotide sequences using, for example, sequencing by synthesis methods. In particular, the present invention contemplates the use of photoprotective buffer mixture of compounds as imaging reagents to improve stability and storage of fluorescent compounds, including but not limited to, nucleotides with fluorescent labels.

(37) In one embodiment, the present invention contemplates compositions comprising photoprotective mixtures as imaging reagents during sequencing-by-synthesis (SBS). In one embodiment, a method comprising imaging occurs during a fluorophore detection step. In one embodiment, a method comprising imaging occurs following nucleotide incorporation. In one embodiment, a photoprotective mixtures comprises compounds such as, but not limited to: carnitine; 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic Acid (trolox); 2,5-dihydroxybenzoic acid (gentisic acid); 3,4-dihydroxybenzoic acid (protocatechuic acid); 3,4-dihydroxybenzoic acid ethyl ester (protocatechuate ethyl ester), 4-hydroxycinnamic acid, 3,4-dihydroxybenzeneacrylic acid, 1,4-diazabicyclo[2.2.2]octane (DABCO), lipoic acid and/or acetyl-carnitine.

(38) In one embodiment, the present invention contemplates a method for sequencing a 150 bp read length. Other significant additional benefits are also provided including, but not limited to: improved manufacturing, storage and quality control processes, improved usability through user friendly kit concept and workflow, improved instrument reliability due to delivery of a single component solution that does not require mixing of individual components through next generation sequencing platform fluidics.

(39) I. Sequencing-By-Synthesis (SBS)

(40) In one embodiment, the present invention contemplates a series of method steps performed by an automated sequencing by synthesis instrument (e.g., a next generation sequencing platform). 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:

(41) 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: 1) Extend A Reagent: Comprises reversibly terminated labeled nucleotides and polymerase. One composition of Extend A may be as follows:

(42) TABLE-US-00001 Component Concentration 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 2) Extend B Reagent: Comprises reversibly terminated unlabeled nucleotides and polymerase, but lacks labeled nucleotide analogues. One composition of Extend B may be as follows:

(43) TABLE-US-00002 Component Concentration 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 3) Wash solution 1 with a detergent (e.g., polysorbate 20) citrate buffer (e.g., saline) 4) Cleave Reagent: One cleaving solution composition may be as follows:

(44) TABLE-US-00003 Component Concentration NaOH (mM) 237.5 TrisHCl (pH 8.0) (mM) 237.5 TCEP (mM) 50 5) Wash solution 2 with a detergent (e.g., polysorbate 20) a tris(hydroxymethyl)-aminomethane (Tris) buffer.
II. Conventional Imaging Solutions

(45) One enzymatic formulation currently being used as an imaging reagent (IB) comprises four-components. These four components comprise HEPES buffer, glucose oxidase, glucose and trolox and are required to be combined to create two separate solutions prior to supporting an SBS method. These two solutions are kept separate throughout the sequencing process to prevent glucose oxidase and glucose from reacting prematurely with oxygen causing degradation of the enzymatic system and elimination of H.sub.2O.sub.2. These two final solutions are mixed during SBS through a mixing valve at every imaging step before introduction into a flow cell. Use of this type of imaging method step, albeit effective, presents challenges in several areas including, but not limited to: i) stability of the manufacturing process; ii) maintaining quality control due to complicated exo/endo specification paradigms; iii) difficult usability due to a complex kit configuration and workflow; iv) limited instrument reliability due to delivery volume failure modes due to mixing valve reliability issues. As seen herein, the conventional or baseline imaging reagent (IB) has been used for performance benchmarking of various embodiments of the presently disclosed photoprotective mixture imaging reagents.

(46) III. Photoprotective Imaging Solutions

(47) In one embodiment, effective imaging solutions and buffer formulations for SBS methods comprise molecular components that ensure photoprotection during light exposure. While not bound by theory, it is believed that they prevent three main phenomena: i) photo-bleaching of nucleotide fluorescent labels; ii) signal quenching; and iii) radically-induced DNA photo-damage and photo-scission. Imaging solutions can be formulated either as either enzymatic systems or mixtures comprising a variety of chemical mixtures such as mixtures including, but not limited to, a molecular oxygen “sink”, an antioxidant/radical scavenger and a singlet oxygen quencher. Some of the components in these mixtures also provide additional protection against oxidative stress and degradation of the imaging solution upon prolonged storage.

(48) Although it is not necessary to understand the mechanism of an invention it is believed that a “mixture” or “cocktail” approach is most suitable for formulating long shelf-life imaging solutions because it best supports a variety of functional aspects pertaining to product design robustness, ranging from functional performance and formulation stability to manufacturability, usability and storage.

(49) In one embodiment, the present invention contemplates a photoprotective mixture as an imaging reagent during a fluorophore detection step following nucleotide incorporation in sequencing-by-synthesis (SBS). These photoprotective mixture imaging reagents comprise an effective antioxidant such as, but not limited to, 2,5-dihydroxybenzoic acid (gentisic acid); 3,4-dihydroxybenzoic acid (protocatechuic acid) or 3,4-dihydroxybenzoic acid ethyl ester (protocatechuate ethyl ester), a fluorescence quenching inhibitor such as, but not limited to, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) and a radical scavenger (e.g., carnitine), an antioxidant and/or a singlet oxygen quencher.

(50) In one embodiment, the photoprotective cocktail comprises a mixture (e.g., imaging reagent SC-P 7B) including the components:

(51) ##STR00001##
In one embodiment, the photoprotective cocktail comprises a mixture (e.g., imaging reagent SC-P 9C) including the components:

(52) ##STR00002##
Photoprotective cocktail mixtures, exemplified by those above, have been designed with properties including, but not limited to, photoprotection, stability, manufacturability, long shelf life storage and usability. These mixtures are also designed for compatibility with off the shelf sequencing kits (i.e., the GeneReader® 1.1 sequencing kit). In particular, carnitine has been bound to induce reduction of oxidative stress and preservation of chemical activity and/or biological function after long term storage of biological fluids and functional buffers. Without being bound by theory, carnitine can conceivably support enhanced storage of complex molecular mixtures due to reduction of oxidative stress caused by molecules including, but not limited to, oxygen, peroxy radicals, or singlet oxygen. Although it is not necessary to understand the mechanism of an invention, it is believed that compounds such as carnitine preserve their protective properties, even upon prolonged storage as a formulation, including but not limited to molecular reduced states and stability against chemical bond scission (e.g., radical- or photo-scission).

(53) The data presented herein demonstrates an interrogation potential for various photoprotective imaging mixtures of compounds as imaging reagents. Additionally, full compatibility with SBS instrument hardware is verified for all components as observed from an inspection of both instrument and liquid waste at the end of sequencing. Improvements of the presently disclosed photoprotective mixture imaging reagents are exemplified with comparative sequencing workflows.

(54) For example, a prospective kit configuration for photoprotective mixture imaging reagents is shown as a single component consumable stored in a kit box compatible with −20° C. storage conditions. See, FIG. 1A-B. An comparative workflow for a conventional imaging reagent kit configuration (FIG. 1A) demonstrates the increased complexity as opposed to the presently disclosed photoprotective imaging reagent kit configuration (FIG. 1B). Although it is not necessary to understand the mechanism of an invention, it is believed that the presently disclosed photoprotective imaging reagent kit configuration greatly improves usability during an SBS method by decreasing the number of components required in the kit and workflow.

(55) Photoprotective imaging reagents as contemplated by the present invention have been tested for long read length sequencing performance (e.g., approximately 150 bp). Some of these tests entailed 157 cycle sequencing and a head-to-head comparison of photoprotective mixture imaging reagents to a conventional imaging reagent (e.g., baseline IB reagent). Studies were performed using Gene Reader instruments and two types of DNA libraries, i.e., NA12878/101X gene panel and NA12878/BRCA gene panel. Sequencing metrics were analyzed to provide comparative system performance indicators, e.g., raw error rate, average read length, output (Gb).

(56) For example, GDP4 Testing was performed using the photoprotective imaging buffer reagent SC-P 7B made in accordance with Example II. The testing was run using an APF protocol v2 comprising 157 cycles and 130 tiles utilizing the SP101x gene panel. Of the four runs (e.g. 8.41) that was performed was deemed to be invalid and retested (noted as “**). It can be seen that the conventional imaging reagent (Baseline IB) and a photoprotective imaging reagent SC-P 7B were comparable across all sequencing metrics. See, FIG. 2 and Table 1.

(57) TABLE-US-00004 TABLE 1 Comparative Sequencing Metrics: Conventional IB (Baseline) versus SC-P 7B IB. Date Run GR Sample ID Output (MFST) Beads/tiles Error rate % Live Mar. 25, 2016 IB 7B 8.5 SP101x 1.77127963 429218.169 0.747583 48% Apr. 15, 2016 IB 7B Retest** 8.41 SP101x 1.904131381 428153.625 0.775358 50% Apr. 5, 2016 IB 7B 8.6 SP101x 1.87737536 440901.246 0.751118 49% Apr. 11, 2016 IB 7B 8.3 SP101x 2.052871269 444626.646 0.728655 51% 1.901 435729 0.75% 49% Mar. 28, 2016 Baseline 8.5 SP101x 1.852687368 434523.031 0.795124 48% Mar. 28, 2016 Baseline 8.41 SP101x 1.808064963 435729 0.77365 49% Apr. 8, 2016 Baseline 8.6 SP101x 1.890117702 438295.854 0.803485 51% Apr. 14, 2016 Baseline 8.3 SP101x 2.023612666 431433.115 0.778109 52% 1.893 434999 0.79% 49% Date % Mapped % Polyclonal % Perfect AVG RL Lead Lag Mar. 25, 2016 29% 36% 0.5955009 103.6339 0.337969 0.1115 Apr. 15, 2016 31% 35% 0.5969785 110.6978 0.405625 0.084068 Apr. 5, 2016 30% 34% 0.5990981 108.7261 0.347992 0.098931 Apr. 11, 2016 31% 35% 0.6067697 113.2278 0.406538 0.117338 30% 35% 59% 110 0.374 0.102 Mar. 28, 2016 29% 36% 0.5857853 112.4375 0.4325 0.123562 Mar. 28, 2016 29% 37% 0.5958026 110.3599 0.424246 0.126323 Apr. 8, 2016 31% 36% 0.583074 108.433 0.332785 0.187277 Apr. 14, 2016 31% 36% 0.5949944 115.0466 0.409185 0.157954 30% 36% 58% 111 0.399 0.149
These data show that the metric, lag, shows the only statistically relevant difference between the two imaging reagents with a 30% lower lag when tested with a photoprotective imaging reagent SC-P 7B. The read length distributions among the four runs were equivalent when tested between the two imaging reagents. See, FIGS. 3A-3D. The raw error plots among the four runs were also equivalent when tested between the two imaging reagents. See, FIGS. 4A-4D.

(58) Testing was also performed using various embodiments of the exemplary photoprotective imaging reagent 9CV2T made in accordance with Example III. Three different versions of the 9CV2T reagents were made. Although it is not necessary to understand the mechanism of an invention, it is believed that at least one of these reagents resulted in an improved signal retention. For example, three of the tested versions of 9CV2T imaging reagents comprised: SC-P9 pH 8.5

(59) TABLE-US-00005 Tris 50 mM L-Carnitine 50 mM Trolox 15 mM Protocatechuic acid 20 mM ethyl ester
or, SC-P9B pH 8.5

(60) TABLE-US-00006 Tris 50 mM L-Carnitine 15 mM Trolox 15 mM Protocatechuic acid 20 mM ethyl ester
and, SC-P9C pH 8.5

(61) TABLE-US-00007 Tris 50 mM L-Carnitine 15 mM Trolox 15 mM Protocatechuic acid ethyl 10 mM ester
A comparison of basic sequencing metrics was made between these three SCRP version 9CV2T imaging reagents and the conventional IB reagent with a sequencing protocol of 137 cycles using a Vaccinia virus (VACV) strain TianTan TP03 genome. See, Table 2.

(62) TABLE-US-00008 TABLE 2 Comparison of Sequencing Metrics Between SC-P Version 9 IBs And A Conventional IB (IB_Baseline). Error Rate Run Name GR/Sample Output (MFST) % Mapped AVG RL (MFST) % Perfect Lead Lag IB_Baseline 8.26/TP03 1.23E+09 32.6% 99.5 0.60% 67.6% 0.421 0.125 IB_SC-P9 8.26/TP03 1.21E+09 33.1% 98.5 0.54% 69.00%  0.407 0.116 IB_SC-P9B 8.26/TP03 1.16E+09 31.8% 98.9 0.54% 69.1% 0.389 0.124 IB_SC-P9C 8.17/TP03 1.12E+09 30.6% 99.7 0.58% 67.5% 0.432 0.093
A relative equivalency was seen between these SC-P version 9CV2T imaging reagents and the baseline imaging reagent with respect to average read length. See, FIG. 5. Such equivalency was also observed for the raw data plots between the SC-P version 9CV2T imaging reagents and the baseline imaging reagent. See, FIGS. 6A-C. These data demonstrate that lowering of concentrations of ionic and aromatic compound (e.g., carnitine and protocatechuic acid, respectively), does improve signal retention. See, FIG. 7. Although it is not necessary to understand the mechanism of an invention, it is believed that this observation is likely due to mitigated signal quenching which usually arises from interactions between fluorophores and aromatic/ionic species in a photoprotective buffer.

(63) The SC-P9C imaging reagent was used in a 157 cycles non-APF/88 tile protocol on a BRCA gene panel (runs 8.17; 8.24; 8.26). The sequencing metrics were compared to a reference imaging IB. See, Table 3.

(64) TABLE-US-00009 TABLE 3 Sequence Metrics Comparison Of SC-P9C To Baseline IB On A BRCA Gene Panel % Output Beads/ % % % Poly- AVG Error Per- Run Condition GR Run date Type (MFST) tile Live Mapped clonal RL (MFST) fect Lead Lag Baseline_BRCA 8.17 Apr. 15, 2016 HG19 BRCA 1.14E+09 426294 47% 26% 41% 115.0 0.72% 58% 0.513 0.060 Baseline_BRCA 8.17 Apr. 15, 2016 HG19 BRCA 1.17E+09 424933 45% 27% 38% 114.0 0.74% 57% 0.466 0.091 Prototype 9C_BRCA 8.26 Apr. 15, 2016 HG19 BRCA 1.09E+09 428096 44% 26% 36% 110.0 0.78% 53% 0.477 0.061 Prototype 9C_BRCA 8.26 Apr. 15, 2016 HG19 BRCA 1.14E+09 422093 45% 26% 36% 112.0 0.79% 54% 0.446 0.084 Prototype 9C_BRCA 8.24 Apr. 15, 2016 HG19 BRCA 1.15E+09 427948 43% 25% 35% 110.0 0.82% 51% 0.444 0.072 Prototype 9C_BRCA 8.24 Apr. 15, 2016 HG19 BRCA 1.18E+09 424163 43% 26% 35% 113.0 0.87% 52% 0.454 0.082

(65) A comparison of the raw error rates demonstrated equivalency between the two imaging reagents. See, FIG. 8. The SC-P9C imaging reagent sequencing metrics were also compared between the HEPES buffer and the TRIS buffer using a 157 cycles non-APF/88 the protocol on a DHMG02 BRCA gene panel. See, Table 4.

(66) TABLE-US-00010 TABLE 4 Sequence Metrics Comparison Of SC-P9C To Baseline IB On A BRCA Gene Panel Comparing HEPES Buffer To TRIS Buffer Run Condition GR Sample ID Run date Type Cycles Output (MFST) Beads/tile Ref_Baseline 8.24 FC1_DHMG02 Apr. 26, 2016 HG19 BRCA 150 1.28E+09 419417 Ref_Baseline 8.24 FC2_DHMG02 Apr. 26, 2016 HG19 BRCA 150 1.34E+09 409874 IB_P9CV2_HEPES 8.26 FC1_DHMG02 Apr. 26, 2016 HG19 BRCA 150 1.37E+09 417428 IB_P9CV2_HEPES 8.26 FC2_DHMG02 Apr. 26, 2016 HG19 BRCA 150 1.35E+09 423869 Proto9C_V2_Tris 8.26 FC1_DHMG02 Apr. 15, 2016 HG19 BRCA 150 1.07E+09 428096 Proto9C_V2_Tris 8.26 FC2_DHMG02 Apr. 15, 2016 HG19 BRCA 150 1.10E+09 422093 Proto9C_V2_Tris 8.24 FC1_DHMG02 Apr. 15, 2016 HG19 BRCA 150 1.03E+09 427948 Proto9C_V2_Tris 8.24 FC2_DHMG02 Apr. 15, 2016 HG19 BRCA 150 1.08E+09 424163 Run Condition % Live % Mapped % Polyclonal AVG RL Error (MFST) % Perfect Lead Lag Ref_Baseline 51% 31% 36% 110.8 0.83% 56% 0.490 0.024 Ref_Baseline 53% 33% 36% 113.2 0.83% 56% 0.490 0.035 IB_P9CV2_HEPES 49% 31% 35% 112.6 0.79% 57% 0.420 0.040 IB_P9CV2_HEPES 50% 32% 35% 114.5 0.76% 58% 0.449 0.046 Proto9C_V2_Tris 44% 26% 36% 109.0 0.96% 51% 0.477 0.061 Proto9C_V2_Tris 45% 26% 36% 111.5 0.94% 52% 0.446 0.084 Proto9C_V2_Tris 43% 25% 35% 108.7 1.03% 50% 0.444 0.072 Proto9C_V2_Tris 43% 26% 35% 112.3 1.02% 50% 0.454 0.082
The data demonstrate that the SC-P 9C imaging reagent formulated in a HEPES buffer outperforms the SC-P 9C imaging reagent formulated in Tris HCl. Similarly, the raw error rate is less using a HEPES buffer as compared to a Tris HCl and is more similar to the baseline IB. See, FIGS. 9A and 9B.

(67) These data demonstrate that photoprotective imaging reagents with protocatechuic ethyl ester is superior to photoprotective imaging reagents with gentisic acid in regards to signal stability and quality. Although it is not necessary to understand the mechanism of an invention, it is believed that signal stability and quality plays a role in sequencing performance quality. Nonetheless, both gentisic- and protocatechuic-based cocktail chemistries (i.e., SC-P 7B and SC-P 9C, respectively) are expected to benchmark competitively in regards to comparative performance for long read sequencing. From a manufacturing perspective, however, photoprotective imaging reagents with protocatechuic ethyl ester may be preferable than photoprotective imaging reagents with gentisic acid due to better flexibility and cost effectiveness of making the formulation.

(68) Further studies were performed to determine if decreasing carnitine concentration in a photoprotective imaging reagent (e.g., between approximately 50 mM to 5 mM) influences sequencing performance. The composition of the tested photoprotective carnitine imaging reagents (e.g., SC-P9, SC-P9B and SC-P9C) are as follows:

(69) Prototype 9

(70) TABLE-US-00011 50 mM Tris buffer pH 8.5 50 mM L-Carnitine 15 mM Trolox 20 mM Protocatechuic Acid Ethyl Ester
Prototype 9B

(71) TABLE-US-00012 50 mM Tris buffer pH 8.5 15 mM L-Carnitine 15 mM Trolox 20 mM Protocatechuic Acid Ethyl Ester

(72) Prototype 9D

(73) TABLE-US-00013 50 mM Tris buffer pH 7.8  5 mM L-Carnitine 15 mM Trolox 20 mM Protocatechuic Acid Ethyl Ester
The sequencing runs (8.17, 8.24, 8.26) setups comprised 132 cycles for both the reference and/or baseline IB reagent and the photoprotective IB reagent using samples NA12878/101X (ID: TP03) or NA12878/BRCA (ID: DHMG02). The data show that 15 mM Carnitine is provides an optimal working concentration based on average read length and signal retention. See, Table 5.

(74) TABLE-US-00014 TABLE 5 ADAM Results From Carnitine Concentration Analysis Run Condition GR Sample ID Run date Type Cycles Output (MFST) Beads/tile % Live IB_baseline_8.26_FC1 8.26 TP03_FC1 Mar. 30, 2016 HG19 101X 125 1.18E+09 446379.5 48.8% IB_baseline_8.26_FC2 8.26 TP03_FC2 Mar. 30, 2016 HG19 101X 125 1.20E+09 414836.1 51.2% IB_baseline_8.17_FC1 8.17 FC1_TP03 Apr. 4, 2016 HG19 101X 125 1.27E+09 426583.5 53.6% IB_baseline_8.17_FC2 8.17 FC2_TP03 Apr. 4, 2016 HG19 101X 125 1.26E+09 426191.8 52.7% IB_SC-P9_FC1 8.26 FC1_TP03 Apr. 4, 2016 HG19 101X 125 1.21E+09 424152.8 52.4% IB_SC-P9_FC2 8.26 FC2_TP03 Apr. 4, 2016 HG19 101X 125 1.22E+09 432521.2 52.8% IB_SC-P9B_FC1 8.26 FC1_TP03 Apr. 11, 2016 HG19 101X 125 1.16E+09 419886.9 49.6% IB_SC-P9B_FC2 8.26 FC2_TP03 Apr. 11, 2016 HG19 101X 125 1.16E+09 414822.6 49.9% Baseline_BRCA_AGR 8.17 1_DHMGO Apr. 15, 2016 HG19 BRCA 125 1.01E+09 426294.1 47.1% Baseline_BRCA_AGR 8.17 2_DHMGO Apr. 15, 2016 HG19 BRCA 125 1.03E+09 424932.8 45.1% IB_Proto90_BRCA_AGR 8.26 1_DHMGO Apr. 19, 2016 HG19 BRCA 125 9.85E+09 428784.6 44.9% IB_Proto90_BRCA_AGR 8.26 2_DHMGO Apr. 19, 2016 HG19 BRCA 125 1.02E+09 430237.1 45.6% Run Condition % BC % mapped G Read Leag Error Rate % Perfect Lead Lag Notes IB_baseline_8.26_FC1 73.0% 30.5% 99.0 0.55% 69.1% 0.415 0.139 baseline 101X IB_baseline_8.26_FC2 73.6% 32.5% 100.9 0.54% 69.3% 0.378 0.149 baseline 101X IB_baseline_8.17_FC1 74.3% 34.1% 99.4 0.58% 67.6% 0.470 0.109 baseline 101X IB_baseline_8.17_FC2 73.6% 33.2% 100.6 0.59% 66.6% 0.423 0.127 baseline 101X IB_SC-P9_FC1 74.8% 33.0% 97.9 0.53% 69.7% 0.409 0.111 50 mM carnitine IB_SC-P9_FC2 75.0% 33.1% 97.0 0.56% 68.3% 0.405 0.122 50 mM carnitine IB_SC-P9B_FC1 73.3% 31.7% 93.6 0.55% 69.0% 0.389 0.126 15 mM carnitine IB_SC-P9B_FC2 73.5% 31.9% 99.2 0.54% 69.2% 0.383 0.123 15 mM carnitine Baseline_BRCA_AGR   71%   26% 103.5 0.68% 64.4% 0.473 0.109 baseline_BRCA Baseline_BRCA_AGR   72%   27% 104.1 0.70% 63.2% 0.431 0.117 baseline_BRCA IB_Proto90_BRCA_AGR   71%   27% 97.2 0.69% 63.6% 0.425 0.101 5 mM carnitine IB_Proto90_BRCA_AGR   71%   27% 99.0 0.68% 63.5% 0.425 0.111 5 mM carnitine
In particular, reduced carnitine concentration lowers data output (MFST) in a concentration-dependent manner in both the 101x gene panel and the BRCA gene panel. See, FIG. 10. This output data was collected under conditions where the percent perfect parameters went unchanged as compared to the reference IB reagent. See, FIG. 11. The average read length, however, was seen to be lower overall when the photoprotective imaging reagents contain carnitine. See, FIG. 12. Nonetheless, there was no effect of photoprotective imaging reagents containing carnitine on percent error rate when compared to the reference IB reagent. See, FIG. 13. There were differences, however, between photoprotective imaging reagents having different carnitine concentrations regarding the raw error rate parameter. For example, the SC-9 IB reagent (50 mM carnitine) and SC-9B IB reagent (15 mM carnitine) had raw error rates similar to the reference IB reagent. See, FIG. 14A. The SC-9D IB reagent (5 mM carnitine), however, showed a higher raw error rate as compared to the reference IB reagent. See, FIG. 14B. This data pattern is seen for the distribution of read lengths between the tested imaging reagents. The SC-9 IB reagent (50 mM carnitine) and SC-9B IB reagent (15 mM carnitine) had read length distributions that were similar to the reference IB reagent. See, FIG. 15A. The SC-9D IB reagent (5 mM carnitine), however, showed a read length distribution that was biased to the early cycles, and somewhat lower, as compared to the reference IB reagent. See, FIG. 15B. Signal retention for all nucleotides was reduced in the presence of carnitine, as shown by the best signal retention with the lower carnitine concentration photoprotective IB reagent (e.g., SC-9B, 15 mM). See, FIG. 16.

(75) Overall, the data presented herein shows that photoprotective SC-P 7B IB reagent and SC-P 9C IB reagent perform similarly to a conventional imaging buffer reagent (e.g., baseline IB) and deliver an average read length minimum requirement that is compatible with state of the art gene readers. Specifically, the data show that photoprotective imaging buffer reagents as contemplated herein are efficient when scanning an average read length of approximately 110 bp as compared to the optimal scanning range of state of the art gene readers of between approximately 110-130 bp.

EXPERIMENTAL

Example I

Photoprotective Imaging Formulation Stability and Preservation

(76) Components in various photoprotective imaging mixtures as described herein have been tested for solubility and stability against precipitation and discoloration in imaging solution formulation using iris Ha as the base buffer. The optimal concentration windows for the various components have been found to be the following: Carnitine (5-50 mM); Trolox (5-15 mM); 2,5-Dihydroxybenzoic Acid (10-50 mM); and 3,4-Dihydroxybenzoic Acid Ethyl Ester (Protocatechuate Ethyl Ester)(10-20 mM).

Example II

Composition and Formulation of Photoprotective Imaging Reagent SC-P 7B

(77) The following example describes the preparation of approximately two hundred (200) milliliters of imaging reagent that would be expected to support a 4FC/157 cycle SBS method. 50 mM Tris buffer: 121.4 g/mol=1.21 g (Sigma: Cat #T1378-1kg) 50 mM L Carnitine: 197.66 g/mol=1.98 g (Sigma: Cat #C02133-100G) 15 mM Trolox: 250.29 g/mol=0.75 g (Sigma: Cat #238813-5G) 50 mM Gentisic Acid 176.1 g/mol=1.76 g (Sigma: G5129-10G) 1. Dissolve Tris Base in 180 mL milliQ water. 2. Add Trolox to the Tris buffer from Step 1. 3. Add L Carnitine to the above solution. 4. Add Gentisic acid sodium salt hydrate and dissolve. 5. Checked pH: ˜4 6. Adjusted pH to 7.8 with 10M NaOH solution, 7. Bring total volume to 200 mL with milliQ water. 8. Filter sterilize. 9. Split imaging buffer into 2 conical tubes (approx. 25 mL aliquots each) for single FC GR run.

Example III

Composition and Formulation of Photoprotective Imaging Reagent 9CV2T

(78) The following example describes the preparation of approximately two hundred (200) milliliters of imaging reagent. 50 mM Tris buffer: 121.4 g/mol=0.607 (Sigma: Cat #T1378-1kg) 15 mM L Carnitine: 197.66 g/mol=0.296 g (Sigma: Cat #C0238-100G) 15 mM Trolox: 250.29 g/mol=0.375 g (Sigma: Cat #238813-5G) 10 mM Protocatechuic Acid Ethyl Ester 182.17 g/mol=182.17 mg milligrams (Sigma: Cat #E24859-5G) 1. Dissolve Tris Base in 75 mL milliQ water. 2. Add Trolox and dissolve completely 3. Added Protocatechuic Acid 4. Add L Carnitine and dissolve completely. 5. Checked pH: 6. Adjusted pH to 8.5 with 10M NaOH. 7. Optional: Sonicate until well mixed. 8. Bring total volume to 100 mL with milliQ water. 9. Filter sterilize.

Example IV

Composition and Formulation of Photoprotective Imaging Reagent 9CV2H

(79) The following example describes the preparation of approximately one hundred (100) milliliters of imaging reagent. 100 mM HEPES: 238.3 gr/mole=2.39 g (Sigma: Cat #H4034) 15 mM L Carnitine: 197.66 g/mol=0.296 g (Sigma: Cat #C0283-100G) 15 mM Trolox: 250.29 g/mol=0.375 g (Sigma: Cat #238813-5G) 10 mM Protocatechuic Acid Ethyl Ester 182.17 g/mol=0.182 g (Sigma: Cat #E24859-5G)
1 Liter 100 mM HEPES 219 gr HEPES Dissolve in MilliQ water—final volume 1 Liter pH was adjusted to 7.0. 1. Obtain 75 mL HEPES buffer (see below). 2. Add Trolox and dissolve completely 3. Added Protocatechuic Acid. 4. Add L Carnitine and dissolve completely. 5. Checked pH: ˜5 6. Adjusted pH to 7.5 with 10M NaOH. 7. Optional: Sonicate until well mixed. 8. Bring total volume to 100 mL with milliQ water. 9. Filter sterilize.