U.S. patent application number 17/471791 was filed with the patent office on 2021-12-30 for photoprotective mixtures as imaging reagents in sequencing-by-synthesis.
The applicant listed for this patent is IsoPlexis Corporation. Invention is credited to Luisa ANDRUZZI, Minakshi GUHA, Dona HEVRONI, Timothy PELLETIER, Michel Georges PERBOST, Austin RICKER.
Application Number | 20210403985 17/471791 |
Document ID | / |
Family ID | 1000005836112 |
Filed Date | 2021-12-30 |
United States Patent
Application |
20210403985 |
Kind Code |
A1 |
ANDRUZZI; Luisa ; et
al. |
December 30, 2021 |
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.
Inventors: |
ANDRUZZI; Luisa; (Concord,
MA) ; PERBOST; Michel Georges; (Belmont, MA) ;
HEVRONI; Dona; (Lexington, MA) ; GUHA; Minakshi;
(Wakefield, MA) ; RICKER; Austin; (Waltham,
MA) ; PELLETIER; Timothy; (Fitchburg, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IsoPlexis Corporation |
Branford |
CT |
US |
|
|
Family ID: |
1000005836112 |
Appl. No.: |
17/471791 |
Filed: |
September 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15799139 |
Oct 31, 2017 |
11162129 |
|
|
17471791 |
|
|
|
|
62419702 |
Nov 9, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12Q 1/6816 20130101; C07C 215/28 20130101; C07B 2200/07 20130101;
G01N 33/582 20130101; C12Q 1/6874 20130101; C07D 311/66
20130101 |
International
Class: |
C12Q 1/6806 20060101
C12Q001/6806; C12Q 1/6816 20060101 C12Q001/6816; C07C 215/28
20060101 C07C215/28; C07D 311/66 20060101 C07D311/66; C12Q 1/6874
20060101 C12Q001/6874; G01N 33/58 20060101 G01N033/58 |
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) an imaging reagent comprising a
photoprotective mixture of compounds, 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
imaging reagent.
2. The method of claim 1, wherein said photoprotective mixture
comprises at least one fluorescence quenching inhibitor.
3. The method of claim 2, wherein said at least one fluorescence
quenching inhibitor is trolox.
4. The method of claim 1, wherein said photoprotective mixture
further comprises at least one antioxidant and at least one radical
scavenger compound.
5. The method of claim 4, wherein said at least one antioxidant is
selected from the group consisting of gentisic acid, protocatechuic
acid and protocatechuate ethyl ester.
6. The method of claim 4, wherein said at least one radical
scavenger compound is carnitine.
7. 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.
8. The method of claim 1, wherein said label is fluorescent.
9. An imaging reagent comprising at least one antioxidant, at least
one fluorescence quenching inhibitor and a buffer.
10. The imaging reagent of claim 9, wherein said at least one
antioxidant comprises compounds selected from the group consisting
of gentisic acid, protocatechuic acid and protocatechuate ethyl
ester.
11. The imaging reagent of claim 9, further comprising at least one
radical scavenger.
12. The imaging reagent of claim 11, wherein said at least one
radical scavenger is carnitine.
13. The imaging reagent of claim 9, wherein said fluorescence
quenching inhibitor is trolox.
14. The imaging reagent of claim 9, wherein said buffer is a TRIS
buffer.
15. The imaging reagent of claim 9, wherein said buffer is a HEPES
buffer.
16. A kit, comprising: i) a first container comprising an imaging
reagent comprising at least one antioxidant, at least one
fluoresence quenching inhibitor and a buffer and ii) a second
container comprising 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.
17. The kit of claim 16, wherein said first container further
comprises at least one radical scavenger.
18. The kit of claim 17, wherein said at least one radical
scavenger is carnitine.
19. The kit of claim 16, wherein said at least one fluorescence
quenching inhibitor is trolox.
20. The kit of claim 16, wherein said buffer is a TRIS buffer.
21. The kit of claim 16, wherein said buffer is a HEPES buffer.
22. A system comprising a solution of primers hybridized to a
template comprising a plurality of nucleotide analogues attached to
a cleavable label and an imaging reagent comprising at least one
antioxidant, at least one fluorescent quenching inhibitor and a
buffer.
23. The system of claim 22, wherein said hybridized primers and
said template are immobilized.
24. The system of claim 22, wherein said hybridized primers and
said template are in a flow cell.
25. The system of claim 22, wherein said imaging agent further
comprises at least one radical scavenger.
26. The system of claim 25, wherein said at least one radical
scavenger is carnitine.
27. The system of claim 22, wherein said at least one fluorescence
quenching inhibitor is trolox.
28. The system of claim 22, wherein said buffer is a TRIS
buffer.
29. The system of claim 22, wherein said buffer is a HEPES
buffer.
30. An imaging reagent comprising: i) a TRIS HCl buffer; ii)
carnitine ranging in concentration between approximately 5-50 mM;
iii) trolox 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.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
15/799,139, filed on Oct. 31, 2017, which claims priority to, and
the benefit of, U.S. Provisional Application No. 62/419,702, filed
on Nov. 9, 2016. The entire contents of each of the
above-identified applications is hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] 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.
BACKGROUND
[0003] Over the past 25 years, the amount of DNA sequence
information that has been generated and deposited into Genbank has
grown exponentially. Traditional sequencing methods (e.g., for
example Sanger sequencing) are being replaced by next-generation
sequencing technologies that use a form of sequencing by synthesis
(SBS), wherein specially designed nucleotides and DNA polymerases
are used to read the sequence of chip-bound, single-stranded DNA
templates in a controlled manner. To attain high throughput, many
millions of such template spots are arrayed across a sequencing
chip and their sequence is independently read out and recorded.
Systems for using arrays for DNA sequencing are known (e.g., Ju et
al., U.S. Pat. No. 6,664,079). However, there is a continued need
for methods and compositions for increasing the efficiency and/or
reagent stability for sequencing nucleic acid sequences with
automated sequencing.
SUMMARY OF THE INVENTION
[0004] 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 reagent mixture of compounds as imaging reagents
to improve stability and storage of fluorescent compounds,
including but not limited to, nucleotides with fluorescent
labels.
[0005] In one embodiment, the present invention contemplates a
photoprotective mixture (e.g., a cocktail) of compounds as an
imaging reagent during a fluorophore detection step following
nucleotide incorporation in sequencing-by-synthesis (SBS). In one
embodiment, the photoprotective mixture comprises at least one
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), at least one fluorescence quenching
inhibitor such as, but not limited to,
6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox)
and at least one radical scavenger such as, but not limited to,
carnitine.
[0006] In one embodiment, the present invention contemplates, a
method of incorporating labeled nucleotides, comprising: a)
providing; i) a plurality of nucleic acid primers and template
molecules, ii) a polymerase, iii) an imaging reagent comprising a
photoprotective mixture of compounds, 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 (e.g., under high stringency) 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 imaging reagent. In one
embodiment, the imaging reagent comprises a fluorescence quenching
inhibitor. In one embodiment, the fluorescence quenching inhibitor
is trolox. In one embodiment, the photoprotective mixture comprises
at least one antioxidant, at least one quenching inhibitor and at
least one radical scavenger compound. In one embodiment, the
antioxidant is selected from the group consisting of gentisic acid,
protocatechuic acid and protocatechuate ethyl ester. In one
embodiment, the radical scavenger compound is carnitine. In one
embodiment, the method further comprises: e) incorporating a second
nucleotide analogue with said polymerase into at least a portion of
said extended primers. In one embodiment, the label is
fluorescent.
[0007] In one embodiment, the present invention contemplates an
imaging reagent comprising at least one antioxidant, at least one
fluorescence quenching inhibitor and a buffer. In one embodiment,
the antioxidant comprises compounds selected from the group
consisting of gentisic acid, protocatechuic acid and
protocatechuate ethyl ester. In one embodiment, the imaging reagent
further comprises a radical scavenger. In one embodiment, the
radical scavenger is carnitine. In one embodiment, the fluorescence
quenching inhibitor is trolox. In one embodiment, the buffer is a
TRIS buffer. In one embodiment, the buffer is a HEPES buffer.
[0008] In one embodiment, the present invention contemplates a kit,
comprising i) a first container comprising an imaging reagent
comprising at least one antioxidant, at least one fluoresence
quenching inhibitor and a buffer; and ii) a second container
comprising 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. In one embodiment,
the imaging reagent further comprises a radical scavenger. In one
embodiment, the radical scavenger is carnitine. In one embodiment,
the fluorescence quenching inhibitor is trolox. In one embodiment,
the buffer is a TRIS buffer. In one embodiment, the buffer is a
HEPES buffer.
[0009] In one embodiment, the present invention contemplates a
system comprising a solution of primers hybridized to a template
comprising a plurality of nucleotide analogues attached to a
cleavable label and an imaging reagent comprising at least one
antioxidant, at least one fluorescent quenching inhibitor and a
buffer. In one embodiment, the hybridized primers and said template
are immobilized. In one embodiment, the hybridized primers and said
template are in a flow cell. In one embodiment, the imaging reagent
further comprises a radical scavenger. In one embodiment, the
radical scavenger is carnitine. In one embodiment, the fluorescence
quenching inhibitor is trolox. In one embodiment, the buffer is a
TRIS buffer. In one embodiment, the buffer is a HEPES buffer.
[0010] In one embodiment, the present invention contemplates an
imaging reagent comprising: i) a TRIS HCl buffer; ii) carnitine
ranging in concentration between approximately 5-50 mM; iii) trolox
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.
Definitions
[0011] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity but also
plural entities and also includes the general class of which a
specific example may be used for illustration. The terminology
herein is used to describe specific embodiments of the invention,
but their usage does not delimit the invention, except as outlined
in the claims.
[0012] The term "about" as used herein, in the context of any of
any assay measurements refers to +/-5% of a given measurement.
[0013] The term "imaging reagent" as used herein, refers to a
mixture of compounds that are capable of enhancing label emission
intensity and/or improving fluorophore detection by at least an
order of magnitude. While not intending to limit the invention to
any particular mechanism, it is believed that the herein described
mixtures enhance signal-to-noise ratios, or reduce photobleaching
and/or fluorophore "blinking." One class of compounds that are
useful in imaging reagents are fluorescence quenching
inhibitors.
[0014] The term "fluorescence quenching inhibitor" as used herein,
refer to a class of compounds that improve the signal quality of
fluorescent labels. Without being bound to any mechanim, it is
believed that such compounds work by reacting with oxidation
compounds that result in a quenching of the fluorescent signal by
non-specific photo-bleaching phenomenon. For example, one such
compound is trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid).
[0015] The term "mixture" or "cocktail" as used herein
interchangeably, refers to a plurality of compounds (generally in a
solution, such as a buffer solution) that together create an
imaging reagent for the purpose of detecting labeled nucleotide
analogues within a nucleotide sequence.
[0016] The term "photoprotective" as used herein, refers to an end
result of an imaging reagent mixture or cocktail that enhances
stability and storage shelf-life of fluorescent compounds. Without
being bound by theory, it is believed that they work by protecting
against: i) photo-bleaching of nucleotide fluorescent labels; ii)
signal quenching; and iii) radically-induced DNA photo-damage and
photo-scission.
[0017] The term "antioxidant compounds" as used herein, refers to a
molecule that inhibits a chemical reaction that can produce free
radicals, For example, many vitamins (e.g., vitamin E and vitamin
C), in addition to certain enzymes (catalase and superoxide
dismutase) are naturally occuring antioxidants. Other chemicals
also have these properties including, but not limited to, gentisic
acid, protocatechuic acid and/or protocatechuate ethyl ester.
[0018] The term "radical scavenger compound" as used herein, refers
to a molecule that remove or de-activate impurities and unwanted
reaction products, for example oxygen. While radical scavenger
compounds have an antioxidant end result, it is believed that they
function by a different mechanism than antioxidant compounds. For
example, one such radical scavenger compound includes, but is not
limited to, tocopherol, carnitine and/or naringenin. Even so, it is
known that some radical scavenger compounds have other biochemical
activities, for example, antioxidant activities and singlet oxygen
quenching.
[0019] The term "buffer" as used herein, refers to a mixture of
basic salts and a hydrogen exchange compound (either a weak acid or
a weak base) that can maintain a stable pH level over a wide range
of environmental conditions (e.g., temperature, salinity),
including changes in hydrogen ion concentration. For example, such
buffers may include, but are not limited to 4-(2-hydroxyethyl)
-1-piperazineethanesulfonic acid (HEPES) buffer and/or
tris(hydroxymethyl)-aminomethane (TRIS) buffer.
[0020] The term "linker" as used herein, refers to any molecule (or
collection of molecules) capable of attaching a label and/or
chemical moiety that is susceptible to cleavage. In one embodiment,
cleavage of the linker may produce toxic radical products. For
example, a linker may include, but is not limited to, a disulfide
linker and/or an azide linker.
[0021] The term "attached" as used herein, refers to any
interaction between a first molecule (e.g., for example, a nucleic
acid) and a second molecule (e.g., for example, a label molecule).
Attachment may be reversible or irreversible. Such attachment
includes, but is not limited to, covalent bonding, ionic bonding,
Van der Waals forces or friction, and the like.
[0022] "Nucleic acid sequence" and "nucleotide sequence" as used
herein refer to an oligonucleotide or polynucleotide, and fragments
or portions thereof, and to DNA or RNA of genomic or synthetic
origin which may be single- or double-stranded, and represent the
sense or antisense strand. Such nucleic acids may include, but are
not limited to, cDNA, mRNA or other nucleic acid sequences.
[0023] The term "an isolated nucleic acid", as used herein, refers
to any nucleic acid molecule that has been removed from its natural
state (e.g., removed from a cell and is, in a preferred embodiment,
free of other genomic nucleic acid).
[0024] In some embodiments, the present invention contemplates
hybridizing nucleic acid together. This requires some degree of
complementarity. As used herein, the terms "complementary" or
"complementarity" are used in reference to "polynucleotides" and
"oligonucleotides" (which are interchangeable terms that refer to a
sequence of nucleotides) related by the base-pairing rules. For
example, the sequence "C-A-G-T," is complementary to the sequence
"G-T-C-A." Complementarity can be "partial" or "total." "Partial"
complementarity is where one or more nucleic acid bases is not
matched according to the base pairing rules. "Total" or "complete"
complementarity between nucleic acids is where each and every
nucleic acid base is matched with another base under the base
pairing rules. The degree of complementarity between nucleic acid
strands has significant effects on the efficiency and strength of
hybridization between nucleic acid strands. This is of particular
importance in amplification reactions, as well as detection methods
which depend upon binding between nucleic acids.
[0025] The terms "homology" and "homologous" as used herein in
reference to nucleotide sequences refer to a degree of
complementarity with other nucleotide sequences. There may be
partial homology or complete homology (i.e., identity). A
nucleotide sequence which is partially complementary, i.e.,
"substantially homologous," to a nucleic acid sequence is one that
at least partially inhibits a completely complementary sequence
from hybridizing to a target nucleic acid sequence. The inhibition
of hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or Northern blot, solution hybridization and the like)
under conditions of low stringency. A substantially homologous
sequence or probe will compete for and inhibit the binding (i.e.,
the hybridization) of a completely homologous sequence to a target
sequence under conditions of low stringency. This is not to say
that conditions of low stringency are such that non-specific
binding is permitted; low stringency conditions require that the
binding of two sequences to one another be a specific (i.e.,
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target sequence which lacks even a
partial degree of complementarity (e.g., less than about 30%
identity); in the absence of non-specific binding the probe will
not hybridize to the second non-complementary target.
[0026] Low stringency conditions comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5 x SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4H2O and 1.85 g/l EDTA,
pH adjusted to 7.4 with NaOH), 0.1% SDS, 5x Denhardt's reagent {50x
Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5
g BSA (Fraction V; Sigma)} and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 5x SSPE, 0.1% SDS
at 42.degree. C. when a probe of about 500 nucleotides in length is
employed. Numerous equivalent conditions may also be employed to
comprise low stringency conditions; factors such as the length and
nature (DNA, RNA, base composition) of the probe and nature of the
target ( DNA, RNA, base composition, present in solution or
immobilized, etc.) and the concentration of the salts and other
components (e.g., the presence or absence of formamide, dextran
sulfate, polyethylene glycol), as well as components of the
hybridization solution may be varied to generate conditions of low
stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, conditions which promote
hybridization under conditions of high stringency (e.g., increasing
the temperature of the hybridization and/or wash steps, the use of
formamide in the hybridization solution, etc.) may also be
used.
[0027] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids using any
process by which a strand of nucleic acid joins with a
complementary strand through base pairing to form a hybridization
complex. Hybridization and the strength of hybridization (i.e., the
strength of the association between the nucleic acids) is impacted
by such factors as the degree of complementarity between the
nucleic acids, stringency of the conditions involved, the Tm of the
formed hybrid, and the G:C ratio within the nucleic acids.
[0028] As used herein the term "hybridization complex" refers to a
complex formed between two nucleic acid sequences by virtue of the
formation of hydrogen bounds between complementary G and C bases
and between complementary A and T bases; these hydrogen bonds may
be further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex may be formed
in solution (e.g., CO t or RO t analysis) or between one nucleic
acid sequence present in solution and another nucleic acid sequence
immobilized to a solid support (e.g., a nylon membrane or a
nitrocellulose filter as employed in Southern and Northern
blotting, dot blotting or a glass slide as employed in in situ
hybridization, including FISH (fluorescent in situ
hybridization)).
[0029] As used herein, the term "Tm " is used in reference to the
"melting temperature." The melting temperature is the temperature
at which a population of double-stranded nucleic acid molecules
becomes half dissociated into single strands. As indicated by
standard references, a simple estimate of the Tm value may be
calculated by the equation: Tm=81.5+0.41 (% G+C), when a nucleic
acid is in aqueous solution at 1M NaCl. Anderson et al.,
"Quantitative Filter Hybridization" In: Nucleic Acid Hybridization
(1985). More sophisticated computations take structural, as well as
sequence characteristics, into account for the calculation of
Tm.
[0030] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. "Stringency" typically occurs in a
range from about
[0031] Tm to about 20.degree. C. to 25.degree. C. below Tm. A
"stringent hybridization" can be used to identify or detect
identical polynucleotide sequences or to identify or detect similar
or related polynucleotide sequences. For example, when fragments
are employed in hybridization reactions under stringent conditions
the hybridization of fragments which contain unique sequences
(i.e., regions which are either non-homologous to or which contain
less than about 50% homology or complementarity) are favored.
Alternatively, when conditions of "weak" or "low" stringency are
used hybridization may occur with nucleic acids that are derived
from organisms that are genetically diverse (i.e., for example, the
frequency of complementary sequences is usually low between such
organisms).
[0032] As used herein, the term "amplifiable nucleic acid" is used
in reference to nucleic acids which may be amplified by any
amplification method. It is contemplated that "amplifiable nucleic
acid" will usually comprise "sample template."
[0033] As used herein, the term "sample template" or (more simply)
"template" refers to nucleic acid originating from a sample which
is analyzed for the presence of a target sequence of interest. In
contrast, "background template" is used in reference to nucleic
acid other than sample template which may or may not be present in
a sample. Background template is most often inadvertent. It may be
the result of carryover, or it may be due to the presence of
nucleic acid contaminants sought to be purified away from the
sample. For example, nucleic acids from organisms other than those
to be detected may be present as background in a test sample.
[0034] "Amplification" is defined as the production of additional
copies of a nucleic acid sequence and is generally carried out
using polymerase chain reaction. Dieffenbach C. W. and G. S.
Dveksler (1995) In: PCR Primer, a Laboratory Manual, Cold Spring
Harbor Press, Plainview, N.Y.
[0035] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and
4,683,202, herein incorporated by reference, which describe a
method for increasing the concentration of a segment of a target
sequence in a mixture of genomic DNA without cloning or
purification. The length of the amplified segment of the desired
target sequence is determined by the relative positions of two
oligonucleotide primers with respect to each other, and therefore,
this length is a controllable parameter. By virtue of the repeating
aspect of the process, the method is referred to as the "polymerase
chain reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified". With PCR, it is possible to amplify a single copy
of a specific target sequence in genomic DNA to a level detectable
by several different methodologies (e.g., hybridization with a
labeled probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of 32P-labeled
deoxynucleotide triphosphates, such as dCTP or dATP, into the
amplified segment). In addition to genomic DNA, any oligonucleotide
sequence can be amplified with the appropriate set of primer
molecules. In particular, the amplified segments created by the PCR
process itself are, themselves, efficient templates for subsequent
PCR amplifications.
[0036] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxy-ribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0037] As used herein, the term "probe" refers; to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, which is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labeled with any "reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0038] The term "label" or "detectable label" are used herein, to
refer to any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. Such labels include biotin for staining with
labeled streptavidin conjugate, magnetic beads (e.g.,
Dynabeads.RTM.), fluorescent dyes (e.g., fluorescein, texas red,
rhodamine, green fluorescent protein, and the like), radiolabels
(e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish
peroxidase, alkaline phosphatase and others commonly used in an
ELISA), and calorimetric labels such as colloidal gold or colored
glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)
beads. Patents teaching the use of such labels include, but are not
limited to, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all herein
incorporated by reference).
[0039] In a preferred embodiment, the label is typically
fluorescent and is linked to the base of the nucleotide. For
cytosine and thymine, the attachment is usually to the 5-position.
For the other bases, a deaza derivative is created and the label is
linked to a 7-position of deaza-adenine or deaza-guanine.
[0040] The labels contemplated in the present invention may be
detected by many methods. For example, radiolabels may be detected
using photographic film or scintillation counters, fluorescent
markers may be detected using a photodetector to detect emitted
light. Enzymatic labels are typically detected by providing the
enzyme with a substrate and detecting, the reaction product
produced by the action of the enzyme on the substrate, and
calorimetric labels are detected by simply visualizing the colored
label.
[0041] The term "luminescence" and/or "fluorescence", as used
herein, refers to any process of emitting electromagnetic radiation
(light) from an object, chemical and/or compound. Luminescence
and/or fluorescence results from a system which is "relaxing" from
an excited state to a lower state with a corresponding release of
energy in the form of a photon. These states can be electronic,
vibronic, rotational, or any combination of the three. The
transition responsible for luminescence can be stimulated through
the release of energy stored in the system chemically or added to
the system from an external source. The external source of energy
can be of a variety of types including, but not limited to,
chemical, thermal, electrical, magnetic, electromagnetic, physical
or any other type capable of causing a system to be excited into a
state higher than the ground state. For example, a system can be
excited by absorbing a photon of light, by being placed in an
electrical field, or through a chemical oxidation-reduction
reaction. The energy of the photons emitted during luminescence can
be in a range from low-energy microwave radiation to high-energy
x-ray radiation. Typically, luminescence refers to photons in the
range from UV to IR radiation.
BRIEF DESCRIPTION OF THE FIGURES
[0042] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0043] FIG. 1A-B presents a comparative workflow between a
conventional imaging buffer configuration and photoprotective
imaging buffer configuration.
[0044] FIG. 1A: An illustrative workflow for a conventional imaging
buffer kit configuration.
[0045] FIG. 1B: A photoprotective imaging buffer kit configuration
comprising new and improved Cleave and Imaging Buffer
Consumables.
[0046] FIG. 2 presents exemplary data showing that SBS metrics are
comparable between a conventional imaging buffer and a
Photoprotective imaging buffer 7B.
[0047] 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.
[0048] FIG. 3A: Run 8.3
[0049] FIG. 3B; Run 8.5
[0050] FIG. 3C; Run 8.6
[0051] FIG. 3D: Run 8.41 (retest)
[0052] 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.
[0053] FIG. 4A: Run 8.3
[0054] FIG. 4B; Run 8.5
[0055] FIG. 4C; Run 8.6
[0056] FIG. 4D: Run 8.41 (retest)
[0057] 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.
[0058] 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.
[0059] FIG. 6A: Photoprotective imaging buffer SC-P 9.
[0060] FIG. 6B: Photoprotective imaging buffer SC-P 9B.
[0061] FIG. 6C: Photoprotective imaging buffer SC-P 9C.
[0062] FIG. 7 presents exemplary data showing the effect of lower
ionic and aromatic compound concentrations in an imaging buffer on
nucleotide signal retention.
[0063] FIG. 8 presents exemplary data showing raw error rates in
sequencing runs using SC-P9C imaging reagent versus a baseline
IB.
[0064] 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.
[0065] FIG. 9A: Results using a HEPES buffer.
[0066] FIG. 9B: Results using a TRIS buffer.
[0067] 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).
[0068] 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).
[0069] 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).
[0070] 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).
[0071] 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).
[0072] FIG. 14A: SC-9 IB and SC9B IB versus reference IB.
[0073] FIG. 14B: SC-9D D3 versus reference IB.
[0074] 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).
[0075] FIG. 15A: SC-9 IB and SC9B IB versus reference IB.
[0076] FIG. 15B: SC-9D D3 versus reference IB.
[0077] 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
[0078] 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 buffer mixture of compounds as imaging reagents
to improve stability and storage of fluorescent compounds,
including but not limited to, nucleotides with fluorescent
labels.
[0079] 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.
[0080] 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.
I. Sequencing-By-Synthesis (SBS)
[0081] 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:
[0082] 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:
TABLE-US-00001 Component Concentration PNSE (% wt/vol) 0.005% Tris
.times. HC1 (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 (.mu.M) 3.83 N3-dTTP (.mu.M) 3.61 N3-dATP (.mu.M) 4.03
N3-dGTP (.mu.M) 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:
TABLE-US-00002 Component Concentration PNSE (% wt/vol) 0.005% Tris
.times. 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 (.mu.M)
21 N3-dTTP (.mu.M) 17 N3-dATP (.mu.M) 21 N3-dGTP (.mu.M) 2
*Alkylated free Cysteine
3) Wash solution 1 with a detergent (e.g., polysorbate 20) citrate
buffer (e.g., saline)
[0083] s4) Cleave Reagent: One cleaving solution composition may be
as follows:
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
[0084] 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
[0085] 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 .
III. Photoprotective Imaging Solutions
[0086] 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.
[0087] 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.
[0088] 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.
[0089] In one embodiment, the photoprotective cocktail comprises a
mixture (e.g., imaging reagent SC-P 7B) including the
components:
##STR00001##
[0090] In one embodiment, the photoprotective cocktail comprises a
mixture (e.g., imaging reagent SC-P 9C) including the
components:
##STR00002##
[0091] 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.RTM. 1.1 sequencing kit). In particular, carnitine has
been found 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).
[0092] 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.
[0093] 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.degree. C. storage conditions. See, FIG. 1. 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.
[0094] 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 D3 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).
[0095] 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.
TABLE-US-00004 TABLE 1 Comparative Sequencing Metrics: Conventional
IB (Baseline) versus C-P 7B IB. Sample Output Date Run GR ID (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 8.41 SP101x
1.904151381 428153.625 0.775358 50% Retest** Apr. 5, 2016 IB 7B 8.6
SP101x 1.87757536 440901.246 0.751118 49% Apr. 11, 2016 IB 7B 8.3
SP101x 2.052871269 444646.646 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 109.6339 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 112.4275 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
0.347992 0.098931 Apr. 14, 2016 31% 36% 0.5949944 115.0466 0.409185
0.157954 30% 36% 58% 111 0.399 0.149
[0096] 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.
[0097] 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
TABLE-US-00005 [0098] Tris 50 mM L-Carnitine 50 mM Trolox 15 mM
Protocatechuic acid 20 mM ethyl ester
or,
SC-P9B pH 8.5
TABLE-US-00006 [0099] Tris 50 mM L-Carnitine 15 mM Trolox 15 mM
Protocatechuic acid 20 mM ethyl ester
and,
SC-P9C pH 8.5
TABLE-US-00007 [0100] Tris 50 mM L-Carnitine 15 mM Trolox 15 mM
Protocatechuic acid ethyl 10 mM ester
[0101] A comparison of basic sequencing metrics was made between
these three SC-P 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.
TABLE-US-00008 TABLE 2 Comparison of Sequencing Metrics Between
SC-P Version 9 IBs And A Conventional IB (IB_Baseline). Output
Error Rate Run Name GR/Sample (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.
[0102] 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.
TABLE-US-00009 TABLE 3 Sequence Metrics Comparison Of SC-P9C To
Baseline IB On A BRCA Gene Panel Output % % % AVG Error % Run
Condition GR Run date Type (MFST) Beads/tile Live Mapped Polyclonal
RL (MFST) Perfect Lead Lag Baseline_BRCA 8.17 Apr. 15, 2016 HG19
1.14E+09 426294 47% 26% 41% 115.0 0.72% 58% 0.513 0.060 BRCA
Baseline_BRCA 8.17 Apr. 15, 2016 HG19 1.17E+09 424933 45% 27% 38%
114.0 0.74% 57% 0.466 0.091 BRCA Prototype 9C_BRCA 8.26 Apr. 15,
2016 HG19 1.09E+09 428096 44% 26% 36% 110.0 0.78% 53% 0.477 0.061
BRCA Prototype 9C_BRCA 8.26 Apr. 15, 2016 HG19 1.14E+09 422093 45%
26% 36% 112.0 0.79% 54% 0.446 0.084 BRCA Prototype 9C_BRCA 8.24
Apr. 15, 2016 HG19 1.15E+09 427948 43% 25% 35% 110.0 0.82% 51%
0.444 0.072 BRCA Prototype 9C_BRCA 8.24 Apr. 15, 2016 HG19 1.18E+09
424163 43% 26% 35% 113.0 0.87% 52% 0.454 0.082 BRCA
A comparision 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 tile
protocol on a DHMG02 BRCA gene panel. See, Table 4.
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 Output Run Condition GR Sample ID Run date Type Cycles
(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 % % % Error % Run
Condition Live Mapped Polyclonal AVG RL (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, FIG. 9A and 9B.
[0103] 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.
[0104] 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:
Prototype 9
TABLE-US-00011 [0105] 50 mM Tris buffer pH 8.5 50 mM L-Carnitine 15
mM Trolox 20 mM Protocatechuic Acid Ethyl Ester
Prototype 9B
TABLE-US-00012 [0106] 50 mM Tris buffer pH 8.5 15 mM L-Carnitine 15
mM Trolox 20 mM Protocatechuic Acid Ethyl Ester
[0107] Prototype 9D
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 D3 reagent and the
photoprotective D3 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.
TABLE-US-00014 TABLE 5 ADAM Results From Carnitine Concentration
Analysis Output Run Condition GR Sample ID Run date Type Cycles
(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_Proto9D_BRCA_AGR 8.26 1_DHMGO Apr. 19, 2016 HG19
BRCA 125 9.85E+08 428784.6 44.9% IB_Proto9D_BRCA_AGR 8.26 2_DHMGO
Apr. 19, 2016 HG19 BRCA 125 1.02E+09 430237.1 45.6% Run Condition %
BC % Mapped AVG RL 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% 100.8
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 camitine IB_SC-P9_FC2 75.0% 33.1%
97.0 0.56% 68.3% 0.405 0.122 50 mM camitine IB_SC-P9B_FC1 73.3%
31.7% 98.6 0.55% 69.0% 0.389 0.126 15 mM camitine IB_SC-P9B_FC2
73.5% 31.9% 99.2 0.54% 69.2% 0.389 0.123 15 mM camitine
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_Proto9D_BRCA_AGR 71% 27% 97.2 0.69% 63.6%
0.425 0.101 5 mM camitine IB_Proto9D_BRCA_AGR 71% 27% 99.0 0.68%
63.5% 0.425 0.111 5 mM camitine
[0108] 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 D3 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 D3 reagent (15 mM carnitine) had raw error rates similar
to the reference D3 reagent. See, FIG. 14A. The SC-9D D3 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
D3 reagent (15 mM carnitine) had read length distributions that
were similar to the reference D3 reagent. See, FIG. 15A. The SC-9D
D3 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.
[0109] 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
[0110] 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 Tris HCl 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
[0111] 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. [0112] 50
mM Tris buffer: 121.4 g/mol =1.21g (Sigma: Cat #T1378-1kg) [0113]
50 mM L Carnitine: 197.66 g/mol =1.98g (Sigma: Cat #CO283-100G)
[0114] 15 mM Trolox: 250.29 g/mol =0.75g (Sigma: Cat #238813-5G)
[0115] 50 mM Gentisic Acid 176.1 g/mol =1.76 g (Sigma: G5129-10G)
[0116] 1. Dissolve Tris Base in 180 mL milliQ water. [0117] 2. Add
Trolox to the Tris buffer from Step 1. [0118] 3. Add L Carnitine to
the above solution. [0119] 4. Add Gentisic acid sodium salt hydrate
and dissolve. [0120] 5. Checked pH: .about.4 [0121] 6. Adjusted pH
to 7.8 with 10M NaOH solution. [0122] 7. Bring total volume to 200
mL with milliQ water. [0123] 8. Filter sterilize. [0124] 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
[0125] The following example describes the preparation of
approximately two hundred (200) milliliters of imaging reagent.
[0126] 50 mM Tris buffer: 121.4 g/mol =0.607g (Sigma: Cat
#T1378-1kg) [0127] 15 mM L Carnitine: 197.66 g/mol =0.296g (Sigma:
Cat #CO238-100G) [0128] 15 mM Trolox: 250.29 g/mol =0.375g (Sigma:
Cat #238813-5G) [0129] 10 mM Protocatechuic Acid Ethyl Ester 182.17
g/mol =182.17 mg milligrams (Sigma: Cat #E24859-5G) [0130] 1.
Dissolve Tris Base in 75 mL milliQ water. [0131] 2. Add Trolox and
dissolve completely [0132] 3. Added Protocatechuic Acid [0133] 4.
Add L Carnitine and dissolve completely. [0134] 5. Checked pH:
[0135] 6. Adjusted pH to 8.5 with 10M NaOH. [0136] 7. Optional:
Sonicate until well mixed. [0137] 8. Bring total volume to 100 mL
with milliQ water. [0138] 9. Filter sterilize.
EXAMPLE IV
Composition and Formulation of Photoprotective Imaging Reagent
9CV2H
[0139] The following example describes the preparation of
approximately one hundred (100) milliliters of imaging reagent.
[0140] 100 mM HEPES: 238.3 gr/mole=2.39g (Sigma: Cat #H4034) [0141]
15 mM L Carnitine: 197.66 g/mol=0.296g (Sigma: Cat #CO283-100G)
[0142] 15 mM Trolox: 250.29 g/mol=0.375g (Sigma: Cat #238813-5G)
[0143] 10 mM Protocatechuic Acid Ethyl Ester 182.17 g/mol=0.182g
(Sigma: Cat #E24859-5G)
1Liter 100 mM HEPES
23.9 gr HEPES
[0144] Dissolve in MilliQ water- final volume 1 Liter pH was
adjusted to 7.0. [0145] 1. Obtain 75 mL HEPES buffer (see below).
[0146] 2. Add Trolox and dissolve completely [0147] 3. Added
Protocatechuic Acid. [0148] 4. Add L Carnitine and dissolve
completely. [0149] 5. Checked pH: .about.5 [0150] 6. Adjusted pH to
7.5 with 10M NaOH. [0151] 7. Optional: Sonicate until well mixed.
[0152] 8. Bring total volume to 100 mL with milliQ water. [0153] 9.
Filter sterilize.
* * * * *