U.S. patent application number 17/445286 was filed with the patent office on 2021-12-16 for compositions and methods for analyzing modified nucleotides.
This patent application is currently assigned to New England Biolabs, Inc.. The applicant listed for this patent is New England Biolabs, Inc.. Invention is credited to Theodore B. Davis, Laurence Ettwiller, Shengxi Guan, Lana Saleh, Zhiyi Sun, Romualdas Vaisvila.
Application Number | 20210388433 17/445286 |
Document ID | / |
Family ID | 1000005808234 |
Filed Date | 2021-12-16 |
United States Patent
Application |
20210388433 |
Kind Code |
A1 |
Vaisvila; Romualdas ; et
al. |
December 16, 2021 |
Compositions and Methods for Analyzing Modified Nucleotides
Abstract
A method for identifying any of the presence, location and
phasing of modified cytosines (C) in long stretches of nucleic
acids is provided. In some embodiments, the method may comprise (a)
reacting a first portion of a nucleic acid sample containing at
least one C and/or at least one modified C with a DNA
glucosyltransferase and a cytidine deaminase to produce a first
product and/or reacting a second portion of the sample with a
dioxygenase, optionally a DNA glucosyltransferase and a cytidine
deaminase to produce a second product and; (b) comparing the
sequences from the first and optionally the second product obtained
in (a), or amplification products thereof, with each other and/or
an untreated reference sequence to determine which Cs in the
initial nucleic acid fragment are modified. A modified TET
methylcytosine dioxygenase with improved efficiency compared to
unmodified TET2 at converting methylcytosine to
carboxymethylcytosine is also provided.
Inventors: |
Vaisvila; Romualdas;
(Ipswich, MA) ; Sun; Zhiyi; (Gloucester, MA)
; Guan; Shengxi; (Stoneham, MA) ; Saleh; Lana;
(Hamilton, MA) ; Ettwiller; Laurence; (Beverly,
MA) ; Davis; Theodore B.; (Boxford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New England Biolabs, Inc. |
Ipswich |
MA |
US |
|
|
Assignee: |
New England Biolabs, Inc.
Ipswich
MA
|
Family ID: |
1000005808234 |
Appl. No.: |
17/445286 |
Filed: |
August 17, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16217819 |
Dec 12, 2018 |
11124825 |
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17445286 |
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15441431 |
Feb 24, 2017 |
10227646 |
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16217819 |
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PCT/US16/59447 |
Oct 28, 2016 |
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15441431 |
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62325626 |
Apr 21, 2016 |
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62300396 |
Feb 26, 2016 |
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62271679 |
Dec 28, 2015 |
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62257284 |
Nov 19, 2015 |
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62248872 |
Oct 30, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6858 20130101; C12Q 1/6827 20130101; C12Y 114/11 20130101;
C12Q 1/6872 20130101; C12Q 2600/154 20130101; C12Q 1/6806 20130101;
C12N 9/0071 20130101 |
International
Class: |
C12Q 1/6858 20060101
C12Q001/6858; C12Q 1/6806 20060101 C12Q001/6806; C12Q 1/6827
20060101 C12Q001/6827; C12N 9/02 20060101 C12N009/02; C12Q 1/6869
20060101 C12Q001/6869; C12Q 1/6872 20060101 C12Q001/6872 |
Claims
1.-8. (canceled)
9. A method comprising: a. determining the location of
substantially all modified cytosines in a test nucleic acid
fragment having a length of at least 2 kb, to obtain a pattern of
cytosine modification; b. comparing the pattern of cytosine
modification in the test nucleic acid fragment with the pattern of
cytosine modification in a reference nucleic acid fragment; and c.
identifying a difference in the pattern of cytosine modification in
the test nucleic acid fragment in cis relative to the reference
nucleic acid fragment.
10. The method of claim 9, wherein the method comprises comparing
the pattern of cytosine modification for the test nucleic acid
fragment, wherein the test nucleic acid is linked, in cis, to a
gene in a transcriptionally active state to the pattern of cytosine
modifications in the same intact nucleic acid fragment that is
linked, in cis, to the same gene in a transcriptionally inactive
state.
11. The method of claim 9, wherein transcription of the gene is
correlated with a disease or condition.
12. The method of claim 9, wherein the method comprises comparing
the pattern of cytosine modification for a nucleic acid fragment
from a patient that has a disease or condition with the pattern of
cytosine modification in the same nucleic acid fragment from a
patient that does not have the disease or condition.
13. The method of claim 9, wherein the method comprises comparing
the pattern of cytosine modification or lack of modification for a
nucleic acid fragment from a patient is undergoing a treatment with
the pattern of cytosine modification or lack of modification in the
same intact nucleic acid fragment from a patient that has not been
treated with the agent.
14. A method according to claim 9, wherein the difference in the
pattern of cytosine modification in the test nucleic acid fragment
relative to the reference nucleic acid fragment corresponds to a
variant single nucleotide polymorphism, an insertion/deletion or a
somatic mutation associated with a pathology.
15. A method according to claim 9, wherein identifying a difference
in the pattern of cytosine modification in the test nucleic acid
fragment relative to the reference nucleic acid fragment further
comprises identifying a difference in the pattern of unmodified
cytosine in cis or a difference in the pattern of modified
cytosines in cis.
Description
CROSS REFERENCE
[0001] This application is a divisional of U.S. application Ser.
No. 15/441,431 filed Feb. 24, 2017 which is a continuation-in-part
of International Application No. PCT/US16/59447 filed Oct. 28, 2016
which claims the benefit of US Provisional Application Nos:
62/248,872, filed Oct. 30, 2015, 62/257,284, filed Nov. 19, 2015
and 62/271,679, filed Dec. 28, 2015, which applications are
incorporated by reference herein.
BACKGROUND
[0002] The ability to phase modified nucleotides (e.g., methylated
or hydroxymethylated nucleotides) in a genome (i.e., determine
whether two or more modified nucleotides are linked on the same
single DNA molecule or on different DNA molecules) can provide
important information in epigenetic studies, particularly for
studies on imprinting, gene regulation, and cancer. In addition, it
would be useful to know which modified nucleotides are linked to
sequence variations.
[0003] Modified nucleotides cannot be phased using conventional
methods for investigating DNA modification because such methods
typically involve bisulfite sequencing (BS-seq). In BS-seq methods,
a DNA sample is treated with sodium bisulfite, which converts
cytosines (C) to uracil (U), but methylcytosine (.sup.mC) remains
unchanged. When bisulfite-treated DNA is sequenced, unmethylated C
is read as thymine (T), and .sup.mC is read as C, yielding
single-nucleotide resolution information about the methylation
status of a segment of DNA.
[0004] However, sodium bisulfite is known to fragment DNA (see,
e.g., Ehrich M 2007 Nucl. Acids Res. 35:e29), making it impossible
to determine whether modified nucleotides are linked on the same
DNA molecule. Specifically, it is impossible for nucleotide
modifications to be phased in the same way that sequence variants
(e.g., polymorphisms) are phased because those methods require
intact, long molecules.
[0005] Moreover, bisulfite sequencing displays a bias toward
cytosine (C) adjacent to certain nucleotides and not others. It
would be desirable to remove the observed bias.
SUMMARY
[0006] Provided herein are methods for phasing modified nucleotides
that do not require bisulfite treatment.
[0007] Further, such methods can be implemented in a way that
distinguishes between .sup.mC and hydroxymethylcytosine (.sup.hmC)
or C, formylcytosine (.sup.fC) and carboxylcytosine (.sup.caC),
providing significant advantages over conventional methods.
[0008] This disclosure provides, among other things, compositions
and methods to detect and phase methylation and/or
hydroxymethylation of nucleotides or unmodified nucleotides in cis
or trans at a single molecule level in long stretches of DNA. In
various embodiments, glucosylation and oxidation reactions overcome
the observed inherent deamination of .sup.hmC and .sup.mC by
deaminases. Deaminases converts .sup.mC to T and C to U while
glucosylhydroxymethylcytosine (.sup.ghmC) and .sup.CaC are not
deaminated. Examples of deaminases include APOBEC (apolipoprotein B
mRNA editing enzyme, catalytic polypeptide-like). Embodiments
utilize enzymes that have substantially no sequence bias in
glycosylation, oxidation and deamination of cytosine. Moreover,
embodiments provide substantially no non-specific damage of the DNA
during the glycosylation, oxidation and deamination reactions.
[0009] In some embodiments, a DNA glucosyltransferase (GT) for
example beta glucosyltransferase (BGT) is utilized for
glucosylating .sup.hmC to protect this modified base from
deamination. However, a person of ordinary skill in the art will
appreciate that other enzymatic or chemical reactions may be used
for modifying the .sup.hmC to achieve the same effect. One
alternative example provided herein is the use of Pyrrolo-dC for
protecting cytosine from being converted to uracil by cytidine
deaminase.
[0010] In general, in one aspect, methods for detecting nucleic
acid (NA) methylation are provided that include subjecting the NA
to enzymatic glucosylation, enzymatic oxidation and enzymatic
deamination where an unmodified C is converted to a U, .sup.mC is
converted to T, an .sup.hmC that is glucosylated (.sup.ghmC) and
remains C and a modified C that is oxidized to .sup.caC remain C.
The majority of modified C are predicted to be .sup.mC. For some
diagnostic purposes, differentiating between .sup.mC and .sup.hmC
is not required. Accordingly, it is sufficient to utilize a single
pathway of oxidation and glucosylation followed by deamination.
Where it is desirable to distinguish .sup.mC from .sup.hmC, this
can be achieved by a performing two different reactions on two
aliquots of the same sample and subsequently comparing the
sequences of the DNA obtained. One reaction utilizes a GT and a
cytidine deaminase while a second reaction utilizes a
methylcytosine dioxygenase and a cytidine deaminase. It has been
found here that the presence of GT in a reaction with a
methylcytosine deoxygenase results in an outcome which shows an
improved conversion rate (greater than 97%, 98% or 99% conversion,
preferably at least 99%) of modified bases and more accurate
mapping than would otherwise be possible. Methylcytosine
dioxygenase variants are described herein which catalyze the
conversion of the .sup.mC to .sup.hmC to .sup.fC and then .sup.caC
with little or no bias caused by neighboring nucleotides. These and
other improved properties of such variants are also described
herein. Methods using enzymes described herein utilizing phasing or
other sequencing methods are more time and sample efficient and
provide improved accuracy for diagnostic sequencing of .sup.mC and
other modified nucleotides.
[0011] In each of these methods, it is desirable to compare the
product of the enzyme reactions with each other and/or an unreacted
sequence. Comparing sequences can be achieved by hybridization
techniques and/or by sequencing. Prior to comparing sequences, it
may be desirable to amplify the NA using PCR or isothermal methods
and/or clone the reacted sequence.
[0012] The NA fragments being analyzed may be DNA, RNA or a hybrid
or chimera of DNA and RNA. The NA fragments may be single stranded
(ss) or double stranded (ds). The NA fragments may be genomic DNA
or synthetic DNA.
[0013] The size of the fragments may be any size but for
embodiments of the present invention that utilize single molecule
sequencing, fragment sizes that are particularly advantageous are
greater than 1 Kb, 2 Kb, 3 kb, 4 kb, 5 kb, 6 Kb, 7 Kb or larger
(for example, preferably greater than 4 kb) with no theoretical
limitation on the upper size although the upper size of the
fragment may be limited by the polymerase in the amplification step
commonly used prior to sequencing if amplification is needed.
[0014] In some cases, the sequences obtained from the reactions are
compared with a corresponding reference sequence to determine: (i)
which Cs are converted into a U in the first product for
differentiating a .sup.mC from a .sup.hmC; and (ii) which Cs are
converted to a U for differentiating an unmodified C from a
modified C in the optional second product. In these embodiments,
the reference sequence may be a hypothetical deaminated sequence, a
hypothetical deaminated and PCR amplified sequence or a
hypothetical non-deaminated sequence for example.
[0015] In any embodiment, the first and second products may be
amplified prior to sequencing. In these embodiments, any U's in the
first and second products may be read as T's in the resultant
sequence reads.
[0016] In any embodiment, the methylcytosine dioxygenase may
convert .sup.mC and .sup.hmC to .sup.caC so that cytidine deaminase
cannot deaminate .sup.mC or .sup.hmC. The methylcytosine
dioxygenase may be a TET protein that enzymatically converts
modified Cs to .sup.caC.
[0017] In any embodiment, the GT may be a DNA
.beta.-glucosyltransferase (.beta.GT) or
.alpha.-glucosyltransferase (aGT) that forms .sup.ghmC so that
substantially no .sup.hmC is deaminated by the cytidine
deaminase.
[0018] In any embodiment, the NA sample may contain at least one
CpG island. In another embodiment, the NA may include at least two
modified Cs with nucleotide neighbors selected from CpG, CpA, CpT
and CpC.
[0019] In any embodiment, the method may comprise determining the
location of the .sup.mC and/or .sup.hmC on a ss of the NA where the
NA is ds.
[0020] In any embodiment, the NA is a fragment of genomic DNA and,
in some cases, the NA may be linked to a transcribed gene (e.g.,
within 50 kb, within 20 kb, within 10 kb, within 5 kb or within 1
kb) of a transcribed gene.
[0021] The method summarized above may be employed in a variety of
applications. A method for sample analysis is provided. In some
embodiments, this method may comprise one or more of the following
steps: (a) determining the location of all modified Cs in a test NA
fragment to identify a pattern for the modified C; (b) comparing
the pattern of C modifications in the test NA fragment with the
pattern of C modifications in a reference NA; (c) identifying a
difference in the pattern of cytosine modifications in the test NA
fragment relative to the reference NA fragment; and (d) determining
a pattern of .sup.hmC in the test NA fragment.
[0022] In some embodiments, this method may comprise comparing the
pattern of C modification or unmodified C for a NA fragment that is
linked, in as, to a gene in a transcriptionally active state to the
pattern of C modifications in the same intact NA fragment that is
linked, in as, to the same gene in a transcriptionally inactive
state. In these embodiments, the level of transcription of the gene
may be correlated with a disease or condition.
[0023] In some embodiments, this method may comprise comparing the
pattern of cytosine modification for a NA fragment from a patient
that has a disease or condition with the pattern of C modification
in the same NA fragment from a patient that does not have the
disease or condition. In other embodiments, the method may comprise
comparing the pattern of cytosine modification for a NA fragment
from a patient is undergoing a treatment with the pattern of C
modification in the same intact NA fragment from a patient that has
not been treated with the agent. In another embodiment, detected
differences in the pattern of C modification in the test NA
fragment relative to the reference NA fragment corresponds to a
variant single nucleotide polymorphism, an insertion/deletion or a
somatic mutation associated with a pathology.
[0024] A variety of compositions are also provided. In some
embodiments, the composition may comprise a NA, wherein the NA
comprises: a) G, A, T, U, C; b) G, A, T, U, .sup.caC and no C
and/or C) G, A, T, U and .sup.ghmC and no C and/or G, A, T, U,
.sup.caC and .sup.ghmC and no C. In some embodiments, the
composition may further comprise a cytidine deaminase or mutant
thereof (as described in U.S. Pat. No. 9,121,061), or a
methylcytosine dioxygenase or mutant thereof as described
below.
[0025] A kit is also provided. In some embodiments, the kit may
comprise a GT, a methylcytosine dioxygenase e.g., a mutant
methylcytosine dioxygenase (TETv as described below) and a cytidine
deaminase, as well as instructions for use. As would be apparent,
the various components of the kit may be in separate vessels.
[0026] In general, in one aspect, a protein is described that
includes an amino acid sequence that is at least 90% identical to
SEQ ID NO:1; and contains SEQ ID NO:2. In one aspect, the protein
is a fusion protein that includes an N-terminal affinity binding
domain. The protein may have methylcytosine dioxygenase activity
where the methylcytosine deoxygenase activity is similarly
effective for NCA, NCT, NCG and NCC in a target DNA. The protein
may be employed in any method herein.
[0027] In any embodiment, the protein may be a fusion protein. In
these embodiments, the variant protein may comprise an N-terminal
affinity binding domain.
[0028] Also provided by this disclosure is a method for modifying a
naturally occurring DNA containing one or more methylated C. In
some embodiments, this method may comprise combining a sample
comprising the DNA with a variant methylcytosine dioxygenase to
make a reaction mix; and incubating the reaction mix to oxidize the
methylated cytosine in the DNA.
[0029] In some embodiments, the reaction mix may further comprising
analyzing the oxidized sample, e.g., by sequencing or mass
spectrometry.
[0030] In some embodiments, the reaction mix may further comprise a
GT.
[0031] In some embodiments, the method may be done in vitro, in a
cell-free reaction.
[0032] In some embodiments, the method may be done in vitro, e.g.,
in cultured cells.
[0033] The above-summarized variant methylcytosine dioxygenase can
be used as a methylcytosine dioxygenase in any of the methods,
compositions or kits described below.
[0034] In general in one aspect, a method is provided for
determining the location of modified cytosines in a nucleic acid
fragment, that includes: (a) reacting a nucleic acid sample
containing at least one C and/or at least one modified C with a
methylcytosine dioxygenase and a DNA glucosyltransferase in a
single buffer either together or sequentially; (b) reacting the
product of (a) with a cytidine deaminase; and (c) comparing the
sequences obtained in (a), or amplification products thereof, with
an untreated reference sequence to determine which Cs in the
initial nucleic acid fragment are modified. In one aspect, the
methylcytosine dioxygenase is an amino acid sequence that is at
least 90% identical to SEQ ID NO:1; and contains the amino acid
sequence of SEQ ID NO:2.
BRIEF DESCRIPTION OF THE FIGURES
[0035] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the office upon
request and payment of the necessary fee.
[0036] Certain aspects of the following detailed description are
best understood when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to scale. On the contrary,
the dimensions of the various features are arbitrarily expanded or
reduced for clarity. Included in the drawings are the following
figures:
[0037] FIG. 1A shows a schematic diagram of a method for protecting
modified Cs from deamination by a cytidine deaminase and a .sup.mC
dioxygenase, for example a TET enzyme such as TETv, that converts
.sup.mC and .sup.hmC (not C) to .sup.caC that are insensitive to
deamination. After methylcytosine dioxygenase treatment,
deamination of unmodified C only occurs resulting in its
replacement by U. From left to right: SEQ ID NO.:20, SEQ ID NO. 20,
SEQ ID NO. 21.
[0038] FIG. 1B shows a second method for protecting .sup.hmC but
not .sup.mC from deamination by APOBEC enzyme. Here .sup.hmc is
glucosylated using a .beta.GT for example T4-.beta.GT or aGT for
example T4-.alpha.GT. C and .sup.mC are modified by a cytidine
deaminase (e.g. deaminase) to a U and a T respectively. From left
to right: SEQ ID NO:20, SEQ ID NO:20, SEQ ID NO:22.
[0039] FIG. 1C is a table showing readouts of bases of a genomic
sample after PCR amplification and Sanger sequencing or NGS
sequencing.
[0040] FIG. 2A-2B shows the methylation and hydroxymethylation
status of mouse genomic DNA.
[0041] FIG. 2A shows the distribution of .sup.mC and .sup.hmC at a
single locus (locus size: 1078 bp) of mouse fibroblast NHI/3T3
genomic DNA following methylcytosine dioxygenase (here TETv) and
cytidine deaminase treatment (according to FIG. 1A).
[0042] FIG. 2B shows the distribution of .sup.hmC at the same locus
as FIG. 3A after GT (here .beta.GT) and cytidine deaminase
treatment (according to FIG. 1B).
[0043] FIG. 2C is a summary of LC-MS data of methylation status of
a locus in genomic DNAs of mouse fibroblasts.
[0044] FIG. 3A-3E shows that ss DNA is not damaged during
preparation and analysis using TETv and/or .beta.GT and cytidine
deaminase in contrast to methods that use conventional bisulfite
treatment (for bisulfite method see for example, Flolmes, et al.
PloS one 9, no. 4 (2014): e93933).
[0045] FIG. 3A shows results obtained with .beta.GT and cytidine
deaminase. Six different fragment sizes (388 bp, 731 bp, 1456 bp,
2018 bp, 3325 bp, and 4229 bp) were analyzed after treatment with a
cytidine deaminase and .beta.GT. Full length fragments in each size
category were amplified. No fragmentation was observed.
[0046] FIG. 3B shows results obtained with TETv and cytidine
deaminase. 6 different fragment sizes (388 bp, 731 bp, 1456 bp,
2018 bp, 3325 bp, and 4229 bp) were analyzed after treatment with a
cytidine deaminase and .beta.GT. Full length fragments in each size
category were amplified. No fragmentation was observed.
[0047] FIG. 3C shows results obtained with bisulfite converted DNA.
6 different fragment sizes (388 bp, 731 bp, 1456 bp, 2018 bp, 3325
bp, and 4229 bp) were analyzed after bisulfite treatment. Full
length fragments in each size category were amplified. When
bisulfite converted DNA was amplified, only the two smallest
fragments were obtained because of the breakdown of the larger
fragments by the bisulfite method.
[0048] FIG. 3D shows results obtained with the primers for 5030 bp
amplicon, and 5378 bp amplicon after treating DNA before
amplification with T4-.beta.GT (.sup.hmC detection) or TETv
(.sup.mC+.sup.hmC detection), and cytidine deaminase (see FIGS. 1A
and 1B). Each amplification is shown in triplicate. No
fragmentation was observed.
[0049] FIG. 3E shows that that a 15 kb fragment of ss DNA
containing .sup.mC/.sup.hmC is not damaged during preparation and
analysis using TETv/.beta.GT/cytidine deaminase enzymes in contrast
to methods that use conventional bisulfite treatment. The light
blue line represents the denatured ss DNA of the 15 kb fragment
which is also the control. The red line is APOBEC deamination on
glucosylated DNA. The dark blue is DNA deamination on TETv oxidized
DNA. And the green is bisulfite treated DNA.
[0050] FIGS. 4A and 4B shows that cytidine deaminase does not
deaminate the modified base-Pyrrolo-dC (Glen Research, Sterling,
Va.). This modified base can be used in Illumina NGS library
construction to protect C in the adapters ligated to the ends of
DNA fragments in the library from deamination prior to cytidine
deaminase treatment.
[0051] FIG. 4A shows the results of treating oligonucleotide
(5'-ATAAGAATAGAATGAATXGTGAAATGAA TATGAAATGAATAGTA-3', X=Pyrrolo-dC,
SEQ ID NO:4) with cytidine deaminase at 37.degree. C. for 16 hours
(upper line (black)). The control (lower line (grey)) is untreated
SEQ ID NO:4. No difference was observed between the sample and the
control confirming that cytidine deaminase does not deaminate
Pyrrolo-dC.
[0052] FIG. 4B shows a chromatogram (LC-MS) of an adaptor
containing Pyrrole dC, with the following sequence, where
X=Pyrrolo-dC.5'/5Phos/GATXGGAAGAGXAXAXGTXTGAAXTXXAGTX/deoxyU/AXAXTXTTTXXX-
TAXAXGAXGXTXTTXXGATCT (SEQ ID NO:5). The LC-MS chromatogram
confirms that all C's are replaced by Pyrrolo-dC, with no trace of
contaminated Cs.
[0053] FIG. 5 shows that the method described in Example 4 that
provides sequences from Next generation sequencing (NGS) using an
Illumina platform as an example of Deaminase-seq provides superior
conversion efficiency compared with BS-seq. Unmethylated lambda DNA
was used as a negative control to estimate the non-conversion error
rate (methylated C calls/total C calls). CD.sup.mC reaction (left
slashes) has the smallest error rate of 0.1% for both CpG and CH
(H=A,C,T) context. Bisulfite conversion using Zymo kit (right
slashes) has 3 times higher error rate than the method shown in
FIGS. 1A and 1B (0.4%), and bisulfite conversion by Qiagen (white)
has even higher error rate of 1.6% for CpG context and 1.5% for CH
context.
[0054] FIG. 6A-6D shows that Deaminase-seq displays no systematic
sequence preference while BS-seq generates a significant amount of
conversion errors most notably in a CA context. Pie charts depict
the numbers and percentages of false positive methylation calls in
each C dinucleotide context in the unmethylated lambda genome by
different methods.
[0055] FIG. 6A shows a pie chart of wild type lambda genome as a
control with the naturally occurring distribution of CT, CA, CG and
CC.
[0056] FIG. 6B shows the representation of .sup.mC in a lambda
genome where every C has been methylated using Deaminase-seq. The
observed distribution matches that found in FIG. 6A.
[0057] FIG. 6C shows the representation of .sup.mC in a lambda
genome where every C has been methylated using BS-seq (Qiagen). The
observed distribution is not consistent with that found in FIG.
6A.
[0058] FIG. 6D shows the representation of .sup.mC in a lambda
genome where every C has been methylated using BS-seq (Zymo). The
observed distribution is not consistent with that found in FIG.
6A.
[0059] FIG. 7 shows that Deaminase-seq (Illumina) covered more CpG
sites and detected more methylated CpG sites than both BS-seq
libraries using the same library analysis and the same number of
sequencing reads demonstrating that Deaminase-seq is a more
efficient and cost effective method than BS-seq.
[0060] FIG. 8A-8C shows that Deaminase-seq provides an even
genome-wide sequence coverage in the mouse genome from Illumina
generated reads of overlapping fragments. Three histograms of CpG
coverage are shown where the 3 methods have the same mean
(5.times.) and median (4.times.) sequencing depth for CpG sites.
However, Deaminase-seq has fewer outliers (sites with very low or
very high copy numbers) when compared with BS-seq kits from Zymo
and Qiagen. Three data sets are shown in which, library size
normalized.
[0061] FIG. 8A shows the distribution of reads for DNA
Deaminase-seq.
[0062] FIG. 8B shows the distribution of reads for BS-seq
(Qiagen).
[0063] FIG. 8C shows the distribution of reads for BS-seq
(Zymo).
[0064] FIG. 9 shows that Deaminase-seq provides higher coverage in
CpG islands than BS-seq for the same number of sequencing reads,
Deaminase-seq gives nearly 2 times as much coverage as BS-seq in
the CpG islands.
[0065] FIG. 10 provides a loci specific map of .sup.hmC on a
genomic fragment from mouse chromosome 8. Deaminase-seq (FIGS. 1A
and 1B) accurately detects .sup.hmC of large fragments (5 Kb) at
base resolution enabling phasing of DNA modifications and phase DNA
modifications together with other genomic features such as SNPs or
variants.
[0066] FIG. 11A-11B shows a .sup.mC and .sup.hmC profile at
single-molecule level across the 5.4 kb region generated by PacBio
sequencing. Each row represents one DNA molecule. Each CpG site in
the 5.4 kb region was represented by a dot. C modification states
were denoted by color.
[0067] FIG. 11A shows that the present method can be used to phase
.sup.mC (red=methylated; blue=unmethylated).
[0068] FIG. 11B shows that the present method can be used to phase
.sup.hmC (red=hydroxymethylated and blue=unmodified).
[0069] FIG. 12A shows an activity comparison of mouse TET2
catalytic domain (TETcd; SEQ ID NO:3) with TETv (SEQ ID NO:1) on
sheared 3T3 genomic DNA.
[0070] FIG. 12B shows activity of TETv on ss and ds genomic (3T3)
DNA is similar.
[0071] FIG. 13 shows that TETv exhibits very low sequence bias and
is context independent for .sup.mC as demonstrated for 5 cell lines
(Arabidopsis, rice, M.Fnu4FI, E14 and Jurkat).
[0072] FIG. 14 shows that TETv does not degrade DNA as determined
from the preservation of supercoiled DNA after enzyme treatment.
Lane 1 is a size ladder. Lane 2 is substrate plasmid only, Lane 3
is supercoiled plasmid+323 pmol of TETv; Lane 4 is supercoiled
plasmid+162 pmol TETv; Lane 5 is supercoiled plasmid+162 pmol TETv;
Lane 6 is Substrate plasmid+323 pmol TETv+BamHI+Mspl; Lane 7 is
Substrate plasmid+162 pmol TETv+BamHI+Mspl; and Lane 8 is Substrate
plasmid+BamHI+Mspl.
[0073] FIG. 15 shows that APOBEC3A can substantially completely
deaminate both C and 5mC.
[0074] FIG. 16 shows that low sequence bias of deaminase-Seq
includes accurate representation of cytosine in cytosine rich
fragments such as CpG islands. Cytosine in CpG islands are
substantially depleted using bisulfite sequencing.
[0075] FIG. 17 shows that the lack of fragmentation using
Deaminase-Seq correlates with a low nucleic acid starting
concentration for detecting the position of modified bases in the
nucleic acid. For example, 1 ng of a genomic DNA library is
sufficient for detecting or mapping normal and modified
cytosine.
[0076] FIG. 18 shows a second example of methylome phasing (also
see FIG. 10 and FIG. 11A-11B) using embodiments of the methods
described herein where the results of methylome phasing using
Deaminase-Seq (SMRT.RTM. sequencing, (Pacific Biosciences, Menlo
Park, Calif.)) of an imprinted gene. The region of imprinting
identified by bisulfite sequencing is relatively short while a
region of greater than twice the length is identified using
Deaminase-Seq. Each red dots on the sequence map correspond to a
modified cytosine.
DEFINITIONS
[0077] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale
& Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, N.Y. (1991) provide one of skill with the general
meaning of many of the terms used herein. Still, certain terms are
defined below for the sake of clarity and ease of reference.
[0078] As used herein, the term "buffering agent", refers to an
agent that allows a solution to resist changes in pH when acid or
alkali is added to the solution. Examples of suitable non-naturally
occurring buffering agents that may be used in the compositions,
kits, and methods of the invention include, for example, Tris,
HEPES, TAPS, MOPS, tricine, or MES.
[0079] The term "non-naturally occurring" refers to a composition
that does not exist in nature.
[0080] Any protein described herein may be non-naturally occurring,
where the term "non-naturally occurring" refers to a protein that
has an amino acid sequence and/or a post-translational modification
pattern that is different to the protein in its natural state. For
example, a non-naturally occurring protein may have one or more
amino acid substitutions, deletions or insertions at the
N-terminus, the C-terminus and/or between the N- and C-termini of
the protein. A "non-naturally occurring" protein may have an amino
acid sequence that is different to a naturally occurring amino acid
sequence (i.e., having less than 100% sequence identity to the
amino acid sequence of a naturally occurring protein) but that that
is at least 80%, at least 85%, at least 90%, at least 95%, at least
97%, at least 98% or at least 99% identical to the naturally
occurring amino acid sequence. In certain cases, a non-naturally
occurring protein may contain an N-terminal methionine or may lack
one or more post-translational modifications (e.g., glycosylation,
phosphorylation, etc.) if it is produced by a different (e.g.,
bacterial) cell. A "mutant" protein may have one or more amino acid
substitutions relative to a wild-type protein and a "fusion"
protein may have one or exogenous domains added to the N-terminus,
C-terminus, and or the middle portion of the protein.
[0081] In the context of a nucleic acid (NA), the term
"non-naturally occurring" refers to a NA that contains: a) a
sequence of nucleotides that is different to a NA in its natural
state (i.e. having less than 100% sequence identity to a naturally
occurring NA sequence), b) one or more non-naturally occurring
nucleotide monomers (which may result in a non-natural backbone or
sugar that is not G, A, T or C) and/or C) may contain one or more
other modifications (e.g., an added label or other moiety) to the
5'-end, the 3' end, and/or between the 5'- and 3'-ends of the
NA.
[0082] In the context of a composition, the term "non-naturally
occurring" refers to: a) a combination of components that are not
combined by nature, e.g., because they are at different locations,
in different cells or different cell compartments; b) a combination
of components that have relative concentrations that are not found
in nature; c) a combination that lacks something that is usually
associated with one of the components in nature; d) a combination
that is in a form that is not found in nature, e.g., dried, freeze
dried, crystalline, aqueous; and/or e) a combination that contains
a component that is not found in nature. For example, a preparation
may contain a "non-naturally occurring" buffering agent (e.g.,
Tris, HEPES, TAPS, MOPS, tricine or MES), a detergent, a dye, a
reaction enhancer or inhibitor, an oxidizing agent, a reducing
agent, a solvent or a preservative that is not found in nature.
[0083] As used herein, the term "composition" refers to a
combination of reagents that may contain other reagents, e.g.,
glycerol, salt, dNTPs, etc., in addition to those listed. A
composition may be in any form, e.g., aqueous or lyophilized, and
may be at any state (e.g., frozen or in liquid form).
[0084] As used herein, the term "location" refers to the position
of a nucleotide in an identified strand in a NA molecule.
[0085] As used herein, the term "phasing" refers to a determination
of the status of two or more nucleotides on a single DNA molecule
or within an allele (i.e. whether the nucleotides are modified or
not, for example, whether the nucleotides such as C are methylated,
hydroxymethylated, formyl modified or carboxylated or unmodified)
are on the same molecule of NA or different homologous chromosomes
from a single cell or from homologous chromosomes from different
cells in a sample noting that in different cells or different
tissues, homologous chromosomes may have a different epigenetic
status.
[0086] As used herein, the term "nucleic acid" (NA) refers to a
DNA, RNA, DNA/RNA chimera or hybrid that may be ss or ds and may be
genomic or derived from the genome of a eukaryotic or prokaryotic
cell, or synthetic, cloned, amplified, or reverse transcribed. In
certain embodiments of the methods and compositions, NA preferably
refers to genomic DNA as the context requires.
[0087] As used herein, the term "modified cytosine" refers to
methylcytosine (.sup.mC), hydroxymethylcytosine (.sup.hmC), formyl
modified, carboxy modified or modified by any other chemical group
that may be found naturally associated with C.
[0088] As used herein, the term "methylcytosine dioxygenase" refers
to an enzyme that converts .sup.mC to .sup.hmC. TET1 (Jin, et al.,
Nucleic Acids Res. 2014 42: 6956-71) is an example of a
methylcytosine dioxygenase, although many others are known
including TET2, TET3 and Naeglaria TET (Pais et al, Proc. Natl.
Acad. Sci. 2015 112: 4316-4321). Examples of methylcytosine
dioxygenases which may be referred to as "oxygenase" are provided
in U.S. Pat. No. 9,121,061. TETv is an example of a methylcytosine
dioxygenase that oxidizes at least 90%, 92%, 94%, 96%, or 98% of
all modified C.
[0089] As used herein, the term "cytidine deaminase" refers to an
enzyme that is capable of deaminating C to form a U. Many cytidine
deaminases are known. For example, the APOBEC family of cytidine
deaminases is described in U.S. Pat. No. 9,121,061. APOBEC 3A
(Stenglein, Nature Structural & Molecular Biology 2010 17:
222-229) is an example of a deaminase. In any embodiment, the
deaminase used may have an amino acid sequence that is at least 90%
identical to (e.g., at least 95% identical to) the amino acid
sequence of GenBank accession number AKE33285.1, which is the human
APOBEC3A. Preferably, the cytidine deaminase converts unmodified
cytosine to uracil with an efficiency of at least 90%, 92%, 94%,
96%, 98% preferably at least 96%.
[0090] As used herein, the term "DNA glucosyltransferase (GT)"
refers to an enzyme that catalyzes the transfer of a .beta. or
.alpha.-D-glucosyl residue UDP-glucose to .sup.hmC residue in DNA.
An example of a GT is T4-.beta.GT. In one example, the use of GT
follows a deoxygenase reaction and ensures that deamination of hmC
is blocked so that less than 10% or 7% or 5% or 3% (preferably less
than 3% of hmC) is converted to U by the deaminase.
[0091] The term "substantially" refers to greater than 50%, 60%,
70%, 80%, or more particularly 90% of the whole.
[0092] As used herein, the term "comparing" refers to analyzing two
or more sequences relative to one another. In some cases, comparing
may be done by aligning or more sequences with one another such
that correspondingly positioned nucleotides are aligned with one
another.
[0093] As used herein, the term "reference sequence" refers to the
sequence of a fragment that is being analyzed. A reference sequence
may be obtained from a public database or it may be separately
sequenced as part of an experiment. In some cases, the reference
sequence may be "hypothetical" in the sense that it may be
computationally deaminated (i.e., to change C's into U's or T's
etc.) to allow a sequence comparison to be made. As used herein,
the terms "G", "A", "T", "U", "C", ".sup.mC", ".sup.caC",
".sup.hmC" and ".sup.ghmC" refer to nucleotides that contain
guanidine (G), adenine (A), thymine (T), uracil (U), cytosine (C),
.sup.mC, .sup.caC, .sup.hmC and .sup.ghmC, respectively. For
clarity, C, .sup.caC, .sup.mC and .sup.ghmC are different
moieties.
[0094] As used herein, the term "no C", in the context of a NA
fragment that contains no C, refers to a NA fragment that contains
no C. Such a NA may contain .sup.caC, .sup.mC and/or .sup.ghmC and
other nucleotides other than C.
[0095] The term "internal" refers to a location within the
polypeptide that is within a region that extends up to amino acids
from either end of the polypeptide.
[0096] The term "repeat" refers to a plurality of amino acids that
are repeated within the polypeptide.
[0097] The term "fusion" refers to a protein having one or
exogenous binding domains added to the N-terminus, C-terminus, and
or the middle portion of the protein. The binding domain is capable
of recognizing and binding to another molecule. Thus, in some
embodiments the binding domain is a histidine tag ("His-tag"), a
maltose-binding protein, a chitin-binding domain, a SNAP-Tag.RTM.
(New England Biolabs, Ipswich, Mass.) or a DNA-binding domain,
which may include a zinc finger and/or a transcription
activator-like (TAL) effector domain.
[0098] As used herein "N-terminal portion of the protein" refers to
amino acids within the first 50% of the protein. As used herein
"C-terminal portion of the protein refers to the terminal 50% of
the protein.
[0099] The term "Next Generation Sequencing (NGS)" generally
applies to sequencing libraries of genomic fragments of a size of
less than 1 kb preferably using an Illumina sequencing platform. In
contrast, single molecule sequencing is performed using a platform
from Pacific Biosystems, Oxford Nanopore, or 10.times. Genomics or
any other platform known in the art that is capable of sequencing
molecules of length greater than 1 kb or 2 kb.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0100] Before the various embodiments are described, it is to be
understood that the teachings of this disclosure are not limited to
the particular embodiments described, and as such can, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
teachings will be limited only by the appended claims.
[0101] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described in any way. While the present teachings are
described in conjunction with various embodiments, it is not
intended that the present teachings be limited to such embodiments.
On the contrary, the present teachings encompass various
alternatives, modifications, and equivalents, as will be
appreciated by those of skill in the art.
[0102] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present teachings, the some exemplary methods and materials are now
described.
[0103] The citation of any publication is for its disclosure prior
to the filing date and should not be construed as an admission that
the present claims are not entitled to antedate such publication by
virtue of prior invention. Further, the dates of publication
provided can be different from the actual publication dates which
can need to be independently confirmed.
[0104] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which can be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present teachings. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0105] All patents and publications, including all sequences
disclosed within such patents and publications, referred to herein
are expressly incorporated by reference.
[0106] Almost all studies on C modification in eukaryotic genomes
have ignored the fact that eukaryotic genomes carry two or more
copies of each chromosome. Thus, most traditional studies on C
modification do not provide any information about linkage between
modified C. For example, methylation studies have traditionally
been done using sodium bisulfite, which converts C into U. However,
as shown below, sodium bisulfite also fragments DNA, thereby making
it difficult, if not impossible, to determine whether two nearby
modified C, are linked on the same DNA molecules or unlinked on
different molecules. The method described herein provides a
solution to this problem.
[0107] In some embodiments, the sequencing may be done in a way
that allows one to determine the identity and location of
unmodified or modified C, as well as whether those unmodified or
modified C that are linked on the same molecule (i.e., "phased").
For example, in some embodiments, the method may comprise reacting
a first portion of a sample that contains relatively long, intact
NA fragments (e.g., at least 1 kb, at least 5 kb, at least 10 kb,
at least 50 kb, up to 100 kb or 200 kb or more in length) with a GT
and a cytidine deaminase to produce a first product. This product
differentiates C and .sup.mC from .sup.hmC as shown in FIG. 1B. A
second portion of the sample may be reacted with a methylcytosine
dioxygenase (and optionally a GT) as shown in FIG. 1A. The
methylcytosine dioxygenase and the GT may be combined in the same
reaction mix or used sequentially in the same or different buffers.
This reaction is followed by a cytidine deaminase reaction to
distinguish between unmodified C and modified C. Depending on the
sequence of the initial fragment (e.g., whether the initial
fragment in FIG. 1B contains G, A, T, C, .sup.mC and, in some
cases, .sup.hmC), the first product may contain G, A, T, U, no C
and .sup.ghmC (if the initial fragment contained .sup.hmC). In FIG.
1A, the second product alone may contain G, A, T, U, .sup.caC and
no C. These enzyme and methods avoid degradation of the NA
substrate and provide improved phasing of modified nucleotide over
long pieces of the genome that are not degraded by the enzymes.
These enzyme and methods achieve sequencing and mapping of modified
nucleotides with minimal bias and improved efficiency.
[0108] After the first and optionally second products are produced,
they may be amplified and/or cloned, and then sequenced using
suitable sequencing method. This may include single molecule
sequencing for phased sequencing, Phased sequencing may be done in
a variety of different ways. In some embodiments, the products may
be sequenced using a long read single-molecule sequencing approach
such as Nanopore sequencing (e.g. as described in Soni, et al Clin
Chem 53:1996-2001 2007, and developed by Oxford Nanopore
Technologies) or Pacific Biosciences' fluorescent base-cleavage
method (which currently have an average read length of over 10 kb,
with some reads over 60 kb). Alternatively, the products may be
sequenced using, the methods of Moleculo (Illumina, San Diego,
Calif.), 10.times. Genomics (Pleasanton, Calif.), or NanoString
Technologies (Seattle, Wash.). In these methods, the sample is
optionally diluted and then partitioned into a number of partitions
(wells of a microtitre plate or droplets in an emulsion, etc.) in
an amount that limits the probability that each partition does not
contain two molecules of the same locus (e.g., two molecules
containing the same gene). Next, these methods involve producing
indexed amplicons of a size that is compatible with the sequencing
platform being used (e.g., amplicons in the range of 200 bp to 1 kb
in length) where amplicons derived from the same partitions are
barcoded with the same index unique to the partition. Finally, the
indexed amplicons are sequenced, and the sequence of the original,
long, molecules can be reconstituted using the index sequences.
Phased sequencing may also be done using barcoded transposons (see,
e.g., Adey Genome Res. 2014 24: 2041-9 and Amini Nat Genet. 2014
46: 1343-9), and by using the "reflex" system of Population
Genetics Technologies (Casbon, Nucleic Acids Res. 2013
41:e112).
[0109] Alternatively, the genome may be fragmented into fragments
of less than 1 kb in size to form a library for Next gen
sequencing. Pyrrolo-dC modified adaptors may be added to the
fragments in the library prior to enzyme treatment according to
FIG. 1A-1B and Example 1. After the enzyme reaction, the adaptor
ligated libraries may be sequenced using an Illumina sequencer.
After the sequences of the first and optionally the second product
are obtained, the sequences are compared with a reference sequence
to determine which C's in the initial NA fragment are modified. A
matrix illustrating an embodiment of this part of the method is
illustrated in FIG. 1C. In some embodiments, this comparing may be
done by comparing the sequences obtained from the first product of
the sample (i.e., the methylcytosine dioxygenase (and optionally
GT) and cytidine deaminase treated portion of the sample) and the
untreated sample and/or second product of the sample (i.e., the GT
and cytidine deaminase treated portion of the sample) with a
corresponding reference sequence (untreated and/or the first
product). Possible outcomes include: [0110] i. The position of a C
in the initial NA fragment is identified by a U in both the first
and second products; [0111] ii. The position of a .sup.mC in the
initial NA fragment is determined by the presence of a C in the
first product or a T in the second product [0112] iii. The position
of a .sup.hmC in the initial NA fragment is determined by the
presence of a C in the second product only.
[0113] It should be noted that should there be no need to
differentiate the .sup.mC from the rarer .sup.hmC, then this
information can be obtained from the second product only (FIG.
1A).
[0114] As would be understood, if the product is cloned, amplified
or sequenced by a polymerase, a "U" will be read as "T". In these
embodiments, nucleotides read as a T in both the first and second
products still indicate Cs that have been changed to Us in the
initial deamination reaction.
[0115] As would be recognized, some of the analysis steps of the
method, e.g., the comparing step, can be implemented on a computer.
In certain embodiments, a general-purpose computer can be
configured to a functional arrangement for the methods and programs
disclosed herein. The hardware architecture of such a computer is
well known by a person skilled in the art, and can comprise
hardware components including one or more processors (CPU), a
random-access memory (RAM), a read-only memory (ROM), an internal
or external data storage medium (e.g., hard disk drive). A computer
system can also comprise one or more graphic boards for processing
and outputting graphical information to display means. The above
components can be suitably interconnected via a bus inside the
computer. The computer can further comprise suitable interfaces for
communicating with general-purpose external components such as a
monitor, keyboard, mouse, network, etc. In some embodiments, the
computer can be capable of parallel processing or can be part of a
network configured for parallel or distributive computing to
increase the processing power for the present methods and programs.
In some embodiments, the program code read out from the storage
medium can be written into memory provided in an expanded board
inserted in the computer, or an expanded unit connected to the
computer, and a CPU or the like provided in the expanded board or
expanded unit can actually perform a part or all of the operations
according to the instructions of the program code, so as to
accomplish the functions described below. In other embodiments, the
method can be performed using a cloud computing system. In these
embodiments, the data files and the programming can be exported to
a cloud computer that runs the program and returns an output to the
user.
[0116] A system can, in certain embodiments, comprise a computer
that includes: a) a central processing unit; b) a main non-volatile
storage drive, which can include one or more hard drives, for
storing software and data, where the storage drive is controlled by
disk controller; c) a system memory, e.g., high speed random-access
memory (RAM), for storing system control programs, data, and
application programs, including programs and data loaded from
non-volatile storage drive; system memory can also include
read-only memory (ROM); d) a user interface, including one or more
input or output devices, such as a mouse, a keypad, and a display;
e) an optional network interface card for connecting to any wired
or wireless communication network, e.g., a printer; and f) an
internal bus for interconnecting the aforementioned elements of the
system.
[0117] The method described above can be employed to analyze
genomic DNA from virtually any organism, including, but not limited
to, plants, animals (e.g., reptiles, mammals, insects, worms, fish,
etc.), tissue samples, bacteria, fungi (e.g., yeast), phage,
viruses, cadaveric tissue, archaeological/ancient samples, etc. In
certain embodiments, the genomic DNA used in the method may be
derived from a mammal, where in certain embodiments the mammal is a
human. In exemplary embodiments, the genomic sample may contain
genomic DNA from a mammalian cell, such as, a human, mouse, rat, or
monkey cell. The sample may be made from cultured cells, formalin
fixed samples or cells of a clinical sample, e.g., a tissue biopsy
(for example from a cancer), scrape or lavage or cells of a
forensic sample (i.e., cells of a sample collected at a crime
scene). In particular embodiments, the NA sample may be obtained
from a biological sample such as cells, tissues, bodily fluids, and
stool. Bodily fluids of interest include but are not limited to,
blood, serum, plasma, saliva, mucous, phlegm, cerebral spinal
fluid, pleural fluid, tears, lactal duct fluid, lymph, sputum,
cerebrospinal fluid, synovial fluid, urine, amniotic fluid, and
semen. In particular embodiments, a sample may be obtained from a
subject, e.g., a human. In some embodiments, the sample analyzed
may be a sample of cell-free DNA obtained from blood, e.g., from
the blood of a pregnant female.
[0118] In some embodiments of the invention, an enzymatic method
has been provided which permits the sequencing of short and long NA
(for example, ss DNA and ds DNA) to discover modified bases and to
determine the phasing of such bases in the genome. Embodiments of
the method may include a composition comprising a mixture of one or
two enzymes where the one, two enzymes are selected from a
methylcytosine dioxygenase and a GT where the cytidine deaminase is
added in a subsequent reaction. The dioxygenase and GT may be
stored in the same or different buffers and combined as desired in
a storage buffer or in a reaction mixture. When added separately to
a reaction mixture, the addition may be sequential or the enzymes
may be added together at the start of the reaction. Embodiments of
the method may utilize two or more enzymes selected from a cytidine
deaminase, a methylcytosine dioxygenase and a GT. Embodiments of
the method may include a methylcytosine dioxygenase and a cytidine
deaminase used sequentially in a reaction mixture; a methylcytosine
dioxygenase and a GT used sequentially or together preferably
followed by a deaminase reaction; or a methylcytosine dioxygenase,
GT and cytidine deaminase used sequentially or together.
[0119] In some embodiments, that utilize a GT, a UDP may be added
to the reaction mixture.
[0120] In one embodiment, the methylcytosine dioxygenase and
optionally the GT may be added to ds DNA in an initial step and
then removed by a proteinase treatment, heat treatment and/or
separation treatment. This may be followed by a cytidine deaminase
reaction with separation and isolation of the deaminated DNA. In
some embodiments, the pH of the cytidine deaminase reaction mixture
is in the range of pH 5.5-8.5, for example pH 6.0-8.0 for example,
pH 6.0, pH 6.3, pH 6.5, pH 6.8, pH 7.0, pH 7.5, or pH 8.0 wherein
the specific activity of the cytidine deaminase is increased at the
lower end of the pH range such as at pH 6.0.
[0121] In one embodiment, concentration ranges of enzymes utilized
in the reaction described for 1 .mu.g DNA include: 0.001-100
micrograms of a methylcytosine dioxygenase such as the Ngo TET
(Pais, supra), TET1, TET2 or TET3 or mutants thereof; 0.001-100
micrograms cytidine deaminase such as APOBEC or Deaminase;
0.001-100 units GT such as T4-.beta.GT or T4-.alpha.GT. When
Pyrollo-dC used in adaptor synthesis, a standard procedure
described in Example 4 is followed. The amount of UDP used follows
the recommendation of the manufacturer.
[0122] The ss DNA product of enzyme reaction or reactions can be
amplified by PCR or isothermal method such ligase mediated
amplification (LMA), helicase dependent amplification (HDA),
rolling circle amplification (RCA), loop mediated amplification
(LAMP), multiple displacement amplification, (MDA); transcription
mediated amplification (TMA), strand displacement amplification
(SDA), nicking enzyme amplification reaction (NEAR).
[0123] The amplified or indeed non-amplified DNA may be sequenced
using any of the sequencing platforms in development or
commercially available such as provided by Illumina, Oxford
Nanopore, or Pacific Biosystems, or methods in development or
commercially available such as Sanger sequencing or any WGS (whole
genome sequencing) method. Long reads are mapped to the genome
using the appropriate algorithm, for example, Bismark (see for
example, Krueger et al. Bioinformatics 27, no. 11 (2011):
1571-1572). The methylation status is called when each reads is
mapped to the targeted region (for example, enhancer and promoter
region).
[0124] Present embodiments provide many advantages over existing
systems that result from factors that include: a lower error rate
in identifying .sup.mC regardless of adjacent nucleotides, and a
lower error rate in detecting low level methylations; no systematic
sequence preference; more consistent genome wide sequencing
coverage; higher coverage in C rich regions and CpG islands;
covering more CpG sites where these may be distributed widely in
the genome portion being analyzed; and accurate detection of
.sup.hmC of large fragments (5 kb) at a base resolution enabling
phasing of DNA modifications and phasing DNA modifications together
with other genomic features such as SNPs or variants.
[0125] In some embodiments, the composition may comprise a NA that
is made up of nucleotides G, A, T, U, .sup.caC, wherein the NA
contains substantially no C. In some embodiments, the composition
may comprise a NA that is made up of nucleotides G, A, T, U and
.sup.ghmC, wherein the NA contains substantially no C. In either
embodiment, the composition may also contain a cytidine deaminase
(e.g., a cytidine deaminase that is at least 90% identical to an
APOBEC cytidine deaminase) and, in certain embodiments, may also
contain a buffering agent and other components (e.g., NaCl) in
amounts that are compatible with cytidine deaminase activity. The
composition may be an aqueous composition.
[0126] Variant. .sup.mC Dioxygenases and Methods for Using the
Same
[0127] A variant methylcytosine dioxygenase is also provided. In
some embodiments, the methylcytosine dioxygenase comprises an amino
acid sequence that is at least 90% identical to (e.g., at least
92%, at least 94%, at least 96%, at least 97%, at least 98%, or at
least 99% identical to) the amino acid sequence of TETv (SEQ ID
NO:1); and contain the amino acid sequence of SEQ ID NO:2. As would
be apparent, this polypeptide has .sup.mC dioxygenase activity. The
TETv sequence is shown below:
TABLE-US-00001 TETv (SEQ ID NO: 1)
GGSQSQNGKCEGCNPDKDEAPYYTHLGAGPDVAAIRTLMEERYGEKGKAI
RIEKVIYTGKEGKSSQGCPIAKWVYRRSSEEEKLLCLVRVRPNHTCETAV
MVIAIMLWDGIPKLLASELYSELTDILGKCGICTNRRCSQNETRNCCCQG
ENPETCGASFSFGCSWSMYYNGCKFARSKKPRKFRLHGAEPKEEERLGSH
LQNLATVIAPIYKKLAPDAYNNQVEFEHQAPDCCLGLKEGRPFSGVTACL
DFSAHSHRDQQNMPNGSTVVVTLNREDNREVGAKPEDEQFHVLPMYIIAP
EDEFGSTEGQEKKIRMGSIEVLQSFRRRRVIRIG DAA
AVQEIEYWSDSEHNFQDPCIGGVAIAPTHGSILIECAKCEVHATTKVNDP
DRNHPTRISLVLYRHKNLFLPKHCLALWEAKMAEKARKEEECGKNGSDHV
SQKNHGKQEKREPTGPQEPSYLRFIQSLAENTGSVTTDSTVTTSPYAFTQ VTGPYNTFV
[0128] TETv is derived from mouse Tet2 catalytic domain and
contains a deletion. The amino acid sequence ELPKSCEVSGQ (SEQ ID
NO:2) is italicized within the sequence of TETv and TETcd sequences
shown above and below.
TABLE-US-00002 TETcd (TET-2 catalytic domain) (SEQ ID. NO. 3)
QSQNGKCEGCNPDKDEAPYYTHLGAGPDVAAIRTLMEERYGEKGKAIRIE
KVIYTGKEGKSSQGCPIAKWVYRRSSEEEKLLCLVRVRPNHTCETAVMVI
AIMLWDGIPKLLASELYSELTDILGKCGICTNRRCSQNETRNCCCQGENP
ETCGASFSFGCSWSMYYNGCKFARSKKPRKFRLHGAEPKEEERLGSHLQN
LATVIAPIYKKLAPDAYNNQVEFEHQAPDCCLGLKEGRPFSGVTACLDFS
AHSHRDQQNMPNGSTVVVTLNREDNREVGAKPEDEQFHVLPMYIIAPEDE
FGSTEGQEKKIRMGSIEVLQSFRRRRVIRIGELPKSCKKKAEPKKAKTKK
AARKRSSLENCSSRTEKGKSSSHTKLMENASHMKQMTAQPQLSGPVIRQP
PTLQRHLQQGORPQQPQPPQPQPQTTPQPQPQPQHIMPGNSQSVGSHCSG
STSVYTRQPTPHSPYPSSAHTSDIYGDTNHVNFYPTSSHASGSYLNPSNY
MNPYLGLLNQNNQYAPFPYNGSVPVDNGSPFLGSYSPQAQSRDLHRYPNQ
DHLTNQNLPPIHTLHQQTFGDSPSKYLSYGNQNMQRDAFTTNSTLKPNVH
HLATFSPYPTPKMDSHFMGAASRSPYSHPHTDYKTSEHHLPSHTIYSYTA
AASGSSSSHAFHNKENDNIANGLSRVLPGFNHDRTASAQELLYSLTGSSQ
EKQPEVSGQDAAAVQEIEYWSDSEHNFQDPCIGGVAIAPTHGSILIECAK
CEVHATTKVNDPDRNHPTRISLVLYRHKNLFLPKHCLALWEAKMAEKARK
EEECGKNGSDHVSQKNHGKQEKREPTGPQEPSYLRFIQSLAENTGSVTTD
STVTTSPYAFTQVTGPYNTFV
[0129] The deleted amino acids correspond to residues 338 to 704
TETcd (shown in italics above). The amino acid sequence ELPKSCEVSGQ
(SEQ ID NO:2) contains 5 amino acids from one side of the junction
and 5 amino acids from the other side of the junction, as shown
above.
[0130] In some embodiments, the variant methylcytosine dioxygenase
may be a fusion protein. In these embodiments, the variant may have
a binding domain that is capable of recognizing and binding to
another molecule. Thus, in some embodiments the binding domain is a
histidine tag ("His-tag") although a maltose-binding protein, a
chitin-binding domain, a SNAP-Tag.RTM. or a DNA-binding domain,
which may include a zinc finger and/or a transcription
activator-like (TAL) effector domain are also examples of binding
moieties.
[0131] Embodiments include a buffered composition containing a
purified TETv. For example, the pH of the buffer in the composition
is pH 5.5-8.5, for example pH 5.5-7.5, pH 7.5-8.0 or pH 8.0. In
various embodiments, the buffered composition may contain glycerol;
and/or contains Fe(II), as cofactor, and .alpha.-ketoglutarate, as
co-substrate, for the enzyme. In some of these embodiments, the
composition contains ATP to allow further oxidation of .sup.hmC to
.sup.fC and .sup.caC; in other embodiments, the composition does
not contain dATP that limits the distribution of the oxidized forms
of .sup.mC.
[0132] Embodiments include an in vitro mixture that includes a
TETv, a .beta.GT, a cytidine deaminase, and/or an endonuclease. The
in vitro mixture may further include a polynucleotide substrate and
at least dATP. The polynucleotide could be ss or ds, a DNA or RNA,
a synthesized oligonucleotide (oligo), chromosomal DNA, or an RNA
transcript. The polynucleotide used could be labeled at one or both
ends. The polynucleotide may harbor a C, .sup.mC, .sup.hmC,
.sup.fC, .sup.caC or .sup.ghmC. In other embodiments, the
polynucleotide may harbor a T, U, hydroxymethyluracil (.sup.hmU),
formyluracil (.sup.fU), or carboxyuracil (.sup.caU).
[0133] Embodiments provide a TETv, which oxidizes .sup.mC to
.sup.hmC, .sup.fC, and/or .sup.caC preferably in any sequence
context with minimal sequence bias and minimal damage to the DNA
substrate compared to BS-seq. TETv may additionally or
alternatively oxidize T to .sup.hmU or .sup.fU with improved
efficiency and reduced bias compared with naturally occurring mouse
TET-2 enzyme, or its catalytic domain (TETcd).
[0134] In an embodiment of the method, C could be distinguished
from .sup.mC by reacting the polynucleotide of interest with a TETv
and a cytidine deaminase wherein only C is converted to U. A
further embodiment includes sequencing the polynucleotide treated
with the .beta.GT and the cytidine deaminase in which C is
converted to U and .sup.mC is converted to a T and comparing the
sequencing results to that of sequencing the untreated
polynucleotide to map .sup.mC and .sup.hmC location in the
polynucleotide.
[0135] In another embodiment of the method, both .sup.mC and
.sup.hmC locations in a polynucleotide are mapped. In this method:
(a) the polynucleotide is untreated; (b) reacted with bisulfite
reagent; or (c) reacted with GT prior to adding a methylcytosine
dioxygenase then treating with bisulfite reagent, (a) through (c)
are sequenced and comparison of the sequencing results enables the
mapping of .sup.mC and .sup.hmC and their differentiation from C:
(a) C, .sup.mC, and .sup.hmC are all sequenced as C; (b) C is
sequenced as C while .sup.mC and .sup.hmC as T; and (c).sup.hmC is
converted to .sup.ghmC and sequenced as C, C is sequenced as C, and
.sup.mC as T.
[0136] In some embodiments, .sup.mC locations in a polynucleotide
are mapped by coupling the oxidation activity of TETv to the
activity of a restriction endonuclease or an AP endonuclease
specific to .sup.hmC or .sup.fC/.sup.caC, respectively.
[0137] In some aspects, .sup.mC, .sup.hmC, or .sup.fC may be mapped
to sites in a polynucleotide using single-molecule sequencing
technologies such as Single Molecule Real-Time (SMRT) Sequencing,
Oxford Nanopore Single Molecule Sequencing (Oxford, UK) or
10.times. Genomics (Pleasanton, Calif.). In some embodiments, the
method may employ TETv, a cytidine deaminase, and/or GT.
[0138] The above-described TETv enzyme can be used as a
methylcytosine dioxygenase in any of the methods, compositions or
kits summarized above and described in greater detail below.
[0139] Kits
[0140] Also provided by the present disclosure are kits for
practicing the subject method as described above. In certain
embodiments, a subject kit may contain: a GT, a methylcytosine
dioxygenase and a cytidine deaminase. The components of the kit may
be combined in one container, or each component may be in its own
container. For example, the components of the kit may be combined
in a single reaction tube or in one or more different reaction
tubes. Further details of the components of this kit are described
above. The kit may also contain other reagents described above and
below that may be employed in the method, e.g., a buffer,
ADP-glucose, plasmids into which NAs can be cloned, controls,
amplification primers, etc., depending on how the method is going
to be implemented.
[0141] In addition to above-mentioned components, the subject kit
may further include instructions for using the components of the
kit to practice the subject method. The instructions for practicing
the subject method are generally recorded on a suitable recording
medium. For example, the instructions may be printed on a
substrate, such as paper or plastic, etc. As such, the instructions
may be present in the kits as a package insert, in the labeling of
the container of the kit or components thereof (i.e., associated
with the packaging or subpackaging) etc. In other embodiments, the
instructions are present as an electronic storage data file present
on a suitable computer readable storage medium, e.g. CD-ROM,
diskette, etc. In yet other embodiments, the actual instructions
are not present in the kit, but means for obtaining the
instructions from a remote source, e.g. via the internet, are
provided. An example of this embodiment is a kit that includes a
web address where the instructions can be viewed and/or from which
the instructions can be downloaded. As with the instructions, this
means for obtaining the instructions is recorded on a suitable
substrate.
[0142] Utility
[0143] In some embodiments, the method can be used to compare two
samples. In these embodiments, the method may be used to identify a
difference in the pattern of C modification in a test NA fragment
relative to the pattern of cytosine modification in a corresponding
reference NA. This method may comprise (a) determining the location
of all modified C in a test NA fragment using the above-described
method to obtain a first pattern of C modification; (b) determining
the location of all modified C in a reference NA fragment using the
above-described method to obtain a first pattern of C modification;
(c) comparing the test and reference patterns of C modification;
and (d) identifying a difference in the pattern of cytosine
modification, e.g., a change in the amount of .sup.mC or .sup.hmC,
in the test NA fragment relative to the reference NA fragment.
[0144] In some embodiments, the test NA and the reference NA are
collected from the same individual at different times. In other
embodiments, the test NA and the reference NA collected from
tissues or different individuals.
[0145] Exemplary NAs that can be used in the method include, for
example, NA isolated from cells isolated from a tissue biopsy
(e.g., from a tissue having a disease such as colon, breast,
prostate, lung, skin cancer, or infected with a pathogen etc.) and
NA isolated from normal cells from the same tissue, e.g., from the
same patient; NA isolated from cells grown in tissue culture that
are immortal (e.g., cells with a proliferative mutation or an
immortalizing transgene), infected with a pathogen, or treated
(e.g., with environmental or chemical agents such as peptides,
hormones, altered temperature, growth condition, physical stress,
cellular transformation, etc.), and NA isolated from normal cells
(e.g., cells that are otherwise identical to the experimental cells
except that they are not immortalized, infected, or treated, etc.);
NA isolated from cells isolated from a mammal with a cancer, a
disease, a geriatric mammal, or a mammal exposed to a condition,
and NA isolated from cells from a mammal of the same species, e.g.,
from the same family, that is healthy or young; and NA isolated
from differentiated cells and NA isolated from non-differentiated
cells from the same mammal (e.g., one cell being the progenitor of
the other in a mammal, for example). In one embodiment, NA isolated
from cells of different types, e.g., neuronal and non-neuronal
cells, or cells of different status (e.g., before and after a
stimulus on the cells) may be compared. In another embodiment, the
experimental material is NA isolated from cells susceptible to
infection by a pathogen such as a virus, e.g., human
immunodeficiency virus (HIV), etc., and the reference material is
NA isolated from cells resistant to infection by the pathogen. In
another embodiment of the invention, the sample pair is represented
by NA isolated from undifferentiated cells, e.g., stem cells, and
NA isolated from differentiated cells.
[0146] In some exemplary embodiments, the method may be used to
identify the effect of a test agent, e.g., a drug, or to determine
if there are differences in the effect of two or more different
test agents. In these embodiments, NA from two or more identical
populations of cells may be prepared and, depending on how the
experiment is to be performed, one or more of the populations of
cells may be incubated with the test agent for a defined period of
time. After incubation with the test agent, the genomic DNA from
one both of the populations of cells can be analyzed using the
methods set forth above, and the results can be compared. In a
particular embodiment, the cells may be blood cells, and the cells
can be incubated with the test agent ex vivo. These methods can be
used to determine the mode of action of a test agent, to identify
changes in chromatin structure or transcription factor occupancy in
response to the drug, for example.
[0147] The method described above may also be used as a diagnostic
(which term is intended to include methods that provide a diagnosis
as well as methods that provide a prognosis). These methods may
comprise, e.g., analyzing C modification from a patient using the
method described above to produce a map; and providing a diagnosis
or prognosis based on the map.
[0148] The method set forth herein may also be used to provide a
reliable diagnostic for any condition associated with altered
cytosine modification. The method can be applied to the
characterization, classification, differentiation, grading,
staging, diagnosis, or prognosis of a condition characterized by an
epigenetic pattern. For example, the method can be used to
determine whether the C modifications in a fragment from an
individual suspected of being affected by a disease or condition is
the same or different compared to a sample that is considered
"normal" with respect to the disease or condition. In particular
embodiments, the method can be directed to diagnosing an individual
with a condition that is characterized by an epigenetic pattern at
a particular locus in a test sample, where the pattern is
correlated with the condition. The methods can also be used for
predicting the susceptibility of an individual to a condition.
[0149] In some embodiments, the method can provide a prognosis,
e.g., to determine if a patient is at risk for recurrence. Cancer
recurrence is a concern relating to a variety of types of cancer.
The prognostic method can be used to identify surgically treated
patients likely to experience cancer recurrence so that they can be
offered additional therapeutic options, including preoperative or
postoperative adjuncts such as chemotherapy, radiation, biological
modifiers and other suitable therapies. The methods are especially
effective for determining the risk of metastasis in patients who
demonstrate no measurable metastasis at the time of examination or
surgery.
[0150] The method can also be used to determining a proper course
of treatment for a patient having a disease or condition, e.g., a
patient that has cancer. A course of treatment refers to the
therapeutic measures taken for a patient after diagnosis or after
treatment. For example, a determination of the likelihood for
recurrence, spread, or patient survival, can assist in determining
whether a more conservative or more radical approach to therapy
should be taken, or whether treatment modalities should be
combined. For example, when cancer recurrence is likely, it can be
advantageous to precede or follow surgical treatment with
chemotherapy, radiation, immunotherapy, biological modifier
therapy, gene therapy, vaccines, and the like, or adjust the span
of time during which the patient is treated.
[0151] In a particular embodiment, a lab will receive a sample
(e.g., blood) from a remote location (e.g., a physician's office or
hospital), the lab will analyze a NA isolated from the sample as
described above to produce data, and the data may be forwarded to
the remote location for analysis.
[0152] Epigenetic regulation of gene expression may involve cis or
trans-acting factors including nucleotide methylation. While
cis-acting methylated nucleotides are remotely positioned in a DNA
sequence corresponding to an enhancer, these sites may become
adjacent to a promoter in a three-dimensional structure for
activating or deactivating expression of a gene. Enhancers can be
megabases away from the corresponding promoter and thus
understanding the relationship between a methylation site in an
enhancer and its impact on a corresponding promoter (phasing) over
long distances is desirable. Phasing the methylation of a distantly
located enhancer to a promoter on which it acts can provide
important insights into gene regulation and mis-regulation that
occurs in diseases such as cancer.
[0153] In order to further illustrate the present invention, the
following specific examples are given with the understanding that
they are being offered to illustrate the present invention and
should not be construed in any way as limiting its scope.
[0154] All references cited herein are incorporated by
reference.
EXAMPLES
Example 1. Enzyme Based Method for Mapping Methylcytosine and
Hydroxymethylcytosine
[0155] Embodiments of methods described herein provide an unbiased
efficient means of mapping .sup.mC and .sup.hmC along long
stretches of genomic DNA. Such methods describe how to protect
biologically relevant DNA modification, such as .sup.mC and
.sup.hmC in DNA deamination reaction in order to detect and read
these modifications. The methods avoid unwanted fragmentation that
arises using chemical methods (such as the bisulfite method). The
enzymatic methods use one or more of the following enzymes: a
cytidine deaminase, a methylcytosine dioxygenase and a GT.
[0156] Examples are provided that utilize a cytidine deaminase
described in U.S. Pat. No. 9,121,061 (specifically APOBEC3A in this
example) although other cytidine deaminases may be used (as
discussed above). The Examples provided herein utilize
Deaminase-seq. Deaminase-seq refers to the pathway that depends on
a deaminase reaction leading to sequencing to detect modified
cytosine. The pathway shown in FIG. 1A may further include a GT
such as .beta.) which may be combined with the methylcytosine
dioxygenase in one reaction mix or added sequentially in one
reaction vessel. A novel methylcytosine dioxygenase is described
herein that provides more efficient and unbiased conversion of
.sup.mC and .sup.hmC to .sup.CaC then does wild type human or mouse
TET proteins. Typically, Deaminase-Seq includes the following
steps: treating genomic DNA or DNA library preparations (such as
Ultra II Library prep with protected adaptors (NEB)), the use of
one or more of TET2 deoxygenase and GT enzymes for example, TET2
deoxygenase followed by GT (BGT) or in parallel with GT, removal of
enzyme activity by for example heat denaturation followed by
deamination using for example Apobec A3A, amplification and then
sequencing in an Illumina sequencer, PacBio sequencer or other
commercially available sequencing device. Further experimental
details for embodiments are provided below.
A. Discrimination of Methylcytosine from Unmodified Cytosine in
Genomic DNA Using an Engineered Methylcytosine Dioxygenase (TETv)
and a Cytidine Deaminase (APOBEC)
[0157] (i) Mouse NIH/3T3 DNA (250 ng) was reacted with TETv (8
.mu.M) in 50 ul Tris buffer at 37.degree. C. for 1 hour and the
oxidized DNA was column purified (Zymo Research, Irvine,
Calif.).
[0158] (ii) The DNA was then heated to 70.degree. C. in presence of
66% of formamide in a thermocycler and then placed on ice. RNase A
(0.2 mg/ml), BSA (10 mg/ml) and cytidine deaminase (0.3 mg/ml) were
added (see also Bransteitter et al. PNAS (2003) vol 100, 4102-4107)
and incubated for 3 hours at 37.degree. C. DNA was column purified
(Zymo Research, Irvine, Calif.). Following PCR with U-bypass DNA
polymerase (New England Biolabs, Ipswich, Mass.) using Primer 1
AATGAAGGAAATGAATTTGGTAGAG (SEQ ID NO:6) and Primer 2 T CCC AA AT AC
AT A AATCC AC ACTT A (SEQ ID NO:7), the products were cloned using
the NEB PCR Cloning Kit (New England Biolabs, Ipswich, Mass.) and
the clones were subjected to Sanger sequencing. Sequencing results
are summarized in FIG. 2A. Empty dots represent unmodified CpG
sites in the PCR fragment, black dots represent .sup.mCpG sites in
the PCR fragment.
B. Discrimination of Hydroxymethylcytosine from Unmodified Cytosine
and Methylcytosine Using T4-8GT (New England Biolabs, Ipswich,
Mass.) and Cytidine Deaminase (i) DNA was reacted with T4-.beta.GT
(20 Units) in the presence of UDP (1 .mu.l) in a volume of 50 .mu.l
at 37.degree. C. for 1 hour and then column purified DNA. The
method followed the steps in (ii) above. Sequencing results are
summarized in FIG. 2B. Empty dots represent unmodified CpG sites in
the PCR fragment, black dots represent .sup.hmCpG sites in the PCR
fragment.
Example 2. ss DNA is not Damaged During Methylcytosine Deoxygenase,
DNA Glucosyltransferase or Cytidine Deaminase Treatment
[0159] The demonstration that DNA damage does not occur during the
analysis of modified bases in ss DNA is a significant advantage of
the current bisulfite method commonly used for methylome analysis
(see FIG. 3A-3E). It is the lack of damage as shown in FIG. 3A-3B,
3D-3E that makes it possible to obtain phase data.
[0160] Mouse E14 genomic DNA was sheared to fragments (Covaris,
Woburn, Mass.) of a size of approximately 15 kb and selected and
purified using AMPure.RTM. XP beads (Beckman Coulter, Brea,
Calif.). The DNA were then treated as follows:
[0161] (a) Control DNA. The 15 kb fragments of DNA was denaturated
to ssDNA at 70.degree. C. in presence of 66% of formamide for 10
minutes.
[0162] (b) Bisulfite converted DNA. The 15 kb fragments of DNA were
treated with sodium bisulfite using EZ DNA Methylation-Gold.TM. Kit
(Zymo Research, Irvine, Calif.), according to the instruction
manual.
[0163] (c) T4-.beta.GT and cytidine deaminase (APOBEC3A) treated
DNA. 15 kb DNA fragments were glucosylated and then deaminated as
described in Example 1.
[0164] (d) TETv and cytidine deaminase (APOBEC3A) treated DNA. 15
kb DNA fragments were treated with TETv, and then deaminated as
described above.
[0165] Initially the DNA from samples (a)-(d) were examined on an
Agilent RNA 6000 pico chip (Agilent, Santa Clara, Calif.). The data
is given in FIG. 3E (y-axis is the fluorescent units while the
X-axis is size (daltons). The light blue line represents the
denatured ss DNA of the 15 kb AMPure size selected fragments, which
is also the control. The red line is APOBEC deamination on
glucosylated DNA. The dark blue is DNA deamination on TETv oxidized
DNA. And the green is bisulfite treated DNA. When comparing to the
control, both cytidine deaminase treated substrates show no
significant difference in size distribution whereas the bisulfite
treated DNA reduced in size greatly, showing significant DNA
degradation.
[0166] The 15 Kb treated DNA from samples (a)-(d) was also PCR
amplified to produce amplicons of 4229 bp, 3325 bp, 2018 bp, 1456
bp, 731 bp and 388 bp using Phusion.RTM. U (ThermoFisher
Scientific, Waltham, Mass.) DNA polymerase.
[0167] Products were analyzed on 1% agarose gels and the results
provided in FIG. 3B-3E. The results show that the treatment of DNA
with cytidine deaminase, GT and the methylcytosine dioxgenase did
not cause detectable fragmentation. In contrast, bisulfite
treatment caused the DNA to fragment to fragments no larger than
731 bp.
TABLE-US-00003 388 (SEQ ID NO: 8)
TAGGATAAAAATATAAATGTATTGTGGGATGAGG (SEQ ID NO: 9)
AAAACATATAACCCCCTCCACTAATAC 731 (SEQ ID NO: 10)
AGATATATTGGAGAAGTTTTGGATGATTTGG (SEQ ID NO: 11)
AAAACATATAACCCCCTCCACTAATAC 1456 (SEQ ID NO: 12)
TAAGATTAAGGTAGGTTGGATTTGG (SEQ ID NO: 13) TCATTACTCCCTCTCCAAAAATTAC
2018 (SEQ ID NO: 14) AAGATTTAAGGGAAGGTTGAATAGG (SEQ ID NO: 15)
ACCTACAAAACCTTACAAACATAAC 3325 (SEQ ID NO: 16)
TGGAGTTTGTTGGGGGGTTTGTTGTTTAAG (SEQ ID NO: 17)
TCTAACCCTCACCACCTTCCTAATACCCAA 4229 (SEQ ID NO: 18)
TGGTAAAGGTTAAGAAGGGAAGATTGTGGA (SEQ ID NO: 19)
AACCCTACTTCCCCCTAACAAATTTTCAAC
Example 3. Synthesis of an Adaptor for NGS Library Construction
where all Cytosines are Protected from Deamination in the Presence
of Cytidine DNA Deaminases
[0168] This example describes the experiment, confirming that
pyrrolo-dC is not a substrate for cytidine deaminase, and may be
used to synthesize a protected adaptor suitable for a sequencing
platform such as Illumina.
[0169] A reaction mixture was made containing 2 .mu.M 44 bp ssDNA
oligonucleotide containing a single Pyrrolo-dC
(5'-ATAAGAATAGAATGAATXGTGAAATGAATATGAAATGAATAGTA-3', X=Pyrrolo-dC)
(SEQ ID NG:4), 50 mM BIS-TRIS pH6.0, 0.1% TritonX-100, 10 .mu.g
BSA, 0.2 .mu.g RNase A, and 0.2 .mu.M purified recombinant cytidine
deaminase. This was incubated at 37.degree. C. for 16 hours. The
DNA was recovered by using DNA Clean and Concentrator.TM. Kit (Zymo
Research, Irvine, Calif.). A mixture of nuclease P1w, Antarctic
phosphatase (and DNase I was used to digest purified ss DNA
substrate to nucleosides. LC-MS was performed on an Agilent 1200
series (G1315D Diode Array Detector, 6120 Mass Detector) (Agilent,
Santa Clara, Calif.) with Waters Atlantis T3 (4.6.times.150 mm, 3
mm, Waters, Milford, Mass.) column with in-line filter and guard
column. The results are shown in FIGS. 4A and 4B. Expected peaks
were observed in each sample, and no changes were detected after
the treatment with cytidine deaminase (MS: m/z=265). Modified
adaptor for NGS library construction was synthesized as 65-mer ss
DNA using standard phosphoramidite chemistry (Glen Research
Sterling, Va.) on an ABI394 Synthesizer (Applied Biosystems, Foster
City, Calif.). Pyrrole phosphoramidite and purification columns
were purchased from Glen Research, Sterling, Va. Oligonucleotide
was deprotected according to the manufacturer's recommendations,
purified using Glen-Pak DMT-ON columns, desalted using Gel-Pak
size-exclusion columns.
[0170] An example of a Pyrrolo dC adaptor sequence is provided
below, where X=Pyrrolo-dC:
TABLE-US-00004 (SEQ ID NO: 5)
5'/5Phos/GATXGGAAGAGXAXAXGTXTGAAXTXXAGTX/deoxy/U/
AXAXTXTTTXXXTAXAXGAXGXTXTTXXGATCT (also see FIGS. 4A and 4B).
Example 4. Whole Genome Methylome Analysis
[0171] To explore whether any sequence bias occurred and also
efficiency of the methodology, mouse ES cell genomic DNA was
sheared to 300 bp fragments with Covaris S2 sonicator (Covaris) for
library preparation with the NEBNext.RTM. Ultra.TM. DNA Library
Prep Kit for Illumina.RTM. according to the manufacturer's
instructions for DNA end repair, methylated adapter ligation, and
size selection. The sample was then denatured by heat. A Pyrrolo-dC
NEBNext adaptor (New England Biolabs, Ipswich, Mass.) was ligated
to the dA-tailed DNA followed by treatment with NEB USER.TM. (New
England Biolabs, Ipswich, Mass.).
TABLE-US-00005 Adaptor Ligation Reaction Component .mu.l dA-tailed
DNA 65 Pyrrolo dC NEBNext adaptor (5 .mu.M) 2 Blunt/TA Ligase
Master Mix 15 Ligation Enhancer 1 Total volume 83
[0172] Three libraries were created. A first library was sodium
bisulfite treated with EZ DNA Methylation-Gold Kit. A second
library was treated with EpiTect.RTM. Bisulfite Kit Cat. No. 59104
(Qiagen, Valencia, Calif.) according to instruction manual. A third
library was treated according to Example 1. The libraries were PCR
amplified using NEBNext Q5.RTM. Uracil PCR Master Mix; NEBNext
Universal PCR Primer for Illumina (15 .mu.M) and NEBNext Index PCR
Primer for Illumina (15 .mu.M) (all commercially available at New
England Biolabs, Ipswich, Mass.).
TABLE-US-00006 TABLE 1 Suggested PCR cycle numbers for mouse ES
cell genomic DNA. DNA input Number of PCR cycles 1 .mu.g 4~7 100 ng
8~10 50 ng 9~11
[0173] The results are shown in FIGS. 5-9.
[0174] Deaminase-seq did not display strong sequence preference
whereas both BS-seq methods produced more non-conversion errors
(FIG. 5). Moreover, Deaminase-seq provided results that accurately
reflected the number of C in a DNA regardless of the nature of the
adjacent nucleotide in contrast to BS-seq which showed significant
biases for CA. (FIG. 6A-6D) With the same normalized library size
of 336 million reads, Deaminase-seq library covered 1.5 million
more CpG dinucleotide sites than both BS-seq libraries and in total
has coverage for 38.0 million single CpG dinucleotide i.e., 89% of
the entire mouse genome (FIG. 7). Deaminase-seq provides a more
even sequencing coverage across the entire genome with few outliers
with very low or very high copy numbers (FIG. 8A-8C). As a result,
Deaminase-seq gives nearly 2 times as many reads as BS-seq in the
CpG islands (FIG. 9), which are among the most important genomic
regions in epigenetic studies.
[0175] A 5.4 kb fragment from glucosylated and deaminated mouse
embryonic stem cell genomic DNA (chromosome 8) was sheared to 300
bp and a library of the fragmented DNA was made using the protocol
described above and sequenced on Illumina sequencer. This method
accurately identified .sup.hmC at single base resolution across the
entire 5.4 kb region (FIG. 10).
Example 5. .sup.mC and .sup.hmC Phasing with SMRT Sequencing
(Pacific Biosystems)
[0176] Embodiments of the methods described have generated phased
genomic maps of epigenetic modifications over regions that are
limited only by the DNA polymerase used to amplify the DNA of
interest. Should amplification not be utilized, whole genomes could
be analyzed using these methods. A typical example is provided
herein with results shown in FIGS. 11A and 11B for a genomic region
of 5.4 Kb.
[0177] Mouse brain genomic DNA was treated as described in FIGS. 1A
and 1B namely by reacting aliquots of the DNA with (a) TETv+
.beta.GT treatment (for .sup.mC/.sup.hmC detection) and (b)
.beta.GT treatment (for .sup.hmC detection) respectively. The
products of these enzyme reactions were deaminated (cytidine
deaminase e.g. APOBEC3A). A 5.4 kb fragment on chromosome 8 was
then amplified from the deaminated DNA by PCR. After purification,
the 5.4 kb amplicons were used to construct PacBio SMRT libraries
following the "Amplicon template preparation and sequencing"
protocol (Pacific Biosystems, Menlo Park, Calif.). One library was
prepared for each modification type and was loaded onto SMRT cell
using the MagBead method. The two libraries were sequenced on a
PacBio RSII machine. Consensus sequences of individual sequenced
molecules (Read of Insert) were generated by the "RS_ReadsOf
Insert" protocol using the SMRT portal and were mapped to the mouse
reference genome using the Bismark algorithm. The modification
states of all the CpG sites across the 5.4 kb were determined for
individual molecule independently. The results show that this 5.4
kb region was heavily methylated across the entire region except
for its 5' end. The molecules can be divided into 2 distinct
populations: either hyper-methylated at 5' end or methylation
depleted at 5' end. In comparison, .sup.hmC exists in a few loci
and is more dynamic between molecules.
Example 6. Methylation Phasing of Long DNA Fragments (More than 10
kb Long) Using DD-Seq and Partitioning Technologies Such as
10.times. Genomics
[0178] ss long converted DNA fragments as describe in Example 5 are
purified and 1 ng of the DNA is subject to 10.times. genomics
GemCode.TM. Platform (10.times. Genomics, Pleasanton, Calif.). DNA
is partitioned into droplets together with droplet-based reagents.
The reagent contains gel beads with millions of copies of an
oligonucleotides and a polymerase that reads through uracil such as
Phusion U. Each oligonucleotide includes the universal Illumina-P5
Adaptor (Illumina, San Diego, Calif.), a barcode, Read 1 primer
site and a semi-random N-mer priming sequence. The partitioning is
done in such a way that statistically, one or several ss converted
long DNA fragments are encapsulated with one bead. The beads
dissolved after partitioning, release the oligonucleotides. The
semi-random N-mer priming sequence anneals randomly on the ss DNA
fragment and polymerase copied the template ss DNA. Droplets are
dissolved, DNA is sheared through physical shearing and after end
repair and dA tailing, and the right adaptor is ligated to the ss
DNA. Amplification of the library is done using the standard
Illumina primers and sequenced using standard Illumina protocol as
well.
Example 7. Activity Comparison of mTET2CD with TETv on Genomic
DNA
[0179] TET2cd (3 .mu.M)(SEQ ID NO: 3) or TETv (SEQ ID NO:1) was
added to 250 ng IMR90 gDNA (human fetal lung fibroblasts) substrate
in a Tris buffer pH 8.0 and the reaction was initiated with the
addition of 50 .mu.M FeSO4. The reaction was performed for 1 hour
at 37.degree. C. Subsequently, the genomic DNA was degraded to
individual nucleotides and analyzed by mass spectrometry.
[0180] The results provided in FIGS. 12A and 12B show that in the
absence of enzyme, .sup.mC is the predominant modified nucleotide
in the DNA with a small amount of .sup.hmC. In the presence of
mTET2CD, some but not all .sup.mC was converted to .sup.hmC and a
subset of these nucleotides were converted to .sup.fC suggesting
incomplete activity and/or bias. In contrast, TETv converted
substantially all the .sup.mC to .sup.caC with very little
intermediate substrate. The results are shown in FIG. 12A.
Example 8: Activity of TETv on ss and ds Mouse Genomic DNA
[0181] Mouse 3T3 gDNA was sheared to 1500 bp and purified using
Qiagen nucleotide purification kit (Qiagen, Valencia, Calif.).
Fragmented gDNA was denatured to form ss fragments by heating at
95.degree. C. for 5 minutes followed by immediate cool down on ice
for 10 minutes. 250 ng sheared 3T3 gDNA substrate was with TETv as
described in Example 8 under similar reaction conditions. Analysis
of modified bases was done according to Example 8. The results are
shown in FIG. 12B.
Example 9: TETv Exhibits Very Low Sequence Bias where Analysis of 5
Genomes Show that the Property is not Substrate Specific
[0182] The reaction was performed according to Example 7 using
genomic DNA from 5 different cell types. Low sequence specificity
is preferable as it denotes lack of sequence bias by the enzyme.
The results are shown in FIG. 13.
Example 10: DNA Treated with Tetv is Intact
[0183] Mspl is sensitive to oxidized forms of .sup.mC but not
.sup.mC. The reaction was performed according to Examples 8. TETv
was used at 3 .mu.M and Hpall plasmid substrate at 100 ng. 20 U of
BamHI (to linearize the plasmid) and 50 U of Mspl in CutSmart.RTM.
buffer (pH 7.9) (New England Biolabs, Ipswich, Mass.) were added
for 1 hour at 37.degree. C. in .mu.L total volume.
[0184] The reaction products were resolved on a 1.8% agarose gel.
The results are shown in FIG. 14.
[0185] It will also be recognized by those skilled in the art that,
while the invention has been described above in terms of preferred
embodiments, it is not limited thereto. Various features and
aspects of the above described invention may be used individually
or jointly. Further, although the invention has been described in
the context of its implementation in a particular environment, and
for particular applications (e.g. epigenetic analysis) those
skilled in the art will recognize that its usefulness is not
limited thereto and that the present invention can be beneficially
utilized in any number of environments and implementations where it
is desirable to examine DNA. Accordingly, the claims set forth
below should be construed in view of the full breadth and spirit of
the invention as disclosed herein
Sequence CWU 1
1
221507PRTArtificial SequenceSynthetic construct 1Gly Gly Ser Gln
Ser Gln Asn Gly Lys Cys Glu Gly Cys Asn Pro Asp1 5 10 15Lys Asp Glu
Ala Pro Tyr Tyr Thr His Leu Gly Ala Gly Pro Asp Val 20 25 30Ala Ala
Ile Arg Thr Leu Met Glu Glu Arg Tyr Gly Glu Lys Gly Lys 35 40 45Ala
Ile Arg Ile Glu Lys Val Ile Tyr Thr Gly Lys Glu Gly Lys Ser 50 55
60Ser Gln Gly Cys Pro Ile Ala Lys Trp Val Tyr Arg Arg Ser Ser Glu65
70 75 80Glu Glu Lys Leu Leu Cys Leu Val Arg Val Arg Pro Asn His Thr
Cys 85 90 95Glu Thr Ala Val Met Val Ile Ala Ile Met Leu Trp Asp Gly
Ile Pro 100 105 110Lys Leu Leu Ala Ser Glu Leu Tyr Ser Glu Leu Thr
Asp Ile Leu Gly 115 120 125Lys Cys Gly Ile Cys Thr Asn Arg Arg Cys
Ser Gln Asn Glu Thr Arg 130 135 140Asn Cys Cys Cys Gln Gly Glu Asn
Pro Glu Thr Cys Gly Ala Ser Phe145 150 155 160Ser Phe Gly Cys Ser
Trp Ser Met Tyr Tyr Asn Gly Cys Lys Phe Ala 165 170 175Arg Ser Lys
Lys Pro Arg Lys Phe Arg Leu His Gly Ala Glu Pro Lys 180 185 190Glu
Glu Glu Arg Leu Gly Ser His Leu Gln Asn Leu Ala Thr Val Ile 195 200
205Ala Pro Ile Tyr Lys Lys Leu Ala Pro Asp Ala Tyr Asn Asn Gln Val
210 215 220Glu Phe Glu His Gln Ala Pro Asp Cys Cys Leu Gly Leu Lys
Glu Gly225 230 235 240Arg Pro Phe Ser Gly Val Thr Ala Cys Leu Asp
Phe Ser Ala His Ser 245 250 255His Arg Asp Gln Gln Asn Met Pro Asn
Gly Ser Thr Val Val Val Thr 260 265 270Leu Asn Arg Glu Asp Asn Arg
Glu Val Gly Ala Lys Pro Glu Asp Glu 275 280 285Gln Phe His Val Leu
Pro Met Tyr Ile Ile Ala Pro Glu Asp Glu Phe 290 295 300Gly Ser Thr
Glu Gly Gln Glu Lys Lys Ile Arg Met Gly Ser Ile Glu305 310 315
320Val Leu Gln Ser Phe Arg Arg Arg Arg Val Ile Arg Ile Gly Glu Leu
325 330 335Pro Lys Ser Cys Glu Val Ser Gly Gln Asp Ala Ala Ala Val
Gln Glu 340 345 350Ile Glu Tyr Trp Ser Asp Ser Glu His Asn Phe Gln
Asp Pro Cys Ile 355 360 365Gly Gly Val Ala Ile Ala Pro Thr His Gly
Ser Ile Leu Ile Glu Cys 370 375 380Ala Lys Cys Glu Val His Ala Thr
Thr Lys Val Asn Asp Pro Asp Arg385 390 395 400Asn His Pro Thr Arg
Ile Ser Leu Val Leu Tyr Arg His Lys Asn Leu 405 410 415Phe Leu Pro
Lys His Cys Leu Ala Leu Trp Glu Ala Lys Met Ala Glu 420 425 430Lys
Ala Arg Lys Glu Glu Glu Cys Gly Lys Asn Gly Ser Asp His Val 435 440
445Ser Gln Lys Asn His Gly Lys Gln Glu Lys Arg Glu Pro Thr Gly Pro
450 455 460Gln Glu Pro Ser Tyr Leu Arg Phe Ile Gln Ser Leu Ala Glu
Asn Thr465 470 475 480Gly Ser Val Thr Thr Asp Ser Thr Val Thr Thr
Ser Pro Tyr Ala Phe 485 490 495Thr Gln Val Thr Gly Pro Tyr Asn Thr
Phe Val 500 505211PRTArtificial SequenceSynthetic construct 2Glu
Leu Pro Lys Ser Cys Glu Val Ser Gly Gln1 5 103871PRTArtificial
SequenceSynthetic construct 3Gln Ser Gln Asn Gly Lys Cys Glu Gly
Cys Asn Pro Asp Lys Asp Glu1 5 10 15Ala Pro Tyr Tyr Thr His Leu Gly
Ala Gly Pro Asp Val Ala Ala Ile 20 25 30Arg Thr Leu Met Glu Glu Arg
Tyr Gly Glu Lys Gly Lys Ala Ile Arg 35 40 45Ile Glu Lys Val Ile Tyr
Thr Gly Lys Glu Gly Lys Ser Ser Gln Gly 50 55 60Cys Pro Ile Ala Lys
Trp Val Tyr Arg Arg Ser Ser Glu Glu Glu Lys65 70 75 80Leu Leu Cys
Leu Val Arg Val Arg Pro Asn His Thr Cys Glu Thr Ala 85 90 95Val Met
Val Ile Ala Ile Met Leu Trp Asp Gly Ile Pro Lys Leu Leu 100 105
110Ala Ser Glu Leu Tyr Ser Glu Leu Thr Asp Ile Leu Gly Lys Cys Gly
115 120 125Ile Cys Thr Asn Arg Arg Cys Ser Gln Asn Glu Thr Arg Asn
Cys Cys 130 135 140Cys Gln Gly Glu Asn Pro Glu Thr Cys Gly Ala Ser
Phe Ser Phe Gly145 150 155 160Cys Ser Trp Ser Met Tyr Tyr Asn Gly
Cys Lys Phe Ala Arg Ser Lys 165 170 175Lys Pro Arg Lys Phe Arg Leu
His Gly Ala Glu Pro Lys Glu Glu Glu 180 185 190Arg Leu Gly Ser His
Leu Gln Asn Leu Ala Thr Val Ile Ala Pro Ile 195 200 205Tyr Lys Lys
Leu Ala Pro Asp Ala Tyr Asn Asn Gln Val Glu Phe Glu 210 215 220His
Gln Ala Pro Asp Cys Cys Leu Gly Leu Lys Glu Gly Arg Pro Phe225 230
235 240Ser Gly Val Thr Ala Cys Leu Asp Phe Ser Ala His Ser His Arg
Asp 245 250 255Gln Gln Asn Met Pro Asn Gly Ser Thr Val Val Val Thr
Leu Asn Arg 260 265 270Glu Asp Asn Arg Glu Val Gly Ala Lys Pro Glu
Asp Glu Gln Phe His 275 280 285Val Leu Pro Met Tyr Ile Ile Ala Pro
Glu Asp Glu Phe Gly Ser Thr 290 295 300Glu Gly Gln Glu Lys Lys Ile
Arg Met Gly Ser Ile Glu Val Leu Gln305 310 315 320Ser Phe Arg Arg
Arg Arg Val Ile Arg Ile Gly Glu Leu Pro Lys Ser 325 330 335Cys Lys
Lys Lys Ala Glu Pro Lys Lys Ala Lys Thr Lys Lys Ala Ala 340 345
350Arg Lys Arg Ser Ser Leu Glu Asn Cys Ser Ser Arg Thr Glu Lys Gly
355 360 365Lys Ser Ser Ser His Thr Lys Leu Met Glu Asn Ala Ser His
Met Lys 370 375 380Gln Met Thr Ala Gln Pro Gln Leu Ser Gly Pro Val
Ile Arg Gln Pro385 390 395 400Pro Thr Leu Gln Arg His Leu Gln Gln
Gly Gln Arg Pro Gln Gln Pro 405 410 415Gln Pro Pro Gln Pro Gln Pro
Gln Thr Thr Pro Gln Pro Gln Pro Gln 420 425 430Pro Gln His Ile Met
Pro Gly Asn Ser Gln Ser Val Gly Ser His Cys 435 440 445Ser Gly Ser
Thr Ser Val Tyr Thr Arg Gln Pro Thr Pro His Ser Pro 450 455 460Tyr
Pro Ser Ser Ala His Thr Ser Asp Ile Tyr Gly Asp Thr Asn His465 470
475 480Val Asn Phe Tyr Pro Thr Ser Ser His Ala Ser Gly Ser Tyr Leu
Asn 485 490 495Pro Ser Asn Tyr Met Asn Pro Tyr Leu Gly Leu Leu Asn
Gln Asn Asn 500 505 510Gln Tyr Ala Pro Phe Pro Tyr Asn Gly Ser Val
Pro Val Asp Asn Gly 515 520 525Ser Pro Phe Leu Gly Ser Tyr Ser Pro
Gln Ala Gln Ser Arg Asp Leu 530 535 540His Arg Tyr Pro Asn Gln Asp
His Leu Thr Asn Gln Asn Leu Pro Pro545 550 555 560Ile His Thr Leu
His Gln Gln Thr Phe Gly Asp Ser Pro Ser Lys Tyr 565 570 575Leu Ser
Tyr Gly Asn Gln Asn Met Gln Arg Asp Ala Phe Thr Thr Asn 580 585
590Ser Thr Leu Lys Pro Asn Val His His Leu Ala Thr Phe Ser Pro Tyr
595 600 605Pro Thr Pro Lys Met Asp Ser His Phe Met Gly Ala Ala Ser
Arg Ser 610 615 620Pro Tyr Ser His Pro His Thr Asp Tyr Lys Thr Ser
Glu His His Leu625 630 635 640Pro Ser His Thr Ile Tyr Ser Tyr Thr
Ala Ala Ala Ser Gly Ser Ser 645 650 655Ser Ser His Ala Phe His Asn
Lys Glu Asn Asp Asn Ile Ala Asn Gly 660 665 670Leu Ser Arg Val Leu
Pro Gly Phe Asn His Asp Arg Thr Ala Ser Ala 675 680 685Gln Glu Leu
Leu Tyr Ser Leu Thr Gly Ser Ser Gln Glu Lys Gln Pro 690 695 700Glu
Val Ser Gly Gln Asp Ala Ala Ala Val Gln Glu Ile Glu Tyr Trp705 710
715 720Ser Asp Ser Glu His Asn Phe Gln Asp Pro Cys Ile Gly Gly Val
Ala 725 730 735Ile Ala Pro Thr His Gly Ser Ile Leu Ile Glu Cys Ala
Lys Cys Glu 740 745 750Val His Ala Thr Thr Lys Val Asn Asp Pro Asp
Arg Asn His Pro Thr 755 760 765Arg Ile Ser Leu Val Leu Tyr Arg His
Lys Asn Leu Phe Leu Pro Lys 770 775 780His Cys Leu Ala Leu Trp Glu
Ala Lys Met Ala Glu Lys Ala Arg Lys785 790 795 800Glu Glu Glu Cys
Gly Lys Asn Gly Ser Asp His Val Ser Gln Lys Asn 805 810 815His Gly
Lys Gln Glu Lys Arg Glu Pro Thr Gly Pro Gln Glu Pro Ser 820 825
830Tyr Leu Arg Phe Ile Gln Ser Leu Ala Glu Asn Thr Gly Ser Val Thr
835 840 845Thr Asp Ser Thr Val Thr Thr Ser Pro Tyr Ala Phe Thr Gln
Val Thr 850 855 860Gly Pro Tyr Asn Thr Phe Val865
870444DNAArtificial SequenceSynthetic
constructmisc_feature(18)..(18)n is Pyrrolo-dC 4ataagaatag
aatgaatngt gaaatgaata tgaaatgaat agta 44565DNAArtificial
SequenceSynthetic constructmisc_feature(4)..(4)n is
Pyrrolo-dcmisc_feature(12)..(12)n is
Pyrrolo-dcmisc_feature(14)..(14)n is
Pyrrolo-dcmisc_feature(16)..(16)n is
Pyrrolo-dcmisc_feature(19)..(19)n is
Pyrrolo-dcmisc_feature(24)..(24)n is
Pyrrolo-dcmisc_feature(26)..(27)n is
Pyrrolo-dcmisc_feature(31)..(31)n is
Pyrrolo-dcmisc_feature(34)..(34)n is
Pyrrolo-dcmisc_feature(36)..(36)n is
Pyrrolo-dcmisc_feature(38)..(38)n is
Pyrrolo-dcmisc_feature(42)..(44)n is
Pyrrolo-dcmisc_feature(47)..(47)n is
Pyrrolo-dcmisc_feature(49)..(49)n is
Pyrrolo-dcmisc_feature(52)..(52)n is
Pyrrolo-dcmisc_feature(54)..(54)n is
Pyrrolo-dcmisc_feature(56)..(56)n is
Pyrrolo-dcmisc_feature(59)..(60)n is Pyrrolo-dc 5gatnggaaga
gnanangtnt gaantnnagt nuanantntt tnnntanang angntnttnn 60gatct
65625DNAArtificial SequenceSynthetic construct 6aatgaaggaa
atgaatttgg tagag 25725DNAArtificial SequenceSynthetic construct
7tcccaaatac ataaatccac actta 25834DNAArtificial SequenceSynthetic
construct 8taggataaaa atataaatgt attgtgggat gagg 34927DNAArtificial
SequenceSynthetic construct 9aaaacatata accccctcca ctaatac
271031DNAArtificial SequenceSynthetic construct 10agatatattg
gagaagtttt ggatgatttg g 311127DNAArtificial SequenceSynthetic
construct 11aaaacatata accccctcca ctaatac 271225DNAArtificial
SequenceSynthetic construct 12taagattaag gtaggttgga tttgg
251325DNAArtificial SequenceSynthetic construct 13tcattactcc
ctctccaaaa attac 251425DNAArtificial SequenceSynthetic constuct
14aagatttaag ggaaggttga atagg 251525DNAArtificial SequenceSynthetic
construct 15acctacaaaa ccttacaaac ataac 251630DNAArtificial
SequenceSynthetic construct 16tggagtttgt tggggggttt gttgtttaag
301730DNAArtificial SequenceSynthetic construct 17tctaaccctc
accaccttcc taatacccaa 301830DNAArtificial SequenceSynthetic
construct 18tggtaaaggt taagaaggga agattgtgga 301930DNAArtificial
SequenceSynthetic construct 19aaccctactt ccccctaaca aattttcaac
302012DNAArtificial SequenceSynthetic construct 20ccgtcggacc gc
122112DNAArtificial SequenceSynthetic construct 21uugtcggauu gc
122212DNAArtificial SequenceSynthetic construct 22uugtcggauu gt
12
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