U.S. patent application number 17/576503 was filed with the patent office on 2022-06-30 for assaying ovarian cyst fluid.
The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Luis Diaz, Kenneth W. Kinzler, Bjorg Kristjansdottir, Nickolas Papadopoulos, Karin Sundfeldt, Bert Vogelstein, Yuxuan Wang.
Application Number | 20220205048 17/576503 |
Document ID | / |
Family ID | 1000006196471 |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220205048 |
Kind Code |
A1 |
Wang; Yuxuan ; et
al. |
June 30, 2022 |
ASSAYING OVARIAN CYST FLUID
Abstract
A diagnostic test for ovarian cysts is based on the detection of
mutations characteristic of the most common neoplasms giving rise
to these lesions. With this test, tumor-specific mutations were
detected in the cyst fluids of 19 of 24 (79%) borderline tumors and
28 of 31 (90%) malignant ovarian cancers. In contrast, we detected
no mutations in the cyst fluids from 10 non-neoplastic cysts and 12
benign tumors. When categorized by the need for exploratory surgery
(i.e., presence of a borderline tumor or malignant cancer), the
sensitivity of this test was 85% and the specificity was 100%.
These tests could inform the diagnosis of ovarian cysts and improve
the clinical management of the large number of women with these
lesions.
Inventors: |
Wang; Yuxuan; (Baltimore,
MD) ; Vogelstein; Bert; (Baltimore, MD) ;
Kinzler; Kenneth W.; (Baltimore, MD) ; Diaz;
Luis; (Ellicot City, MD) ; Papadopoulos;
Nickolas; (Towson, MD) ; Sundfeldt; Karin;
(Gothenburg, SE) ; Kristjansdottir; Bjorg;
(Gothenburg, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
|
|
Family ID: |
1000006196471 |
Appl. No.: |
17/576503 |
Filed: |
January 14, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15749887 |
Feb 2, 2018 |
11286531 |
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PCT/US2016/046453 |
Aug 11, 2016 |
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17576503 |
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62203573 |
Aug 11, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/156 20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886 |
Goverment Interests
[0002] This invention was made with government support under CA
043460, 057345, and 062924 awarded by National Institutes of
Health. The government has certain rights in the invention.
Claims
1-34. (canceled)
35. A method, comprising: testing ovarian cyst fluid for a mutation
in at least one gene mutated in a type II ovarian neoplasm, wherein
said at least one gene comprises TP53, wherein the step of testing
employs a step of adding a unique identifier (UID) to a plurality
of TP53 template DNA molecules in the ovarian cyst fluid, and
wherein the method has a sensitivity level of at least 70%.
36. The method of claim 35, wherein said at least one gene further
comprises KRAS.
37. The method of claim 35, wherein the step of testing employs
TP53-specific reagents.
38. The method of claim 35, wherein the step of testing employs
TP53 mutation-specific reagents.
39. The method of claim 35, wherein the testing is performed on the
ovarian cyst fluid and on a sample selected from the group
consisting of cyst wall and normal, non-ovarian tissue.
40. The method of claim 35, wherein the ovarian cyst fluid is
obtained by needle aspiration of an ovarian cyst.
41. The method of claim 35, wherein the ovarian cyst fluid is
obtained prior to any surgical removal of the ovarian cyst.
42. The method of claim 35, wherein the ovarian cyst fluid is
obtained after surgical removal of the ovarian cyst and recurrence
of the ovarian cyst.
43. The method of claim 35, wherein a copy number variation, a loss
of heterozygosity, or both, is determined for said at least one
gene.
44. The method of claim 35, wherein a point mutation, a
rearrangement, a frameshift, or combinations thereof, is determined
for said at least one gene.
45. The method of claim 35, wherein the method has a sensitivity
level of at least 85%.
46. The method of claim 35, wherein the method has a specificity
level of at least 90%.
47. The method of claim 35, wherein the ovarian neoplasm needs
surgery.
Description
[0001] This application is a Continuation of U.S. application Ser.
No. 15/749,887, filed Feb. 2, 2018, which is a National Stage
application under 35 U.S.C. .sctn. 371 of International Application
No. PCT/US2016/046453, having an International Filing Date of Aug.
11, 2016, which claims the benefit of U.S. Provisional Application
No. 62/203,573, filed Aug. 11, 2015, each of which is incorporated
herein by reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention is related to the area of DNA analysis. In
particular, it relates to analysis of genes in clinical
samples.
BACKGROUND OF THE INVENTION
[0004] Ovarian cancer is the most lethal gynecologic malignancy,
with 21,980 estimated new cases and 14,270 estimated deaths in the
United States in 2014. Approximately 1.3% of women will be
diagnosed with ovarian cancer during their lifetime (1). These
cancers commonly present as an adnexal mass with cystic components,
but are not associated with specific symptoms. As a result,
two-thirds of ovarian cancers are diagnosed at late stage (Stage
III and IV), when the 5-year survival is less than 30% (1).
[0005] Complicating the diagnosis of ovarian cancer is the fact
that ovarian cysts are common in women of all ages, with a
prevalence of 35% and 17% in pre- and post-menopausal women,
respectively (2). These cysts are frequently benign and found
incidentally on routine imaging (2). Though malignancy is an
unusual cause of the cysts, 30% of the cysts exhibit radiographic
features suspicious for malignancy, such as solid areas or mass
(2). In addition to the anxiety that such findings provoke, many
women undergo unnecessary surgery for cysts that are not malignant
and may not be responsible for the symptoms they have. For example,
only 5% of 570 women in a large ovarian cancer screening randomized
trial who underwent surgical evaluation actually had a malignancy
(3). Compounding this issue is the fact that surgery for ovarian
cysts requires general anesthesia and is associated with
significant morbidity, causing serious complications in 15% of
women. These complications include damage to nerves and ureters,
bleeding, infection, perforation of adjacent viscera, as well as
hormonal and fertility loss (in the case of bilateral oophorectomy)
(4). Even minimal procedures such as ovarian cystectomy can affect
fertility in premenopausal women by decreasing follicular response
and oocyte number (5, 6). If a preoperative test could be performed
that indicated whether the cystic lesion was benign or malignant,
unnecessary surgery and its associated complications could be
avoided in a large number of patients, particularly women of
reproductive age who wish to preserve their fertility, as well as
women whose medical comorbidities or functional status makes
anesthesia and surgery hazardous.
[0006] Ovarian cysts and tumors are classified as non-neoplastic,
benign, borderline, or malignant based on microscopic examination
after surgical removal (FIG. 1). Non-neoplastic cysts are by far
the most common class of ovarian cyst. They are frequently
functional in pre-menopausal women, arising when an egg is not
released properly from either the follicle or corpus luteum and
usually resolve spontaneously within several months (7). Benign
cystic tumors, such as cystadenomas and cystadenofibromas, rarely
progress to malignancy (8, 9). No genetic alterations have yet been
identified in either non-neoplastic cysts or in benign cystic
tumors (9). Neither of these cyst types requires surgery unless
they are symptomatic or have undergone torsion (8). These cysts can
be easily sampled with ultrasound-guided fine-needle aspiration in
an outpatient setting without the need for anesthesia (10).
[0007] At the other end of the spectrum are epithelial ovarian
cancers, which are potentially lethal and unequivocally require
surgery. A dualistic model has been proposed to classify these
neoplasms (11). Type I tumors are composed of low-grade serous,
low-grade endometrioid, clear cell, and mucinous carcinomas. They
are clinically indolent, frequently diagnosed at early stage (Stage
I or II), and develop from well-established precursor lesions
("borderline" or "atypical proliferative" tumors, as described
below) (12). Type I cancers commonly exhibit mutations in KRAS,
BRAF, CTNNB1, PIK3CA, PTEN, ARID1A, or PPP2R1A (11). In contrast,
type II tumors are generally high-grade serous carcinomas. They are
highly aggressive, most often diagnosed in late stage (Stage III or
IV), and have suggested origins from the distal fallopian tube
(13). Type II cancers almost always harbor TP53 mutations (14).
Also unlike type I cancers, which are relatively chemo-resistant
and more often treated only with surgical excision, type II cancers
respond to conventional chemotherapy, particularly after maximal
debulking to reduce tumor burden (15, 16).
[0008] "Borderline" or "atypical proliferative" tumors lie in the
middle of this spectrum, between the malignant cancers and the
relatively harmless (non-neoplastic or benign) lesions. They are
distinguished from carcinomas by the absence of stromal invasion
and are precursors of type I cancers. In light of their potential
for malignancy, the standard of care for borderline tumors is
surgical excision. Following surgery, the prognosis is excellent
compared to ovarian cancers, with 5-year survival rates over 85%
(17). A minor but significant portion of borderline tumors recur
after surgery, however, and a subset of the recurrences are found
to have advanced to type I cancers (18). This progression is
consistent with molecular findings: serous borderline tumors
typically exhibit mutations in BRAF or KRAS, like their malignant
counterparts (low-grade serous carcinoma) (19, 20). The presence of
a BRAF mutation in a borderline tumor is associated with better
prognosis and a low probability of progression to carcinoma (21).
In contrast, KRAS mutations are associated with the progression to
type I cancers (22).
[0009] The examination of fluids from pancreatic, renal, and
thyroid cysts is routinely used in clinical management (23-25). The
fluids have historically been studied by cytology to identify
malignant cysts. Ovarian cysts share many features with these other
types of cysts, in that they are common, often diagnosed
incidentally, and are nearly always benign. However, aspiration of
ovarian cyst fluid for cytology is not standard-of-care. From a
historical perspective, the difference in diagnostic management
probably lies in the fact that cytology has not proven to be very
informative for ovarian cysts, particularly for distinguishing
benign vs. borderline tumors (26, 27).
[0010] More recently, genetic analysis of specific types of cyst
fluids has been considered as an aid to cytology, given that
conventional cytology often has limited sensitivity and specificity
(28). Based on the emerging success of the molecular genetic
evaluation of other types of cysts, we reasoned that a similar
approach could be applied to ovarian cysts. Evaluation of DNA from
cells and cell fragments shed into the cyst fluid would presumably
allow the identification of tumor-specific mutations. Unlike other,
conventional markers of neoplasia such as CA-125, cancer gene
mutations are exquisitely specific indicators of a neoplastic
lesion (29). Moreover, the type of mutation can in some cases
indicate the type of neoplastic lesion present (30). Yamada et al.
have demonstrated that mutations can be detected in the cystic
fluid of ovarian tumors by querying exons 4 to 9 of TP53, achieving
sensitivities of 12.5% and 10%, for borderline and malignant
tumors, respectively (31). Extremely sensitive methods for mutation
detection, capable of identifying one mutant template allele among
thousands of normal templates in a panel of genes, have recently
been developed (32-34). In this study, we here applied one of these
technologies to determine whether mutations could be identified in
ovarian cyst fluids, and if so, whether they provided information
that could in principle be used in diagnosis and management.
[0011] Because there is currently no reliable way to determine
whether an ovarian cyst is malignant prior to surgical excision,
many women undergo unnecessary, invasive surgeries for
non-malignant lesions. There is a need in the art for techniques to
determine whether surgery is required or unnecessary.
SUMMARY OF THE INVENTION
[0012] According to one aspect of the invention a method is
provided in which ovarian cyst fluid is tested for mutations in a
panel of genes frequently mutated in ovarian neoplasms, wherein the
panel comprises BRAF, KRAS, and TP53.
[0013] According to another aspect of the invention a method is
provided in which ovarian cyst fluid is tested for mutations in a
panel of genes frequently mutated in ovarian neoplasms, wherein the
panel comprises BRAF, KRAS, TP53, and one or more of AKT1, APC,
BRCA1, BRCA2, CDKN2A, EGFR, FBXW7, FGFR2, MAPK1, NRAS, PIK3R1, and
POLE.
[0014] According to another aspect of the invention a method is
provided in which ovarian cyst fluid is tested for mutations in a
panel of genes frequently mutated in ovarian neoplasms, wherein the
panel comprises BRAF, KRAS, TP53, and one or more of CTNNB1,
PIK3CA, PTEN, ARID1A, and PPP2R1A.
[0015] According to an additional aspect of the invention a method
is provided in which ovarian cyst fluid is tested for mutations in
a panel of genes frequently mutated in ovarian neoplasms, wherein
the panel comprises BRAF, KRAS, TP53, AKT1, APC, BRCA1, BRCA2,
CDKN2A, EGFR, FBXW7, FGFR2, MAPK1, NRAS, PIK3R1, and POLE.
[0016] According to an additional aspect of the invention a method
is provided in which ovarian cyst fluid is tested for mutations in
a panel of genes frequently mutated in ovarian neoplasms, wherein
the panel comprises BRAF, KRAS, TP53, CTNNB1, PIK3CA, PTEN, ARID1A,
and PPP2R1A.
[0017] According to an additional aspect of the invention a method
is provided in which ovarian cyst fluid is tested for mutations in
a panel of genes frequently mutated in ovarian neoplasms, wherein
the panel comprises BRAF, KRAS, TP53, AKT1, APC, BRCA1, BRCA2,
CDKN2A, EGFR, FBXW7, FGFR2, MAPK1, NRAS, PIK3R1, POLE, CTNNB1,
PIK3CA, PTEN, ARID1A, and PPP2R1A.
[0018] These and other embodiments which will be apparent to those
of skill in the art upon reading the specification provide the art
with powerful methods for assessing ovarian cysts without recourse
to unnecessary surgeries.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1. Schematic showing classes of ovarian cysts and the
diagnostic potential of the cyst fluid. Ovarian cysts and tumors
are currently classified according to microscopic evaluation after
surgical removal. The majority of ovarian cysts are non-neoplastic
(often "functional" in premenopausal women). Ovarian tumors with
combined cystic and solid components are either benign tumors,
borderline tumors, or malignant cancers (type I or II). Only cysts
associated with borderline tumors and cancers require surgical
excision. We show here that the DNA purified from cyst fluid can be
analyzed for somatic mutations commonly found in their associated
tumors. The type of mutation detected not only indicates the type
of tumor present but also could inform management.
[0020] FIGS. 2A-2B. Mutant allele fractions. (FIG. 2A)
Classification by tumor type. No mutations were found in the DNA of
non-neoplastic or benign cysts (red). Of the cysts that required
surgery (blue), the median mutant allele fraction was higher in the
cyst fluids associated with type II cancer (60.3%) than type I
(7.8%) or borderline tumors (2.4%). (FIG. 2B) Classification by
tumor stage. The DNA from cyst fluids of late-stage cancers had a
higher median mutant allele fraction (51.2%) than those of
early-stage cancers (7.4%) or borderline tumors (2.4%). Horizontal
bars depict median and IQR.
[0021] FIGS. 3A-3D. (Table 1.) Patient demographics. The clinical
characteristics of patients in this study and their tumor
characteristics are indicated.
[0022] FIG. 4A-4E. (Table 2.) Mutations identified in tumors and
cyst fluids. The mutations, mutant allele fractions, and amount of
DNA recovered from cyst fluids are indicated.
[0023] FIG. 5 (Table 3.) Detection of tumor-specific mutations in
cyst fluid. The fraction of samples detected and the median
fraction of mutant alleles are indicated, grouped by cyst type,
cancer stage, and the need for surgery.
[0024] FIG. 6 (Table 4.) Multivariate analysis for markers
associated with need for surgery. The presence of a mutation, cyst
DNA amount, and common serum biomarkers for ovarian cancer were
analyzed for association with cysts that require surgical removal
(Firth's penalized likelihood logistic regression).
[0025] FIG. 7A-7C (Fig. S1.) Markers associated with the need for
surgery. Cyst DNA amount and levels of commonly used ovarian cancer
serum biomarkers are plotted according to the cyst type and need
for surgery. (FIG. 7A) The amounts of DNA in cyst fluids was
generally higher in cysts requiring surgery (blue) than those that
do not (red), but no significant correlation was found (p=0.69).
(FIG. 7B) CA-125 levels were significantly higher in cysts that
required surgery than those that do not (p=0.01). (FIG. 7C) Serum
HE4 levels was not correlated with the need for surgery (p=0.92).
P-values were calculated using Firth's penalized likelihood
logistic regression in a multivariate model (See Example 1).
[0026] FIGS. 8A-8H (Table S1) Primer sequences used in multiplex
assay; Forward primers (SEQ ID NO: 1-133); Reverse primers (SEQ ID
NO: 134-266).
[0027] FIG. 9A-9B Mutated genes found in the cyst fluid samples.
FIG. 9A shows non-neoplastic, benign, and borderline. FIG. 9B shows
malignant Type I and malignant Type II. Yellow boxes represent
mutations with mutant allele frequency (MAF) between 0.1% and 1%;
orange boxes represent mutations with MAF between 1 and 10%; red
boxes represent mutations with MAF greater than 10% (* indicates
patients with insufficient DNA for analysis; ** indicates patients
with two detected mutations).
DETAILED DESCRIPTION OF THE INVENTION
[0028] The inventors have developed an assay for testing cyst
fluids. Cyst fluids are typically aspirated by a needle, preferably
a fine needle. The aspiration can be performed under the guidance
of a radiological technique, such as ultrasound. Other guidance
techniques can be used as convenient. Cyst fluids can typically be
collected from any type of ovarian cyst or cystic neoplasm, and the
term "cyst" is used here to refer to all types of ovarian growths
with a cystic component.
[0029] Non-neoplastic ovarian cysts typically do not require
surgical removal and do not display mutations. In contrast, ovarian
cysts that are associated with malignancy do require surgical
removal and frequently display mutations; these mutations can
further indicate the type and severity of the disease. Testing for
a panel that includes markers for a broad range of ovarian cysts
permits the identification of cyst type and prognosis. It also
permits a clinical decision to surgically remove or not.
[0030] Other markers and clinical indication can be used in
combination with the ovarian cyst fluid assay results. Plasma
markers such as CA-125 and HE4 can be assessed in patient plasma.
Other protein or genetic markers can be used in conjunction with
the ovarian cyst fluid assay. Other clinical indicators, including
radiological findings and physical findings may be used in
conjunction with the ovarian cyst fluid assay.
[0031] Testing may be performed using any technique that is
targeted for particular genes. These are not techniques that screen
for any and all gene mutations. Rather, they are designed to detect
mutations in certain predetermined genes. In some cases they are
designed to detect certain mutations or mutations in certain
codons. Any analytic technique can be used for detecting mutations
as is convenient, efficient, and sufficiently sensitive to detect
mutations in ovarian cyst fluid. The assays may be hybridization
based, such as using specific probes or specific primers. The
assays may employ labeled probes or primers. The assays may employ
labeled secondary reagents that permit the primary reagents to be
detected. Such labels include radiolabels, fluorescent labels,
enzymatic labels, chromophores, and the like.
[0032] A variety of different mutation types can be detected and
may be useful in providing prognosis or management decisions. Such
mutations include LOH, point mutations, rearrangements,
frameshifts, point mutations, and copy number variations. Specific
detection techniques for these mutation types or generic detection
techniques may be used. It may be desirable to use control samples
from other parts of the patient's body, such as a body fluid, like
plasma, saliva, urine, feces, and the like. Alternatively other
control samples may include tissues such as normal tissue from a
non-ovary, or cells or tissues from the ovarian cyst wall.
[0033] Cyst fluid may be obtained by any technique known in the
art, including but not limited to needle aspiration. The aspiration
may optionally be guided by a radiological technique such as
ultrasound. Cyst fluid may be aspirated before or after initial
surgical removal or subsequent surgical removal.
[0034] In some embodiments, primers will incorporate unique
identification DNA sequence (UID) which are molecular barcodes.
These can be randomly generated and attached to templates as a
means to reduce errors arising from amplification and sequencing.
Probes, primers, and UIDs can incorporate non-naturally occurring
modifications to DNA sequences, by internucleotide linkage
modifications, by sugar modifications, and by nucleobase
modifications. For example, phosphorothioate (PS) linkages can be
used in which sulfur substitutes for one nonbridging phosphate
oxygen. This imparts resistance to nuclease degradation. Other
modifications which can be used include N3' phosphoramidate (NP)
linkages, Boranophosphate internucleotide linkages,
Phosphonoacetate (PACE) linkages, Morpholino phosphoramidates,
Peptide nucleic acid (PNA), 2'-O-Me nucleoside analog, 2'F-RNA
modification, 2'-deoxy-2'-fluoro-.beta.-D-arabino nucleic acid
(2'F-ANA) modification and Locked nucleic acid (LNA).
[0035] Other techniques which are unbiased toward particular genes
can be used as well for assessing genes of interest in cyst fluid.
Such techniques include whole-genome or whole exome techniques.
These may include assessments by nucleotide sequencing. The
nucleotide sequencing may be redundant nucleotide sequencing.
Targeted sequencing methods can be used as well.
[0036] The methods described here achieve high degrees of
sensitivity and specificity. Sensitivity may be at least 15%, at
least 20%, at least 25%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 85%, at least 90%, at least 95% for
borderline and malignant tumors. Specificity may be at least 15%,
at least 20%, at least 25%, at least 50%, at least 60%, at least
70%, at least 80%, at least 85%, at least 90%, at least 95% for
borderline and malignant tumors.
[0037] Removal of ovarian cyst fluid assay from the body can be
accomplished before any surgery occurs. Thus the results of the
assay can help guide the decision to perform surgery. If surgery
has occurred to remove the ovarian cyst, and if it returns, a
sample of ovarian cyst fluid may be obtained from the body at that
time. The assays will typically be performed in a clinical
laboratory on samples that have been removed by a skilled
clinician, such as an interventional radiologist or a surgeon. The
samples may be assayed immediately or they may suitable stored and
or shipped for testing. It is possible that DNA will be extracted
from the sample prior to shipping it to a laboratory for testing.
Results will generally be communicated back from the assaying
laboratory to the clinician for communication to a patient. Results
may be recorded in paper or electronic medical records.
[0038] Ovarian cancer is the most lethal gynecologic cancer in
women. However screening is not recommended by the U.S. Preventive
Services Task Force using current diagnostic approaches, which too
frequently lead to "important harms, including major surgical
interventions in women who do not have cancer" (Moyer and Force,
2012). We have demonstrated here that driver mutations in ovarian
tumors are also present in their associated cyst fluids. Moreover,
the mutant allele frequencies in the cyst fluids are relatively
high (median 12.6%, IQR of 2.7% to 40.2%), facilitating their
detection. There were no mutations detected in the cyst fluids that
were not also present in the tumors, and vice versa. Also
importantly, no mutation was identified in non-neoplastic cysts or
cysts associated with benign tumors. Overall, mutations were
detected in a major fraction (87%) of cysts requiring surgery but
not in any cyst that did not require surgery.
[0039] Our results demonstrate that mutations present in ovarian
tumors are also present in their associated cyst fluids. Moreover,
the mutant allele frequencies in the cyst fluids are relatively
high (median 12.6%, IQR of 2.7% to 40.2%), facilitating their
detection. There were no mutations detected in the cyst fluids that
were not also present in the tumors, and vice versa. And most
importantly, mutations were detected in a major fraction (85%) of
cysts requiring surgery but not in any cyst that did not require
surgery (Tables 2 and 3).
[0040] Although most (85%) of the 55 cysts requiring surgery had
detectable mutations in their fluidic compartment, eight did not.
All of these eight cysts occurred in borderline tumors or type I
cancers, while mutations were always (100%) detectable in type II
cancers (Tables 2 and 3). There are two potential explanations for
our failure to detect mutations in these eight cysts. First, it is
possible that the mutant DNA concentration in these cysts was below
the level of technical sensitivity of our assay (.about.0.1% mutant
allele fraction). We excluded this possibility by evaluating the
tumors themselves: no mutations were detected in any of the tumors
from these 8 patients. The second, and therefore more likely
explanation, is that our panel of 133 amplicons, containing regions
of 17 genes, was not adequate to capture the mutations that were
present. Unlike type II cancers, which nearly always contain TP53
mutations (94% of the type II cancers we studied, for example), the
genomic landscapes of type I cancers and borderline tumors are more
heterogeneous and not as well studied (II). Further genetic
evaluation of these tumors should facilitate the incorporation of
additional amplicons in the panel to reach higher sensitivities.
Nevertheless, the 100% sensitivity for type II cancers in our study
is highly encouraging, given that these cancers account for over
90% of ovarian cancer deaths.
[0041] One limitation of our study is the number of patients
evaluated. Though excision of ovarian cysts is one of the most
commonly performed surgical procedures, banking of cyst fluids is
not common, even in academic centers. Thus, we only had relatively
small numbers (n=22) of non-neoplastic cysts and benign tumors
available for study. Even so, the differences in genetic
alterations among the various cyst types were striking (Tables 2
and 3). Our study will hopefully stimulate collection and analyses
of ovarian cyst fluids that will be able to establish smaller
confidence limits around the sensitivities and specificities
reported in the current study.
[0042] A potential clinical limitation of our approach is the
concern by gynecologists that needle puncture of a malignant
ovarian cyst leads to seeding of the peritoneum. This concern is
based on inconclusive evidence about the dangers of cyst rupture
during surgery and is, at best, controversial (40). Moreover,
leakage is expected to be much less likely when a tiny needle is
inserted into the cyst under ultrasound-guidance than when cysts
are manipulated during surgery. The idea that malignant cysts might
shed cancer cells if needle-punctured also seems incongruent with
the widespread practice of laparoscopic removal of ovarian cysts
(41). Laparoscopic removal of a cyst carries a risk of cyst
rupture, perhaps higher than needling (42). Finally, malignant
pancreatic cysts are at least as dangerous as malignant ovarian
cysts, yet the standard-of-care for pancreatic cysts involves
repeated sampling of cyst fluid through endoscopic ultrasound over
many years (43, 44). Though pancreatic cysts and ovarian cysts lie
in different anatomical compartments, it is encouraging that
aspiration of pancreatic cysts is not associated with an increased
risk of mortality in patients with pancreatic cancer (45). Finally,
recent advancements in methods to plug biopsy tracts, using
materials such as absorbable gelatin slurry and torpedo, can
further decrease the risk of tumor spillage associated with
fine-needle aspirations (46, 47). On the basis of these
observations and recent developments, we believe that
ultrasound-guided aspiration of ovarian cyst fluids would likely be
a safe and well-tolerated procedure.
[0043] As noted in the background of the invention section above,
seven to ten patients with benign ovarian cyst lesions undergo
surgery for each case of ovarian cancer found (48). In addition to
the psychological impact a potential diagnosis of cancer has on
patients, surgery for benign lesions entails considerable cost and
morbidity. OVA-1 is the only FDA-cleared test to date that aims to
distinguish benign versus malignant adnexal mass. It measures
levels of five serum markers (CA-125, (3-2 microglobulin,
apolipoprotein A1, prealbumin, and transferrin) and is used to
stratify patients who should consult a gynecologic oncologist
rather than a general gynecologist for surgery. However the test
has a specificity of 43% for ovarian cancer, which is even lower
than that of CA-125 alone (49). While the test might encourage
patients with suspected ovarian cancer to seek specialized care, it
would not decrease the number of unnecessary surgeries for women
with benign adnexal masses.
[0044] This study was driven by the need for a biomarker that would
help distinguish malignant ovarian tumors from benign lesions and
thereby reduce the number of unnecessary surgeries. Such
distinction is often difficult based on symptoms and conventional
diagnostic criteria. For example, in a large study of 48,053
asymptomatic postmenopausal women who underwent ultrasound
examination by skilled sonographers, 8 (17%) of the 47 ovarian
cancers that were identified occurred in women with persistently
normal sonographic findings (Sharma et al., 2012). All eight cases
were type II cancers, highlighting the potential utility of an
additional assay to detect this highly lethal and aggressive type
of ovarian cancer. On the other hand, of the 4367 women with
abnormal sono graphic findings, less than 1% of cases proved to
have malignancy upon surgery. Furthermore, of the 32 women with
borderline or Type I cancers diagnosed, 22 (69%) had a serum CA-125
level within the clinically accepted normal range (.ltoreq.35
units/mL). In our study, 18 of 18 (100%) type II cancers were
detectable by virtue of the mutations found in cyst fluid DNA while
none of the 18 benign or non-neoplastic cyst fluid contained such
mutations. It is also important to note that the readout of our
assay is quantitative and not dependent on the skill level of the
reader (in contrast to sonography). Finally, the procedure can be
performed minimally invasively in an outpatient setting. The goal
of our test is not to replace clinical, radiologic, or sonographic
evaluation but to augment them with molecular genetic markers.
[0045] Our study, though only proof-of-principle, illustrates one
route to improve management of patients with ovarian cysts. Genetic
analysis is not the only such route; proteomics could also provide
clues to the correct diagnosis (50, 51). One can easily imagine how
such additional information could be used to inform clinical
practice in conjunction with current diagnostic methods. For
example, if a cyst contained low amounts of DNA, no detectable
mutations, and if the patient had low CA-125 levels, our data
suggest that it is very unlikely to be a borderline tumor or
malignant lesion. Either no surgery, or laparoscopic rather than
open surgery, could be recommended for that patient, even if there
was some solid component upon imaging. The option to avoid surgery
would be particularly valuable for pre-menopausal women who
generally have a low risk of ovarian cancer and might wish to
preserve their fertility, as well as patients who are poor surgical
candidates. However, our assay in its current format cannot
completely rule out malignancy because a fraction of early-stage
cancer patients did not have detectable mutations in their cysts.
Therefore, patients whose clinical and functional status allows
them to undergo surgery and anesthesia might still choose to have a
surgical procedure. On the other hand, a minimally invasive test
that provides additional, orthogonal information to patients and
surgeons could inform their decision about the advisability of
surgery.
[0046] Our data suggest that a cyst without any solid component
upon imaging, and thereby unlikely via conventional criteria to be
malignant, should be removed promptly if the cyst fluid contained a
TP53 mutation. Radical, rather than conservative, surgery might be
appropriate due to the high likelihood of an aggressive type II
cancer. In contrast, if a BRAF mutation was identified, the lesion
is presumably a borderline or low-grade tumor; thus conservative
rather than radical surgery might be sufficient. Lastly, given that
certain types of ovarian cancers (type II) tend to respond well to
chemotherapy while others (type I) are relatively chemo-resistant,
knowing the type of cancer present prior to surgery based on the
mutation profile could help guide decisions regarding the use of
neoadjuvant chemotherapy. Validation of these data in a much
larger, prospective trial will of course be required before
incorporation of this approach into clinical practice.
[0047] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
EXAMPLE 1
Materials and Methods
Patient Samples
[0048] Cyst fluids were collected prospectively from 77 women
presenting with a suspected ovarian tumor. Patients were diagnosed
by transvaginal sonography or computed tomography and admitted for
surgical removal of the cyst by gynecologic oncology surgeons at
Sahlgrenska University Hospital, Gothenburg, Sweden. The study was
approved by the ethical board of Gothenburg University and patients
provided written consent. According to the approved protocol,
ovarian cyst fluids were collected after removal of the cyst from
the abdomen. All samples were immediately put in 4.degree. C. for
15-30 minutes, centrifuged for 10 minutes at 500 g, and aliquoted
into Eppendorf tubes. The fluids were transferred to -80.degree.
C., within 30-60 minutes after collection. All histology was
reviewed by board-certified pathologists (Table 1).
[0049] Plasma HE4 concentrations were determined using a commercial
HE4 EIA assay (Fujirebio Diagnostics) and plasma CA-125 levels were
measured using the Architect CA 125 II (Abbott Diagnostics, USA).
DNA was purified from tumor tissue (either freshly-frozen, or
formalin-fixed and paraffin-embedded) after microdissection to
remove neoplastic components. DNA was purified from tumors and from
cyst fluids using an AllPrep DNA kit (Qiagen) according to the
manufacturer's instructions. Purified DNA from all samples was
quantified as previously described (52).
Statistical Analysis
[0050] A Wilcoxon rank-sum test was used to compare the amount of
DNA in the cancers and borderline tumors with the amount of DNA in
the simple cysts and benign tumors. The fraction of samples
detected by tumor-specific mutations in the cyst fluid, as well as
their 95% confidence intervals, was calculated for each tumor type
(Table 3). When the presence of a mutation in the cyst fluid was
used to predict the need for surgery, the sensitivity and
specificity of the test, as well as their 95% confidence intervals,
were calculated. Firth's penalized likelihood logistic regression
was used to quantify the association between molecular features of
cyst fluids and the need for surgery (Table 4) in a multivariate
model. The model predictors included the presence of mutation,
log10 (ng) of cyst DNA and indicators for normal CA-125 and HE4
values. Normal CA-125 values were defined as <35 U/mL and normal
HE4 values were defined as <92 pmol/L and <121 pmol/L for
pre- and post-menopausal women, respectively. Statistical analyses
were performed using the R statistical package (version 3.1.2).
Unless noted otherwise, all patient-related values are reported as
means.+-.SD.
Mutation Detection and Analysis
[0051] DNA from either cyst fluids or tumors was used for multiplex
PCR, as previously described (34). One-hundred-and-thirty-three
primer pairs were designed to amplify 110 to 142 bp segments
containing regions of interest from the following 17 genes: AKT1,
APC, BRAF, CDKN2A, CTNNB1, EGFR, FBXW7, FGFR2, KRAS, MAPK1, NRAS,
PIK3CA, PIK3R1, POLE, PPP2R1A, PTEN, and TP53. Primer sequences are
listed in Table S1. These primers were used to amplify DNA in 25
.mu.L reactions as previously described (34). For each sample,
three multiplex reactions, each containing non-overlapping
amplicons, were performed. Reactions were purified with AMPure XP
beads (Beckman Coulter) and eluted in 100 .mu.L of Buffer EB
(Qiagen). A fraction (2.5 .mu.L) of purified PCR products were then
amplified in a second round of PCR, as described (34). The PCR
products were purified with AMPure and sequenced on an Illumina
MiSeq instrument.
[0052] We used Safe-SeqS, an error-reduction technology for
detection of low frequency mutations as described to distinguish
better between genuine mutations in the samples and artifactual
variants arising from sequencing and sample preparation steps,
(34). High quality sequence reads were selected based on quality
scores, which were generated by the sequencing instrument to
indicate the probability a base was called in error. The
template-specific portion of the reads was matched to reference
sequences. Reads from a common template molecule were then grouped
based on the unique identifier sequences (UIDs) that were
incorporated as molecular barcodes. Artifactual mutations
introduced during the sample preparation or sequencing steps were
reduced by requiring a mutation to be present in >90% of reads
in each UID family (i.e., to be scored as a "supermutant"). In
addition, DNA from normal individuals was used as a control to
identify potential false positive mutations (see main text). Only
supermutants in samples with frequencies far exceeding their
frequencies in control DNA samples (i.e., >mean+5 standard
deviations) were scored as positive.
EXAMPLE 2
Characteristics of the Tumors and Cyst Fluid Samples
[0053] DNA was isolated from surgically excised ovarian cysts of 77
women. Ten of them had non-neoplastic cysts, 12 had benign tumors,
24 had borderline tumors, and 31 had cancers (13 Type I and 18 Type
II). Age, histopathologic diagnosis, stage, and other clinical
information are provided in Table 1. The median amount of DNA
recovered from the cysts was 222 ng (interquartile range (IQR) of
53 to 3120 ng) (Table 2). There was no significant difference in
the amounts of DNA between borderline tumors and type I or type II
cancers (Table 2). However, the borderline tumors and cancers
contained significantly more DNA than the non-neoplastic cysts or
benign tumors (4453.+-.6428 ng vs. 62.+-.64 ng; p<0.001,
Wilcoxon rank-sum test).
EXAMPLE 3
A Multiplex PCR-Based Test to Identify Tumor-Specific Mutations in
Cyst Fluid Samples
[0054] We designed a multiplex PCR-based test that could
simultaneously assess the regions of 17 genes frequently mutated in
ovarian tumors. The amount of DNA shed from neoplastic cells was
expected to be a minor fraction of the total DNA in the cyst fluid,
with most DNA emanating from normal cells. We therefore used a
sensitive detection method, called Safe-SeqS (Safe-Sequencing
System), to identify mutations in cyst fluid samples (34). In
brief, primers were designed to amplify 133 regions, covering 9054
distinct nucleotide positions within the 17 genes of interest
(Table S1). Three multiplex PCR reactions, each containing
non-overlapping amplicons, were then performed on each sample. One
primer in each pair included a unique identifier (UID) for each
template molecule, thereby drastically minimizing the error rates
associated with PCR and sequencing, as described previously (34)
(Table S1). Under the conditions used in the current experiments,
mutations present in >0.1% of template molecules could generally
be reliably determined. We could not perform sequencing on five
cysts (two simple cysts, two cystadenomas, one borderline tumor)
because there was insufficient DNA (<3 ng recovered), and these
were scored in a conservative fashion, as "negative" for mutations.
When this test was applied to the 22 cyst fluids obtained from
patients with simple cysts (n=9) or benign tumors (n=13), no
mutations were identified (Tables 2 and 3). This was in stark
contrast to the fluids obtained from the 18 patients with type II
cancers, all of which were found to contain a mutation (Tables 2
and 3). Ten (77%) of the 13 cyst fluids from patients with type I
cancers and 19 (79%) of the 24 cyst fluids from patients with
borderline tumors contained at least one detectable mutation. When
categorized by the need for surgery (i.e., presence of a borderline
tumor or a type I or type II cancer), the sensitivity of this test
was 85% (47 of 55 cysts; 95% confidence interval of 73% to 94%) and
the specificity was 100% (95% confidence interval of 78% to 100%;
Table 3).
[0055] Ovarian cancers are generally detected only late in the
course of disease, explaining the poor prognosis of patients.
Accordingly, only 11 of the 31 cysts associated with cancers in our
study had early (Stage I or II) disease (Table 1). As expected,
most of these were type I carcinomas (n=8). Nevertheless, it was
encouraging that mutant DNA could be detected in nine (82%) of
these 11 patients (Table 3). Mutations could be detected in 95% of
the 20 patients with Stage III or IV cancers (Table 3).
[0056] A variety of control experiments were performed to confirm
the integrity of these results. One informative positive control
was provided by the results of sequencing of DNA from the tumors,
using the identical method used to analyze DNA from the cyst
fluids. Fifty-three of the 55 borderline and malignant cases had
tumor available for this purpose. Every mutation identified in a
tumor was found in its cyst fluid, and vice versa. As expected, the
mutant allele frequencies in the tumors were often, but not always,
higher than in the cyst fluid (Table 2). As another positive
control, we used an independent PCR and sequencing reaction to
confirm each of the cyst fluid mutations listed in Table 2. This
validated not only the presence of a mutation, but also confirmed
its fractional representation. The median relative difference
between the fractions of mutant alleles in replicate experiments
was 7.0% (IQR of 3.5% to 8.9%). Finally, four patients were found
to have two independent mutations (Table 2). For example, the cyst
fluid of patient OVCYST 081, who had high-grade endometrioid
carcinoma, had a missense mutation (R280K) in TP53 plus an in-frame
deletion of PIK3R1 at codons 458 and 459 of PIK3R1. The TP53
mutation was found in 3.0% of alleles while the PIK3R1 mutation was
found in 3.7% of the alleles analyzed. Similar mutant allele
frequencies among completely different mutations in the cyst fluid
of three other patients provided further indicators of
reproducibility (Table 2). All genetic assays were performed in a
blinded manner, with the operator unaware of the diagnoses of the
patients from whom the cyst fluids were obtained.
[0057] In addition to DNA from normal individuals used as controls,
additional negative controls were provided by the simple cysts and
benign tumors. Using the identical assay, none of the DNA from
their cyst fluids contained detectable mutations (Table 2). A final
control was provided by the borderline and malignant tumors
themselves. In general, only one or two of the 9054 base-pairs (bp)
queried were mutated in any one tumor (Table 2). The other 9000 bp
could then be independently queried in the corresponding cyst
fluid, and none of these positions were found to be mutated.
EXAMPLE 4
Relationship Between the Type of Tumor Present and the Type of
Mutation Found in the Associated Cyst Fluid Sample
[0058] The mutant allele fractions in the cyst fluids tended to be
higher in the type II cancers (median of 60.3%) than the type I
cancers (median of 7.8%) or borderline tumors (median of 2.4%),
though there was considerable overlap (Tables 2 and 3). On the
other hand, the type of mutation varied considerably among these
cysts. In type I tumors, the genes mutated were BRAF (n=1), KRAS
(n=5), NRAS (n=1), PIK3R1 (n=1), PPP2R1A (n=1), PTEN (n=1), or TP53
(n=3). Two distinct mutations were found per sample in three type I
cancers. One type I cancer had a BRAF mutation. This BRAF mutation
(V600_S605>D) is unusual that it resulted from an in-frame
deletion/insertion rather than the base substitution (V600E)
characteristic of the vast majority of BRAF mutations reported in
the literature. This mutation has been observed in a papillary
thyroid cancer and a cutaneous melanoma (35, 36). The deletion
results in loss of a phosphorylation site in the activation loop of
BRAF, while the insertion of an aspartic acid has been suggested to
increase BRAF kinase activity by mimicking an activating
phosphorylation (37). In contrast, all but one type II cancers (94%
of 18) had mutations in TP53; the only exception was OVCYST 081, a
high-grade endometrioid carcinoma. The borderline tumors were
distinguished by yet a different pattern from that of the either
type I or type II cancers. Of the 19 mutations in borderline
tumors, 12 (63%) were at BRAF V600E, never observed in type I or
type II cancers, and the remainder were at KRAS 12 or 61 (Table
2).
EXAMPLE 5
Markers Associated with the Need for Surgery
[0059] A multivariate analysis was used to identify the most
informative molecular features of cyst fluids and to compare them
to the commonly used serum biomarkers for ovarian cancer, HE4
(human epididymis protein 4) and CA-125 (38, 39) (Table 4). We
defined "informative" as indicating a need for surgery (i.e.,
borderline tumors or type I or II cancers). The amount of DNA in
cyst fluids was generally, but not significantly, higher in the
cysts requiring surgery (p=0.69, Table 4), though there were many
cysts not requiring surgery that had higher DNA levels than cysts
requiring surgery (Figure S1A). Similarly, the serum CA-125 levels
were significantly higher in cysts requiring surgery (p=0.01, Table
4), but there were many cysts not requiring surgery that had higher
levels than those requiring surgery (Figure S1B). Serum HE4 levels
were not correlated with cyst type (P=0.92, Table 4; Figure S1C).
On the other hand, the presence of a mutation was highly
informative for the presence of a cyst requiring surgery in the
multivariate analysis, as no mutations were found in cysts not
requiring surgery (P<0.001, Table 4).
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Ho, S. Y. Leung, S. T. Yuen, B. L. Weber, H. F. Seigler, T. L.
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J. Raycraft, M. Hayden-Ledbetter, J. A. Ledbetter, M. Schummer, M.
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Sequence CWU 1
1
266147DNAArtificial SequenceForward and reverse primers 1cacacaggaa
acagctatga ccatgcaaca tgactgtcct ttcacca 47245DNAArtificial
SequenceForward and reverse primers 2cacacaggaa acagctatga
ccatgggcaa ctaccatcca gcaac 45352DNAArtificial SequenceForward and
reverse primers 3cacacaggaa acagctatga ccatggtatt ctaatttggc
ataaggcata ga 52449DNAArtificial SequenceForward and reverse
primers 4cacacaggaa acagctatga ccatgtgatg gttatggtaa aagaggtca
49553DNAArtificial SequenceForward and reverse primers 5cacacaggaa
acagctatga ccatgggatg ataatgatgg agaactagat aca 53647DNAArtificial
SequenceForward and reverse primers 6cacacaggaa acagctatga
ccatgtcgat ttgtttctga accattg 47750DNAArtificial SequenceForward
and reverse primers 7cacacaggaa acagctatga ccatgtttgt tggtctctct
tcttcttcat 50846DNAArtificial SequenceForward and reverse primers
8cacacaggaa acagctatga ccatgcggtt ttactgcttt gtccag
46954DNAArtificial SequenceForward and reverse primers 9cacacaggaa
acagctatga ccatggaaaa acatattgga gtatcttcta caca
541045DNAArtificial SequenceForward and reverse primers
10cacacaggaa acagctatga ccatggtgct gtgacactgc tggaa
451145DNAArtificial SequenceForward and reverse primers
11cacacaggaa acagctatga ccatgagaat cagccaggca caaag
451246DNAArtificial SequenceForward and reverse primers
12cacacaggaa acagctatga ccatggctcc gttcagagtg aaccat
461345DNAArtificial SequenceForward and reverse primers
13cacacaggaa acagctatga ccatgagcac tcaggctgga tgaac
451447DNAArtificial SequenceForward and reverse primers
14cacacaggaa acagctatga ccatggggaa tgaaacagaa tcagagc
471549DNAArtificial SequenceForward and reverse primers
15cacacaggaa acagctatga ccatgcaacc tgttttgtga tggtagaag
491644DNAArtificial SequenceForward and reverse primers
16cacacaggaa acagctatga ccatggggtc gggtagagga ggtg
441742DNAArtificial SequenceForward and reverse primers
17cacacaggaa acagctatga ccatggaccc cgccactctc ac
421845DNAArtificial SequenceForward and reverse primers
18cacacaggaa acagctatga ccatggccat ggaaccagac agaaa
451945DNAArtificial SequenceForward and reverse primers
19cacacaggaa acagctatga ccatgctgga tcccagaagg tgaga
452045DNAArtificial SequenceForward and reverse primers
20cacacaggaa acagctatga ccatgtccct ggtgtcagga aaatg
452148DNAArtificial SequenceForward and reverse primers
21cacacaggaa acagctatga ccatgttgtt tttctgtttc tccctctg
482245DNAArtificial SequenceForward and reverse primers
22cacacaggaa acagctatga ccatggcaga gtatttgggc gaatg
452352DNAArtificial SequenceForward and reverse primers
23cacacaggaa acagctatga ccatgtttac ctctattgtt ggatcatatt cg
522449DNAArtificial SequenceForward and reverse primers
24cacacaggaa acagctatga ccatgggaaa taaatgtgat ttgccttct
492546DNAArtificial SequenceForward and reverse primers
25cacacaggaa acagctatga ccatgacacc cccaggattc ttacag
462645DNAArtificial SequenceForward and reverse primers
26cacacaggaa acagctatga ccatgccccc tccatcaact tcttc
452750DNAArtificial SequenceForward and reverse primers
27cacacaggaa acagctatga ccatgcaatg aattaaggga aaatgacaaa
502848DNAArtificial SequenceForward and reverse primers
28cacacaggaa acagctatga ccatggcatg ccaatctctt cataaatc
482948DNAArtificial SequenceForward and reverse primers
29cacacaggaa acagctatga ccatggggtt ttgggctgat attaaaac
483049DNAArtificial SequenceForward and reverse primers
30cacacaggaa acagctatga ccatgtgttt ccatgtcagc tattttgtt
493152DNAArtificial SequenceForward and reverse primers
31cacacaggaa acagctatga ccatgtgcag taagagattg ttctatgaaa gg
523245DNAArtificial SequenceForward and reverse primers
32cacacaggaa acagctatga ccatgtttct tttgcctgca ggatt
453347DNAArtificial SequenceForward and reverse primers
33cacacaggaa acagctatga ccatgcctga attgtagcaa tcaccaa
473445DNAArtificial SequenceForward and reverse primers
34cacacaggaa acagctatga ccatggatga agatttgccc catca
453545DNAArtificial SequenceForward and reverse primers
35cacacaggaa acagctatga ccatgcccat cccaggagct tactt
453648DNAArtificial SequenceForward and reverse primers
36cacacaggaa acagctatga ccatgttccc ttctgagagt gtcagtgt
483743DNAArtificial SequenceForward and reverse primers
37cacacaggaa acagctatga ccatgagcca caggctccca gac
433854DNAArtificial SequenceForward and reverse primers
38cacacaggaa acagctatga ccatgaatag ttgttttaga agatatttgc aagc
543952DNAArtificial SequenceForward and reverse primers
39cacacaggaa acagctatga ccatgaagat tcaggcaatg tttgttagta tt
524054DNAArtificial SequenceForward and reverse primers
40cacacaggaa acagctatga ccatgtttct tattctgagg ttatcttttt acca
544148DNAArtificial SequenceForward and reverse primers
41cacacaggaa acagctatga ccatggcaat tcactgtaaa gctggaaa
484249DNAArtificial SequenceForward and reverse primers
42cacacaggaa acagctatga ccatgtcaat ttggcttctc ttttttttc
494350DNAArtificial SequenceForward and reverse primers
43cacacaggaa acagctatga ccatgaggca tttcctgtga aataatactg
504451DNAArtificial SequenceForward and reverse primers
44cacacaggaa acagctatga ccatgtctat gtgatcaaga aatcgatagc a
514551DNAArtificial SequenceForward and reverse primers
45cacacaggaa acagctatga ccatgtgggt tttcatttta aattttcttt c
514652DNAArtificial SequenceForward and reverse primers
46cacacaggaa acagctatga ccatgggtcc attttcagtt tattcaagtt ta
524746DNAArtificial SequenceForward and reverse primers
47cacacaggaa acagctatga ccatgccttc caatggatcc actcac
464844DNAArtificial SequenceForward and reverse primers
48cacacaggaa acagctatga ccatgagccc cctagcagag acct
444944DNAArtificial SequenceForward and reverse primers
49cacacaggaa acagctatga ccatgagctc ccagaatgcc agag
445047DNAArtificial SequenceForward and reverse primers
50cacacaggaa acagctatga ccatggccct gactttcaac tctgtct
475144DNAArtificial SequenceForward and reverse primers
51cacacaggaa acagctatga ccatggccat ggccatctac aagc
445245DNAArtificial SequenceForward and reverse primers
52cacacaggaa acagctatga ccatggtgga aggaaatttg cgtgt
455345DNAArtificial SequenceForward and reverse primers
53cacacaggaa acagctatga ccatgtgtga tgatggtgag gatgg
455446DNAArtificial SequenceForward and reverse primers
54cacacaggaa acagctatga ccatgtgcct cttgcttctc ttttcc
465547DNAArtificial SequenceForward and reverse primers
55cacacaggaa acagctatga ccatgaagaa gaaaacggca ttttgag
475645DNAArtificial SequenceForward and reverse primers
56cacacaggaa acagctatga ccatggttcc gagagctgaa tgagg
455745DNAArtificial SequenceForward and reverse primers
57cacacaggaa acagctatga ccatggccac ctgaagtcca aaaag
455846DNAArtificial SequenceForward and reverse primers
58cacacaggaa acagctatga ccatgtcctt gtagccaatg aaggtg
465945DNAArtificial SequenceForward and reverse primers
59cacacaggaa acagctatga ccatgcccaa ggcatctcat cgtag
456047DNAArtificial SequenceForward and reverse primers
60cacacaggaa acagctatga ccatgtgtta cccagctcct cttcatc
476146DNAArtificial SequenceForward and reverse primers
61cacacaggaa acagctatga ccatggccaa agtcatggaa gaagtg
466249DNAArtificial SequenceForward and reverse primers
62cacacaggaa acagctatga ccatgaagtc ggaaaattca aataggaca
496352DNAArtificial SequenceForward and reverse primers
63cacacaggaa acagctatga ccatgaagat gatgaaagta agttttgcag tt
526448DNAArtificial SequenceForward and reverse primers
64cacacaggaa acagctatga ccatgagatg agcagttgaa ctctggaa
486546DNAArtificial SequenceForward and reverse primers
65cacacaggaa acagctatga ccatgatttt ggacagcagg aatgtg
466647DNAArtificial SequenceForward and reverse primers
66cacacaggaa acagctatga ccatgtcaat aggctgatcc acatgac
476748DNAArtificial SequenceForward and reverse primers
67cacacaggaa acagctatga ccatgttcct tcatcacaga aacagtca
486852DNAArtificial SequenceForward and reverse primers
68cacacaggaa acagctatga ccatgcttgc aaagtttctt ctattaacca ag
526946DNAArtificial SequenceForward and reverse primers
69cacacaggaa acagctatga ccatgggtca gctgaagatc ctgtga
467045DNAArtificial SequenceForward and reverse primers
70cacacaggaa acagctatga ccatgggtgc tcagacaccc aaaag
457145DNAArtificial SequenceForward and reverse primers
71cacacaggaa acagctatga ccatgcatgc caccaagcag aagta
457244DNAArtificial SequenceForward and reverse primers
72cacacaggaa acagctatga ccatggagcc tcgatgagcc attt
447352DNAArtificial SequenceForward and reverse primers
73cacacaggaa acagctatga ccatgaggac ctattagatg attcagatga tg
527453DNAArtificial SequenceForward and reverse primers
74cacacaggaa acagctatga ccatgtgttt tcctttactt actacacctc aga
537541DNAArtificial SequenceForward and reverse primers
75cacacaggaa acagctatga ccatggggga gagcaggcag c 417646DNAArtificial
SequenceForward and reverse primers 76cacacaggaa acagctatga
ccatgtggct ctgaccattc tgttct 467743DNAArtificial SequenceForward
and reverse primers 77cacacaggaa acagctatga ccatgcttcc tggacacgct
ggt 437844DNAArtificial SequenceForward and reverse primers
78cacacaggaa acagctatga ccatgtgtgc cagggacctt acct
447945DNAArtificial SequenceForward and reverse primers
79cacacaggaa acagctatga ccatgcgatc tgcacacacc agttg
458046DNAArtificial SequenceForward and reverse primers
80cacacaggaa acagctatga ccatggaagt cccaaccatg acaaga
468148DNAArtificial SequenceForward and reverse primers
81cacacaggaa acagctatga ccatgttgag acaggccagt gtttacat
488245DNAArtificial SequenceForward and reverse primers
82cacacaggaa acagctatga ccatggctgg gcatcactgt aaacc
458346DNAArtificial SequenceForward and reverse primers
83cacacaggaa acagctatga ccatggcagc cagaaatgtt ttggta
468448DNAArtificial SequenceForward and reverse primers
84cacacaggaa acagctatga ccatgttctc ccttctcagg attcctac
488546DNAArtificial SequenceForward and reverse primers
85cacacaggaa acagctatga ccatgacatt caacccacac aagagg
468645DNAArtificial SequenceForward and reverse primers
86cacacaggaa acagctatga ccatggatgt ggctcgccaa ttaac
458746DNAArtificial SequenceForward and reverse primers
87cacacaggaa acagctatga ccatgttatt ccagacgcat ttccac
468848DNAArtificial SequenceForward and reverse primers
88cacacaggaa acagctatga ccatgtttga tgacattgca tacattcg
488948DNAArtificial SequenceForward and reverse primers
89cacacaggaa acagctatga ccatgtcagg gaagaagtga atgaaaaa
489050DNAArtificial SequenceForward and reverse primers
90cacacaggaa acagctatga ccatgtctag gatcaagttg tcaaagaaga
509148DNAArtificial SequenceForward and reverse primers
91cacacaggaa acagctatga ccatgccaaa tgaaaaggac agctattg
489251DNAArtificial SequenceForward and reverse primers
92cacacaggaa acagctatga ccatgttgac agtagaagaa gattggaaga a
519345DNAArtificial SequenceForward and reverse primers
93cacacaggaa acagctatga ccatggtctg tgtggtgccc agttt
459447DNAArtificial SequenceForward and reverse primers
94cacacaggaa acagctatga ccatgacatg gggatgatct cactctt
479551DNAArtificial SequenceForward and reverse primers
95cacacaggaa acagctatga ccatggctgc atatttcaga tatttctttc c
519654DNAArtificial SequenceForward and reverse primers
96cacacaggaa acagctatga ccatgcagta agatacagtc tatcgggttt aagt
549747DNAArtificial SequenceForward and reverse primers
97cacacaggaa acagctatga ccatgaaacc caaaatctgt tttccaa
479853DNAArtificial SequenceForward and reverse primers
98cacacaggaa acagctatga ccatggcgct atgtgtatta ttatagctac ctg
539945DNAArtificial SequenceForward and reverse primers
99cacacaggaa acagctatga ccatgtgtgg tctgccagct aaagg
4510051DNAArtificial SequenceForward and reverse primers
100cacacaggaa acagctatga ccatgtttgg gtaaatacat tcttcatacc a
5110152DNAArtificial SequenceForward and reverse primers
101cacacaggaa acagctatga ccatgtttaa caaaaaatga tcttgacaaa gc
5210245DNAArtificial SequenceForward and reverse primers
102cacacaggaa acagctatga ccatgtagag gagccgtcaa atcca
4510346DNAArtificial SequenceForward and reverse primers
103cacacaggaa acagctatga ccatggcaat ggatgatttg atgctg
4610446DNAArtificial SequenceForward and reverse primers
104cacacaggaa acagctatga ccatggcatt gaagtctcat ggaagc
4610545DNAArtificial SequenceForward and reverse primers
105cacacaggaa acagctatga ccatgctccg tcatgtgctg tgact
4510643DNAArtificial SequenceForward and reverse primers
106cacacaggaa acagctatga ccatggtccc caggcctctg att
4310747DNAArtificial SequenceForward and reverse primers
107cacacaggaa acagctatga ccatgtggct ctgactgtac caccatc
4710844DNAArtificial SequenceForward and reverse primers
108cacacaggaa acagctatga ccatgcgtgt ttgtgcctgt cctg
4410948DNAArtificial SequenceForward and reverse primers
109cacacaggaa acagctatga ccatgtttta tcacctttcc ttgcctct
4811042DNAArtificial SequenceForward and reverse primers
110cacacaggaa acagctatga ccatgccctg gctccttccc ag
4211146DNAArtificial SequenceForward and reverse primers
111cacacaggaa acagctatga ccatgatgtc atctctcctc cctgct
4611249DNAArtificial SequenceForward and reverse primers
112cacacaggaa acagctatga ccatgtgatt atgtttttga caccaatcg
4911345DNAArtificial SequenceForward and reverse primers
113cacacaggaa acagctatga ccatggagaa cgcggaattg gtcta
4511450DNAArtificial SequenceForward and reverse primers
114cacacaggaa acagctatga ccatggttct atgccttatg ccaaattaga
5011545DNAArtificial SequenceForward and reverse primers
115cacacaggaa acagctatga ccatgagccg acctagccca taaaa
4511646DNAArtificial SequenceForward and reverse primers
116cacacaggaa acagctatga ccatgatgtg gttggaactt gaggtg
4611747DNAArtificial SequenceForward and reverse primers
117cacacaggaa acagctatga ccatgccaat ggttcagaaa caaatcg
4711852DNAArtificial SequenceForward and reverse primers
118cacacaggaa acagctatga ccatgaagaa gagagaccaa caaattatag ca
5211947DNAArtificial SequenceForward and reverse primers
119cacacaggaa acagctatga ccatgccgaa catatgtctt caagcag
4712054DNAArtificial SequenceForward and reverse primers
120cacacaggaa acagctatga ccatggatgt agttcattat catctttgtc atca
5412145DNAArtificial SequenceForward and reverse primers
121cacacaggaa acagctatga ccatgcagga gaccccactc atgtt
4512246DNAArtificial SequenceForward and reverse primers
122cacacaggaa acagctatga ccatgctcaa acagctcaaa ccaagc
4612345DNAArtificial SequenceForward and reverse primers
123cacacaggaa acagctatga ccatgtttgc cacggaaagt actcc
4512448DNAArtificial SequenceForward and reverse primers
124cacacaggaa acagctatga ccatgaaaac caagagaaag aggcagaa
4812542DNAArtificial SequenceForward and reverse primers
125cacacaggaa acagctatga ccatgagcct tcggctgact gg
4212641DNAArtificial SequenceForward and reverse primers
126cacacaggaa
acagctatga ccatgccgag tggcggagct g 4112741DNAArtificial
SequenceForward and reverse primers 127cacacaggaa acagctatga
ccatgaggag ctgggccatc g 4112853DNAArtificial SequenceForward and
reverse primers 128cacacaggaa acagctatga ccatgagata atattgaagc
tgtagggaaa aaa 5312946DNAArtificial SequenceForward and reverse
primers 129cacacaggaa acagctatga ccatgtttga agaacagtgc cagacc
4613053DNAArtificial SequenceForward and reverse primers
130cacacaggaa acagctatga ccatggaaaa cacaacatga atataaacat caa
5313150DNAArtificial SequenceForward and reverse primers
131cacacaggaa acagctatga ccatggctac ctgttaaaga atcatctgga
5013246DNAArtificial SequenceForward and reverse primers
132cacacaggaa acagctatga ccatgtgact gctcttttca cccatc
4613344DNAArtificial SequenceForward and reverse primers
133cacacaggaa acagctatga ccatgctgca ccagcagctc ctac
4413455DNAArtificial SequenceForward and reverse primers
134cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntagac caattccgcg ttctc
5513560DNAArtificial SequenceForward and reverse primers
135cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncttct gtcttcctga
gaggtatgaa 6013658DNAArtificial SequenceForward and reverse primers
136cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgtgt gacagatgag agaaatgc
5813761DNAArtificial SequenceForward and reverse primers
137cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgcac tatgtatttt
atgggctagg 60t 6113854DNAArtificial SequenceForward and reverse
primers 138cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngtttg ggtcttgccc
atct 5413961DNAArtificial SequenceForward and reverse primers
139cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncctgt ttatactgag
agcactgatg 60a 6114059DNAArtificial SequenceForward and reverse
primers 140cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcaaa atgtaagcca
gtctttgtg 5914156DNAArtificial SequenceForward and reverse primers
141cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncgtca tgtggatcag cctatt
5614257DNAArtificial SequenceForward and reverse primers
142cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccatc caagttctgc acagagt
5714355DNAArtificial SequenceForward and reverse primers
143cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncagac gacacaggaa gcaga
5514455DNAArtificial SequenceForward and reverse primers
144cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaacat gagtggggtc tcctg
5514555DNAArtificial SequenceForward and reverse primers
145cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaggag gtggtggagg tgttt
5514657DNAArtificial SequenceForward and reverse primers
146cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggacc taagcaagct gcagtaa
5714763DNAArtificial SequenceForward and reverse primers
147cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcaat atcatcatca
tctgaatcat 60cta 6314855DNAArtificial SequenceForward and reverse
primers 148cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccatg ccaacaaagt
catca 5514952DNAArtificial SequenceForward and reverse primers
149cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctccc gctgcagacc ct
5215053DNAArtificial SequenceForward and reverse primers
150cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngctcc tcagccaggt cca
5315155DNAArtificial SequenceForward and reverse primers
151cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncctca ggattgcctt tacca
5515254DNAArtificial SequenceForward and reverse primers
152cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncccac acagcaaagc agaa
5415357DNAArtificial SequenceForward and reverse primers
153cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngcagc atgtcaagat cacagat
5715455DNAArtificial SequenceForward and reverse primers
154cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctgca acatgaccca tcaaa
5515557DNAArtificial SequenceForward and reverse primers
155cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntctgg tgtcagagat ggagatg
5715663DNAArtificial SequenceForward and reverse primers
156cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgact gaatataaac
ttgtggtagt 60tgg 6315763DNAArtificial SequenceForward and reverse
primers 157cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntttca gtgttactta
cctgtcttgt 60ctt 6315855DNAArtificial SequenceForward and reverse
primers 158cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncgcct gtcctcatgt
attgg 5515955DNAArtificial SequenceForward and reverse primers
159cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngaaaa agccgaaggt cacaa
5516060DNAArtificial SequenceForward and reverse primers
160cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctcca ttttagcact
tacctgtgac 6016155DNAArtificial SequenceForward and reverse primers
161cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntccaa agcctcttgc tcagt
5516257DNAArtificial SequenceForward and reverse primers
162cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccata tttcccatct cgatgaa
5716360DNAArtificial SequenceForward and reverse primers
163cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgtaa ttttttccct
acagcttcaa 6016455DNAArtificial SequenceForward and reverse primers
164cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngggtc tggcactgtt cttca
5516556DNAArtificial SequenceForward and reverse primers
165cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntactc agctgcctgc ttcttc
5616659DNAArtificial SequenceForward and reverse primers
166cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnacgta tgaacagcat taaaccaga
5916755DNAArtificial SequenceForward and reverse primers
167cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntctcc cggacaagaa aagtg
5516855DNAArtificial SequenceForward and reverse primers
168cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggca tttgacattg agacg
5516953DNAArtificial SequenceForward and reverse primers
169cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggtg atgcccactc tgc
5317058DNAArtificial SequenceForward and reverse primers
170cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgaca gaaaggtaaa gaggagca
5817158DNAArtificial SequenceForward and reverse primers
171cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnttaat ggtggctttt tgtttgtt
5817255DNAArtificial SequenceForward and reverse primers
172cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngcaat taaatttggc ggtgt
5517359DNAArtificial SequenceForward and reverse primers
173cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncatga ttgtcatctt cacttagcc
5917457DNAArtificial SequenceForward and reverse primers
174cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggtc cttacttccc catagaa
5717562DNAArtificial SequenceForward and reverse primers
175cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncactg gtctataatc
cagatgattc 60tt 6217655DNAArtificial SequenceForward and reverse
primers 176cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgaac ttgtcttccc
gtcgt 5517758DNAArtificial SequenceForward and reverse primers
177cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaagta tcggttggct ttgtcttt
5817860DNAArtificial SequenceForward and reverse primers
178cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaggtt cattgtcact
aacatctggt 6017957DNAArtificial SequenceForward and reverse primers
179cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctgac accactgact ctgatcc
5718053DNAArtificial SequenceForward and reverse primers
180cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnactgc cttccgggtc act
5318154DNAArtificial SequenceForward and reverse primers
181cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncagcc caacccttgt cctt
5418255DNAArtificial SequenceForward and reverse primers
182cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggga agggacagaa gatga
5518353DNAArtificial SequenceForward and reverse primers
183cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggggg tgtggaatca acc
5318454DNAArtificial SequenceForward and reverse primers
184cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaccag ccctgtcgtc tctc
5418555DNAArtificial SequenceForward and reverse primers
185cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncttaa cccctcctcc cagag
5518656DNAArtificial SequenceForward and reverse primers
186cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcatc ttgggcctgt gttatc
5618756DNAArtificial SequenceForward and reverse primers
187cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngcgga gattctcttc ctctgt
5618855DNAArtificial SequenceForward and reverse primers
188cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccagc caaagaagaa accac
5518955DNAArtificial SequenceForward and reverse primers
189cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntagga aggcagggga gtagg
5519054DNAArtificial SequenceForward and reverse primers
190cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngaggc tgtcagtggg gaac
5419156DNAArtificial SequenceForward and reverse primers
191cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnagggt ctgacgggta gagtgt
5619258DNAArtificial SequenceForward and reverse primers
192cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaggac agtcatgttg ccagtatt
5819358DNAArtificial SequenceForward and reverse primers
193cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaagtt cctggatttt ctgttgct
5819455DNAArtificial SequenceForward and reverse primers
194cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngtgta tgggcagcag agctt
5519559DNAArtificial SequenceForward and reverse primers
195cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgacc tcttttacca taaccatca
5919663DNAArtificial SequenceForward and reverse primers
196cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggtg tatctagttc
tccatcatta 60tca 6319758DNAArtificial SequenceForward and reverse
primers 197cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccttg attgtctttg
ctcacttt 5819859DNAArtificial SequenceForward and reverse primers
198cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnttctt gacacaaaga ctggcttac
5919962DNAArtificial SequenceForward and reverse primers
199cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgata agcctaccaa
ttatagtgaa 60cg 6220056DNAArtificial SequenceForward and reverse
primers 200cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgatt ctgcctcttg
gcatta 5620161DNAArtificial SequenceForward and reverse primers
201cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgatt acatcctatt
tcatcttcag 60c 6120256DNAArtificial SequenceForward and reverse
primers 202cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcgct cctgaagaaa
attcaa 5620355DNAArtificial SequenceForward and reverse primers
203cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctggc aatcgaacga ctctc
5520456DNAArtificial SequenceForward and reverse primers
204cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngcagc ttgcttaggt ccactc
5620559DNAArtificial SequenceForward and reverse primers
205cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggtt ttcatttgat tctttaggc
5920657DNAArtificial SequenceForward and reverse primers
206cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggtgg aggtaatttt gaagcag
5720757DNAArtificial SequenceForward and reverse primers
207cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaactg ttcaaactga tgggacc
5720854DNAArtificial SequenceForward and reverse primers
208cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncacct cctctacccg accc
5420952DNAArtificial SequenceForward and reverse primers
209cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngggtc gggtgagagt gg
5221053DNAArtificial SequenceForward and reverse primers
210cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngcagg taccgtgcga cat
5321154DNAArtificial SequenceForward and reverse primers
211cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggaga agctcccaac caag
5421254DNAArtificial SequenceForward and reverse primers
212cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngcatc tgcctcacct ccac
5421355DNAArtificial SequenceForward and reverse primers
213cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncggac actcaaagtg tggaa
5521455DNAArtificial SequenceForward and reverse primers
214cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncagtc tctggatccc acacc
5521555DNAArtificial SequenceForward and reverse primers
215cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcttc cctctctcca ccaga
5521656DNAArtificial SequenceForward and reverse primers
216cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnactgc catcgactta cattgg
5621756DNAArtificial SequenceForward and reverse primers
217cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgtac tggtccctca ttgcac
5621856DNAArtificial SequenceForward and reverse primers
218cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnatcta tgtccctgaa gcagca
5621955DNAArtificial SequenceForward and reverse primers
219cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngattg tcagtgcgct tttcc
5522057DNAArtificial SequenceForward and reverse primers
220cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnacatt cacgtaggtt gcacaaa
5722160DNAArtificial SequenceForward and reverse primers
221cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngatcc aatccatttt
tgttgtccag 6022258DNAArtificial SequenceForward and reverse primers
222cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncagct atattccctg gcttacct
5822355DNAArtificial SequenceForward and reverse primers
223cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctggg atgtgcgggt atatt
5522456DNAArtificial SequenceForward and reverse primers
224cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnttctt tctcattgcc ttcacg
5622556DNAArtificial SequenceForward and reverse primers
225cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggtc tctcgtcttt ctcagc
5622655DNAArtificial SequenceForward and reverse primers
226cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngactg cccacaggaa ggtaa
5522755DNAArtificial SequenceForward and reverse primers
227cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntactt ccggaacctg tgctc
5522862DNAArtificial SequenceForward and reverse primers
228cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncatca tcaatattgt
tcctgtatac 60gc 6222963DNAArtificial SequenceForward and reverse
primers 229cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntttta aacttttctt
ttagttgtgc 60tga 6323055DNAArtificial SequenceForward and reverse
primers 230cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaggca caagaggccc
tagat 5523156DNAArtificial SequenceForward and reverse primers
231cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncgcca ctgaacattg gaatag
5623262DNAArtificial SequenceForward and reverse primers
232cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngttct gtttgtggaa
gaactctact 60tt 6223356DNAArtificial SequenceForward and reverse
primers 233cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntctgc acgctctata
ctgcaa 5623455DNAArtificial SequenceForward and reverse primers
234cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnacaag tcaacaaccc ccaca
5523560DNAArtificial SequenceForward and reverse primers
235cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctgat cttcatcaaa
aggttcattc 6023653DNAArtificial SequenceForward and reverse primers
236cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncggtg taggagctgc tgg
5323755DNAArtificial SequenceForward and reverse primers
237cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccctt cccagaaaac ctacc
5523857DNAArtificial SequenceForward and reverse primers
238cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncaaca agatgttttg ccaactg
5723957DNAArtificial SequenceForward and reverse primers
239cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncgaaa agtgtttctg tcatcca
5724053DNAArtificial SequenceForward and reverse primers
240cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngtggc aagtggctcc tga
5324155DNAArtificial SequenceForward and reverse primers
241cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngcttc ttgtcctgct tgctt
5524260DNAArtificial SequenceForward and reverse primers
242cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncaaga cttagtacct
gaagggtgaa 6024355DNAArtificial SequenceForward and reverse primers
243cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncttct ccccctcctc tgttg
5524456DNAArtificial SequenceForward and reverse primers
244cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnagtct gagtcaggcc cttctg
5624557DNAArtificial SequenceForward and reverse primers
245cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngaaga ggagctgggt
aacactg
5724655DNAArtificial SequenceForward and reverse primers
246cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccatg actttggcaa tctgg
5524755DNAArtificial SequenceForward and reverse primers
247cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggatt caatcgaggg tttca
5524855DNAArtificial SequenceForward and reverse primers
248cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgaag gactttgcct tccag
5524961DNAArtificial SequenceForward and reverse primers
249cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngaccc aaacacataa
tagaagatga 60a 6125063DNAArtificial SequenceForward and reverse
primers 250cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcttc agagtaacgt
tcactataat 60tgg 6325160DNAArtificial SequenceForward and reverse
primers 251cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaaaat gactgtttct
gtgatgaagg 6025255DNAArtificial SequenceForward and reverse primers
252cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnagcct tttgaggctg accac
5525360DNAArtificial SequenceForward and reverse primers
253cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngctga cctagttcca
atcttttctt 6025456DNAArtificial SequenceForward and reverse primers
254cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnatgcc acttaccatt ccactg
5625555DNAArtificial SequenceForward and reverse primers
255cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnagcat ctggaagaac ctgga
5525658DNAArtificial SequenceForward and reverse primers
256cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncccat tgtcattttc ctgaactg
5825757DNAArtificial SequenceForward and reverse primers
257cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnttggc atggcagaaa taataca
5725855DNAArtificial SequenceForward and reverse primers
258cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggcct ccgaccgtaa ctatt
5525954DNAArtificial SequenceForward and reverse primers
259cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncacca gcgtgtccag gaag
5426060DNAArtificial SequenceForward and reverse primers
260cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnacaaa ttctcagatc
atcagtcctc 6026155DNAArtificial SequenceForward and reverse primers
261cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaaaac tcacctggga tgtgc
5526256DNAArtificial SequenceForward and reverse primers
262cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnagcac aagaacaagg gaaaca
5626358DNAArtificial SequenceForward and reverse primers
263cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncatta ttgctatggg atttcctg
5826458DNAArtificial SequenceForward and reverse primers
264cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggaag gatgagaatt tcaagcac
5826555DNAArtificial SequenceForward and reverse primers
265cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcatc tggacctggg tcttc
5526655DNAArtificial SequenceForward and reverse primers
266cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncagaa tgcaagaagc ccaga
55
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