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OMTN, Volume 26
Supplemental information
Preclinical characterization of antagomiR-218
as a potential treatment for myotonic dystrophy
Estefanía Cerro-Herreros, Irene González-Martínez, Nerea Moreno, Jorge Espinosa-Espinosa, Juan M. Fernández-Costa, Anna Colom-Rodrigo, Sarah J. Overby, DavidSeoane-Miraz, Javier Poyatos-García, Juan J. Vilchez, Adolfo López de Munain, MiguelA. Varela, Matthew J. Wood, Manuel Pérez-Alonso, Beatriz Llamusí, and Rubén Artero
Supplemental Figures
Figure S1. Predicted binding sites of miR-218 to Mbnl1 and MBNL1 3’UTR. (A) Scale
representation of Mbnl1 (up) and MBNL1 (down) 3’ UTR and predicted miR-218 binding sites
according to miRanda, TargetScan and miRDB algorithms. (B) Conservation of miR-218 binding
sites in humans and mice. The miR-218 seed region is highlighted in blue. A single sequence
difference between humans and mice is shown in red. (C) Gaussia luciferase levels, relative to
internal control secreted alkaline phosphatase (SEAP), are shown normalized to levels in HeLa
cells co-transfected with an MBNL1 3’-UTR reporter construct and a scramble control agomiR
(SC). AgomiR-218 manages to reduce the reporter activity by 50%, while a similar construct
containing a perfect match for miR-218 serves as a positive control. Statistical analysis used
Student´s t-test. p < 0.05, **p < 0.01, **p < 0.001.
Figure S2. Subcutaneous or intravenous delivery of antagomiR-218 shows similar results
in HSALR mice. (A) Graphical representation of the administration protocol. Relative expression
levels of miR-218 (B,B’), Mbnl1 transcripts (C,C’), and Mbnl1 protein (D,D’) quantified from
(B,C,D) gastrocnemius and (B´,C’,D’) quadriceps muscles after subcutaneous or intravenous
injection. U1 and U6 snRNA expression levels served as endogenous controls for miR-218, while
Gapdh was the reference for Mbnl1 transcript data. Mbnl1 protein expression was quantified by
ELISA and was normalized to total protein. (E) Representative western blots to detect Mbnl1,
Histone H3, and Gapdh proteins from the gastrocnemius (upper half of panel) and quadriceps
(lower half of panel) muscles treated with PBS, scrambled antagomiR (SC), or antagomiR-218 by
subcutaneous or intravenous injection. Histone H3 and Gapdh were used as the loading controls
for the nuclear and cytoplasmic fractions, respectively. Mice were evaluated for grip strength (F),
represented as a percentage of normal force, and myotonia grade (G) before sacrifice. N value
indicates the number of mice used in each experiment. Statistical comparisons (Student’s t-test)
were performed against PBS-treated HSALR values (indicated by a black dashed line). Results
between mice treated with subcutaneous or intravenous antagomiR-218 were also compared in
all panels using the same test. p value: ns=non significant, * p<0.05, ** p<0.01, *** p<0.001, ****
p<0.0001.
Figure S3. The HSALR transgene does not significantly change its expression upon
treatment with increasing concentrations of antagomiR-218. (A) Bar graph representing the
expression of the HSALR transgene in the indicated muscles relative to the Gapdh endogenous
control and normalized to the expression in quadriceps in the PBS-treated HSALR control (black
dashed line). The quantification was performed from muscles of mice treated with 3 mg/kg, 12.5
mg/kg, and 40 mg/kg of SC negative control or antagomiR-218 by subcutaneous injection. None
of the comparisons to the reference values (black dashed line) revealed a statistically significant
difference using a Student’s t-test. (B-E) Linear correlation of Mbnl1 transcript and protein levels
with antagomiR-218 dose in HSALR mice. Relative Mbnl transcript (B,C) or protein (D,E)
expression levels at the indicated doses of antagomiR-218 in gastrocnemius (B,D) and
quadriceps (C,E) muscles. The tables underneath the graphs include the Pearson correlation
coefficients, p value, and slope of the adjustment for SC and antagomiR-218 treatments. *
indicates p<0.05. Correlations were generally high, with coefficients ranging from 0.79 to 0.99 for
the protein vs. antagomiR-218 linear adjustment. (F) Relative expression of miR-218 from
gastrocnemius and quadriceps muscles relative to Scramble. N indicates the number of mice that
were processed. All statistical comparisons were performed against the data obtained in PBS-
treated HSALR mice (black dashed line) using Student’s t-test. p values: * p<0.05, ** p<0.01, ***
p<0.001, **** p<0.0001.
Figure S4. The response of Mbnl1-dependent splicings to increasing concentrations of
antagomiR-218 in HSALR muscles. (A,B) Nfix and (C,D) Clcn1 exon 7 inclusion was quantified
from (A,C) gastrocnemius and (B,D) quadriceps of HSALR mice after subcutaneous administration
of PBS, SC, or antagomiR-218 at the indicated concentrations. Control procedure PBS1
corresponds to 12.5 mg/kg treatment, while PBS2 serves as procedural control for 3 mg/kg and
40 mg/kg treatments. Normal exon inclusion levels are provided by the FVB control injected with
vehicle only (PBS). Individual percentages of exon inclusion (Ψ) are shown in the y-axis. The
mean of each treatment was compared to its corresponding PBS control with a Student’s t-test.
p-values: * p<0.05, ** p<0.01, *** p<0.001.
Figure S5. Time response study of Mbnl1-dependent splicings after treatment with
antagomiRs. (A,B) Nfix and (C,D) Clcn1 exon 7 inclusion was quantified from (A,C)
gastrocnemius and (B,D) quadriceps muscles of HSALR mice after treatment with PBS, SC, or
antagomiR-218 at the indicated days post-injection. Normal exon inclusion levels are provided by
the FVB control. Individual data points are indicated. Statistical comparisons (Student’s t-test)
were performed with PBS-treated HSALR controls. P values: * p<0.05, ** p<0.01, *** p<0.001.
Figure S6. Biodistribution and activity of antagomiR-218 in selected mouse tissues, and
the pattern of expression of miR-218 in the indicated human samples. (A,B) Detection of
Cy3-labeled antagomiR-218 in kidney (A) and liver (B) cryosections of HSALR treated mice (n = 1).
Myonuclei were counterstained with DAPI (blue). Scale bar = 50 μm. Relative expression of (C)
miR-218, (D) Mbnl1 transcripts, and (E) Mbnl1 protein from brain and heart relative to U1 and U6
snRNA, Gapdh, and total protein, respectively. miR-218 and transcripts were quantified by RT-
qPCR, while protein levels were determined by ELISA. N indicates the number of mice used for
the experiment. All statistical comparisons were performed against the data obtained in PBS-
treated HSALR mice (black dashed line) using Student’s t-test. p values: * p<0.05, ** p<0.01. (F)
Representation of miR-218 levels in human tissues and organs from public qPCR database
miRNAMap.
Figure S7. miR-218 levels from all muscle biopsies do not correlate with the age or sex of
the patients. Pearson correlation coefficient of miR-218 expression against the age of the donor
(A), and levels of miR-218 in male and female patients (B). (A,B) Values are shown relative to a
normal miR-218 expression.
Figure S8. miR-218 levels are good predictors of the DM1 condition. The area under the
curve (AUC) of an empirical receiver operating characteristics (ROC) curve provides a useful
metric of the accuracy of a predictor test. Whereas an AUC value close to 1 indicates an excellent
diagnostic test, a curve that lies close to the diagonal (AUC = 0.5) has no information content and
no diagnostic utility 1. According to 2 an AUC between 0.8–0.9 is regarded as having a good
predictive value.
Figure S9. AntagomiR-218 significantly rescues defective cell fusion and delayed
differentiation of DM1 myoblasts. DM1 myoblasts were transfected with the indicated range of
concentrations of antagomiR. (A) The cell fusion index was determined in Desmin-stained
preparations four days after inducing transdifferentiation. (B) The same preparations were
analyzed to obtain the percentage of Desmin-positive cells normalized to the total number of cells
(detected by DAPI). As procedural controls, healthy and DM1 myoblasts were transfected with
transfection reagent only. Individual data points are indicated. Mean values were compared with
a two-tailed unpaired Student’s t-test. P values: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.
Figure S10. DMPK and SCN4A transcript levels are significantly reduced in the disease
and antagomiR-218 treated cells. (A-C) Box plot representation of DMPK transcript
quantification relative to GAPDH from RNA isolated using a conventional Trizol extraction (A) and
a Hot Trizol protocol separating the nuclear (B) and cytosolic (C) fractions. (D) Box plot
representation of log2 of reads per million of SCN4A. Experiments were carried out in healthy
control (CNT) and DM1 myoblasts treated with the scramble antagomiR (SC) or antagomiR-218
(218). The line marks the mean and the box the upper and lower values of the standard error from
the mean. Means were compared using a Student’s t-test. p values: * p<0.05; ** p<0.01; ***
p<0.001.
Supplemental Tables
Table S1. Statistical analysis of blood parameters from the indicated experiments. Serum
biochemistry profile for the intravenous (IV) vs subcutaneous (Sub) (Tab A), dose-response (Tab
C), and time-response (Tab E) experiments. Shown are the mean and the standard error of the
mean (SEM) of each of the parameters of the indicated groups so that the statistical test compares
the administration route, dose, or time-response between the scramble and antagomiR-218
treated mice and FVB controls when included in the experiment. White blood cells (WBC) count
for the IV vs Sub (Tab B), dose-response (Tab D), and time-response (Tab F) experiments.
Shown are the mean percentage and the SEM of each of the cell types, doses, and experimental
groups indicated so that the statistical test compares the administration route, dose, or time-
response between the SC, and antagomiR-218 treated mice, and FVB controls when included in
the experiment. Tabs B,D,F also includes a compositional data dendrogram for the administration
route (Tab B), dose (Tab D), or time (Tab F) at time points T1: 4 days, T2: 15 days, T3: 30 days,
and T4: 45 days. Abbreviations used: PBS, SC, and 218 indicate HSALR mouse injected with PBS,
or negative control (SC) or antagomiR-218, respectively. FVB controls were mock-treated with
PBS. P values: *p<0.05, **p<0.01, ***p<0.001.
Table S2. Basic medical characterization of the healthy and DM1 donors of the muscle
biopsies. All DM1 patients were molecularly confirmed by detecting the mutation, but the size of
the expansion was only available for a subset of them from southern blot determinations, which
were given as the size of the restriction fragment containing the expansion (Valencia cohort) or
estimated number of CTG repeats (Biodonostia cohort). Age was at the time the biopsy was taken.
Table S3. Differentially expressed genes. Results obtained by using R Package edgeR on gene
count for the comparison of healthy vs DM1 cells treated with SC negative control (HvsD), healthy
control cells vs DM1 cells treated with antagomiR-218 (Hvs218), and DM1 control cells vs DM1
cells treated with antagomiR-218 (Dvs218). Additionally, miR-218 targets were annotated using
the mirWalk (update Mar/2020) database.
Table S4. Gene Ontology terms significantly rescued in the comparison between healthy
and DM1 cells treated with antagomiR-218. Functional annotation of totally recovered genes
(50-150% of normal expression) according to the R package clusterProfiler. Both Gene Ontology
and Reactome databases were used. Gene Ontology results were simplified using the simplify
module included in the package.
Table S5. Raw data from the indicated mice and experiment. The table shows the data from
individual mice for RT-qPCR, ELISA, myotonia grade, and percentage normal force (PNF)
determinations from the administration route (Tab A), dose-response (Tab B), and time-response
(Tab C) experiments. Nfix and Clcn1 percentage spliced-in (PSI) of the indicated muscle is also
provided for the dose and time-response studies (Tabs B and C). Tab D shows the concentration
of antagomiR-218 in six different tissues. Tab E shows the data from individual mice for RT-qPCR
and ELISA in the brain and heart. Tab F shows mean values of Mbnl1 immunofluorescence
(arbitrary units) and percentage of cells containing at least one CUG foci (according to FISH data)
from muscle sections of the indicated mice, treatments, and doses.
Supplemental Methods
Method S1. Sequences of mutated and perfect match 3’UTR of Mbnl1.
DELETIONS
>>M1-218-Site1 CCACAAGTATGTTACCCAGATGTAGAGCTGTCGTCACAAAACAATCATACAAAGAGGAAAGGACAGTGTGCTTGATTAGAGTAAGGACGACGTCATTAGCCATATTGTATATACCGTCAAGCAACACATACAAAAATCCCTCAGCCACAAGACATCCACATATTGCATGTTAACCAGAAGAAACGACAACATGGGAACCTGCTGCACACTGTTGCCTACACACTTTGTACATTCAGTTGGTATTTGTGCTGAGGTGATATTCCTATCTAAAACAACAACATTGTCTTTCTTTTGTAGCACAGAGTTATGCATTAAAATATGCATACGTAATTAGTTTCCTATATATTCATGCCATCTTGAAAAGACAGACTATGGTGTGACCATGATTCTATTATGTATTGGTACGTCTGTAGACCAAGATATAATTTTTTAAAAATAAGTTTATTTCTTTCAAGGTTTACAAGTAACCAAGGTGCACCTTGTATTTAAAATCGCCGTTAGAGCTGAGAGCGCGCATGCAGAGTCATTTTTGTTTGAGAGTAATATTTTTACTGTAATAGATTGTACGACATGGTGAGGGAGGGAACTGACAGATGAATGTGCCAAGCAAAACCACAACTGTGTATATTTTAAAGCACACCATGGCTTTAAGTACCATGTTGTTAAGGATTCTCATGAAGTGCCATAGACTGTACATCAAATTAGAGTATTATTTCTTCAGTGTTATTGTTTCTGGAGCCACATTTTGTTGCTTATTTGCTAGTACTAATCAATCAAAGGGCACCATTCTTTTCTTTTTTGTTTTTGAAACCAAAGCTGTCTCAGAAATGGCCAATTTAACTTTACAGTAACAATAGACAGCACAACACAAACTCAATACAGATAACCTTTCACATACTGGAGATATATATGATAGATATATAAAATTATTTTAATGCATTGTAGTGTAATATTTATGCATACTCTACTATATAACATGTTATTCAAAAGGGATATGCCATTTCTGAGACACAATAACAAAAAATGTTTGAGGAAATTATTTTGCTTCTATTTATAGCCTCTGTCAAAAGTCAAAAGACTATAAATGCTTTGCAGAAATGGGTTCACGTTTGCTTAAACGCTTCATCACAGTCACATTCAAAATAGTGACTCTAAACAAAGAGAACAGCACTGTCATCAGATGCATGATAAACCAAAATATGAAAATGGGAAATGTTTAATTAACCTAGTAATTGGGTGGGTTAAGTACATGGGTGAATTTTATATGTGATTCTTTTGTTCAGATTAACTGCTTATAGCCTTAGAAAGCCTTTTAAAAAATTTTAAAAATAGATGTGCATTCAGTTTTTAAGAATGGATTCATCCAAAGGAATTCCCCTTTTTTGTGGTTTGGATGTTGCAGCTAGGAAAGGCTATTTTTGCTCTGTTCAGCAGTTCTAAAATCGCTGAGTAGGGGCCAGGTCACTGGCAGTTCTAGTGTGGAATGGGAGAAGTGAGAGTTCTGTTATAGAACTTTCCATACTTCCAAGTTTACTGCAAGTTTTTATGCTTGAGAGAGATGCTTTCTAATATAAGACTGATGTGTTGATTTTCCTGATTGTACTGTACATCTATTAAAGCCTTAGATTATTACATTACGGGTTGGAACCCATACCAATGTAATTTCAATCGTGTTAAGAGAGTAATGGTGACTTCACATGTTATTGTAGTTAGTTACGTTATAGAATATTACTTATTTTTCTTGTTAAAATGTAGTTTTTCATTTCCTACATTTATTGGATTTTCATTTTCTATTAACAGTTGAATACCATTTCAGTTTTTAGACTATTGTTTTATTAGATTTTACCAATGAATTTTTCAAAATACAAAAAAATTAAAGTAGTTTTTTCTTCATAACATACTCAGTTTTAAATTACATGTAGTGTCATATGAATATCCGTATTATTGTTAACTAAATGATTTATATTTTACTGATTTAATATTACAGTGTAAGAATGTCAGTCATTGTTCTTGTCTAGTTTTCATTAAAAGAACAAAGATCTTTTATATGGATATCTTATAAATATATAATCATTGCTAAGTAAGAAGTTAAGTTGTTGCTATGGCAACAATCCTGGCAGACAATTGAGTAATATTTTGATGATTTATTTTGTTTGTAATTAGTTATTATGAGAAGATCTAGATCCTAGATATTAGAATAAAATTTATTTTCTACTGTATCCATTTCAAATGTTAAAGTATTGTTTAATATTTTTGAAATCCCTGAATATCAGGCCTTGTTATAAATAAGCTGCATAATCAATAAATAGAACAAGGGACTTTTTGTTGATAATCCAAATACTCAAAGTTTACGTAATGAGAATTTTAGCGTGTGTGCAAACTCTTGAGGGTTGATGATGCTGCAATTTAGCATGTTGGAAAGTCTAGAGAGAAGGTTGACTTTTTGCACTTCTGTATATAGTCAAAAGAGAGAAACCTGTATAATAGCAAGATCTTATTTTGAATAAAAACGTCTATAATTACAAGGAGTTTTGTTAAGGCTAATGAAATGACAGACTGAGCAAAATTGCTTGCAAAAGTGGCACAGAGT
TAGCACTCCATACCCTTCAAACACGTCGCTTTGCTTTTTGTGGACAGCTTGTAGTTTGCCAGGATTTTTCAGCTGGAAAGATTTGCCATCCTTCCAAGATCTCATGACTGACAAAACTCCATTGGGCCAAATCTGCCTGAAGATCATTACCAAAAAATAGCAGGTACTTCAGCCACTAAGATGAAATCATGGATCAGATATCCCTTACATTGTTTTCAAAACTACTGCATGTTTAAAACTTCAACAAAAAGAGAGAAAGAACTATGCTAAGGACATATATTATTCAGATCGATATCTACCAATTTCAGTGGTTTAATGTTCACAAAATGAAATCTTGAAAATAACTATTGACTTTCACAAAATTTTAACCATAAACAGGCAAACCAAACAGCACACCTGTAGTTGTTCTGTGATTGTTTTTTAATTGCTGTAGATCATGTTCTTTCCGCAGGTGGAAAAAAAAAAAAAAAAAAAAAAAGAAGTTCAAATTTCACAGTTTTAATTTTCAACTCAGAAGCAAAAGAGCAAAATGTGACAATGGCCACTTGTTTAATGACTTGGTTGCCCAGCTGTCACTGCAGCTGGCTACTGATGTTGCACTTACCAGCAACCCACCCACCTTCATCTGCCGAAAGGACAGTGAGCTTGGTTTTACGATTATGTAATCACAACTTACTTTCTGCTTGTAGTGGCTTAAAATTATGTATTTTGTCTAGGGCTGCAATTTGTTTTATGCTTACTTTATTATTACTGCAGTAGTTGACTTTGCTGTATGGAAAAATAAAGCGAAATTGCCCTAATAAAACTTCTCTTTCTTAAGTATAAAAAAAAAAAAAAAAAAA
>>M1-218-Site2 CCACAAGTATGTTACCCAGATGTAGAGCTGTCGTCACAAAACAATCATACAAAGAGGAAAGGACAGTGTGCTTGATTAGAGTAAGGACGACGTCATTAGCCATATTGTATATACCGTCAAGCAACACATACAAAAATCCCTCAGCCACAAGACATCCACATATTGCATGTTAACCAGAAGAAACGACAACATGGGAACCTGCTGCACACTGTTGCCTACACACTTTGTACATTCAGTTGGTATTTGTGCTGAGGTGATATTCCTATCTAAAACAACAACATTGTCTTTCTTTTGTAGCACAGAGTTATGCATTAAAATATGCATACGTAATTAGTTTCCTATATATTCATGCCATCTTGAAAAGACAGACTATGGTGTGACCATGATTCTATTATGTATTGGTACGTCTGTAGACCAAGATATAATTTTTTAAAAATAAGTTTATTTCTTTCAAGGTTTACAAGTAACCAAGGTGCACCTTGTATTTAAAATCGCCGTTAGAGCTGAGAGCGCGCATGCAGAGTCATTTTTGTTTGAGAGTAATATTTTTACTGTAATAGATTGTACGACATGGTGAGGGAGGGAACTGACAGATGAATGTGCCAAGCAAAACCACAACTGTGTATATTTTAAAGCACACCATGGCTTTAAGTACCATGTTGTTAAGGATTCTCATGAAGTGCCATAGACTGTACATCAAATTAGAGTATTATTTCTTCAGTGTTATTGTTTCTGGAGCCACATTTTGTTGCTTATTTGCTAGTACTAATCAATCAAAGGGCACCATTCTTTTCTTTTTTGTTTTTGAAACCAAAGCTGTCTCAGAAATGGCCAATTTAACTTTACAGTAACAATAGACAGCACAACACAAACTCAATACAGATAACCTTTCACATACTGGAGATATATATGATAGATATATAAAATTATTTTAATGCATTGTAGTGTAATATTTATGCATACTCTACTATATAACATGTTATTCAAAAGGGATATGCCATTTCTGAGACACAATAACAAAAAATGTTTGAGGAAATTATTTTGCTTCTATTTATAGCCTCTGTCAAAAGTCAAAAGACTATAAATGCTTTGCAGAAATGGGTTCACGTTTGCTTAAACGCTTCATCACAGTCACATTCAAAATAGTGACTCTAAACAAAGAGAACAGCACTGTCATCAGATGCATGATAAACCAAAATATGAAAATGGGAAATGTTTAATTAACCTAGTAATTGGGTGGGTTAAGTACATGGGTGAATTTTATATGTGATTCTTTTGTTCAGATTAACTGCTTATAGCCTTAGAAAGCCTTTTAAAAAATTTTAAAAATAGATGTGCATTCAGTTTTTAAGAATGGATTCATCCAAAGGAATTCCCCTTTTTTGTGGTTTGGATGTTGCAGCTAGGAAAGGCTATTTTTGCTCTGTTCAGCAGTTCTAAAATCGCTGAGTAGGGGCCAGGTCACTGGCAGTTCTAGTGTGGAATGGGAGAAGTGAGAGTTCTGTTATAGAACTTTCCATACTTCCAAGTTTACTGCAAGTTTTTATGCTTGAGAGAGATGCTTTCTAATATAAGACTGATGTGTTGATTTTCCTGATTGTACTGTACATCTATTAAAGCCTTAGATTATTACATTACGGGTTGGAACCCATACCAATGTAATTTCAATCGTGTTAAGAGAGTAATGGTGACTTCACATGTTATTGTAGTTAGTTACGTTATAGAATATTACTTATTTTTCTTGTTAAAATGTAGTTTTTCATTTCCTACATTTATTGGATTTTCATTTTCTATTAACAGTTGAATACCATTTCAGTTTTTAGACTATTGTTTTATTAGATTTTACCAATGAATTTTTCAAAATACAAAAAAATTAAAGTAGTTTTTTCTTCATAACATACTCAGTTTTAAATTACATGTAGTGTCATATGAATATCCGTATTATTGTTAACTAAATGATTTATATTTTACTGATTTAATATTACAGTGTAAGAATGTCAGTCATTGTTCTTGTCTAGTTTTCATTAAAAGAACAAAGATCTTTTATATGGATATCTTATAAATATATAATCATTGCTAAGTAAGAAGTTAAGTTGTTGC
TATGGCAACAATCCTGGCAGACAATTGAGTAATATTTTGATGATTTATTTTGTTTGTAATTAGTTATTATGAGAAGATCTAGATCCTAGATATTAGAATAAAATTTATTTTCTACTGTATCCATTTCAAATGTTAAAGTATTGTTTAATATTTTTGAAATCCCTGAATATCAGGCCTTGTTATAAATAAGCTGCATAATCAATAAATAGAACAAGGGACTTTTTGTTGATAATCCAAATACTCAAAGTTTACGTAATGAGAATTTTAGCGTGTGTGCAAACTCTTGAGGGTTGATGATGCTGCAATTTAGCATGTTGGAAAGTCTAGAGAGAAGGTTGACTTTTTGCACTTCTGTATATAGTCAAAAGAGAGAAACCTGTATAATAGCAAGATCTTATTTTGAATAAAAACGTCTATAATTACAAGGAGTTTTGTTAAGGCTAATGAAATGACAGACTGAGCAAAATTGCTTGCAAAAGTGGCACAGAGTTAGCACTCCATACCCTTCAAACACGTCGCTTTGCTTTTTGTGGACAGCTTGTAGTTTGCCAGGATTTTTCAGCTGGAAAGATTTGCCATCCTTCCAAGATCTCATGACTGACAAAACTCCATTGGGCCAAATCTGCCTGAAGATCATTACCAAAAAATAGCAGGTACTTCAGCCACTAAGATGAAATCATGGATCAGATATCCCTTACATTGTTTTCAAAACTACTGCATGTTTAAAACTTCAACAAAAAGAGAGAAAGAACTATGCTAAGGACATATATTATTCAGATCGATATCTACCAATTTCAGTGGTTTAATGTTCACAAAATGAAATCTTGAAAATAACTATTGACTTTCACAAAATTTTAACCATAAACAGGCAAACCAAACAGCACACCTGTAGTTGTTCTGTGATTGTTTTTTAATTGCTGTAGATCATGTTCTTTCCGCAGGTGGAAAAAAAAAAAAAAAAAAAAAAAGAAGTTCAAATTTCACAGTTTTAATTTTCAACTCAGAAGCAAAAGAGCAAAATGTGACAATGGCCACTTGTTTAATGACTTGGTTGCCCAGCTGTCACTGCAGCTGGCTACTGATGTTGCACTTACCAGCAACCCACCCACCTTCATCTGCCGAAAGGACAGTGAGCTTGGTTTTACGATTATGTAATCACAACTTACTTTCTGCTTGTAGTGGCTTAAAATTATGTATTTTGTCTAGGGCTGCAATTTGTTTTATGCTTACTTTATTATTACTGCAGTAGTTGACTTTGCTGTATGGAAAAATAAAGCGAAATTGCCCTAATAAAACTTCTCTTTCTTAAGTATAAAAAAAAAAAAAAAAAAA
>>M1-218-Site3 CCACAAGTATGTTACCCAGATGTAGAGCTGTCGTCACAAAACAATCATACAAAGAGGAAAGGACAGTGTGCTTGATTAGAGTAAGGACGACGTCATTAGCCATATTGTATATACCGTCAAGCAACACATACAAAAATCCCTCAGCCACAAGACATCCACATATTGCATGTTAACCAGAAGAAACGACAACATGGGAACCTGCTGCACACTGTTGCCTACACACTTTGTACATTCAGTTGGTATTTGTGCTGAGGTGATATTCCTATCTAAAACAACAACATTGTCTTTCTTTTGTAGCACAGAGTTATGCATTAAAATATGCATACGTAATTAGTTTCCTATATATTCATGCCATCTTGAAAAGACAGACTATGGTGTGACCATGATTCTATTATGTATTGGTACGTCTGTAGACCAAGATATAATTTTTTAAAAATAAGTTTATTTCTTTCAAGGTTTACAAGTAACCAAGGTGCACCTTGTATTTAAAATCGCCGTTAGAGCTGAGAGCGCGCATGCAGAGTCATTTTTGTTTGAGAGTAATATTTTTACTGTAATAGATTGTACGACATGGTGAGGGAGGGAACTGACAGATGAATGTGCCAAGCAAAACCACAACTGTGTATATTTTAAAGCACACCATGGCTTTAAGTACCATGTTGTTAAGGATTCTCATGAAGTGCCATAGACTGTACATCAAATTAGAGTATTATTTCTTCAGTGTTATTGTTTCTGGAGCCACATTTTGTTGCTTATTTGCTAGTACTAATCAATCAAAGGGCACCATTCTTTTCTTTTTTGTTTTTGAAACCAAAGCTGTCTCAGAAATGGCCAATTTAACTTTACAGTAACAATAGACAGCACAACACAAACTCAATACAGATAACCTTTCACATACTGGAGATATATATGATAGATATATAAAATTATTTTAATGCATTGTAGTGTAATATTTATGCATACTCTACTATATAACATGTTATTCAAAAGGGATATGCCATTTCTGAGACACAATAACAAAAAATGTTTGAGGAAATTATTTTGCTTCTATTTATAGCCTCTGTCAAAAGTCAAAAGACTATAAATGCTTTGCAGAAATGGGTTCACGTTTGCTTAAACGCTTCATCACAGTCACATTCAAAATAGTGACTCTAAACAAAGAGAACAGCACTGTCATCAGATGCATGATAAACCAAAATATGAAAATGGGAAATGTTTAATTAACCTAGTAATTGGGTGGGTTAAGTACATGGGTGAATTTTATATGTGATTCTTTTGTTCAGATTAACTGCTTATAGCCTTAGAAAGCCTTTTAAAAAATTTTAAAAATAGATGTGCATTCAGTTTTTAAGAATGGATTCATCCAAAGGAATTCCCCTTTTTTGTGGTTTGGATGTTGCAGCTAGGAAAGGCTATTTTTGCTCTGTTCAGCAGTTCTAAAATCGCTGAGTAGGGGCCAGGTCACTGGCAGTTCTAGTGTGGAATGGGAGAAGTGAGAGTTCTGTTATAGAACTTTCCATACTTCCAAGTTTACTGCAAGTTTTTATGCTTGAGAGAGATGCTTTCTAATATAAGACTGATGTGTTGATTTTCCTGATTGTACTGTACAT
CTATTAAAGCCTTAGATTATTACATTACGGGTTGGAACCCATACCAATGTAATTTCAATCGTGTTAAGAGAGTAATGGTGACTTCACATGTTATTGTAGTTAGTTACGTTATAGAATATTACTTATTTTTCTTGTTAAAATGTAGTTTTTCATTTCCTACATTTATTGGATTTTCATTTTCTATTAACAGTTGAATACCATTTCAGTTTTTAGACTATTGTTTTATTAGATTTTACCAATGAATTTTTCAAAATACAAAAAAATTAAAGTAGTTTTTTCTTCATAACATACTCAGTTTTAAATTACATGTAGTGTCATATGAATATCCGTATTATTGTTAACTAAATGATTTATATTTTACTGATTTAATATTACAGTGTAAGAATGTCAGTCATTGTTCTTGTCTAGTTTTCATTAAAAGAACAAAGATCTTTTATATGGATATCTTATAAATATATAATCATTGCTAAGTAAGAAGTTAAGTTGTTGCTATGGCAACAATCCTGGCAGACAATTGAGTAATATTTTGATGATTTATTTTGTTTGTAATTAGTTATTATGAGAAGATCTAGATCCTAGATATTAGAATAAAATTTATTTTCTACTGTATCCATTTCAAATGTTAAAGTATTGTTTAATATTTTTGAAATCCCTGAATATCAGGCCTTGTTATAAATAAGCTGCATAATCAATAAATAGAACAAGGGACTTTTTGTTGATAATCCAAATACTCAAAGTTTACGTAATGAGAATTTTAGCGTGTGTGCAAACTCTTGAGGGTTGATGATGCTGCAATTTAGCATGTTGGAAAGTCTAGAGAGAAGGTTGACTTTTTGCACTTCTGTATATAGTCAAAAGAGAGAAACCTGTATAATAGCAAGATCTTATTTTGAATAAAAACGTCTATAATTACAAGGAGTTTTGTTAAGGCTAATGAAATGACAGACTGAGCAAAATTGCTTGCAAAAGTGGCACAGAGTTAGCACTCCATACCCTTCAAACACGTCGCTTTGCTTTTTGTGGACAGCTTGTAGTTTGCCAGGATTTTTCAGCTGGAAAGATTTGCCATCCTTCCAAGATCTCATGACTGACAAAACTCCATTGGGCCAAATCTGCCTGAAGATCATTACCAAAAAATAGCAGGTACTTCAGCCACTAAGATGAAATCATGGATCAGATATCCCTTACATTGTTTTCAAAACTACTGCATGTTTAAAACTTCAACAAAAAGAGAGAAAGAACTATGCTAAGGACATATATTATTCAGATCGATATCTACCAATTTCAGTGGTTTAATGTTCACAAAATGAAATCTTGAAAATAACTATTGACTTTCACAAAATTTTAACCATAAACAGGCAAACCAAACAGCACACCTGTAGTTGTTCTGTGATTGTTTTTTAATTGCTGTAGATCATGTTCTTTCCGCAGGTGGAAAAAAAAAAAAAAAAAAAAAAAGAAGTTCAAATTTCACAGTTTTAATTTTCAACTCAGAAGCAAAAGAGCAAAATGTGACAATGGCCACTTGTTTAATGACTTGGTTGCCCAGCTGTCACTGCAGCTGGCTACTGATGTTGCACTTACCAGCAACCCACCCACCTTCATCTGCCGAAAGGACAGTGAGCTTGGTTTTACGATTATGTAATCACAACTTACTTTCTGCTTGTAGTGGCTTAAAATTATGTATTTTGTCTAGGGCTGCAATTTGTTTTATGCTTACTTTATTATTACTGCAGTAGTTGACTTTGCTGTATGGAAAAATAAAGCGAAATTGCCCTAATAAAACTTCTCTTTCTTAAGTATAAAAAAAAAAAAAAAAAAA
>>M1-218-Site4 CCACAAGTATGTTACCCAGATGTAGAGCTGTCGTCACAAAACAATCATACAAAGAGGAAAGGACAGTGTGCTTGATTAGAGTAAGGACGACGTCATTAGCCATATTGTATATACCGTCAAGCAACACATACAAAAATCCCTCAGCCACAAGACATCCACATATTGCATGTTAACCAGAAGAAACGACAACATGGGAACCTGCTGCACACTGTTGCCTACACACTTTGTACATTCAGTTGGTATTTGTGCTGAGGTGATATTCCTATCTAAAACAACAACATTGTCTTTCTTTTGTAGCACAGAGTTATGCATTAAAATATGCATACGTAATTAGTTTCCTATATATTCATGCCATCTTGAAAAGACAGACTATGGTGTGACCATGATTCTATTATGTATTGGTACGTCTGTAGACCAAGATATAATTTTTTAAAAATAAGTTTATTTCTTTCAAGGTTTACAAGTAACCAAGGTGCACCTTGTATTTAAAATCGCCGTTAGAGCTGAGAGCGCGCATGCAGAGTCATTTTTGTTTGAGAGTAATATTTTTACTGTAATAGATTGTACGACATGGTGAGGGAGGGAACTGACAGATGAATGTGCCAAGCAAAACCACAACTGTGTATATTTTAAAGCACACCATGGCTTTAAGTACCATGTTGTTAAGGATTCTCATGAAGTGCCATAGACTGTACATCAAATTAGAGTATTATTTCTTCAGTGTTATTGTTTCTGGAGCCACATTTTGTTGCTTATTTGCTAGTACTAATCAATCAAAGGGCACCATTCTTTTCTTTTTTGTTTTTGAAACCAAAGCTGTCTCAGAAATGGCCAATTTAACTTTACAGTAACAATAGACAGCACAACACAAACTCAATACAGATAACCTTTCACATACTGGAGATATATATGATAGATATATAAAATTATTTTAATGCATTGTAGTGTAATATTTATGCATACTCTACTATATAACATGTTATTCAAAAGGGATATGCCATTTCTGAGACACAATAACAAAAAATGTTTGAGGAAATTATTTTGCTTCTATTTATAGCCTCTGTCAAAAGTCAAAAGACTATAAATGCTTTGCAGAAATGGGTTCACGTTTGCTTAAACG
CTTCATCACAGTCACATTCAAAATAGTGACTCTAAACAAAGAGAACAGCACTGTCATCAGATGCATGATAAACCAAAATATGAAAATGGGAAATGTTTAATTAACCTAGTAATTGGGTGGGTTAAGTACATGGGTGAATTTTATATGTGATTCTTTTGTTCAGATTAACTGCTTATAGCCTTAGAAAGCCTTTTAAAAAATTTTAAAAATAGATGTGCATTCAGTTTTTAAGAATGGATTCATCCAAAGGAATTCCCCTTTTTTGTGGTTTGGATGTTGCAGCTAGGAAAGGCTATTTTTGCTCTGTTCAGCAGTTCTAAAATCGCTGAGTAGGGGCCAGGTCACTGGCAGTTCTAGTGTGGAATGGGAGAAGTGAGAGTTCTGTTATAGAACTTTCCATACTTCCAAGTTTACTGCAAGTTTTTATGCTTGAGAGAGATGCTTTCTAATATAAGACTGATGTGTTGATTTTCCTGATTGTACTGTACATCTATTAAAGCCTTAGATTATTACATTACGGGTTGGAACCCATACCAATGTAATTTCAATCGTGTTAAGAGAGTAATGGTGACTTCACATGTTATTGTAGTTAGTTACGTTATAGAATATTACTTATTTTTCTTGTTAAAATGTAGTTTTTCATTTCCTACATTTATTGGATTTTCATTTTCTATTAACAGTTGAATACCATTTCAGTTTTTAGACTATTGTTTTATTAGATTTTACCAATGAATTTTTCAAAATACAAAAAAATTAAAGTAGTTTTTTCTTCATAACATACTCAGTTTTAAATTACATGTAGTGTCATATGAATATCCGTATTATTGTTAACTAAATGATTTATATTTTACTGATTTAATATTACAGTGTAAGAATGTCAGTCATTGTTCTTGTCTAGTTTTCATTAAAAGAACAAAGATCTTTTATATGGATATCTTATAAATATATAATCATTGCTAAGTAAGAAGTTAAGTTGTTGCTATGGCAACAATCCTGGCAGACAATTGAGTAATATTTTGATGATTTATTTTGTTTGTAATTAGTTATTATGAGAAGATCTAGATCCTAGATATTAGAATAAAATTTATTTTCTACTGTATCCATTTCAAATGTTAAAGTATTGTTTAATATTTTTGAAATCCCTGAATATCAGGCCTTGTTATAAATAAGCTGCATAATCAATAAATAGAACAAGGGACTTTTTGTTGATAATCCAAATACTCAAAGTTTACGTAATGAGAATTTTAGCGTGTGTGCAAACTCTTGAGGGTTGATGATGCTGCAATTTAGCATGTTGGAAAGTCTAGAGAGAAGGTTGACTTTTTGCACTTCTGTATATAGTCAAAAGAGAGAAACCTGTATAATAGCAAGATCTTATTTTGAATAAAAACGTCTATAATTACAAGGAGTTTTGTTAAGGCTAATGAAATGACAGACTGAGCAAAATTGCTTGCAAAAGTGGCACAGAGTTAGCACTCCATACCCTTCAAACACGTCGCTTTGCTTTTTGTGGACAGCTTGTAGTTTGCCAGGATTTTTCAGCTGGAAAGATTTGCCATCCTTCCAAGATCTCATGACTGACAAAACTCCATTGGGCCAAATCTGCCTGAAGATCATTACCAAAAAATAGCAGGTACTTCAGCCACTAAGATGAAATCATGGATCAGATATCCCTTACATTGTTTTCAAAACTACTGCATGTTTAAAACTTCAACAAAAAGAGAGAAAGAACTATGCTAAGGACATATATTATTCAGATCGATATCTACCAATTTCAGTGGTTTAATGTTCACAAAATGAAATCTTGAAAATAACTATTGACTTTCACAAAATTTTAACCATAAACAGGCAAACCAAACAGCACACCTGTAGTTGTTCTGTGATTGTTTTTTAATTGCTGTAGATCATGTTCTTTCCGCAGGTGGAAAAAAAAAAAAAAAAAAAAAAAGAAGTTCAAATTTCACAGTTTTAATTTTCAACTCAGAAGCAAAAGAGCAAAATGTGACAATGGCCACTTGTTTAATGACTTGGTTGCCCAGCTGTCACTGCAGCTGGCTACTGATGTTGCACTTACCAGCAACCCACCCACCTTCATCTGCCGAAAGGACAGTGAGCTTGGTTTTACGATTATGTAATCACAACTTACTTTCTGCTTGTAGTGGCTTAAAATTATGTATTTTGTCTAGGGCTGCAATTTGTTTTATGCTTACTTTATTATTACTGCAGTAGTTGACTTTGCTGTATGGAAAAATAAAGCGAAATTGCCCTAATAAAACTTCTCTTTCTTAAGTATAAAAAAAAAAAAAAAAAAA
PERFECT MATCH
>>M1-218-Site1 AAAACAACAACATTGTCTTTCTTTTGTAGCACAGA ACATGGTTAGATCAAGCACAA >>M1-218-Site1 CCACAAGTATGTTACCCAGATGTAGAGCTGTCGTCACAAAACAATCATACAAAGAGGAAAGGACAGTGTGCTTGATTAGAGTAAGGACGACGTCATTAGCCATATTGTATATACCGTCAAGCAACACATACAAAAATCCCTCAGCCACAAGACATCCACATATTGCATGTTAACCAGAAGAAACGACAACATGGGAACCTGCTGCACACT
GTTGCCTACACACTTTGTACATTCAGTTGGTATTTGTGCTGAGGTGATATTCCTATCTAAAACAACAACATACATGGTTAGATCAAGCACAAAGTTATGCATTAAAATATGCATACGTAATTAGTTTCCTATATATTCATGCCATCTTGAAAAGACAGACTATGGTGTGACCATGATTCTATTATGTATTGGTACGTCTGTAGACCAAGATATAATTTTTTAAAAATAAGTTTATTTCTTTCAAGGTTTACAAGTAACCAAGGTGCACCTTGTATTTAAAATCGCCGTTAGAGCTGAGAGCGCGCATGCAGAGTCATTTTTGTTTGAGAGTAATATTTTTACTGTAATAGATTGTACGACATGGTGAGGGAGGGAACTGACAGATGAATGTGCCAAGCAAAACCACAACTGTGTATATTTTAAAGCACACCATGGCTTTAAGTACCATGTTGTTAAGGATTCTCATGAAGTGCCATAGACTGTACATCAAATTAGAGTATTATTTCTTCAGTGTTATTGTTTCTGGAGCCACATTTTGTTGCTTATTTGCTAGTACTAATCAATCAAAGGGCACCATTCTTTTCTTTTTTGTTTTTGAAACCAAAGCTGTCTCAGAAATGGCCAATTTAACTTTACAGTAACAATAGACAGCACAACACAAACTCAATACAGATAACCTTTCACATACTGGAGATATATATGATAGATATATAAAATTATTTTAATGCATTGTAGTGTAATATTTATGCATACTCTACTATATAACATGTTATTCAAAAGGGATATGCCATTTCTGAGACACAATAACAAAAAATGTTTGAGGAAATTATTTTGCTTCTATTTATAGCCTCTGTCAAAAGTCAAAAGACTATAAATGCTTTGCAGAAATGGGTTCACGTTTGCTTAAACGCTTCATCACAGTCACATTCAAAATAGTGACTCTAAACAAAGAGAACAGCACTGTCATCAGATGCATGATAAACCAAAATATGAAAATGGGAAATGTTTAATTAACCTAGTAATTGGGTGGGTTAAGTACATGGGTGAATTTTATATGTGATTCTTTTGTTCAGATTAACTGCTTATAGCCTTAGAAAGCCTTTTAAAAAATTTTAAAAATAGATGTGCATTCAGTTTTTAAGAATGGATTCATCCAAAGGAATTCCCCTTTTTTGTGGTTTGGATGTTGCAGCTAGGAAAGGCTATTTTTGCTCTGTTCAGCAGTTCTAAAATCGCTGAGTAGGGGCCAGGTCACTGGCAGTTCTAGTGTGGAATGGGAGAAGTGAGAGTTCTGTTATAGAACTTTCCATACTTCCAAGTTTACTGCAAGTTTTTATGCTTGAGAGAGATGCTTTCTAATATAAGACTGATGTGTTGATTTTCCTGATTGTACTGTACATCTATTAAAGCCTTAGATTATTACATTACGGGTTGGAACCCATACCAATGTAATTTCAATCGTGTTAAGAGAGTAATGGTGACTTCACATGTTATTGTAGTTAGTTACGTTATAGAATATTACTTATTTTTCTTGTTAAAATGTAGTTTTTCATTTCCTACATTTATTGGATTTTCATTTTCTATTAACAGTTGAATACCATTTCAGTTTTTAGACTATTGTTTTATTAGATTTTACCAATGAATTTTTCAAAATACAAAAAAATTAAAGTAGTTTTTTCTTCATAACATACTCAGTTTTAAATTACATGTAGTGTCATATGAATATCCGTATTATTGTTAACTAAATGATTTATATTTTACTGATTTAATATTACAGTGTAAGAATGTCAGTCATTGTTCTTGTCTAGTTTTCATTAAAAGAACAAAGATCTTTTATATGGATATCTTATAAATATATAATCATTGCTAAGTAAGAAGTTAAGTTGTTGCTATGGCAACAATCCTGGCAGACAATTGAGTAATATTTTGATGATTTATTTTGTTTGTAATTAGTTATTATGAGAAGATCTAGATCCTAGATATTAGAATAAAATTTATTTTCTACTGTATCCATTTCAAATGTTAAAGTATTGTTTAATATTTTTGAAATCCCTGAATATCAGGCCTTGTTATAAATAAGCTGCATAATCAATAAATAGAACAAGGGACTTTTTGTTGATAATCCAAATACTCAAAGTTTACGTAATGAGAATTTTAGCGTGTGTGCAAACTCTTGAGGGTTGATGATGCTGCAATTTAGCATGTTGGAAAGTCTAGAGAGAAGGTTGACTTTTTGCACTTCTGTATATAGTCAAAAGAGAGAAACCTGTATAATAGCAAGATCTTATTTTGAATAAAAACGTCTATAATTACAAGGAGTTTTGTTAAGGCTAATGAAATGACAGACTGAGCAAAATTGCTTGCAAAAGTGGCACAGAGTTAGCACTCCATACCCTTCAAACACGTCGCTTTGCTTTTTGTGGACAGCTTGTAGTTTGCCAGGATTTTTCAGCTGGAAAGATTTGCCATCCTTCCAAGATCTCATGACTGACAAAACTCCATTGGGCCAAATCTGCCTGAAGATCATTACCAAAAAATAGCAGGTACTTCAGCCACTAAGATGAAATCATGGATCAGATATCCCTTACATTGTTTTCAAAACTACTGCATGTTTAAAACTTCAACAAAAAGAGAGAAAGAACTATGCTAAGGACATATATTATTCAGATCGATATCTACCAATTTCAGTGGTTTAATGTTCACAAAATGAAATCTTGAAAATAACTATTGACTTTCACAAAATTTTAACCATAAACAGGCAAACCAAACAGCACACCTGTAGTTGTTCTGTGATTGTTTTTTAATTGCTGTAGATCATGTTCTTTCCGCAGGTGGAAAAAAAAAAAAAAAAAAAAAAAGAAGTTCAAATTTCACAGTTTTAATTTTCAACTCAGAAGCAAAAGAGCAAAATGTGACAATGGCCACTTGTTTAATGACTTGGTTGCCCAGCTGTCACTGCAGCTGGCTACTGATGTTGCACTTACCAGCAACCCACCCACCTTCATCTGCCGAAAGGACAGTGAGCTTGGTTTTACGATTATGTAATCACAACTTACTTTCTGCTTGTAGTGGCTTAAAATTATGTATTTTGTCTAGGGCTGCAATTTGTTTTATGCTTACTTTATTATTACTGCAGTAGTTGACTTTGCT
GTATGGAAAAATAAAGCGAAATTGCCCTAATAAAACTTCTCTTTCTTAAGTATAAAAAAAAAAAAAAAAAAA
>>M1-218-Site2 AAAACCACAACTGTGTATATTTTAAAGCACACCATGGCTT ACATGGTTAGATCAAGCACAA >>M1-218-Site2 CCACAAGTATGTTACCCAGATGTAGAGCTGTCGTCACAAAACAATCATACAAAGAGGAAAGGACAGTGTGCTTGATTAGAGTAAGGACGACGTCATTAGCCATATTGTATATACCGTCAAGCAACACATACAAAAATCCCTCAGCCACAAGACATCCACATATTGCATGTTAACCAGAAGAAACGACAACATGGGAACCTGCTGCACACTGTTGCCTACACACTTTGTACATTCAGTTGGTATTTGTGCTGAGGTGATATTCCTATCTAAAACAACAACATTGTCTTTCTTTTGTAGCACAGAGTTATGCATTAAAATATGCATACGTAATTAGTTTCCTATATATTCATGCCATCTTGAAAAGACAGACTATGGTGTGACCATGATTCTATTATGTATTGGTACGTCTGTAGACCAAGATATAATTTTTTAAAAATAAGTTTATTTCTTTCAAGGTTTACAAGTAACCAAGGTGCACCTTGTATTTAAAATCGCCGTTAGAGCTGAGAGCGCGCATGCAGAGTCATTTTTGTTTGAGAGTAATATTTTTACTGTAATAGATTGTACGACATGGTGAGGGAGGGAACTGACAGATGAATGTGCCAAGCAAAACCACAACACATGGTTAGATCAAGCACAACATGGCTTTAAGTACCATGTTGTTAAGGATTCTCATGAAGTGCCATAGACTGTACATCAAATTAGAGTATTATTTCTTCAGTGTTATTGTTTCTGGAGCCACATTTTGTTGCTTATTTGCTAGTACTAATCAATCAAAGGGCACCATTCTTTTCTTTTTTGTTTTTGAAACCAAAGCTGTCTCAGAAATGGCCAATTTAACTTTACAGTAACAATAGACAGCACAACACAAACTCAATACAGATAACCTTTCACATACTGGAGATATATATGATAGATATATAAAATTATTTTAATGCATTGTAGTGTAATATTTATGCATACTCTACTATATAACATGTTATTCAAAAGGGATATGCCATTTCTGAGACACAATAACAAAAAATGTTTGAGGAAATTATTTTGCTTCTATTTATAGCCTCTGTCAAAAGTCAAAAGACTATAAATGCTTTGCAGAAATGGGTTCACGTTTGCTTAAACGCTTCATCACAGTCACATTCAAAATAGTGACTCTAAACAAAGAGAACAGCACTGTCATCAGATGCATGATAAACCAAAATATGAAAATGGGAAATGTTTAATTAACCTAGTAATTGGGTGGGTTAAGTACATGGGTGAATTTTATATGTGATTCTTTTGTTCAGATTAACTGCTTATAGCCTTAGAAAGCCTTTTAAAAAATTTTAAAAATAGATGTGCATTCAGTTTTTAAGAATGGATTCATCCAAAGGAATTCCCCTTTTTTGTGGTTTGGATGTTGCAGCTAGGAAAGGCTATTTTTGCTCTGTTCAGCAGTTCTAAAATCGCTGAGTAGGGGCCAGGTCACTGGCAGTTCTAGTGTGGAATGGGAGAAGTGAGAGTTCTGTTATAGAACTTTCCATACTTCCAAGTTTACTGCAAGTTTTTATGCTTGAGAGAGATGCTTTCTAATATAAGACTGATGTGTTGATTTTCCTGATTGTACTGTACATCTATTAAAGCCTTAGATTATTACATTACGGGTTGGAACCCATACCAATGTAATTTCAATCGTGTTAAGAGAGTAATGGTGACTTCACATGTTATTGTAGTTAGTTACGTTATAGAATATTACTTATTTTTCTTGTTAAAATGTAGTTTTTCATTTCCTACATTTATTGGATTTTCATTTTCTATTAACAGTTGAATACCATTTCAGTTTTTAGACTATTGTTTTATTAGATTTTACCAATGAATTTTTCAAAATACAAAAAAATTAAAGTAGTTTTTTCTTCATAACATACTCAGTTTTAAATTACATGTAGTGTCATATGAATATCCGTATTATTGTTAACTAAATGATTTATATTTTACTGATTTAATATTACAGTGTAAGAATGTCAGTCATTGTTCTTGTCTAGTTTTCATTAAAAGAACAAAGATCTTTTATATGGATATCTTATAAATATATAATCATTGCTAAGTAAGAAGTTAAGTTGTTGCTATGGCAACAATCCTGGCAGACAATTGAGTAATATTTTGATGATTTATTTTGTTTGTAATTAGTTATTATGAGAAGATCTAGATCCTAGATATTAGAATAAAATTTATTTTCTACTGTATCCATTTCAAATGTTAAAGTATTGTTTAATATTTTTGAAATCCCTGAATATCAGGCCTTGTTATAAATAAGCTGCATAATCAATAAATAGAACAAGGGACTTTTTGTTGATAATCCAAATACTCAAAGTTTACGTAATGAGAATTTTAGCGTGTGTGCAAACTCTTGAGGGTTGATGATGCTGCAATTTAGCATGTTGGAAAGTCTAGAGAGAAGGTTGACTTTTTGCACTTCTGTATATAGTCAAAAGAGAGAAACCTGTATAATAGCAAGATCTTATTTTGAATAAAAACGTCTATAATTACAAGGAGTTTTGTTAAGGCTAATGAAATGACAGACTGAGCAAAATTGCTTGCAAAAGTGGCACAGAGTTAGCACTCCATACCCTTCAAACACGTCGCTTTGCTTTTTGTGGACAGCTTGTAGTTTGCCAGGATTTTTC
AGCTGGAAAGATTTGCCATCCTTCCAAGATCTCATGACTGACAAAACTCCATTGGGCCAAATCTGCCTGAAGATCATTACCAAAAAATAGCAGGTACTTCAGCCACTAAGATGAAATCATGGATCAGATATCCCTTACATTGTTTTCAAAACTACTGCATGTTTAAAACTTCAACAAAAAGAGAGAAAGAACTATGCTAAGGACATATATTATTCAGATCGATATCTACCAATTTCAGTGGTTTAATGTTCACAAAATGAAATCTTGAAAATAACTATTGACTTTCACAAAATTTTAACCATAAACAGGCAAACCAAACAGCACACCTGTAGTTGTTCTGTGATTGTTTTTTAATTGCTGTAGATCATGTTCTTTCCGCAGGTGGAAAAAAAAAAAAAAAAAAAAAAAGAAGTTCAAATTTCACAGTTTTAATTTTCAACTCAGAAGCAAAAGAGCAAAATGTGACAATGGCCACTTGTTTAATGACTTGGTTGCCCAGCTGTCACTGCAGCTGGCTACTGATGTTGCACTTACCAGCAACCCACCCACCTTCATCTGCCGAAAGGACAGTGAGCTTGGTTTTACGATTATGTAATCACAACTTACTTTCTGCTTGTAGTGGCTTAAAATTATGTATTTTGTCTAGGGCTGCAATTTGTTTTATGCTTACTTTATTATTACTGCAGTAGTTGACTTTGCTGTATGGAAAAATAAAGCGAAATTGCCCTAATAAAACTTCTCTTTCTTAAGTATAAAAAAAAAAAAAAAAAAA
>>M1-218-Site3 TTTAACTTTACAGTAACAATAGACAGCACAACACAAAC ACATGGTTAGATCAAGCACAA >>M1-218-Site3 CCACAAGTATGTTACCCAGATGTAGAGCTGTCGTCACAAAACAATCATACAAAGAGGAAAGGACAGTGTGCTTGATTAGAGTAAGGACGACGTCATTAGCCATATTGTATATACCGTCAAGCAACACATACAAAAATCCCTCAGCCACAAGACATCCACATATTGCATGTTAACCAGAAGAAACGACAACATGGGAACCTGCTGCACACTGTTGCCTACACACTTTGTACATTCAGTTGGTATTTGTGCTGAGGTGATATTCCTATCTAAAACAACAACATTGTCTTTCTTTTGTAGCACAGAGTTATGCATTAAAATATGCATACGTAATTAGTTTCCTATATATTCATGCCATCTTGAAAAGACAGACTATGGTGTGACCATGATTCTATTATGTATTGGTACGTCTGTAGACCAAGATATAATTTTTTAAAAATAAGTTTATTTCTTTCAAGGTTTACAAGTAACCAAGGTGCACCTTGTATTTAAAATCGCCGTTAGAGCTGAGAGCGCGCATGCAGAGTCATTTTTGTTTGAGAGTAATATTTTTACTGTAATAGATTGTACGACATGGTGAGGGAGGGAACTGACAGATGAATGTGCCAAGCAAAACCACAACTGTGTATATTTTAAAGCACACCATGGCTTTAAGTACCATGTTGTTAAGGATTCTCATGAAGTGCCATAGACTGTACATCAAATTAGAGTATTATTTCTTCAGTGTTATTGTTTCTGGAGCCACATTTTGTTGCTTATTTGCTAGTACTAATCAATCAAAGGGCACCATTCTTTTCTTTTTTGTTTTTGAAACCAAAGCTGTCTCAGAAATGGCCAATTTAACTTTAACATGGTTAGATCAAGCACAACACAAACTCAATACAGATAACCTTTCACATACTGGAGATATATATGATAGATATATAAAATTATTTTAATGCATTGTAGTGTAATATTTATGCATACTCTACTATATAACATGTTATTCAAAAGGGATATGCCATTTCTGAGACACAATAACAAAAAATGTTTGAGGAAATTATTTTGCTTCTATTTATAGCCTCTGTCAAAAGTCAAAAGACTATAAATGCTTTGCAGAAATGGGTTCACGTTTGCTTAAACGCTTCATCACAGTCACATTCAAAATAGTGACTCTAAACAAAGAGAACAGCACTGTCATCAGATGCATGATAAACCAAAATATGAAAATGGGAAATGTTTAATTAACCTAGTAATTGGGTGGGTTAAGTACATGGGTGAATTTTATATGTGATTCTTTTGTTCAGATTAACTGCTTATAGCCTTAGAAAGCCTTTTAAAAAATTTTAAAAATAGATGTGCATTCAGTTTTTAAGAATGGATTCATCCAAAGGAATTCCCCTTTTTTGTGGTTTGGATGTTGCAGCTAGGAAAGGCTATTTTTGCTCTGTTCAGCAGTTCTAAAATCGCTGAGTAGGGGCCAGGTCACTGGCAGTTCTAGTGTGGAATGGGAGAAGTGAGAGTTCTGTTATAGAACTTTCCATACTTCCAAGTTTACTGCAAGTTTTTATGCTTGAGAGAGATGCTTTCTAATATAAGACTGATGTGTTGATTTTCCTGATTGTACTGTACATCTATTAAAGCCTTAGATTATTACATTACGGGTTGGAACCCATACCAATGTAATTTCAATCGTGTTAAGAGAGTAATGGTGACTTCACATGTTATTGTAGTTAGTTACGTTATAGAATATTACTTATTTTTCTTGTTAAAATGTAGTTTTTCATTTCCTACATTTATTGGATTTTCATTTTCTATTAACAGTTGAATACCATTTCAGTTTTTAGACTATTGTTTTATTAGATTTTACCAATGAATTTTTCAAAATACAAAAAAATTAAAGTAGTTTTTTCTTCATAACATACTCAGTTTTAAATTACATGTAGTGTCATATGAATATCCGTATTATTGTTAACTAAATGAT
TTATATTTTACTGATTTAATATTACAGTGTAAGAATGTCAGTCATTGTTCTTGTCTAGTTTTCATTAAAAGAACAAAGATCTTTTATATGGATATCTTATAAATATATAATCATTGCTAAGTAAGAAGTTAAGTTGTTGCTATGGCAACAATCCTGGCAGACAATTGAGTAATATTTTGATGATTTATTTTGTTTGTAATTAGTTATTATGAGAAGATCTAGATCCTAGATATTAGAATAAAATTTATTTTCTACTGTATCCATTTCAAATGTTAAAGTATTGTTTAATATTTTTGAAATCCCTGAATATCAGGCCTTGTTATAAATAAGCTGCATAATCAATAAATAGAACAAGGGACTTTTTGTTGATAATCCAAATACTCAAAGTTTACGTAATGAGAATTTTAGCGTGTGTGCAAACTCTTGAGGGTTGATGATGCTGCAATTTAGCATGTTGGAAAGTCTAGAGAGAAGGTTGACTTTTTGCACTTCTGTATATAGTCAAAAGAGAGAAACCTGTATAATAGCAAGATCTTATTTTGAATAAAAACGTCTATAATTACAAGGAGTTTTGTTAAGGCTAATGAAATGACAGACTGAGCAAAATTGCTTGCAAAAGTGGCACAGAGTTAGCACTCCATACCCTTCAAACACGTCGCTTTGCTTTTTGTGGACAGCTTGTAGTTTGCCAGGATTTTTCAGCTGGAAAGATTTGCCATCCTTCCAAGATCTCATGACTGACAAAACTCCATTGGGCCAAATCTGCCTGAAGATCATTACCAAAAAATAGCAGGTACTTCAGCCACTAAGATGAAATCATGGATCAGATATCCCTTACATTGTTTTCAAAACTACTGCATGTTTAAAACTTCAACAAAAAGAGAGAAAGAACTATGCTAAGGACATATATTATTCAGATCGATATCTACCAATTTCAGTGGTTTAATGTTCACAAAATGAAATCTTGAAAATAACTATTGACTTTCACAAAATTTTAACCATAAACAGGCAAACCAAACAGCACACCTGTAGTTGTTCTGTGATTGTTTTTTAATTGCTGTAGATCATGTTCTTTCCGCAGGTGGAAAAAAAAAAAAAAAAAAAAAAAGAAGTTCAAATTTCACAGTTTTAATTTTCAACTCAGAAGCAAAAGAGCAAAATGTGACAATGGCCACTTGTTTAATGACTTGGTTGCCCAGCTGTCACTGCAGCTGGCTACTGATGTTGCACTTACCAGCAACCCACCCACCTTCATCTGCCGAAAGGACAGTGAGCTTGGTTTTACGATTATGTAATCACAACTTACTTTCTGCTTGTAGTGGCTTAAAATTATGTATTTTGTCTAGGGCTGCAATTTGTTTTATGCTTACTTTATTATTACTGCAGTAGTTGACTTTGCTGTATGGAAAAATAAAGCGAAATTGCCCTAATAAAACTTCTCTTTCTTAAGTATAAAAAAAAAAAAAAAAAAA
>>M1-218-Site4 TAAACAGGCAAACCAAACAGCACACCTGTAGT ACATGGTTAGATCAAGCACAA >>M1-218-Site4 CCACAAGTATGTTACCCAGATGTAGAGCTGTCGTCACAAAACAATCATACAAAGAGGAAAGGACAGTGTGCTTGATTAGAGTAAGGACGACGTCATTAGCCATATTGTATATACCGTCAAGCAACACATACAAAAATCCCTCAGCCACAAGACATCCACATATTGCATGTTAACCAGAAGAAACGACAACATGGGAACCTGCTGCACACTGTTGCCTACACACTTTGTACATTCAGTTGGTATTTGTGCTGAGGTGATATTCCTATCTAAAACAACAACATTGTCTTTCTTTTGTAGCACAGAGTTATGCATTAAAATATGCATACGTAATTAGTTTCCTATATATTCATGCCATCTTGAAAAGACAGACTATGGTGTGACCATGATTCTATTATGTATTGGTACGTCTGTAGACCAAGATATAATTTTTTAAAAATAAGTTTATTTCTTTCAAGGTTTACAAGTAACCAAGGTGCACCTTGTATTTAAAATCGCCGTTAGAGCTGAGAGCGCGCATGCAGAGTCATTTTTGTTTGAGAGTAATATTTTTACTGTAATAGATTGTACGACATGGTGAGGGAGGGAACTGACAGATGAATGTGCCAAGCAAAACCACAACTGTGTATATTTTAAAGCACACCATGGCTTTAAGTACCATGTTGTTAAGGATTCTCATGAAGTGCCATAGACTGTACATCAAATTAGAGTATTATTTCTTCAGTGTTATTGTTTCTGGAGCCACATTTTGTTGCTTATTTGCTAGTACTAATCAATCAAAGGGCACCATTCTTTTCTTTTTTGTTTTTGAAACCAAAGCTGTCTCAGAAATGGCCAATTTAACTTTACAGTAACAATAGACAGCACAACACAAACTCAATACAGATAACCTTTCACATACTGGAGATATATATGATAGATATATAAAATTATTTTAATGCATTGTAGTGTAATATTTATGCATACTCTACTATATAACATGTTATTCAAAAGGGATATGCCATTTCTGAGACACAATAACAAAAAATGTTTGAGGAAATTATTTTGCTTCTATTTATAGCCTCTGTCAAAAGTCAAAAGACTATAAATGCTTTGCAGAAATGGGTTCACGTTTGCTTAAACGCTTCATCACAGTCACATTCAAAATAGTGACTCTAAACAAAGAGAACAGCACTGTCATCAGATGCATGATAAACCAAAATATGAAAATGGGAAATGTTTAATTAACCTAGTAATTGGGTGGGTTAAGTACATGGGTGAATT
TTATATGTGATTCTTTTGTTCAGATTAACTGCTTATAGCCTTAGAAAGCCTTTTAAAAAATTTTAAAAATAGATGTGCATTCAGTTTTTAAGAATGGATTCATCCAAAGGAATTCCCCTTTTTTGTGGTTTGGATGTTGCAGCTAGGAAAGGCTATTTTTGCTCTGTTCAGCAGTTCTAAAATCGCTGAGTAGGGGCCAGGTCACTGGCAGTTCTAGTGTGGAATGGGAGAAGTGAGAGTTCTGTTATAGAACTTTCCATACTTCCAAGTTTACTGCAAGTTTTTATGCTTGAGAGAGATGCTTTCTAATATAAGACTGATGTGTTGATTTTCCTGATTGTACTGTACATCTATTAAAGCCTTAGATTATTACATTACGGGTTGGAACCCATACCAATGTAATTTCAATCGTGTTAAGAGAGTAATGGTGACTTCACATGTTATTGTAGTTAGTTACGTTATAGAATATTACTTATTTTTCTTGTTAAAATGTAGTTTTTCATTTCCTACATTTATTGGATTTTCATTTTCTATTAACAGTTGAATACCATTTCAGTTTTTAGACTATTGTTTTATTAGATTTTACCAATGAATTTTTCAAAATACAAAAAAATTAAAGTAGTTTTTTCTTCATAACATACTCAGTTTTAAATTACATGTAGTGTCATATGAATATCCGTATTATTGTTAACTAAATGATTTATATTTTACTGATTTAATATTACAGTGTAAGAATGTCAGTCATTGTTCTTGTCTAGTTTTCATTAAAAGAACAAAGATCTTTTATATGGATATCTTATAAATATATAATCATTGCTAAGTAAGAAGTTAAGTTGTTGCTATGGCAACAATCCTGGCAGACAATTGAGTAATATTTTGATGATTTATTTTGTTTGTAATTAGTTATTATGAGAAGATCTAGATCCTAGATATTAGAATAAAATTTATTTTCTACTGTATCCATTTCAAATGTTAAAGTATTGTTTAATATTTTTGAAATCCCTGAATATCAGGCCTTGTTATAAATAAGCTGCATAATCAATAAATAGAACAAGGGACTTTTTGTTGATAATCCAAATACTCAAAGTTTACGTAATGAGAATTTTAGCGTGTGTGCAAACTCTTGAGGGTTGATGATGCTGCAATTTAGCATGTTGGAAAGTCTAGAGAGAAGGTTGACTTTTTGCACTTCTGTATATAGTCAAAAGAGAGAAACCTGTATAATAGCAAGATCTTATTTTGAATAAAAACGTCTATAATTACAAGGAGTTTTGTTAAGGCTAATGAAATGACAGACTGAGCAAAATTGCTTGCAAAAGTGGCACAGAGTTAGCACTCCATACCCTTCAAACACGTCGCTTTGCTTTTTGTGGACAGCTTGTAGTTTGCCAGGATTTTTCAGCTGGAAAGATTTGCCATCCTTCCAAGATCTCATGACTGACAAAACTCCATTGGGCCAAATCTGCCTGAAGATCATTACCAAAAAATAGCAGGTACTTCAGCCACTAAGATGAAATCATGGATCAGATATCCCTTACATTGTTTTCAAAACTACTGCATGTTTAAAACTTCAACAAAAAGAGAGAAAGAACTATGCTAAGGACATATATTATTCAGATCGATATCTACCAATTTCAGTGGTTTAATGTTCACAAAATGAAATCTTGAAAATAACTATTGACTTTCACAAAATTTTAACCATAAAACATGGTTAGATCAAGCACAACTGTAGTTGTTCTGTGATTGTTTTTTAATTGCTGTAGATCATGTTCTTTCCGCAGGTGGAAAAAAAAAAAAAAAAAAAAAAAGAAGTTCAAATTTCACAGTTTTAATTTTCAACTCAGAAGCAAAAGAGCAAAATGTGACAATGGCCACTTGTTTAATGACTTGGTTGCCCAGCTGTCACTGCAGCTGGCTACTGATGTTGCACTTACCAGCAACCCACCCACCTTCATCTGCCGAAAGGACAGTGAGCTTGGTTTTACGATTATGTAATCACAACTTACTTTCTGCTTGTAGTGGCTTAAAATTATGTATTTTGTCTAGGGCTGCAATTTGTTTTATGCTTACTTTATTATTACTGCAGTAGTTGACTTTGCTGTATGGAAAAATAAAGCGAAATTGCCCTAATAAAACTTCTCTTTCTTAAGTATAAAAAAAAAAAAAAAAAAA
References:
1. Warnock, DG and Peck, CC (2010). A roadmap for biomarker qualification. Nat. Biotechnol. 28: 444–445.
2. Xia, J, Broadhurst, DI, Wilson, M and Wishart, DS (2013). Translational biomarker discovery in clinical metabolomics: an introductory tutorial. Metabolomics 9: 280–299.