Methylation Sequence Data Analysis with
Mutation Surveyor Software
One of the methods of deducing the methylation state of a DNA
molecule is the use of bisulfite treatment. Bisulfite treatment
of DNA samples converts C to T in a DNA sequence, without converting
methylated C bases in CpG sites. DNA sequencing is an excellent
way to measure DNA methylation state. Mutation Surveyor software
is a powerful software tool using unique comparison technology
to detect nucleotide changes between two sequence traces.
In this application of our software, a GenBank sequence is used
as a “ruler” to report nucleotide changes, including
methylations and mutations. The reference trace is physically
compared to the sample traces to find nucleotide differences.
Mutation Surveyor automatically creates a synthetic reference
trace from the GenBank sequence text or derivatives of the sequence
As previously mentioned, we use the GenBank sequence as a ruler
for methylation studies. Therefore, the appropriate GenBank file
must be input into the Reference Dialog Box. A reference trace
is then synthesised from the derivative of the GenBank file(s).
Do NOT input a reference file in the box labeled Reference
Input the sample trace files into the box labeled Sample Files.
The user can choose the method to modify the GenBank sequence
based on your interest. The modifications are categorised as CG
to TG, or CA to TA, or CC to TC, or CT to TT, or any combination
above. If the user is not sure which one to choose, please use
the Auto Methylation setting. In the Auto Methylation mode, Mutation
Surveyor chooses the best conditions so that the number of mutations
is minimised. The Auto Methylation mode will simplify data analysis.
Procedures to Analyze DNA Methylation Sequence Data:
1. Set Mutation Surveyor software to Methylation application
in the Process > Options > Others: Methylation.
If methylation is NOT checked, the software will process the
data as normal for mutation detection.
The GenBank file must be the forward strand so that CpG is well
defined. If only the reverse complement sequence is available,
it must first be converted to forward strand using
Tools: Seq File Editor and saved as reverse complement.
Modification of GenBank files should be carefully considered.
The modifications are categorized as CG to TG, or CA to TA, or
CC to TC, or CT to TT, or any combination above. The reference
synthetic trace is created from the sequence after modification
parameters are set.
a) Set by User means that user can choose the modification methods.
b) Auto Methylation allows computer to modify the sequence text,
minimizing the number of nucleotide changes (mutations) detection.
Figure 1. Methylation Options. Auto Methylation
modifies the GenBank sequence to create the synthetic reference
trace with minimum number of nucleotide changes. The user may
also set the sequence modification.
2. Interface and data Interpretation:
Figure 2. The top panel is the modified reference
trace synthesized from the derivative GenBank file. The blue lines
above the text indicate that the GenBank text was C (blue colour)
prior to modification. The second panel is the sample trace. The
third panel is the mutation detection panel.
3. Analysis Output:
Click the icon
for the customer output and select the template: Methylation.
Figure 3. Report Table: M means methylated
and U means unmethylated. S stands for success of the conversion
from C to T, and I represents incomplete conversion.
Saving: Click on disk icon to save report in
a text format (.txt) which can be exported to Excel or other program
4. Graphical output of methylation results:
To review global graphical results in text format, click
icon in the Analysis output report.
Figure 4. Graphic output of methylation results.
Red color lines represent methylated sites and blue color lines
represent unmethylated locations.
Download the Mutation
Surveyor Sequence Analysis Package PDF