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Answers
to General Software Questions
1a. What does Mutation Surveyor do?
Mutation Surveyor detects
mutations and SNPs with DNA sequencing trace data. The variations of sample
DNA sequence traces are compared to wildtype and GenBank sequence traces.
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1b. What types of mutations
does the software detect?
The software detects
homozygous and heterozygous point mutations and indels.
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Heterozygous Point Mutation G/AG
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Homozygous Point Mutation G/A
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Deletion G
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Heterozygous Insertion A
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1c. What is the procedure to
produce results?
There are only three simple
steps to produce results using the automatic software:
a. Data Entry 
b. Automated analysis 
c. Reporting 
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1d. What are the advantages of the software?
The software has many
advantages for mutation analysis: the software detects real mutations and
ignores base calling errors; the analysis is automated, making analysis a
simple, fast process; the software is 99% accurate in detecting mutations;
the software uses direct trace comparison for mutation detection; there are
different reporting options to meet specific research or clinical needs.
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1e. What is the difference
between Mutation Surveyor and Mutation Explorer?
Mutation Surveyor is designed
for researchers to tweak mutation detection, whereas Mutation Explorer is
designed for clinical diagnostics where consistency is critical. The
parameters are fixed in Explorer.
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1f. How do I obtain a copy of the software?
You can obtain a demo version
(6 lanes) of the software by downloading it from our website. In order to
obtain a 30 day trial version, you need to register with our company and
you will be given a password with which you can download the trial version.
If you prefer a hard copy, we can Federal Express a trial version of the
software anywhere in North America
overnight.
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1g. Can the software detect somatic mutations?
Yes the software can detect somatic mutations. Prior to running the data go
to Process>Options>Display and check the box labeled, "Check 2D Small Peaks
(Mosaic)." The software will place a green line above the somatic mutation
position.
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Answers
to Data Entry Questions
2a. Which types of files do I
input?
There are a few options for data input:
a. If you do not have a GenBank sequence for the gene
of interest, simply enter your trace samples in the third window and click
“OK” and the software will automatically
download the sequence from the database.
b. Enter a GenBank file in the first panel and trace samples in the third
panel to compare the trace files to the GenBank sequence text.
c. You may input all of the sample
traces in the reference panel and the software will choose the best quality
trace as a reference.
d. Use your own reference samples and enter them in
the second window along with your trace samples in the third window (and
GenBank sequence in the first window: Optional).
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2b. How do I add forward and
reverse samples?
Data analysis is most accurate
when both forward and reverse sequence traces are used.
a. Software assumes that the filenames
with only _F and _R or _2F and _2R differences are from the same sample.
b.
If the filenames of both forward and reverse do not follow any
rules, you may construct a 2D text file containing the sample filenames. The text file shall contain
many columns and rows, and each row represents filenames of a sample. The
text file must be in the same directory as the trace file. You may check
“Load 2D Match” adjacent to “OK” in the open-data-file dialog box.
c. To sort forward and reverse
trace files, go to 2D Filename Match Editor
in the Tools menu or the ND Filename Match Editor
if you have more than 2 primers. Load the data, and the Editor will sort forward and reverse samples
according to the chosen model. Save the 2D files as text files (*.txt) and
check Load 2D Match when adding the
samples for analysis.
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2c. How do I analyze more than
400 samples?
If you have over 400 samples
or data from a whole gene, it is necessary to utilize the Whole Gene Data feature of the software. Analyze
the samples in projects of 400 or less samples. In order to merge the
project files, use the Open Whole Gene Data
option under the File menu. This feature displays mutation analysis of a
whole gene.
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2d. How do I import files that were generated using
the SeqScape autoanalysis?
Currently Mutation Surveyor cannot read files that were generated using the
SeqScape auto-analysis software. However, it is possible to export files
into standard data collection software rather than allowing the samples to
be analyzed with SeqScape’s autoanalysis program as they are generated. By
exporting the files to standard data collection software, it is possible to
import the files into both SeqScape and Mutation Surveyor for analysis.
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Answers
to Data Analysis Questions
3a. In the Graphic Analysis
Display (GAD), how do I distinguish between real and unreal mutations in
the electropherograms?
Scroll through each mutation and delete those that
only appear in one direction (Forward or Reverse) and not in both
directions. Mutations that appear in both directions and are written in
blue are most likely real and should be kept. If the quality of the sample
lane is poor (0-10) and a 1 directional mutation is written in red, either
delete the mutation or the entire lane by right clicking on Lane
Quality.
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Real Mutation
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Unreal Mutation (Dye-blob)
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Unreal Mutation
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Unreal Mutation
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3b. How do I verify each
mutation?
To verify the existing
mutations, open the 2D Output Table and
check the frequency of each mutation located below the list of mutations.
If the mutation frequency is high, the mutation is most likely real. If a
mutation is written in blue, the confidence is high, but if it is written
in red, the confidence is low and should be checked by double-clicking the
cell to bring up the electropherogram. If the background of the cell is
shaded in purple, the mutation has been reported to the database.
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3c. How do I prevent the software
from missing mutations?
In order to prevent the software from missing
mutations, it is important to specify the directional parameters of the
analysis by clicking the 2 Directional icon  if you are using
bi-directional data and the 1 directional icon  if you are
working with samples in one direction.
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3d. What does “Added by
Computer” in the mutation comments box signify?
The software adds mutations in
two different instances:
a.
When the dropping factor of a
mutation is > .15 and the intensity is >500, yet the score does not
meet the detection threshold of 5.00, the software adds this mutation,
because the low score is most likely due to mobility shift.
b.
When the data is noisy, but
the frequency of the mutation is high (>10%).
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3e. What does “Deleted by
Computer” in the mutation comments box signify?
The software deletes false
positive mutations when they exist only in 1 direction with lower local
quality than the other direction for 2 directional
data.
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3f. Which type of settings should
I use for analysis
Since the software remembers the last used settings, it is best to click the
default settings for all tabs under Process >> Options. This will prevent
the software from missing mutations as a result of incorrect parameter
settings.
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3g. How can I exclude poor
quality data from the mutation report?
You may exclude poor quality data from the
report either before or after data analysis. To exclude prior to analysis,
go to Process>Options>Output and set the Lane Quality Threshold to 5 for
example. The software will then reject lanes with a quality score (signal to
noise ratio) below 5 from the statistical mutation frequency calculations.
You may also delete lanes with poor quality data after running the analysis
by right clicking directly above the electropherogram where it says
“Quality.” The mutations in this lane will then be excluded from the overall
mutation frequency calculations.
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3h. How do I detect all
possible mutations?
First, under Process à
Options, choose the Mutation tab, then click on “High Sensitivity”. Second,
analyze with 1D parameters, by clicking on the  icon
in the toolbar and ensuring that the colors are gray and blue, rather than
red and blue ,
which indicates 2D settings. By using high sensitivity and 1D settings, you
will detect mutations in areas of high noise, although you will need to
review the called mutations because you may have more false positives using
this method.
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3i.
How do I reduce false
positives?
First, under Process à
Options, choose the Mutation tab, then click on “Medium Sensitivity”.
Second, analyze with 2D parameters, by clicking on the icon
in the toolbar and ensuring that the colors are red and blue, rather than
gray and blue ,
which indicates 2D settings. By using medium sensitivity and 2D settings,
you will miss mutations in regions of high noise.
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Answers
to System Tools Questions
4a. How do I sort forward and
reverse trace files?
To sort forward and reverse
trace files use the 2D Filename Match in the Tools menu or the ND Filename
Match if you have more than 1 primer. After the Filename Match Editor sorts
the samples, save as a text file. Before entering the samples in the Open
Files window, check the Load 2D Match box and then input the text file. The
same should be done for your reference traces.
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4b. How do I group sample
files together for whole gene analysis?
To group sample files for
whole gene analysis, open the 2D Output Table and click the Whole Gene
Output Icon. Select Filename Match and enter desired characters. This
function will group samples with the same designated characters together in
the output reports. For example, if you select characters 1-7, the software
will group all samples with identical characters 1-7 in the output report:
PCR.s01f14
PCR.s01R14
PCR.s05f14
PCR.s05R14
PCR.s07f14
PCR.s.07R14
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4c. When viewing files in the
SEQ and GBK File Editors, how do I change the numbering for the coding
region start point?
In order to change the
numbering for the coding region in the SEQ and GBK File Editors, select
Adjust and choose the desired number for the first coding base. If you
enter 1, the number of the first base in the coding sequence will be 1.
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4d. How do I download GenBank
sequences (GBK Files)?
To download GenBank Files go to the NCBI (National
Center for Biotechnology
Information) website at www.ncbi.nlm.nig.gov.
i) Search Nucleotide for [gene name] and click GO.
ii) After locating mRNA of the Gene, click on Links
(far right of screen) and select Map Viewer.
iii) Scroll to the right of the screen and locate
highlighted gene. Click dl to download the contig sequence.
iv) Change the sequence format to GenBank and the
Display link.
v) In order to view all features of the gene sequence,
click Features, check the SNP box, and click Set.
 vi) To save the GenBank sequence, slick Save As in the
File menu, and save as a text file
(*.txt).
vii) Import the GenBank text file into the GBK File
Editor and the save it as a GBK file for subsequent analysis.
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4e. Why do I get
a warning when I copy/paste sequences into the SEQ
File Editor?
When you copy/paste sequences into the SEQ File Editor, you need to enter the specific
numbering for the “Number of the First Base”
and “Coding Sequence” and then click Refresh. If you would like the coding sequence
to begin at number 1, click on Adjust and
have the “Number of the Coding Base” start
at 1.
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4f. How can I change
the reading frame for the reference trace?
The user can change the reading frame for the reference trace by opening the
.seq file in the SEQ File Editor (Tools Menu) and specify the reading frame
(1, 2, 3). Save the file and then input as the reference in data entry. The
user may also specify the reading frame for .gbk files in Features 2 of the
GBK File Editor.
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Answers
to Data Output Questions
5a. How many different output
reports are available?
The software has various
output reports available including 1Directional, 2 Directional, Whole Gene
Data, Bentley (shows flanking sequence around mutations), Pretty Base
(displays the alleles at mutation sites), graphic display, and sequence
text report.
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5b. How do I save the data
output?
To save the reports, we
suggest you to save them as text, htm files, then open in Excel, where it
is possible to manipulate the report before printing.
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5c. Is it possible to view only the Region of Interest
or Exon in the output reports?
Go to Process >>
Options >> Output and select either “Region
of Interest Only” or “Exon Only”.
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5d. How do I make my comments
appear in the output reports?
To view comments in the Output
Tables Go to Process >> Options >>
Output and check the Comments box.
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5e. How do I print the mutation report?
To print the mutation reports, it is suggested that
you save the report as either a text file (*.txt) or Excel file (*.xls),
open in an Excel spreadsheet, and print using Excel.
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5f. How do I
print a clinical report?
To print a clinical report, click on the print
icon  and when the page
opens, click the print sample Icon  , and a page will open that displays electropherograms of
all the mutations for that sample.
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Answers
to Mutation and Quality Score Questions
6a. How is the mutation score
calculated for each peak?
Our mutation score is from the
theoretical calculation of signal to noise ratio with the normal
distribution. Signal is defined as a peak in the mutation electropherogram
and noise is defined as the smaller peaks surrounding the mutation in the
electropherogram. Mutation Score = -10 log[erfc(s/n)] of a math function.
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6b. Which individual factors
does the “Lane Quality” value reflect and how is it calculated?
The Lane Quality is a
measurement of the average signal to noise ratio. A lane quality score of
20 signifies that there is 5% noise in that lane. (n/s = 1/20 = .05)
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Answers
to Process Options Questions
7a. What is the function of “Exclude 1st Base Difference” in the Options
window?
When unselected, the software uses 300 as starting
point and sorts lanes with a starting base
difference greater than 300 bases into two contigs. For example, the
reference covers 1 -- 700 bases, sample one covers 10 to 600 base, and the
2nd sample covers 500-900 bases, then the software would sort them into the
same contig. However, it would be very difficult to find mutations, because
of the fat peaks at the end of the sequences with which is compared to the
front sharp peaks. If this option is selected to 100, the software would
sorts the two samples into two contigs.
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7b. What does the “BasePatch” option do when checked?
The “BasePatch”
option corrects for basecalling errors caused by poor mobility shift (Adds
mutations where the mutation threshold for score is unmet due to mobility
shift but overlap and dropping factor are sufficient).
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7c. What does Score Trim option do?
Score trimming is designed to eliminate false positives from regions of low
quality. If the score trimming is set to 15 (similar to a phred score) for
example, and 7 out of 9 consecutive bases have a score below 15, then the
software will ignore these bases for mutation calling.
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7d.
What does End Trim option do?
End trimming allows the user to trim or cut
the beginning and the end of traces in order to eliminate poor quality data.
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