1. General Software Questions

1. 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.

2. What types of mutations does the software detect?

   The software detects homozygous and heterozygous point mutations and indels.

   Heterozygous Point Mutation G/AG

   

   Homozygous Point Mutation G/A

   

   Deletion G

   

   Heterozygous Insertion A

   
3. What is the procedure to produce results?

   There are only three simple steps to produce results using the automatic software:
   I. Data Entry
   II. Automated analysis
   III. Reporting

4. 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.

5. 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.

6. 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.

7. 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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2. Data Entry Questions

1. Which types of files do I input?

   There are a few options for data input:
   I. 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.
   II. 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.
   III. You may input all of the sample traces in the reference panel and the software will choose the best quality trace as a reference.
   IV. 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).

2. How do I add forward and reverse samples?

   Data analysis is most accurate when both forward and reverse sequence traces are used.
   I. Software assumes that the filenames with only _F and _R or _2F and _2R differences are from the same sample.
   II. 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.
   III. 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.

3. 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.

4. 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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3. Data Analysis Questions

1. In the Graphic Analysis Display, 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.

   The software detects homozygous and heterozygous point mutations and indels.

   Real Mutation

   

   Unreal Mutation (Dye-blob)

   

   Unreal Mutation (Spike)

   

   Unreal Mutation (False-position indel)

   
2. 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.

3. 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.

4. What does "Added by Computer" in the mutation comments box signify?

   The software adds mutations in two different instances:
   I. 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.
   II. When the data is noisy, but the frequency of the mutation is high (>10%).

5. 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.

6. 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.

7. 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.

8. 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.

9. 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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4. System Tools Questions

1. How do I sort forward and reverse trace files?

   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
2. How do I group sample files together for whole gene analysis?

   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.

3. 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.

4. How do I download GenBank sequences (GBK Files)?

   To download GenBank Files, follow this guide.

5. 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.

6. 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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5. Data Output Questions

1. 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.

2. 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.

3. 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".

4. 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.

5. 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.

6. 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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6. Mutation and Quality Score Questions

1. 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.

2. 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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7. Process Options Questions

1. What is the function of "Exclude 1st Base Difference" in the Options dialog box?

   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.

2. 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).

3. 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.

4. 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.