Sequencing Analysis Software v6.0, Initial License - FAQs

The .ab1 files from a CD are locked (read-only), so you won't be able to analyze them. To work with files from a CD:

1. Drag the .ab1 files from the CD to the hard drive (not the desktop).
2. Select the files, right-click and select Properties.
3. De-select the Read Only check box and click OK.
4. The .ab1 files can now be added to Sequencing Analysis Software and analyzed.

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In the Sequencing Analysis software, after Analyzing your samples, the BC check box will change color, reflecting the status of the analysis.

Green: Analysis was successful.

Yellow: Poor quality data (if using KB Basecaller, a partial output file is available).

Red: Analysis Failed

The PP and P check boxes will only contain Green or Red status colors.

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In the Sequencing Analysis software, in order to display the data, the check box in the Show column needs to be checked next to the samples you wish to display. The "Show" button was designed to automatically check the show boxes for selected samples. You can select the samples by clicking on the row number. Holding down the shift key while clicking on the row number will allow you to select continuous samples, holding the control key while clicking the row number will allow you to select individual samples.

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Yes, bases can be edited in the Sequencing Analysis software. You can insert or delete bases in your electropherogram by simply typing in the change or highlighting the base and pressing the delete key. After making the changes, you must save the sample file in order for those changes to be reflected in your .seq files. Re-analyzing the samples after making any changes will remove the changes made to the sample file.

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In the Sequencing Analysis software, you can zoom in and out of the data or choose to view the full view of the samples by going to the View menu and selecting one of the display options or by clicking on the appropriate display button. When you zoom in or out, all samples that are being displayed at that time will be affected.

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You can choose which columns you would like in the Analysis Report by right-clicking on a column header in the table and select or deselect the column headings you would like or would like to remove. Once you have the columns, you can re-arrange the order they appear in the table by holding down the Control key and dragging the column to the new location on the table where you would like it to appear.

For more information about the Analysis Report, please see Chapter 7 in the ABI PRISMSequencing Analysis v.5.1 Software for WindowsXP and 2000 Platforms User Guide, Rev.B (p/n 4346366).

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In the Sequencing Analysis software, the Signal to Noise Ratio is the average of the signal intensity of the "A", "C", "G", or "T" base divided by the average noise for that base.

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The Post Processing step, within the Sequencing Analysis software, is where the Clear Range is calculated based on the Quality Values and user-defined parameters. Post Processing can only be done on samples that have been basecalled.

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In the Sequencing Analysis software, to change the Length of Read, go to the Analysis Menu and select Display Settings. In the Report Display section, you can define the range for the Short, Medium, and Long ranges by moving the slider bar to the left or right.

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In the Sequencing Analysis software, the length of read is the range of high-accuracy or high quality bases as determined by the quality values. The length of read (LOR) can be seen in the Analysis Report. Only samples analyzed with the KB Basecaller will generate LOR values.

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When editing sample files by inserting or deleting bases in Sequencing Analysis software, the change will be reflected in the .seq file after the changes have been saved. It is not necessary to re-analyze the samples. In fact, re-analyzing the samples will remove any changes made to the sample file. If the changes had been made and saved and you inadvertently re-analyzed the samples, do not save the file. Remove it from the sample manager without saving and add it to the sample manager again to restore it to its last saved version.

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There are different ways to override the Analysis Defaults in the Sequencing Analysis software, but keep in mind that if you do, it will apply to all samples analyzed from that point forward, until you remove the override. To override the Analysis Defaults, you can either:

a) Go to the Tools menu and select Options. In the File Format Tab, you can select the "Override the sample's Analysis Protocol and set to:" radial button and make your choices there.

or

b) Go to the Analysis menu and select Analysis Defaults. This display can also be brought up by going to the File Menu, selecting Add Sample(s), and choosing the Analysis Defaults button on the bottom of the display. You can then select the "Override the sample's Analysis Protocol and set to:" radial button and make your choices there.

Note: The display from the Tools menu (a) and the display from the Analysis menu (b) are 2 different displays. Changes made to one will not be shown in the other, but when the data is analyzed, both will be used in producing additional file format files.

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In the Sequencing Analysis software, there are 2 ways that you can generate .phd and .scf files:

Analysis Protocol: you can select which of the files you would like to produce in the General tab.

Tools Menu: if you select Options, you have the ability to override the Analysis Protocol to generate any of the files. Keep in mind that if you utilize this option, it will override all Analysis Protocols and produce these files.

Note: When creating a new Analysis Protocol, the default is to have each sample generate a .seq and .phd.1 file after basecalling. If you do not wish to have these files generated, uncheck the option in the Analysis Protocol.

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In the Sequencing Analysis software, a standard chromatogram format (.scf) file is compatible with Staden package.

Note: When standard chromatogram file format is created, the .scf extension is not appended to the file name. However, the file format is correct.

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In the Sequencing Analysis software, Phred (.phd.1) files are used in some downstream data analysis programs and contain a header with data description, revised base calls, assigned quality values, and peak location for the entire sequence. The file can be opened with any text editor, but contains no raw data.

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In the Sequencing Analysis software, you can override the Analysis Protocol for specific samples by first selecting the samples that you would like to override (to do so, click on the row number of the selected sample). Then go to the Analysis Protocol Manager, select the desired Analysis Protocol, and click on the "Apply to Selected Samples" button. Once the changes have been made, select the BC column and re-analyze your samples

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In the Sequencing Analysis software, the Analysis Protocol can be changed specifically for a sample while leaving the master protocol intact, or you can change or create a new Protocol in the Analysis Manager and apply it to all samples.

Analysis Protocol: If changing the Analysis Protocol for 1 sample, you can make the change in the Analysis Protocol by selecting the row number for the sample you wish to change, going to the Analysis menu and selecting Analysis Protocol. Select the appropriate tab, make the changes there and click OK when complete. The changes made will affect the Analysis Protocol only for that sample. If you wish to revert to the original settings, select the row number of the sample, go to the Analysis menu and select "Apply Pre-Analysis settings". This will change the mobility file to the Pre-Analysis settings. Change the basecaller to the appropriate settings and re-analyze the samples.

Analysis Manager: If changing the Analysis Protocol for multiple samples, you can do so by applying a new Analysis Protocol or changing an existing one. To do so, go to the Analysis Menu and select Analysis Protocol Manager. Click once on the Protocol you wish to modify and select Edit, or select New from the menu choices on the bottom of the panel. Go to the appropriate tab and make the changes. If creating a new Analysis Protocol, make any other appropriate changes to the other tabs. When finished, press OK. At this point you can either select "Apply to All Samples", which will apply the changes to all samples in the Sample Manager, or "Apply to Selected Samples", which will apply the changes to the samples that had been selected.

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Within the Sequencing Analysis software, you can set your Analysis Protocol to detect mixed bases if you are analyzing the data with the KB Basecaller. To do so, go to the Mixed Bases tab and select Use Mixed Base Identification. This will allow the software to assign a pure base (A, C, G, T) or an IUB code to the peaks. For Pure Base calling only, make sure this option is deselected.

You can determine when the software assigns an IUB code by adjusting how high the second peak has to be before the software calls it. You can do so by moving the slider line on the graphic up or down or by entering a percentage in the value box.

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In the Sequencing Analysis software , the clear range is the region of sequence that remains after excluding the low-quality or error-prone sequence at both the 5' and 3' ends. In the Electropherogram and Sequence views, the excluded data is displayed in gray. The bases outside the clear range cannot be edited.

The Clear Range is determined by the user and is set up in the Analysis Protocol under the Clear Range Tab. The end user can set the clear range by Quality Value, Number of Bases, or Number of N’s. If multiple criteria are used, the software will select the most stringent in determining the Clear Range.

Clear Range determination is a Post Processing feature. When analyzing samples, select the BC column to call the bases and assign quality values and the PP column to determine the Clear Range.

Note: Clear Range will only be determined if the data has been analyzed with the KB Basecaller.

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The Mobility File can be changed in a number of locations in the Sequencing Analysis software :

Sample Manager: Change the Mobility file in the DyeSet/Primer column by selecting another Mobility File from the pull down menu. You can make the same change for all the samples by making the change in the first sample, then click on the column name to select the column. Go to Edit and select Fill Down.

Analysis Protocol: If changing the Mobility File for 1 sample, you can make the change in the Analysis Protocol by selecting the row number for the sample you wish to change, going to the Analysis menu and selecting Analysis Protocol. Select the Basecalling tab and make the change there.

Analysis Manager: If changing the Mobility File for multiple samples, you can do that by applying a new Analysis Protocol or changing an existing one. To do so, go to the Analysis Menu and select Analysis Protocol Manager. Click once on the Protocol you wish to modify and select Edit, or select New from the menu choices on the bottom of the panel. Go to the Basecalling tab and make the changes. If creating a new Analysis Protocol, make any other appropriate changes to the other tabs. When finished, press OK. At this point you can either select "Apply to All Samples", which will apply the changes to all samples in the Sample Manager, or "Apply to Selected Samples", which will apply the changes to the samples that had been selected.

Once the changes have been made, select the BC column and re-analyze your samples.

Note: When changing the Mobility File, make sure that the Mobility File and the Basecaller match. For example, if using the KB Basecaller, make sure that you are using the KB Mobility Files. If they do not match, analysis might fail.

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Within the Sequencing Analysis software, the Basecaller can be changed in a number of locations:

Sample Manager: Change the Basecaller in the Basecaller column by selecting another Basecaller from the pull down menu. You can make the same change for all the samples by making the change in the first sample, then click on the column name to select the column. Go to Edit and select Fill Down.

Analysis Protocol: If changing the Basecaller for 1 sample, you can make the change in the Analysis Protocol by selecting the row number for the sample you wish to change, going to the Analysis menu and selecting Analysis Protocol. Select the Basecalling tab and make the change there.

Analysis Manager: If changing the Basecaller for multiple samples, you can do that by applying a new Analysis Protocol or changing an existing one. To do so, go to the Analysis Menu and select Analysis Protocol Manager. Click once on the Protocol you wish to modify and select Edit, or select New from the menu choices on the bottom of the panel. Go to the Basecalling tab and make the changes. If creating a new Analysis Protocol, make any other appropriate changes to the other tabs. When finished, press OK. At this point you can either select "Apply to All Samples", which will apply the changes to all samples in the Sample Manager, or "Apply to Selected Samples", which will apply the changes to the samples that had been selected.

Note: Whenever a change is made to the Basecaller, you must select the appropriate mobility file as well. Analysis with a KB Basecaller and ABI mobility file can result in a failed analysis. Analysis with an ABI Basecaller and KB mobility file might pass analysis, but the data might appear to have overlapping peaks or peaks within peaks, giving the appearance of poor quality data.

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In the Sequencing Analysis software, an Analysis Protocol contains all of the settings necessary for basecalling and post-processing the data. A protocol is stored in the sample file once it has been applied to the sample file and saved. A sample must have an Analysis Protocol made up for it prior to analysis. If a protocol is not attached to a sample file, a default protocol can be applied.

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In the Sequencing Analysis software, the differences in the ABI and KB Basecallers are the following:

The ABI Basecaller analyzes data using an algorithm from previous versions of the ABI PRISMSequencing Analysis Software.

The KB Basecaller has the following features:
* Calculation of mixed bases: Mixed bases are one-base positions that contain two bases. These bases are assigned the appropriate IUB code.
* Calculation and display of quality values (QVs) for pure and mixed bases. The QV is a per-base estimate of the basecaller accuracy.
* Option to call Ns when the quality threshold is not met.
* Option to process data with true or flat profile.

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In the Sequencing Analysis software, the selection of the profiles within the Analysis Protocol will determine how your processed data will appear in the Electropherogram view after analysis. These options are only available if you are using the KB Basecaller. The ABI Basecaller is True Profile.

True Profile: After analysis, the data is displayed uniformly so that the average height of peaks in the region of strongest signal is about equal to a fixed value. The profile of the processed traces will be very similar to that of the raw traces. So after analysis, you should see differences in the peak heights in the analyzed data based on the signal intensity of the raw data and how strong the signal is of a particular base compared to the strongest signal in the run.

Flat Profile: After analysis, the data is displayed as processed traces scaled semi-locally so that the average height of peaks in any region is about equal to a fixed value. The profile of the processed traces will be flat on an intermediate scale (greater than about 40 bases). So after analysis, the peaks in the electropherogram view should appear to be about the same height, regardless of the intensity of the peak in the raw data.

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Within the Sequencing Analysis software, multiple samples can be simultaneously moved horizontally. When displaying multiple samples in the Electropherogram view, a master horizontal scroll bar will appear on the bottom of the display. Moving this scroll bar will move the scroll bars on all of the individual samples at the same time and rate.

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To display the data for select samples in the Sequencing Analysis software, peform the following:

1. While holding down the Control key, select the row of the samples you wish to view.
2. Press the Show button to display the data for the selected samples.

To undo the action, select the Show button again or, if you wish to view a specific sample, double click on the row number of the sample you wish to view.

To display the data for non-consecutive samples in the Sample Navigator view, click on the button to switch to Sample Navigator view or go to the View menu and select Sample Navigator. Then follow the above steps.

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To display the data for consecutive samples in the Sequencing Analysis software, perform the following steps:

1. Click the empty box above Row 1 to select all of the samples (you can also hold the shift key while dragging the mouse down the row if you wish to only show a few of the consecutive samples).
2. Press the Show button to display the data for all of the samples.

To undo the action, select the Show button again or, if you wish to view a specific sample, double click on the row number of the sample you wish to view.

To display the data for consecutive samples in the Sample Navigator view, click on the button to switch to Sample Navigator view or go to the View menu and select Sample Navigator. Then follow the above steps.

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You can add the sample files to the Sample Manager by either:
* Double clicking on the sample file name
* Go to the File menu and select Add Samples (or click on the Add Samples button)
* Drag the files to the shortcut icon
* Select the files, right click, and select "Open with SeqA5App".

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The different User Categories in the Sequencing Analysis software and their privileges are the following:

Analyst
* View, customize, export, and print the Analysis Report.
* Add and remove samples to and from the Sample Manager.
* Print Sample files views.
* Change the Basecaller, Mobility, and Matrix (where applicable) files in the Sample Manager.
* Insert, delete, or change bases in the electropherogram or sequence views.
* Text Searches.
* Edit the Sample Name.

Scientist
Can do everything in the Analyst category, plus:
* Create or edit an existing Analysis Protocol (either from the Analysis menu or from the Analysis Defaults) and apply it to a set of samples.
* Delete an Analysis Protocol.
* Set the Clear Range determination.
* Edit the Display Settings.

Admin

Can do everything the Scientist and Analyst can do, plus:
* Create or inactivate User Accounts.
* Import or export User Accounts.
* Turn the Timeout feature On or Off.
* Turn the Audit Trail feature On or Off.

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The following capillary electrophoresis secondary analysis software are compatible with Windows 10 (and also Windows 7):

-Sequencing Analysis Software v7
-SeqScape Software v4
-Variant Reporter Software v3
-GeneMapper Software v6
-Minor Variant Finder v1.2
-MicrobeBridge v1.1

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Please visit our Applied Biosystems Genetic Analyzer Freeware and Patches website: https://www.thermofisher.com/sangerpatches

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The Sequencing Analysis Software 6 is compatible with the following data files:

-310 PC, Data Collection Software v.3.0, 3.1
-3100 series, Data Collection Software v.1.0, 1.1, 2.0
-3130 series, Data Collection Software v.3.0, 3.1, 4
-3730 series, Data Collection Software v.3.0, 3.1, 4
-3500 series, Data Collection Software v.1.0, 1.1, 2

For more information, please click here for the Sequencing Analysis Software 6 datasheet(https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Sequencing-Analysis-Software-6-datasheet.pdf).

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The computer requirements are as follows:

-Minimum: 2 GHz CPU or faster
-2 GB RAM
-1 GB hard disk space for application

Recommended CPU: Core 2 Duo E8400/3.0 GHz
-4 GB RAM
-1 GB hard disk space

For the operating system, Sequencing Analysis Software 6 requires Windows 7 Professional, 32-bit edition, service pack 1. Please click here (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Sequencing-Analysis-Software-6-datasheet.pdf) for the Sequencing Analysis Software 6 datasheet.

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This software has been extensively tested on Windows 7 SP1 (32-bit version). While there are some indications that the software will run normally on the 64-bit version of Windows 7 SP1 operating system, the 64-bit configuration is not officially supported. It is strongly recommended that this software be run on Windows 7 SP1 (32-bit).

If installing on the instrument computer, the update will require a new computer and upgrading the Data Collection Software as well. Please click here for the Sequencing Analysis Software 6 datasheet (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/Sequencing-Analysis-Software-6-datasheet.pdf).

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Software Support Center.