Sequencing Analysis Software v5.4 Initial License

You can make a matrix from running the Sequencing Standard or by running Matrix Standards. To make the Matrix:

(1) After the run, open the DNA Sequencing Analysis Software.

(2) Go to the File menu and select Add Sample(s). Select the file(s) of the standards that you ran, click on the “Add Selected Samples” button, then click the OK box.

(3) After the samples have been added to the Sample Manager, click the Show box, and check the raw data. Peak height should be greater than 200 rfu and the baseline needs to be stable.

(4) Go to the “Tools” menu and select “Make Matrix”. Select the number of files you will be generating the matrix from (e.g., if you are generating it from a sample file, select 1; if from Matrix Standards, select 4).

(5) Click on the “…” box to the right of each line to browse to the sample file(s). Select the file and click “Open”. Do this for all of the samples you will use for the Matrix.

(6) In the “Specify the path for the new matrix file” area, name your matrix. The location the file should be stored in should be the default location and in D:\AppliedBio\Shared\Analysis\Basecaller\Matrix. Note:The matrix file needs to be in both locations in order to autoanalyze the data. If you attempt to point the preferences to look for the matrix in D:\AppliedBiosystems\SeqA5.X\AppSeqA\bin\Basecaller\Matrix, the entire pathway will be written to the .ab1 file for the matrix name and autoanalysis will fail.

(7) Click the “Make Matrix” button. The matrix will either be made correctly or it will generate an error message with specific information on why it failed.

(8) Either copy and paste the Matrix file from the default location to D:\AppliedBio\Shared\Analysis\Basecaller\Matrix or make the matrix 2, the second being saved to that location.

In order to autoanalyze samples on the 310 Genetic Analyzer, there are three things that need to be done:

(1) Copy the Matrix and Mobility files from the Matrix and Mobility folders located at D:\AppliedBiosystems\SeqA5.X\AppSeqA\bin\Basecaller and paste them into the Matrix and Mobility folders at D:\AppliedBio\Shared\Analysis\Basecaller. If you have not made a matrix yet, you will need to do so prior to running samples.
(2) Open Sequencing Analysis, create an Analysis Protocol, and set it as the Analysis Default.
(3) Set up the Preferences to point to the mobility and matrix files in the local directory and in the General Settings tab, point the Autoanalyze with menu to D:\AppliedBiosystems\SeqA5.4\AppSeqA\Automation310.exe

Once the files are copied and the preferences set, you will be ready to run. At the end of the run, the sample files (.ab1) will be analyzed, but you will not see Sequencing Analysis open. The sample files are analyzed with a non-visible version of Sequencing Analysis.

The improved basecalling algorithm (the KB Basecaller) is designed to accurately call more bases out of sequencing data, and reduce manual data review time by providing per-base quality value (QV) assignment, which is a measure of basecaller accuracy. The KB Basecaller supports most chemistries and run modules available on the Applied Biosystems 3130 Series Genetic Analyzers running Data Collection v3.0 and v.3.1 Software, and is also integrated in the Applied Biosystems Sequencing Analysis v. 5.X Software and SeqScape v2.X Software. In previous versions of Data Collection Software, resolution performance for all run modules was calibrated with a 98.5% basecalling accuracy (1.5% probability of error) specification using the ABI Basecaller. Utilizing the KB Basecaller v1.2 release for Data Collection v3.0 Software, resolution performance for the all the run modules was calibrated with a quality score cutoff of QV20 (99% basecalling accuracy or a 1% probability of error) using a sliding window of 20 bases from the 3' end. Therefore, while shorter read length specifications are generated with the KB Basecaller, the calibrations are of higher basecalling accuracy. In this release, we are using the same specifications for 98.5% basecalling accuracy, which is on the conservative end, to keep the specifications uniform.

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

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