Data Retrieval, Evaluation and Troubleshooting
Downloading data from the FTP site
To gain access to your data, click onto the Client FTP login and type in your Institute's username and password (available from the Facility).
Files are retained on the FTP site for a period of three months and all data is backed up to permanent archive.
Sequencing files created
Two file types are generated for each sample:.ab1 file (raw data trace, electropherogram, annotation of the sample) and .seq file (text file of the sequence).
It is essential to check base calling accuracy using the electropherogram as reference.
Manual editing may be required, especially at the end of the run.
Programs to view sequence files
Sequence Scanner (Windows) is free viewing software provided by Applied Biosystems that enables you to view, edit, print and export sequence data and graphically express results in report format.Chromas Lite (Windows) is freeware enabling you to view, print, export, copy and reverse/complement sequence data and search for sequences by exact matching or redundant codes. FinchTV enables you to view chromatogram files on Mac OSX and Windows. 4Peaks (Mac) enables you to view and edit sequence files without prior file conversion.
Quality Values (QV) range from 1 to 50+ and are assigned to each base, indicating the probability of error. The calculated QV is viewed using QV bars above each base call and can be viewed using the Applied Biosystems Sequence Scanner program. The DNA sequencing analysis v5.3 program used within the Centre assigns an "N" to any base with a QV < 15. pGEM and LongRead controls routinely generate 950 to 1000 QV ≥ 20 base pairs.UPAC/IUB Codes are assigned to mixed bases within the sequence. The code is defined by the International Union of Biochemistry.
Data evaluation and troubleshooting resources
- Qiagen Guide to Template Purification and DNA Sequencing Second Edition
- Applied Biosystems DNA Sequencing by Capillary Electrophoresis Chemistry Guide Second Edition (Refer Chapter 8: Troubleshooting)