Retrieving Data

When retrieving data in dnalims, you are able to download analyzed sequencing data produced from both ABI Sequecing Analysis (ABI) and Phred basecalling programs. These two programs use different algorithms to produce nucleic acid base calls from raw, unanalyzed, fluorescent data for different end uses. Both analyzed sequencing files, also referred to as "trace files," can be viewed in ABI's Sequence Scanner. Both text files can be opened in a word processor program. "Sequencer Output" is ABI-generated trace and text data only.

The "Phred Output" section uses standard, DNA sequencing industry terms to describe how data can be displayed. You can download your data in Fasta, Qual, Phred, and SCF file formats. Fasta displays sequence text. Qual displays Phred quality scores per base (maximum is 99). The display is a string of quality values with no associated base call. Phred displays capillary run information, base calls, quality scores, and laser scan numbers generated from recording the fluorescent marker as it passes by a CCD camera. The first three "Phred Output" files listed (Fasta, Qual, and Phred) can be opened in a word processor program. The SCF file format is a Phred generated trace file viewable in Chromas. The Phred trace is the default file in the display window. Having your data read by two different base calling programs allows you to choose which one is right for you.

You should examine both trace files to determine which program is right for your research needs. Phred is generally better at base calling. However, it will assign bases to peaks generated from low quality sequencing reactions (see troubleshooting guide) that may only be noise. Phred rarely inserts an "N" (ambiguous base) and may mislead a new user into thinking the sequencing reactions worked well. Phred detects blobs and spikes, but will probably call them incorrectly. Phred excels at accurately determining bases near the end of a sequencing run (i.e. after 600 bp in the trace). For these reasons, it is very important to scrutinize the chromatogram for quality, rather than blindly rely on text file information.

Links

Articles by Phil Green:

• Base-Calling of Automated Sequencer Traces Using Phred. I. Accuracy Assessment
• Base-Calling of Automated Sequencer Traces Using Phred. II. Error Probabilities