From a sequence, software is used to generate a trace file (also known as a chromatogram.) Three segments of a sequence that was generated from the AraC/LacZ PCR product are shown in the frame at the right.

These segments display the full output generated by software from Applied Biosystems Inc., (ABI). (ABI is a major producer of dye terminator sequencing machines and reagents.) The traces are produced by software interpretation of the original gel file. The software utilizes spectral information of the dye terminators to isolate an intensity trace for each of the four terminators (and thus for each of the four bases).

In order to automatically read the trace, the computer analyzes the periodicity to determine the spacing between the bases. It then proceeds to each anticipated peak and determines which dye (i.e. base) has the greatest intensity, and "calls" the base for that position. Such automated base calling is generally very good, especially for stretches of high quality sequence.

Examine these segments to determine how the trace file can be deciphered into DNA sequence. It is often neccesary to manually check the precision of the base calling software. The program can be confused in certain circumstances - for example, if a position contains signal of equal intensity for two dyes, or if a very strong peak neighbors a weak one. It is possible for the researcher to identify miscalled or ambiguous bases, and manually correct the error. In this case the machine called all the bases correctly.

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Part I-A. Viewing a DNA Sequence

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Part I-C. "Calling" Your Own DNA Sequence