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Download Chromas.
Download the example sequence file by right-clicking on the link and saving the file to your desktop.
Open Chromas.
Select FILE --> OPEN --> example_trace.ab1
Recall how sequencing machines operate and how the program has predicted which of the four nucleotides is at that point in the sequence based on flourescent trace peaks. Notice how messy the trace looks. Now scroll to the right and notice how clean the trace becomes at about 110... and then how the last few nucleotides at about 488 become difficult to assign.
Select EDIT --> COPY SEQUENCE --> FASTA FORMAT
Open Notepad (Notepad is the simple ascii text editor that comes with Windows) or TextEdit (the simple ascii text editor that comes on a Mac). We always use simple text editors rather than WORD because there must be no hidden characters inserted into our sequence files.
Select EDIT --> PASTE
Be careful with the next two steps... usually at least one end of every sequence trace is "chemically messy." We cannot be certain of those nucleotides and Chromas also has doubts, so it designates them "N" instead of A, G, C or T. Often, when we look at the second trace generated by the sequencing primer in the other direction we will be able to assign a nucleotide to the "N." The GOAL of this step is to prepare the largest possible chunk of clean sequence containing only A's, G's, T's and C's.
Use Ctrl-F to find the "N's," at the beginning of the sequence and delete the sequence up to the final "N" in that group (see example below).
Then use Ctrl-F to find the next "N," and delete the sequence after that N as was done in the example below:
>Example raw sequence from trace file CGGGNNGGGCCGGCGCCGGGNAACCGNCCACTNNCACTCCTCGCCGGGGGGGTNCGCCTC CNNTCCANGACGCANCCNTGGGTACGCTCTATNTTGAANAGCATCNACAATCGTATGCGG GACCGTGTTTTTCTTGGTACAACTGCGGGAATATTACTGAAACTCCTACACTATTGCAGA TAGGGTTTATGGTAGGGTTTTCTATTTACTAACTGGATTCCATGGGATACATGTTGTCGT AGGGACTATTTGGCTAATGGTAAGGTTAGTTCGACTATGACGCGGGGAGTTTTCTAGTCA ACGACACTTTGGGTTTGAGGCTTGTATTTGGTACTGACATTTTGTAGATGTGGTATGAGT GGCATTGTGGTGCTTAGTGTACGTGTGGTTTGGAGGATGATTATATATGTGATGGTTTAA GATATGGGACGGGGATGTTTATACATTTAAGTATCCGGATGCCAAACCTTCCTGGTACGC TTATTGNAAANAGCATANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNBECOMES
>Example raw sequence from trace file ACAATCGTATGCGG GACCGTGTTTTTCTTGGTACAACTGCGGGAATATTACTGAAACTCCTACACTATTGCAGA TAGGGTTTATGGTAGGGTTTTCTATTTACTAACTGGATTCCATGGGATACATGTTGTCGT AGGGACTATTTGGCTAATGGTAAGGTTAGTTCGACTATGACGCGGGGAGTTTTCTAGTCA ACGACACTTTGGGTTTGAGGCTTGTATTTGGTACTGACATTTTGTAGATGTGGTATGAGT GGCATTGTGGTGCTTAGTGTACGTGTGGTTTGGAGGATGATTATATATGTGATGGTTTAA GATATGGGACGGGGATGTTTATACATTTAAGTATCCGGATGCCAAACCTTCCTGGTACGC TTATTG
Then select FILE --> SAVE AS --> example_fasta.txt
