How do I edit a Sanger trace (chromatogram) file?
SnapGene can read ABI trace files (.ab1), SCF (.scf) or ZTR (.ztr) format trace files.
Nucleotide base calls associated with Sanger trace files are called by the DNA Sequencing Instrument software. Ambiguous or heterozygous peaks or compressed regions may sometimes be called incorrectly.
SnapGene does not parse or recall sequence traces. You can correct miscalls by manually editing a trace file.
- Miscalls due to compression of peaks are more likely at the start of a sequence
- Miscalls due to merged peaks are more likely towards the end of a sequence
De novo assembly of overlapping Sanger traces via Tools → Assemble Contigs (see De Novo Assembly of Sanger Sequences) maybe more reliable if you edit and correct Sanger reads prior to assembly.
Open a Trace file
Open a trace file in SnapGene.
Click the "Show quality values" to see the graph of "Phred" quality score for each based call. Quality values may assist in assessing the reliability each peak call.
Use the scroll bar to visually examine the sequence.
Correct Error Due to Compressions
In this example compression near the start of a sequence of what is likely separate A and T peaks has been called as a single N†.
Select the "N" and type the first replacement to open the "Insert Bases" dialog. Enter the replacement sequence.
Click Insert to complete the edit.
Use lower case when manually editing so that the edits can be identified at a later date.
† Base calls associated with Sanger traces are called by the Applied Biosystems Sequencing Instrument software.
The edit will be added to the sequence.
Correct Heterozygous Positions
Select a mixed position that have been called as a N.
Hover over over the nucleotide position to see the raw values for the nucleotide calls. In this example, we see the peak comprises almost equally sized A and G peaks.
Type the replacement IUPAC code for the mixed position. Click Insert to replace the N with the IUPAC ambiguity code "R" (A or G). see View the Codes for more information.
Correct all ambiguous/heterozygous positions.
Click Tools → Letter Codes to see a summary of IUPAC codes used to describe ambiguous nucleotide positions.
Manually Delete Low Quality or Unwanted Ends
Click and drag on the sequence to select a region for removal , then click Edit → Delete Bases (or hit the "delete" key) to remove the base calls.
The selected sequence will be removed from the trace file.
The SnapGene Align to Reference Sequence and Assemble Contigs tools automatically trims and/or hides low quality ends of traces. In most cases you should not need to manually trim ends.
Save the Edited Trace File
Click File → Save to save the edited trace to an appropriate location on your computer.
Note that edited trace files must be saved in .SCF or .ZTR format to retain the edited sequence.