How do I show Sanger sequence quality scores in my alignment or assembly?
Create or View a "Map to Reference" or Assembly File
See the following lessons to learn how to create a "Map to Reference" alignment or create a de novo assembly using "Assemble Contigs"
View a "Map to Reference" or "Assemble Contigs" assembly file and switch to Sequence view. In the example below, created using "Assemble contigs", there is a disagreement that need to be investigated/resolved between the forward and reverse sequence reads obtained by Sanger sequencing of a clonal sequence.
Show Sanger Quality data
The Sanger Sequencer machine software "calls" the peaks and associated quality values found in .ab1 (and .scf) files. To learn more see the Sequence Analysis Software User Guide.
To show quality scores for the "called" peaks, click Aligned Sequences → Show Quality Values for Sequences Traces.
- The quality information, shown as a bar for each "called" peak is displayed below the sequence trace.
- In this example the top forward read has a region of very low quality, which results in a 3 nucleotide insertion, and 6 nucleotide disagreement compared to the reverse read.
- The bottom forward read has relatively high quality values in the corresponding region.
Hover over a peak or quality bart to see the "called" peak height and the associated quality score.
Looking closely at the peaks in the top forward read, there are clearly miscalled bases based on peak spacing (blue arrows) compared to flanking bases (blue arrows), and visual identification of the probable peaks (magenta arrows) show that the miscalls are likely due to a region of spurious mis-spaced low level A peaks in the region.
Correct the Miscalled Read
In this example, sequencing both strands allows identification of the correct sequence based on the higher quality bases calls in the bottom reverse strand.
The "Assemble Contigs" tool uses the CAP3 algorithm for assembly, and the resulting sequence is the consensus of the assembly.
- Although the reads disagree, the CAP3 algorithm has correctly called the consensus using the quality information.
- If you want to correct the miscalled read, select the region that needs correction, in the dialog that opens, type the replacement sequence, then click Insert/Delete.
The corrected read is now in agreement with the top read and the "consensus" sequence.
Save to retain the changes in the aligned read.