How do I align Sanger traces to a plasmid sequence to confirm cloning or mutagenesis results?
To watch a video on "aligning reads to a reference sequence" – see Aligning to a Reference DNA Sequence.
The "Align Sanger Reads to a Reference" tool is an alignment tool. It is not an Map to reference assembly tool. All sequences/reads are aligned independently to the reference sequence.
Create or Open a Reference Sequence
Open a reference sequence, or use the SnapGene cloning tools to create your predicted reference sequence.
In this example we validate a plasmid created by In-Fusion cloning. The predicted plasmid, YIpLac211-TPM1-mxGFP, has been generated in SnapGene using the SnapGene In-fusion cloning tool.
Import and Align Sanger Traces to the Reference
In Sequence view or Map view click the "Show Alignment" button on the Side toolbar.
If the reference sequence has no aligned sequences then clicking the "Show Alignment" button the side toolbar will open a dialog to allow you to add sequences for alignment.
- Select "Align imported sequences" then click Align to browse to and select sequences for alignment.
- Select "Align copied sequence" the click Align to align a sequence that has been copied to the clipboard.
- Select "Align Open sequences:" then click Align to align sequence files that are already open in SnapGene.
Alternatively, click Cancel to dismiss the dialog and drag and drop the trace files into the "Align with" field.
In Map view, aligned traces will be depicted as arrows above the reference map. See How do I Interpret the Align to Reference Map View? for more information.
In this example, 4 primers have been used to sequence across the msGFP insert and the vector/insert fusion boundaries.
During import, low quality sequence trace ends will be hidden based on settings defined in Preferences → Traces and Alignments and, if possible, the traces will be aligned with the reference.
Validate the Aligned Sequences
Switch to Sequence view to view and edit the assembly. The top panel #1 shows the reference and features. The bottom panel #2 shows the reference (Original Sequence) and any aligned sequences within the field of view.
Click the triangle in the bottom panel to jump to the first discrepancy in the alignment.
View the Sequence and Trace/Chromatogram
Click on the disclosure triangle to expand the trace view.
Option- click (macOS) or ALT-click (Windows) on a disclosure triangle to show or hide all traces at once
The expanded trace view provides controls to change display of the peaks, and provides information on trace length and orientation, and a summary of differences compared to the reference.
Edit the Traces or Reference
Correct Miscall Errors in Traces
Select the sequence to be corrected and type to add sequence, or hit delete to remove sequence. In the above example, the sequencer has miscalled a compression of two A peaks as a single A, so we have selected the gap and typed "a". Click OK to accepted the insertion of an "a".
Adjust Hiding of Chromatogram Ends
If nucleotide calls near trace ends are unreliable, then drag the slider to manually hide further nucleotides from the end of the trace.
You can also adjust all ends by clicking Align Sequences → Hide Ends of Aligned Sequence Traces and choosing a higher or lower stringency. Choose "No Hidden Bases" to reinstate all hidden bases.
Save the File if the Clone is 100% Identical to the Reference
Once all traces are corrected then if all nucleotide positions are in agreement with the reference then do the following.
1. Closed the "Align with" panel.
2. Show the Description panel and check the option "Confirmed experimentally". A green check mark will then appear next to the sequence length indicator showing that the sequence has been confirmed by sequencing.
3. Save the Sequence.
Save the File if the Clone Differs from the Reference
In some cases the clonal sequence may differ from the reference, for example, if mutagenesis of the amplified insert had been performed prior to fusion/ligation, or if an unwanted misincorporation error occurred during PCR amplification of the insert or vector.
If sequencing confirms the sequenced region differs from the reference then you can create a new sequence that replaces a portion of the reference with the selected read/s.
In this example, Sanger sequencing has confirmed a single point mutation at codon 2 of mxGFP, resulting in an amino acid change in the gene product (G → T = amino acid change Glu [E] → Asp [D]).
Select one or more reads that validate any differences with the reference by shift-clicking on the arrows. Arrows will appear blue for selected reads.
Click Aligned Sequences → Replace Original with Aligned → Make New File....
Confirm the sequences that will replace corresponding regions in the reference, enter an appropriate name for the new sequence, then click OK. The sequence will open as a new (unsaved) document.
Examination of the new file in Sequence view will show that clonal differences in the sequence trace are now incorporated into the new sequence. Click File → Save, or File → Save to Collection and save the new sequence to an appropriate location.