How do I align a "whole plasmid sequencing" read (fastq or fasta) to a reference plasmid sequence?
Use SnapGene to validate a plasmid construct by aligning a "whole plasmid sequencing" read (a .fastq or .fasta consensus sequence generated using Oxford Nanopore Minion technology) to a reference plasmid sequence.
Create or Open a Reference Plasmid Sequence
Open a reference plasmid, or use the SnapGene cloning tools to create your predicted reference plasmid.
Your plasmid reference sequence must be a circular sequence to ensure proper alignment. If your reference is a linear sequence file then circularize and save before you perform the alignment - see Circularize or Linearize a DNA Sequence
In this example we validate a plasmid created by In-Fusion cloning. The expected sequence of pET-26b_xynB has been generated in SnapGene using the SnapGene In-fusion cloning tool.
If you are validating a plasmid by Sanger sequencing then see Align Sanger Reads to a Reference Sequence.
Align a "Whole Plasmid Sequencing" read to the Reference plasmid
Option 1: Align via the Project Side Panel
Open a Project folder that contains the reference sequence and the "whole plasmid sequencing" read.
Select the Reference plasmid and one or more reads in the side panel, then click "Align to Reference" in the main window.
Option 2: Align via the Side Toolbar
View the Reference plasmid in Map View or Sequence View.
Click the "Align to Reference" button in the side toolbar, or alternatively, click menu Tools → Align to Reference DNA Sequence.
- Select "Align imported sequences" then click Align to browse to and select one or more reads 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 one or more sequence files that are already open in SnapGene.
Alternatively, click Cancel to dismiss the dialog and drag and drop the read fastq or fasta files into the "Align with" field.
Validate the Aligned Sequence
In this example, a fastq "whole plasmid sequencing" read is aligned to the reference plasmid.
- In Map view, an aligned read will be depicted as an arrow above the reference map. The start (+1) position of whole plasmid read is usually arbitrary, and so will most likely be different to the reference +1 position. The end/start boundaries of the read with respect to the reference will be marked as shown.
- Any disagreements between the reference and the read will be marked on the line. See How do I Interpret the Align to Reference Map View? for more information.
View the Alignment
Switch to Sequence view to view and, if required, edit the alignment.
- The top panel shows the reference sequence with features.
- The bottom panel shows the reference (Original Sequence) aligned with any aligned reads within the field of view.
Click on the disclosure triangle to expand the read view.
Click the "Show Quality data" button to view quality information associated with the read (fastq only).
If multiple reads have been aligned, option- click (macOS) or ALT-click (Windows) on any disclosure triangle to expand/collapse all reads at once
The expanded read view provides controls to display features or quality information, and provides information on trace length and orientation, and a summary of differences compared to the reference.
View Mismatches
Click the arrows to jump to any differences detected between the aligned read and the reference.
Differences will be highlighted. Use the quality information to assess the reliability of the read at the mismatch position.
In the above example the mismatched base (A→G) is relatively low quality so may not be a "real" difference. Consider confirming this difference is real by Sanger sequencing.
Click the "Show with Annotations" button to view how the observed difference, if real, would affect translated features.
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. 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.
Create a New 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 event has occurred during PCR amplification of the insert or vector.
If "whole plasmid sequencing" confirms the clonal sequence differs from the reference then you can create a new sequence that replaces some or all of the reference with the selected read sequence.
Select the appropriate read in the "Align with:" panel.
Click "Aligned Sequences" → Replace Original with Aligned → Make New File.
Enter an appropriate name for the new sequence file.
Click Save and save the new sequence to an appropriate location on your computer.