Pairwise and multiple sequence alignment can be performed pre and post analysis. Alignment can be performed using the Align operation under the Post-processing tab. We currently support the iteration-based alignment method, MUSCLE (multiple sequence comparison by log-expectation) for sequence alignment.
To align sequences in the Files table, select more than one sequence and Post-processing followed by Align in the dropdown.
To align sequences within a Result document, select a Biologics Annotator Result document, select two or more sequences in the Sequence Table and click Post-processing followed by Align in the dropdown.
You can choose to either align the whole sequence or just a region of your sequences. To align the entire sequence, click Entire sequence under Region to align. This operation will align all the selected sequences from the 5' end to the 3' end.
To align a region, click Extract region with name and select the appropriate region name in the drop-down. This operation will align the sequences from the 5' end to the 3' end of the selected region.
To align a region found in both the heavy and light chains, select Filter by and select from either heavy chain or light chain from the dropdown. For example, to align the heavy CDR3 regions of the selected sequences, select Extract region with name: CDR3 and Filter by: Heavy Chain in the respective dropdowns.
In the absence of annotations within the selected sequences, you will see the following message:
You can also align translated sequences by selecting the Translate nucleotide sequence(s) prior to alignment option. This operation will translate the nucleotide sequence according to the selected genetic code and translation frame before aligning the sequences. The available genetic codes are obtained from NCBI.
The standard start codon, AUG codes for Methionine in eukaryotes and a modified Met (fMet) in prokaryotes. Alternative start codons are still translated as Met when they are located at the start of a coding sequence. When Consider Alternative Start Codons is selected, you can select from the following options:
Auto detect: Alternative start codons are translated as M when the annotation is of type CDS, ORF or gene
Always consider: Alternative start codons are translated as M regardless of annotation type
Always ignore: Alternative start codons are not translated as M
Alignment tree file
All alignment document are outputted as Tree files and the trees are build using the Neighbour-joing algorithm (Saitou & Nei 1987).
To view your aligned sequences, select your Tree file in the Files table. The default view of sequence alignment is a Tree view of your aligned sequences. To view the alignment in a sequence view, click Sidebar in the right Sequence Viewer panel and uncheck Show Tree.
To include external assay or metadata in the alignment document as a heatmap, you will need to first import the assay data into the results table prior to alignment. Learn more on how to add assay data to your results in the following article.
Upon importing assay data, select the sequences you would like to align and proceed with the alignment operation as usual. To view the metadata in the tree or alignment view, click Sidebar in the right Sequence Viewer panel and select the assay data you would like to be included. To view the values of the metadata, click Show values for metadata and hover over the heatmap to see the details of the metadata.