The Basic Local Alignment Search Tool (BLAST) identifies regions of similarity between sequences by comparing nucleotide and protein sequences to calculate the statistical significance of matches. You can use BLAST to infer functional and evolutionary relationships between sequences and identify members of gene families. Benchling's BLAST algorithms allow you to find DNA and amino acid (AA) sequences within Benchling based on their similarity to an input sequence. The search results display matching sequences and their similarity scores, which are determined by the selected algorithm.
Search sequences using BLAST
- To launch blast you have two options:
- Open a sequence, right-click a sequence selection and select Run Benchling BLAST
Click the Global Search (lens) icon in the left-side menu to open global search, then click the DNA helix in the search bar to open the BLAST modal
- In the BLAST modal, select the BLAST algorithm you're using, then select the input sequence using one of these methods. Note that multiple sequences can be queried at the same time by adding multiple sequences using methods below:
- Select the entities in Benchling
- Enter or paste the sequences in the text box
- Upload a file of the sequences, using an approved format
- To customize your query you can select the parameter values. View the list of parameters and their effects at the National Library of Medicine website.
- The modal will automatically select the recommended values based on the input sequences. For example, when running blastp for an amino acid shorter than 30 residues, the program is automatically set to blastp-short, which is optimized for shorter sequences. The parameter recommendation is the same as that on NCBI BLAST.
- Feel free to try different general and scoring parameters. You can always use the Reset to algorithm defaults to reset them. Please note that the program is not included in the reset.
- Click Run BLAST. The modal will close and the search launches in the global search panel. Note that some search features, like filters, are disabled when using BLAST.
- Use the information to the right of the Authors column to compare the similarities of each returned sequence to the input sequence and select the best candidate for your next steps.
BLAST result attributes
The BLAST search results offer two views for most algorithms: Table View and Alignment View.
Table view
The table view resembles other search result views in Benchling. It lists all hit sequences in a table, displaying BLAST result attributes (e.g., query coverage, bit score) and attributes of the hits (e.g., ID, modified timestamp). In this view, you can select which columns to include, change display preferences like sorting order, and perform bulk operations on multiple selected hit sequences.
- Filter columns by clicking on the leftmost icon in the table header:
- Change sort order, display density, and results per page:
- Select multiple hit sequences and perform bulk operations:
Alignment view
The alignment view provides detailed information about the hit sequence. It lists each High Scoring Pair (HSP) separately and combines both the graphic summary and the flat query-anchored alignment view from NCBI BLAST.
You can switch to the alignment view by clicking the Alignment view button.
Under the alignment view, BLAST result attributes such as hit range, percent identity, and E-value are displayed on the left side of the table. These attribute columns can be filtered in the same manner as in the Table View by clicking on the leftmost icon in the table header.
Under the alignment view, clicking between any nucleotide or amino acid will introduce a vertical guideline across sequences.
Bar graph vs pairwise Alignment
The query and hit sequences are listed in the Sequence column to the right of the alignment view table. The width of this column adjusts based on the screen size and the number of attribute columns on the left. If more space is needed in the Sequence column, you can shrink or filter out the attribute columns. The Sequence column will disappear if the available screen space becomes too narrow after the attribute columns. You can restore it by resizing or filtering out attribute columns.
You can toggle between two visualization modes for the Sequence column: Bar graph alignment and Pairwise alignment.
- Bar graph alignment score: The bar graphs are colored according to each HSP's alignment score. Hover over each segment to view the actual score. The alignment score reflects the similarity between the query sequence and the HSP sequence, with higher scores indicating greater similarity. The alignment score legend is only visible in the bar graph view.
- Pairwise alignment coloring scheme: By default, the pairwise alignment view highlights mismatched nucleotides or residues with a light red background. This is the only coloring scheme for nucleotide sequences. For amino acid sequences, more coloring schemes are available. When in the pairwise alignment view for amino acids, a View menu appears with a dropdown of four coloring schemes: Mismatch (default), Rasmol, Polarity, and Hydrophobicity.
You can zoom in and out on the sequences using the + and - buttons on the zoom level slider, or by dragging the slider directly. When the zoom level on the slider is over halfway, the bar graph view automatically switches to pairwise view.
Select and compare multiple results
In the alignment view, you can select one or multiple hit sequences to focus on them. When any hit sequences are selected, a Compare # results button will appear above the table. Clicking this button will hide all other results, including all HSPs for the selected hits.
Multi-Query BLAST
When multiple sequences are queried simultaneously, an additional dropdown list of sequences appears above the result table. You can click this dropdown to select and view the results for each queried sequence.
Limitations
- BLAST queries for long sequences (e.g. over 20K) may timeout and fail.
- BLAST queries for highly repetitive sequences may fail or require careful search parameter tuning.
- The alignment view is unavailable for tblastx.
Frequently asked questions
Q: What BLAST algorithms are available?
A: Benchling currently supports the following BLAST algorithms:
- blastn
- blastp
- blastx
- tblastn
- tblastx
Q: Why does a blastp query return nothing, while there are matches in my Benchling Registry?
A: For complex BLAST searches, such as those involving an amino acid sequence with high repetition, the results can be sensitive to the parameter values. If a blastp query yields no results, try using a different scoring matrix.
Q: Why does my BLAST search say that it times out?
A: The general Benchling search system currently has a one-minute limitation. If a BLAST query cannot be completed within this minute, it will time out. Timeouts can occur for long query sequences or with complex BLAST algorithms like tblastx. While Benchling plans to support longer sequences and more complex queries in the future, in the interim, you can run BLAST with shorter sequences.
Q: Why is the alignment view not available for tblastx?
A: The alignment view is currently unavailable for tblastx queries, and the toggle for this algorithm is disabled. This is because each tblastx query performs multiple searches, one for each reading frame. The results are more complex than those of other BLAST algorithms and require a more specialized user experience for easy digestion.
Q: Can I use BLAST to find sequences in external databases?
A: No. BLAST currently only searches for sequences in Benchling that you have permissions to access.