How to read BLAST results
BLAST finds sequences similar to yours in a database. Understanding four numbers in the results table — E-value, percent identity, query cover and max score — covers most decisions.
The primer tool's "Search this primer with BLAST" button opens blastn with the sequence filled in, and the cloning helper offers reverse complement, translation and ORF finding for sequence checks.
Open the cloning helper →1. Choosing a program
| Situation | Program |
|---|---|
| What is my sequence? (near-identical hits, sequencing checks) | blastn → megablast (default, fast) |
| Similar genes in other species | blastn → dc-megablast or blastn |
| Short sequences such as primers (≈ 30 nt or less) | blastn (auto-adjusts for short queries) — for primer pairs Primer-BLAST is better |
| Protein sequences | blastp · translated searches blastx / tblastn |
2. Database
- core_nt / nt: the broad default, for unknown sequences.
- RefSeq RNA / genomic: curated reference sequences only — cleaner results.
- If you know the species, enter it under Organism to make the search faster and easier to read.
3. The numbers in the results table
| Number | Meaning | How to judge |
|---|---|---|
| E-value | How many hits this good you would expect by chance | Smaller is more certain; below 1e-50 is essentially the same sequence. Short queries get larger E-values even when identical |
| Percent identity | Share of identical bases in the aligned part | 99–100% for sequencing checks; 80–95% for the same gene in other species |
| Query cover | Share of your sequence that aligned | 100% identity with 30% cover means only part matches |
| Max / Total score | Alignment score (longer and more exact = higher) | Total above Max means the match is in several pieces (e.g. exons) |
Always read identity together with cover. "100% identity" may cover only a short stretch.
4. Two everyday uses
Checking Sanger sequencing
Trim the low-quality first and last 20–50 nt, then use "Align two or more sequences" (BLAST 2 sequences) against your designed sequence. At any mismatch, look at the chromatogram (.ab1) to see whether the peak is clean — a real mutation or a base-calling error.
What is this colony or PCR product?
Search with megablast and check identity and cover of the top hit. If E. coli or vector sequence comes first, suspect contamination or an empty vector.
The primer tool's "Search this primer with BLAST" button opens blastn with the sequence filled in, and the cloning helper offers reverse complement, translation and ORF finding for sequence checks.
Open the cloning helper →