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# BLAST: Introduction
> The [`BLAST` (Basic Local Alignment Search Tool) software suite](https://blast.ncbi.nlm.nih.gov/Blast.cgi) provides a set of tools for comparing a biological [query sequence](glossary.qmd#query-sequence) to the sequences in a database, and returning those sequences from the database that resemble the query sequence above a defined threshold similarity level.
::: { .callout-note }
The original `BLAST` paper is one of the most highly cited publications of all time, and has [over 110,000 citations in the literature](https://scholar.google.co.uk/scholar?cites=13256359684734893683&as_sdt=2005&sciodt=0,5&hl=en).
:::
::: { .callout-tip collapse="true"}
## A brief history of `BLAST`
- 1990: `BLAST` is first described (@Altschul1990-ar).
- 1997: A refined version of `BLAST` is published, introducing a new way of managing gapped alignments and `PSI-BLAST` - a method of compiling a _profile_ of similar sequences to make searching more sensitive (@Altschul1997-vt).
- 2000: The `MegaBLAST` algorithm for fast alignment-based searching of large nucleotide sequences is proposed, and incorporated into the `BLAST` suite (@Zhang2000-wn).
- 2009: A completely rewritten version of the software suite, `BLAST+` is released. This improved performance and changed many features of the algorithms used for searching and building databases (@Camacho2009-za).
**The latest updates to the `BLAST` software are described on the [BLAST news page](https://blast.ncbi.nlm.nih.gov/doc/blast-news/2023-BLAST-News.html)**
:::
## Performing a `BLAST` search
This section describes a general `BLAST` query using the [NCBI BLAST server](https://blast.ncbi.nlm.nih.gov/Blast.cgi). It is intended as a reference guide for you to return to as you get used to querying `BLAST` through this interface.
::: { .callout-caution }
There are implementations of `BLAST` and other sequence search methods at many other databases, and they may present a different interface and choice of options, or even a totally different search method. For example, the `RCSB-PDB` protein structure database offers a [sequence search page](https://www.rcsb.org/search/advanced/sequence) which uses the `mmseqs2` search algorithm (@Steinegger2017-sy).
:::
### Navigate to the NCBI `BLAST` webserver
Open a web browser and navigate to the [NCBI BLAST webserver](https://blast.ncbi.nlm.nih.gov/Blast.cgi). You should see a landing page that resembles @fig-ncbi-blast-landing.
{#fig-ncbi-blast-landing width="80%"}
::: { .callout-tip collapse="true" }
## You can use an NCBI account to save searches
Note that there is a `Log in` button at the top right of the landing page. If you have a suitable account with NCBI/NIH, NCBI maintains a record of your searches and history so you can store your searches and retrieve them later.
- [Sign up](https://account.ncbi.nlm.nih.gov/signup/) for a free NCBI account (you _do not_ need this for the workshop)
:::
### Select the `BLAST` tool you want to use
The `BLAST` suite provides search tools for finding matches to a query in a database. The query can be either a nucleotide or a protein sequence, and the database being searched can contain either protein sequences or nucleotide sequences. `BLAST` provides four different programs to carry out these combinations of search.
| Query type | nucleotideDB | proteinDB |
|---|:-:|:-:|
| nucleotide | `blastn` | `blastx` |
| protein | `tblastn` | `blastp` |
: The four main `BLAST` programs, and the combination of query/database sequence type they are used for {#tbl-blast-programs .striped .hover tbl-width="75%"}
::: { .callout-note collapse="true" }
## Specialised `BLAST` tools
The NCBI `BLAST` webserver provides specialised search options with specific combinations of parameters and databases pre-selected to support particular kinds of search (@fig-blast-specialised).
{#fig-blast-specialised width="80%"}
:::
Select `Nucleotide BLAST` from the NCBI landing page, to get to the `blastn` search page (@fig-ncbi-blastn).
{#fig-ncbi-blastn width="80%"}
### Enter the query sequence
Copy the DNA sequence below, and paste it into the box marked **
Enter accession number(s), gi(s), or FASTA sequence(s)** at the NCBI search page (@fig-ncbi-blastn-query).
```text
ATGCGTCGAGGGCGTCTGCTGGAGATCGCCCTGGGATTTACCGTGCT
TTTAGCGTCCTACACGAGCCATGGGGCGGACGCCAATTTGGAGGC
TGGGAACGTGAAGGAAACCAGAGCCAGTCGGGCC
```
{#fig-ncbi-blastn-query width="80%"}
### Set appropriate parameter choices
::: { .callout-caution }
If you make no more changes to the parameter settings for your search, the default options will be used. Your query will be made against the `core_nt` core nucleotide collection, a very large database. Due to the size of the database, the search may take a relatively long time.
:::
::: { .callout-tip collapse="true"}
## Improving your searches by changing parameters
You can make your `BLAST` searches quicker, and more relevant to your biological question, if you can use information about your sequence and the type of organism you want to search.
NCBI `BLAST` offers a number of smaller specialised databases with particular sequence types (e.g. RNA databases, sequences of protein structures, etc.) (@fig-ncbi-specialised-db).
{#fig-ncbi-specialised-db width="80%"}
You can also narrow down the search by specifying an organism, or other taxonomic rank, using the `Organism` field (@fig-ncbi-organism).
{#fig-ncbi-organism width="80%"}
:::
Restrict the sequences being searched by typing "Homo sapiens" in the `Organism` field and selecting the appropriate option from the drop-down list (@fig-ncbi-homo-sapiens).
{#fig-ncbi-homo-sapiens width="80%"}
### Run the `BLAST` search
Click on the `BLAST` button (@fig-ncbi-blast-button).
{#fig-ncbi-blast-button width="80%"}
### Wait for the search to complete
While the search runs, you will see a holding page that updates you with progress (@fig-ncbi-blast-progress)
{#fig-ncbi-blast-progress width="80%"}
::: { .callout-tip collapse="true" }
## SPOILER!
When the search is complete, you will see a `blastn` results page similar, but not identical, to @fig-ncbi-blast-results.
{#fig-ncbi-blast-results width="80%"}
:::
## Next Steps
::: { .callout-note title="Well done!"}
After completing this section you should:
- be able to carry out a `BLAST` search at the NCBI `BLAST` service
- be able to choose the correct database for your search type
- be able to modify search parameters, suitable for your biological question
Now you are ready to move on to using `BLAST`, which uses _local_ sequence alignments to search large databases of reference sequences to find the best matches to an input query sequence.
:::
You will next be presented with three scenarios, representing real uses of `BLAST` in research or clinical settings. Please work through the scenarios, completing the assessed sections and - if you have time - attempting the _stretch activities_.
::: { .callout-important }
There is a formative assessment [on MyPlace]({{< var myplace.bm214 >}}) for each scenario, which you should complete as part of the workshop.
- [Scenario 1 Formative Quiz]({{< var myplace.quiz1 >}})
- [Scenario 2 Formative Quiz]({{< var myplace.quiz2 >}})
- [Scenario 3 Formative Quiz]({{< var myplace.quiz3 >}})
:::