Sequence Alignment: BLAST

Transcription

1 E S S E N T I A L S O F N E X T G E N E R A T I O N S E Q U E N C I N G W O R K S H O P 2015 U N I V E R S I T Y O F K E N T U C K Y A G T C Class 6 Sequence Alignment: BLAST Be able to install and use the Basic Local Alignment Search Tool (BLAST ) to align and compare sequences Search the NCBI non-redundant BLAST database with a query file blast/moter_retrotransposons.fasta blast/morepeats.fasta blast/magnaporthe_oryzae_70-15_8_supercontigs.fasta blast/moters.nrblastn blast/morepeats.moryzae_genomeblastn1 blast x64/db/moryzae_genome.fasta 6.1 Installing BLAST First, we will download the BLAST binaries directly from the NCBI website. Go the NCBI homepage at Click the Data and Software link (left-hand panel) Click the Downloads tab Click the BLAST (Stand-alone) link Click the ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ link under BLAST+ executables. Note that the latest executable for Linux is: ncbi-blast x64-linux.tar.gz If we were to click on this link, it would download the file to the machine that we are working on not the Linux server where the program needs to be installed. Essentials of Next Generation Sequencing 2015 Page 1 of 5

2 Instead, we will copy the link to the file to make downloading it via the command line easier. We have a 64-bit ( x64 ) Linux system, so right-click the ncbi-blast x64-linux.tar.gz link and select Copy Link Address or the equivalent in your browser. Use PuTTY to connect to your server via SSH. We will now use the command line to download the latest BLAST executables from the NCBI FTP server straight into to your home directory on csurs11. The program we use for downloading from the web is wget. Type wget followed by a space and then paste the copied link (right-click when using PuTTY). The command should look like the following: wget \ ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ncbi-\ blast x64-linux.tar.gz After it finishes downloading, unpack the executables: tar zxvpf ncbi-blast x64-linux.tar.gz Add the directory with the executables to your system PATH: PATH=/home/yourusername/ncbi-blast /bin:$PATH Make sure to replace yourusername with your actual user name (i.e. daniel.harris)! Tell the computer to use the new path: export PATH Essentials of Next Generation Sequencing 2015 Page 2 of 5

3 6.2 Run a Local Remote BLAST Search Use the command line to search the NCBI non-redundant BLAST database with a query file blast/moter_retrotransposons.fasta blast/moters.nrblastn Now, use your locally installed blastn program to search the NCBI database using the query file MoTeR_retrotransposons.fasta: blastn db nr query blast/moter_retrotransposons.fasta out \ BLAST takes several parameters here: -db: -query: -out: -evalue: -outfmt: -remote: blast/moters.nrblastn evalue 1e-20 outfmt 1 remote specifies the database to be searched (we will use the NCBI nr database) specifies the local query sequence file (full path required) name of output file tells program to only report matches with specified value specifies format of output (values can range from 0 to 11). Possible formats are listed below: 0 = pairwise 1 = query-anchored showing identities 2 = query-anchored no identities 3 = flat query-anchored, show identities 4 = flat query-anchored, no identities 5 = XML Blast output 6 = tabular 7 = tabular with comment lines 8 = Text ASN.1 9 = Binary ASN.1 10 = Comma-separated values 11 = BLAST archive format (ASN.1) tells the program to search a remote (NCBI) database You may want to experiment with this parameter to see which format suits your needs. Examine the BLAST outputs with less. Essentials of Next Generation Sequencing 2015 Page 3 of 5

4 6.3 Create and Search a Custom BLAST Database Create a BLAST nucleotide database from the genome assembly and perform a query against it blast/morepeats.fasta blast/magnaporthe_oryzae_70-15_8_supercontigs.fasta blast/morepeats.moryzae_genomeblastn1 blast /db/moryzae_genome.fasta Now we will create a BLAST database from our existing genome data so we can perform searches against it. First, we will need to tell BLAST where to look for your custom database with an.ncbirc file. Use the nano text editor to create a file named.ncbirc (yes, the prefix period should be included) inside your home directory. Here s a good example where a Word document will not work. The file should contain the text: [BLAST] BLASTDB=/home/yourusername/ncbi-blast /db Again, be sure to replace yourusername with your actual user name (i.e. first.last) Create a subdirectory named db within the ncbi-blast directory. Copy the BLAST/magnaporthe_oryzae_70-15_8_supercontigs.fasta genome file into your newly created db directory. Change to the db directory, and use makeblastdb to create a new BLAST database: makeblastdb in magnaporthe_oryzae_70-15_8_supercontigs.fasta \ dbtype nucl out Moryzae_genome.fasta Now change back to your home directory and run a blastn search using the sequence in MoRepeats.fasta as the query and your new genome as the database: blastn db Moryzae_genome.fasta query blast/morepeats.fasta out \ blast/morepeats.moryzae_genomeblastn1 evalue 1e-20 outfmt 1 Examine your output file with less. Essentials of Next Generation Sequencing 2015 Page 4 of 5

5 Now is a good time to try running the search using a few different output format options (0 through 11). Try outfmt 6 for example. blastn db Moryzae_genome.fasta query blast/morepeats.fasta out \ blast/morepeats.moryzae_genomeblastn6 evalue 1e-20 outfmt 6 Note that BLAST comes in many flavors, not just blastn. blastn: nucleotide-nucleotide alignment blastp: protein-protein alignment blastx: does six-frame translation of query nucleotide sequence and aligns against a protein database and many more! Essentials of Next Generation Sequencing 2015 Page 5 of 5