Questions
If you have questions about the lab after the course, you are welcome to contact me: martin.norling@nbis.se
Many of you may have used BLAST before - either of the web or on the command line - but it’s quite useful so we’ll go through it again!
BLAST can be quite slow though, especially when running towards one of the more general databases! Therefore we offer a choice of two databases: either use /sw/data/uppnex/blast_databases/nt to get general, “real”, results - or use /proj/g2016024/nobackup/illumina_assembly/blastdb/bacterial to run against a small bacterial database. The expected times to query an assembly against the two databases are about 5 minutes for nt and about 2 seconds for the small bacterial database.
To run blast you need to load the module load blast/2.4.0+ database, then run blastn -h to get help on how to run nucleotide blast. Try different values of the argument -outfmt to get different kinds of output. The options are:
With more detail available here.
In the read preparation you attempted to verify that only reads from the intended organism went into the assembly. We continue this verification here, by classifying the contigs and plotting them on a GC vs. coverage blob plot.
To do the classification – we will need taxonomy information! This can be downloaded (using the command wget) from NCBI, at this location: ftp://ftp.ncbi.nlm.nih.gov/pub/taxonomy/taxdump.tar.gz. This file needs to be unpacked, so use the command $ tar xzf taxdump.tar.gz to unpack the contents.
We also need a BAM file of mapped reads. You already made on of these in the assessment tutorial though, so you’re set! If not, just go back there for instructions on how to make one.
The third requirement is to actually have a BLAST classification of the contigs. We will use blastn, towards either of the databases mentioned in the BLAST part. Even though we just ran BLAST, in this case we will need a quite specific command to get a file that’s useful for blobtools, so use this command:
blastn -task megablast \
-query [your assembly file].fasta \
-db path-to-db \
-outfmt '6 qseqid staxids bitscore std sscinames sskingdoms stitle' \
-culling_limit 5 \
-num_threads 8 \
-evalue 1e-25 \
-out [output_name].blast.out
Now we can finally run blobtools! The uppmax module for blobtools is called blobtools/0.9.17. Sadly, the uppmax module is suffering from a small, but devastating, permission problem right now though, so it won’t work. We’ve put a second version that should work at /proj/g2016024/nobackup/illumina_assembly/blobtools/blobtools though! You’ll still need to load the module to get the dependencies right, but then use the full path to this version of the tool instead of the module version.
Blobtools works by using a creating a blob database - then you can query the database to get information and plots. But first - run blobtools –help to get more information on how to run blobtools.
Continue by creating a database using blobtools create. Use blobtools create –help to get help on the syntax. Now supply the assembly, BWA mapping, and blast result, as well as the nodes.dmp and names.dmp from the downloaded taxdump. Name the output based on the assembly you used.
You can now run blobtools view to create an output table, and blobtools blobplot to create a blobplot.