Test Drive¶
In this test drive, we will first download a bacterial genome and FASTQ files of Illumina reads. Then, we will use breseq to predict mutations that are present in the re-sequencing data relative to this reference genome.
1. Download data files¶
First, create a directory called tutorial.
Reference sequence¶
breseq needs the reference sequence in Genbank format. In this example, the reference sequence is Escherichia coli B strain REL606. The Genbank (Refseq) accession number is: NC_012967 . You can search for this sequence at http://www.ncbi.nlm.nih.gov/ or follow this direct link.
Once the sequence is displayed, you will want to select “Show sequence” from the Display options on the right then click update view. Finally, use the send menu to choose “Complete Record” and Destination: “File” and “Genbank (Full)”. It should start downloading a file called “sequence.gb”. Rename this to NC_012967.gbk after it downloads.
Read files¶
We’re going to use Illumina genome re-sequencing data from a strain that evolved for 20,000 generations in a long-term evolution experiment [Barrick2009a]. This data is available in the European Nucleotide Archive (ENA). Go to http://www.ebi.ac.uk/ and search for the accession number: SRR030257. Then click on the accession number to open the record and download the two data files using the links in the ‘ftp’ column.
Move all three of these files into the tutorial directory that you created.
2. Run breseq¶
Check to be sure that you have changed into the tutorial directoryand that you have all of the input files (and have uncompressed them).
>>> ls
NC_012967.gbk SRR030257_1.fastq SRR030257_2.fastq
Now, run breseq:
>>> breseq -r NC_012967.gbk SRR030257_1.fastq SRR030257_2.fastq
The first named argument (-r) is the reference sequence. If you had multiple reference sequences, you could input multiple ones (e.g., -r NC_012967.gbk -r plasmid.gbk).
The unnamed arguments at the end of the command line are the read files. You can input as many as you need to and mix FASTQ files from different sequencing technologies (Illumina and 454).
Warning
Running breseq on a full data set like this is not fast. Depending on your computer, this could take several hours. If you want to speed things up, you might only include one of the two read files on the command line.
