Tutorial 5: Masking using a regions file

When working with a complex sequence, SEISMIC-RNA allows to select distinct parts of it for analyzing.

In the case that, for example, a pair of primers was used to create an amplicon, it is possible to mask the positions of the primers using a regions file (see Metadata for Regions). Conceptually, this prevents the lack of mutations in the primer sequence regions to be incorrectly considered low reactivity when folding.

Download example files

Paired-end FASTQ files were generated using seismic sim. The files, with their corresponding reference fasta file and the regions file, can be downloaded here:

If you have wget, you can download the tutorial data simply by typing

wget https://raw.githubusercontent.com/rouskinlab/seismic-rna/main/src/userdocs/tutorials/regions-file/data.tar

Otherwise, click this link to download the tutorial data: https://raw.githubusercontent.com/rouskinlab/seismic-rna/main/src/userdocs/tutorials/regions-file/data.tar

To ensure the download is complete and not corrupted, verify that the SHA-256 checksum is 214e5fe859291a5f18bc67519047f58cb8ab4b2a7d56158ce7e6b4ed7fde3f3d by typing this command:

shasum -a 256 data.tar

If this command prints a different checksum, then retry the download. If the problem persists, then raise an issue (see Bugs and Requests).

After downloading and verifying the data, untar the data by typing

tar xvf data.tar

and then navigate into the data directory:

cd data

Run the SEISMIC-RNA workflow

The regions file can be included using --regions-file. This will mask the sequences determined by the file, and then use the rest for folding (for which the flags --fold and --draw are added):

seismic wf fq/Regions_Ref.fa -x fq/ --regions-file fq/regions_file.csv --fold --draw

Output

Given that the primer sequences were masked, the model shows no reactivity at the first nor last 20 bases: