Example 2) Clustering

In this second example, basic instructions to do clustering on paired-end FASTQs are provided.

Download example file

Paired-end FASTQ files were generated using seismic sim. The files, with their corresponding reference fasta file, can be downloaded here: https://raw.githubusercontent.com/rouskinlab/seismic-rna/main/src/userdocs/examples/Clustering/fq/Clustering.zip

Run the SEISMIC-RNA workflow

For this example, the entire workflow (seismic wf) will be run adding a flag for clustering (--cluster), and -x for paired-end FASTQs. There is no need to provide the FASTQ files one by one, SEISMIC-RNA will find them with only giving it the directory:

seismic wf fq/sim_clustering.fa -x fq/ --cluster

Output

Aside from the default outputs already described in Example 1) Running a sample with a single-end FASTQ, clustering will provide additional tables, reports, and plots:

A stacked barplot depicting the abundance of each cluster found (abundance_clustered):
The plots that were shown in Example 1, are also provided for each cluster, i.e.

the barplot with the mutation rate per base in the unmasked positions (profile_filtered_m-ratio):

Further analysis

SEISMIC-RNA also allows for further analysis. For instance, a rolling correlation plot can be done, comparing the clusters. For that, the seismic graph function can be used:

seismic graph corroll /out/sim_clustering_ref/cluster/sim_clustering_ref/full/cluster-position-table.csv --compself

Where corroll indicated the type of plot (rolling correlation, see Commands, Arguments, Options for more information), and --compself indicated that the comparison ought to be done of the clusters in the same table (as opposed to comparing samples in different tables).