This is a sample workflow of using the riboplot and ribocount programs which are part of the riboplot suite.
Both these tools are available under "RiboSeq Analysis --> Riboplot" on RiboGalaxy.
The FASTQ files were then uploaded to the RiboGalaxy FTP server and then uploaded to RiboGalaxy using the "Upload File" tool under "Get Data". The format was selected as fastqsanger.
riboplot and ribocount require Ribo-Seq data to be aligned to a transcriptome using Bowtie version 1.
This can be done on RiboGalaxy itself by following the steps below
This can be done using Cutadapt and Remove rRNA using Bowtie tools on RiboGalaxy (under Pre-processing tools).Example:
The FASTQ reads in the sample data were pre-processed using Cutadapt to remove the
adaptor sequence (ATCTCGTATGCCGTCTTCTGCTTG) and then rRNA.
Upload a FASTA format file of the transcriptome.
If you do not have a FASTA format file of the transcriptome, please follow the Alignments to
a transcriptome section of the RiboGalaxy Help page to obtain a FASTA format file of the
Then use "Transcriptome Mapping --> Align to transcriptome using Bowtie" to align reads
to the transcriptome.
Optional for riboplot: If you wish to plot RNA coverage, you will need to align RNA-Seq
data as above for Ribo-Seq.
The Zebrafish transcriptome as downloaded and the reads were aligned to it using the
and the selecting the "Convert Format" tab. Click "Convert" using the default option
"Convert SAM to BAM".
Using the "Sort Data --> Sort BAM dataset" tool using the default sort by option.
To plot and output read counts for a single transcript, select the riboplot tool from the menu and provide the following inputs (Figure 1)
Figure 1: Input options for riboplot
Description of options:
Figure 2: Plot of Ribo-Seq read counts and RNA coverage data with ORF architecture
Figure 3: A CSV file will also be included in the output containing Ribo-Seq read counts in 3 frames for the given transcript
To output read counts for all transcripts in an alignment, select the ribocount tool from the RiboPlot suite (Figure 4).
Figure 4: Input options for ribocount
Description of options
Figure 5: Summary of the ribocount run
As indicated, please download and extract the ribocount_output.zip file and open the index.html in a browser.
Total reads for each transcript will be displayed in a table along with the name of the transcript and a link to
the CSV file containing the read counts in 3 frames for each position in the transcript (Figure 6).
Figure 6: HTML page with results of the ribocount run for all transcripts in an alignment.