CSI-UTR is a perl-based model that detects differential expression of 3-prime UTRs.
The paper is titled Detection of Differentially
Expressed Cleavage Site Intervals Within 3' Untranslated Regions Using CSI-UTR Reveals Regulated Interaction Motifs
The tool is free to download
and documentation was used as a reference
to create the nextflow pipeline flow of this module
CSI-UTR requires exactly two distinct conditions to be provided as inputs. Furthermore, at least two replicates need to be provided per condition otherwise the tool errors out. The test data provided in APAeval only has one replciate per condition. As such, additional test datasets of two distinct conditions and two replicates per condition are provided here.
Additionally, CSI-UTR annotation and bed files have to be downloaded. In the same folder above, there are folders called annotations and locations that contain CSI annotation and bed files needed to run the tool for Mm10 genome, the genome used for APAeval test data.
Hence, to run CSI-UTR with APAeval test data, download all the folders and files in the folder above and place them under a data/ folder in CSI-UTR working directory.
CSI-UTR is compatible with the following genomes: Hg38, Mm10, Rn6, and Rn6_extended. For each genome, CSI annotation and bed files are required to be downloaded from CSI-UTR github repo.
An example sample sheet is available at samplesheet_example_files.csv. Each row in the samplesheet has four
columns:
- sample: name of the sample to be included as prefix in identification challenge output file name(e.g control_replicate1)
- condition: name of the condition (e.g control)
- replicate: replicate number of this condition (e.g 1)
- bam: BAM input file for the sample
- bai: BAI index file for sample's bam input
It is important to name samples of the same condition with the exact condition name under the condition
column in the samplesheet since samples are grouped per condition to be processed in the differential step.
To run CSI-UTR with test data provided for APAeval, check the path to CSI-UTR with pwd and replace
the path_to in samplesheet_example_files.csv with the path
from the pwd command.
When using your own data and input file instead of the provided test data and sample sheet, make sure to include in the input file you are using the absolute paths to the files, with the column names following the column names above.
This workflow uses docker containers. To run with docker, make sure that docker is installed and running
(e.g. to ensure docker is running, run the command docker --help and a help message should be printed).
Additionally, make sure that line 49 in CSI-UTR/nextflow.config file docker.enabled=true is uncommented while line
51 singularity.enabled=true is commented out
To run with singularity, comment out line 49 in Dapars/nextflow.config file docker.enabled=true and make sure that line
51 singularity.enabled=true is uncommented
Parameters used to run the CSI-UTR are specified in conf/modules.config file. Parameters relevant to the workflow itself are:
run_differential- true/false, whether to run to obtain differential challenge output.output_dir- name of the folder that the final output files are going to be in, located under CSI-UTR/results/csi-utr/identification_out_suffx- suffix of the output file for identification challenge ending with .bed, final output file name is prefixed with sample name obtained from the input sample sheetdifferential_out- name of differential challenge output file ending in .tsvdifferential_type- which differential expression approach to use and report in the differential challenge output. Must be one of 'DEXSeq', 'PAIRWISE' or 'WITHIN_UTR' (case sensitive). Consult publication for definitions of these approachesgenome_file- absolute path from the CSI-UTR folder to the input GTF annotation file can be obtained by replacingpath_towith the path to CSI-UTR, and if using your own genome file, make sure to use the absolute path to your genome fileCSI_bed_file- absolute path from the CSI-UTR folder to the downloaded CSI bed file can be obtained by replacingpath_towith the path to CSI-UTRCSI_annotation_file- absolute path from the CSI-UTR folder to the downloaded CSI annotation file can be obtained by replacingpath_towith the path to CSI-UTRgenome- the genome of the samples, value could be Hg38, Mm10, Rn6, or Rn6_extendedr- read lengthcoverage_cut- coverage cutoffusage_cut- usage cutoffp- p-value significance cutoffq- FDR significance cutoff
- Set the 'run_differential' parameter in conf/modules.config to true
- Change 'differential_out' parameter in conf/modules.config to the desired file name that ends with '.tsv'
- Ensure the sample sheet contains exactly two distinct conditions in the condition column and at least two replicates per condition. An example input file is samplesheet_example_files.csv
- Run the nextflow pipeline with nextflow main.nf --input samplesheet_example_files.csv
CSI-UTR identification run results in a file located under CSi-UTR/results/csi_utr/[output_dir]/[sample_name]_[identification_out_suffix] CSI-UTR differential run results in a file located under CSi-UTR/results/csi_utr/[output_dir]/[differential_out]
- CSI-UTR uses a pre-provided set of polyA site annotations and does not perform de-novo polyA site identification. As such it does not qualify for the identification challenge.
- It is not possible to obtain quantification challenge output data since the TPM value in the output file is provided per CSI region and not per APA site. Hence, this tool is not compatible for quantification challenge.
- CSI-UTR needs exactly two distinct conditions with at least 2 replicates per condition. If less than 2 replicates are provided per condition, the tool will error out.
If you have any question or comment about CSI-UTR, please contact the corresponding author, Dr. Eric Rouchka ([email protected]).