DaPars is a python-based model that directly infers the dynamic alternative polyadenylation (APA) usage by comparing standard RNA-seq. Given the annotated gene model, DaPars can infer the de novo proximal APA sites as well as the long and short 3’UTR expression levels. Finally, it identifies the dynamic APA usages between two conditions.
The paper is titled Dynamic analyses of alternative polyadenylation from
RNA-seq reveal a 3′-UTR landscape across seven tumour types
The application download is free to download
and documentation was used as a reference
to create the nextflow pipeline flow of this module
An example sample sheet is available at samplesheet_example_files.csv. Each row in the samplesheet has four
columns:
- sample: name of the sample for logs (e.g control_replicate1)
- condition: name of the condition (e.g Control)
- 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 DaPars with test data provided for APAeval, check the path to DaPars 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 four files, with the four 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 Dapars/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 two steps of DaPars are specified in conf/modules.config file. Parameters relevant to the workflow itself are:
run_identification- set to true to obtain identification challenge output. Specifying any other value will throw an error.run_relative_usage_quantification- set to true to obtain relative expression quantification challenge output. Specifying any other value will throw an error.run_differential- set to true to obtain differential challenge output. Specifying any other value will throw an error.output_dir- name of the folder that the final output files are going to be in, located under Dapars/results/dapars/output_file- name of the output file for the current run ending with .bed if running identification and .tsv if running differentialgenome_file- absolute path from the DaPars folder to the input GTF annotation file can be obtained by replacingpath_towith the path to DaPars, and if using your own genome file, make sure to use the absolute path to your genome file
- 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. An example input file is samplesheet_example_files.csv
- Set the 'run_identification' parameter in conf/modules.config to true
- Change 'identification_out_suffix' parameter in conf/modules.config to the desired file suffix that ends with '.bed'
- Sample name for each row in the sample sheet should be unique
- Every sample (every row) in the sample sheet will run through the identification workflow
- Set the 'run_relative_usage_quantification' parameter in conf/modules.config to true
- Change 'relative_usage_quantification_out_suffix' parameter in conf/modules.config to the desired file suffix that ends with '.bed'
- Sample name for each row in the sample sheet should be unique
- Every sample (every row) in the sample sheet will run through the relative usage quantification workflow
Once parameters have been set in conf/modules.config file, run the nextflow pipeline with
nextflow main.nf --input samplesheet_example_files.csv.
When using the default output_dir parameter value in conf/modules.config, DaPars store results under DaPars/results/dapars/challenge_outputs folder. For identification and relative usage quantification outputs, the files have sample names as prefixes to differentiate the different runs. The differential output file will stay as the name specified in modules.config file.
- Make sure that the input bam files have leading 'chr' in the chromosome column. Otherwise, once
converted to input bedgraph file for DaPars, the workflow will exit with an error
This is because DaPars checks for the leading 'chr' in the process and would error out otherwise.
Hence, we've added a step to check for this leading 'chr' early in the workflow to prevent having to
go through the entire workflow before erroring out for efficiency. - DaPars' documentation specifies that a gene symbol file
is required. The gene symbol file consists of a column of transcript id and another column of gene symbol. This
file is then used to include the gene symbol for each row in the output file under
Genecolumn, for example looks likeENSMUST00000203335.1|Wnk1|chr6|-. However, since the differential output file requires a gene id instead of a gene symbol, this workflow extracts a gene symbol file required by DaPars by populating it with transcript id and gene id, such that theGenecolumn in Dapars' output file looks likeENSMUST00000203335.1|ENSMUSG00000045962.16|chr6|-.The gene id for each row can then be easily extracted for differential output file by taking the second item from the output above.
If you have any question or comment about DaPars, please post on DaPars Google Groups (https://groups.google.com/u/1/g/DaPars) or the author, Dr. Zheng Xia ([email protected]).