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| This project contains the framework of the circular RNA toolbox ``circtools``. | ||
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| # Installation | ||
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| This package is written in python3 (3.4). It has a number of external dependencies, mostly standard bioinformatics tools: | ||
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| * [bedtools (>= 2.26.0)](http://bedtools.readthedocs.io/en/latest/content/installation.html) [RBP enrichment module, installed automatically] | ||
| * [R (>= 3.3)](https://www.digitalocean.com/community/tutorials/how-to-install-r-on-ubuntu-16-04-2) | ||
| [Primer design module] | ||
| * [OligoArrayAux](http://unafold.rna.albany.edu/?q=DINAMelt/OligoArrayAux) | ||
| [required by DECIPHER Bioconductor package for annealing efficiency estimations, installed automatically] | ||
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| Installation is managed through `python3 setup.py install`. No sudo access is required if the installation is executed with ``--user`` which will install the package in a user-writeable folder. The binaries should be installed to ``/home/$user/.local/bin/`` in case of Debian-based systems. | ||
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| ``Circtools`` was developed and tested on Debian Jessie. | ||
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| The installation requires running python on the command line: | ||
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| ``` | ||
| git clone git@github.com:dieterich-lab/circtools.git | ||
| cd circtools | ||
| python3 setup.py install --verbose --user | ||
| ``` | ||
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| The installation procedure will automatically install two dependencies: [DCC](https://github.com/dieterich-lab/DCC) and [FUCHS](https://github.com/dieterich-lab/FUCHS). The primer-design module as well as the exon analysis and circRNA testing module require a working installation of [R](https://cran.r-project.org/) with [BioConductor](https://www.bioconductor.org/install/). All R packages required are automatically installed during the setup. | ||
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| # Usage | ||
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| Circtools currently offers four modules: | ||
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| ``` | ||
| $ circtools | ||
| Available commands: | ||
| enrich: circular RNA RBP enrichment scan | ||
| primer: circular RNA primer design tool | ||
| detect: circular RNA detection with DCC | ||
| reconstruct: circular RNA reconstruction with FUCHS | ||
| circtest: circular RNA statistical testing | ||
| exon: circular RNA alternative exon analysis | ||
| quickcheck: circular RNA sequencing library quick checks | ||
| circtools: a modular, python-based framework for circRNA-related tools that | ||
| unifies several functions in single command line driven software. | ||
| positional arguments: | ||
| command Command to run | ||
| optional arguments: | ||
| -h, --help show this help message and exit | ||
| -V, --version show program's version number and exit | ||
| ``` | ||
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| ### detect | ||
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| The ``detect`` command is an interface to [DCC](https://github.com/dieterich-lab/DCC), also developed at the Dieterich lab. Please see the corresponding [manual](https://github.com/dieterich-lab/DCC) on the GitHub project for instructions how to run DCC. The parameters supplied to circtools will be directly passed to DCC. | ||
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| ### reconstruct | ||
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| The ``reconstruct`` command is an interface to [FUCHS](https://github.com/dieterich-lab/FUCHS). FUCHS is employing DCC-generated data to reconstruct circRNA structures. Please see the corresponding [manual](https://github.com/dieterich-lab/FUCHS) on the GitHub project for instructions how to run FUCHS. All parameters supplied to circtools will be directly passed to FUCHS. | ||
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| ### primer | ||
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| The ``primer`` command is used to design and visualize primers required for follow up wet lab experiments to verify circRNA candidates. The full documentation for the ``primer`` module can be found in its own [manual](R/circtools/vignettes/plot-transcripts.md). | ||
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| ### enrich | ||
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| The ``enrichment`` module may be used to identify circRNAs enriched for specific RNA binding proteins (RBP) based on DCC-identified circRNAs and processed [eCLIP](http://www.nature.com/nmeth/journal/v13/n6/full/nmeth.3810.html) data. For K526 and HepG2 cell lines plenty of this data is available through the [ENCODE](https://www.encodeproject.org/search/?type=Experiment&assay_title=eCLIP) | ||
| project. The enrich module understands the following options: | ||
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| ``` | ||
| usage: circtools [-h] -c CIRC_RNA_INPUT -b BED_INPUT -a ANNOTATION -g | ||
| GENOME_FILE [-o OUTPUT_DIRECTORY] [-i NUM_ITERATIONS] | ||
| [-p NUM_PROCESSES] [-t TMP_DIRECTORY] [-T THRESHOLD] | ||
| [-P PVAL] [-H HAS_HEADER] [-F OUTPUT_FILENAME] | ||
| [-I INCLUDE_FEATURES] | ||
| circular RNA RBP enrichment tools | ||
| optional arguments: | ||
| -h, --help show this help message and exit | ||
| Required options: | ||
| -c CIRC_RNA_INPUT, --circ-file CIRC_RNA_INPUT | ||
| Path to the CircRNACount file generated by DCC | ||
| -b BED_INPUT, --bed-input BED_INPUT | ||
| One or more BED files containing features to overlap | ||
| -a ANNOTATION, --annotation ANNOTATION | ||
| Genome reference annotation file used to not shuffle | ||
| into intragenic regions | ||
| -g GENOME_FILE, --genome GENOME_FILE | ||
| Genome file for use with bedtools shuffle. See | ||
| bedtools man page for details. | ||
| Additional options: | ||
| -o OUTPUT_DIRECTORY, --output OUTPUT_DIRECTORY | ||
| The output folder for files created by circtest | ||
| [default: .] | ||
| -i NUM_ITERATIONS, --iterations NUM_ITERATIONS | ||
| Number of iterations for CLIP shuffling [default: | ||
| 1000] | ||
| -p NUM_PROCESSES, --processes NUM_PROCESSES | ||
| Number of threads to distribute the work to | ||
| -t TMP_DIRECTORY, --temp TMP_DIRECTORY | ||
| Temporary directory used by pybedtools | ||
| -T THRESHOLD, --threshold THRESHOLD | ||
| p-value cutoff | ||
| -P PVAL, --pval PVAL p-value cutoff | ||
| -H HAS_HEADER, --header HAS_HEADER | ||
| Defines if the circRNA input file has a header line | ||
| [default: no] | ||
| -F OUTPUT_FILENAME, --output-filename OUTPUT_FILENAME | ||
| Defines the output file prefix [default: output] | ||
| -I INCLUDE_FEATURES, --include-features INCLUDE_FEATURES | ||
| Defines the the features which should be used for | ||
| shuffling. May be specified multiple times. [default: | ||
| all - shuffle over the whole genome] | ||
| ``` | ||
| ### circtest | ||
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| The ``circtest`` command is an interface to [CircTest](https://github.com/dieterich-lab/CircTest). The module a a very convenient way to employ statistical testing to circRNA candidates generated with DCC without having to write an R script for each new experiment. For detailed information on the implementation itself take a look at the [CircTest documentation](https://github.com/dieterich-lab/CircTest). In essence, the module allows dynamic grouping of the columns (samples) in the DCC data. | ||
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| ``` | ||
| circtools circtest --help | ||
| usage: circtools [-h] -d DCC_DIR -l CONDITION_LIST -c CONDITION_COLUMNS -g | ||
| GROUPING [-r NUM_REPLICATES] [-f MAX_FDR] [-s FILTER_SAMPLE] | ||
| [-C FILTER_COUNT] [-o OUTPUT_DIRECTORY] [-n OUTPUT_NAME] | ||
| [-p MAX_PLOTS] [-a LABEL] | ||
| circular RNA statistical testing - Interface to https://github.com/dieterich- | ||
| lab/CircTest | ||
| optional arguments: | ||
| -h, --help show this help message and exit | ||
| Required: | ||
| -d DCC_DIR, --DCC DCC_DIR | ||
| Path to the detect/DCC data directory | ||
| -l CONDITION_LIST, --condition-list CONDITION_LIST | ||
| Comma-separated list of conditions which should be | ||
| comparedE.g. "RNaseR +","RNaseR -" | ||
| -c CONDITION_COLUMNS, --condition-columns CONDITION_COLUMNS | ||
| Comma-separated list of 1-based column numbers in the | ||
| detect/DCC output which should be compared; e.g. | ||
| 10,11,12,13,14,15 | ||
| -g GROUPING, --grouping GROUPING | ||
| Comma-separated list describing the relation of the | ||
| columns specified via -c to the sample names specified | ||
| via -l; e.g. -g 1,2 and -r 3 would assign sample1 to | ||
| each even column and sample 2 to each odd column | ||
| Processing options: | ||
| -r NUM_REPLICATES, --replicates NUM_REPLICATES | ||
| Number of replicates used for the circRNA experiment | ||
| [Default: 3] | ||
| -f MAX_FDR, --max-fdr MAX_FDR | ||
| Cut-off value for the FDR [Default: 0.05] | ||
| -p PERCENTAGE, --percentage PERCENTAGE | ||
| The minimum percentage of circRNAs account for the | ||
| total transcripts in at least one group. [Default: | ||
| 0.01] | ||
| -s FILTER_SAMPLE, --filter-sample FILTER_SAMPLE | ||
| Number of samples that need to contain the amount of | ||
| reads specified via -C [Default: 3] | ||
| -C FILTER_COUNT, --filter-count FILTER_COUNT | ||
| Number of CircRNA reads that each sample specified via | ||
| -s has to contain [Default: 5] | ||
| Output options: | ||
| -o OUTPUT_DIRECTORY, --output-directory OUTPUT_DIRECTORY | ||
| The output directory for files created by circtest | ||
| [Default: .] | ||
| -n OUTPUT_NAME, --output-name OUTPUT_NAME | ||
| The output name for files created by circtest | ||
| [Default: circtest] | ||
| -p MAX_PLOTS, --max-plots MAX_PLOTS | ||
| How many of candidates should be plotted as bar chart? | ||
| [Default: 50] | ||
| -a LABEL, --label LABEL | ||
| How should the samples be labeled? [Default: Sample] | ||
| ``` | ||
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| Two samples, each with RNaseR+/- Treatment, three replicates each distributed through the columns specified via -c: | ||
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| ``` | ||
| circtools circtest -d DCC_DIR | ||
| -l Sample_1_RNaseR-,Sample_1_RNaseR+,Sample_2_RNaseR-,Sample_2_RNaseR+ | ||
| -c 4,6,8,10,12,14,16,17,18,19,20,21 | ||
| -g 1,1,1,2,2,2,3,3,3,4,4,4 | ||
| ``` | ||
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| ### exon | ||
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| The exon module of circtools employs the [ballgown R package](https://www.bioconductor.org/packages/release/bioc/html/ballgown.html) to combine data generated with DCC and circtest with ballgown-compatible `stringtie` output or cufflinks output converted via [tablemaker](https://github.com/leekgroup/tablemaker) in order get deeper insights into differential exon usage within circRNA candidates. | ||
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| ``` | ||
| circtools exon --help | ||
| usage: circtools [-h] -d DCC_DIR -l CONDITION_LIST -c CONDITION_COLUMNS -g | ||
| GROUPING -r REPLICATES -b BALLGOWN_DATA -G GTF_FILE -C | ||
| CIRCTEST_FILE [-H HAS_HEADER] [-o OUTPUT_DIRECTORY] | ||
| [-n OUTPUT_PREFIX] | ||
| circular RNA exon usage analysis | ||
| optional arguments: | ||
| -h, --help show this help message and exit | ||
| Required: | ||
| -d DCC_DIR, --DCC DCC_DIR | ||
| Path to the detect/DCC data directory | ||
| -l CONDITION_LIST, --condition-list CONDITION_LIST | ||
| Comma-separated list of conditions which should be | ||
| comparedE.g. "RNaseR +","RNaseR -" | ||
| -c CONDITION_COLUMNS, --condition-columns CONDITION_COLUMNS | ||
| Comma-separated list of 1-based column numbers in the | ||
| detect/DCC output which should be compared; e.g. | ||
| 10,11,12,13,14,15 | ||
| -g GROUPING, --grouping GROUPING | ||
| Comma-separated list describing the relation of the | ||
| columns specified via -c to the sample names specified | ||
| via -l; e.g. -g 1,2 and -r 3 would assign sample1 to | ||
| each even column and sample 2 to each odd column | ||
| -r REPLICATES, --replicates REPLICATES | ||
| Comma-separated list describing the relation of the | ||
| samples specified via -g to the sample names specified | ||
| via -l; e.g. -g 1,2 and -r 3 would assign sample1 to | ||
| each even column and sample 2 to each odd column | ||
| -b BALLGOWN_DATA, --ballgown-data BALLGOWN_DATA | ||
| Path to the ballgown data directory | ||
| -G GTF_FILE, --gtf-file GTF_FILE | ||
| Path to the GTF file containing the employed genome | ||
| annotation | ||
| -C CIRCTEST_FILE, --circtest-output CIRCTEST_FILE | ||
| Path to the CircTest CSV file containing the CircTest | ||
| results | ||
| Additional options: | ||
| -H HAS_HEADER, --has-header HAS_HEADER | ||
| Do the CircTest result files have a header? [Default: | ||
| No] | ||
| Output options: | ||
| -o OUTPUT_DIRECTORY, --output-directory OUTPUT_DIRECTORY | ||
| The output directory for files created by circtest | ||
| [Default: .] | ||
| -n OUTPUT_PREFIX, --output-prefix OUTPUT_PREFIX | ||
| The output name (prefix) for files created by circtest | ||
| [Default: exon_analysis] | ||
| ``` | ||
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| ### quickcheck | ||
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| The quickcheck module of circtools is an easy way to check the results of a DCC run for problems and to quickly assess the number of circRNAs in a given experiment. The module needs the mapping log files produced by STAR as well as the directory with the DCC results. The module than generates a series of figures in PDF format to assess the results. | ||
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| ``` | ||
| usage: circtools [-h] -d DCC_DIR -s STAR_DIR -l CONDITION_LIST -g GROUPING | ||
| [-o OUTPUT_DIRECTORY] [-n OUTPUT_NAME] | ||
| circular RNA sequencing library quality assessment | ||
| optional arguments: | ||
| -h, --help show this help message and exit | ||
| Required: | ||
| -d DCC_DIR, --DCC DCC_DIR | ||
| Path to the detect/DCC data directory | ||
| -s STAR_DIR, --star STAR_DIR | ||
| Path to the base STAR data directory containing sub- | ||
| folders with per-sample mappings | ||
| -l CONDITION_LIST, --condition-list CONDITION_LIST | ||
| Comma-separated list of conditions which should be | ||
| comparedE.g. "RNaseR +","RNaseR -" | ||
| -g GROUPING, --grouping GROUPING | ||
| Comma-separated list describing the relation of the | ||
| columns specified via -c to the sample names specified | ||
| via -l; e.g. -g 1,2 and -r 3 would assign sample1 to | ||
| each even column and sample 2 to each odd column | ||
| Output options: | ||
| -o OUTPUT_DIRECTORY, --output-directory OUTPUT_DIRECTORY | ||
| The output directory for files created by circtest | ||
| [Default: ./] | ||
| -n OUTPUT_NAME, --output-name OUTPUT_NAME | ||
| The output name for files created by circtest | ||
| [Default: quickcheck] | ||
| ``` |
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