# nf-core/circrna **circRNA quantification, differential expression analysis and miRNA target prediction of RNA-Seq data** ## Introduction **nf-core/circrna** is a best-practice analysis pipeline for the quantification, miRNA target prediction and differential expression analysis of circular RNAs in paired-end RNA sequencing data. The pipeline is built using [Nextflow](https://www.nextflow.io), a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It comes with docker containers making installation trivial and results highly reproducible. ## Quick Start 1. Install [`Nextflow`](https://www.nextflow.io/docs/latest/getstarted.html#installation) (`>=21.04.0`) 2. Install any of [`Docker`](https://docs.docker.com/engine/installation/), [`Singularity`](https://www.sylabs.io/guides/3.0/user-guide/), [`Podman`](https://podman.io/), [`Shifter`](https://nersc.gitlab.io/development/shifter/how-to-use/) or [`Charliecloud`](https://hpc.github.io/charliecloud/) for full pipeline reproducibility _(please only use [`Conda`](https://conda.io/miniconda.html) as a last resort; see [docs](https://nf-co.re/usage/configuration#basic-configuration-profiles))_ 3. Download the pipeline and test it on a minimal dataset with a single command: ```bash nextflow run nf-core/circrna -profile test, ``` > Please check [nf-core/configs](https://github.com/nf-core/configs#documentation) to see if a custom config file to run nf-core pipelines already exists for your Institute. If so, you can simply use `-profile ` in your command. This will enable either `docker` or `singularity` and set the appropriate execution settings for your local compute environment. 4. Start running your own analysis! ```bash nextflow run nf-core/circrna -profile --module 'circrna_discovery, mirna_prediction, differential_expression' --tool 'circexplorer2' --input 'samples.csv' --input_type 'fastq' --phenotype 'phenotype.csv' ``` Refer to [usage documentation](https://nf-co.re/circrna/usage) for exapanded details on running each analysis module. ## Pipeline Summary 1. Input type conversion [`SamToFastq`](https://gatk.broadinstitute.org/hc/en-us/articles/360036485372-SamToFastq-Picard-) 2. Raw read quality control [`FastQC`](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) 3. Adapter trimming + read filtering [`BBDUK`](https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/) 4. circRNA quantification 1. [`STAR`](https://github.com/alexdobin/STAR) -> [`CIRCexplorer2`](https://circexplorer2.readthedocs.io/en/latest/) 2. [`STAR`](https://github.com/alexdobin/STAR) -> [`circRNA finder`](https://github.com/orzechoj/circRNA_finder) 3. [`STAR`](https://github.com/alexdobin/STAR) -> [`DCC`](https://github.com/dieterich-lab/DCC) 4. [`HISAT2`](http://daehwankimlab.github.io/hisat2/) -> [`CIRI2`](https://sourceforge.net/projects/ciri/files/CIRI2/) -> [`BWA`](http://bio-bwa.sourceforge.net/) -> [`CIRIquant`](https://github.com/Kevinzjy/CIRIquant) 5. [`Bowtie2`](http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) -> [`find circ`](https://github.com/marvin-jens/find_circ) 6. [`Bowtie`](http://bowtie-bio.sourceforge.net/index.shtml) -> [`MapSplice`](http://www.netlab.uky.edu/p/bioinfo/MapSplice2) 7. [`Segemehl`](https://www.bioinf.uni-leipzig.de/Software/segemehl/) -> [`Segemehl`](https://www.bioinf.uni-leipzig.de/Software/segemehl/) 5. circRNA filtering 1. Filter candidate circRNAs by number of reads spanning back-splice junction 6. circRNA annotation 1. Annotate candidates as circRNA, ciRNA or EI-circRNA 2. Calculate mature spliced length 3. Export mature spliced length as FASTA file 4. Annotate parent gene, underlying transcripts. 5. Export information as customised BED12 file 7. circRNA count matrix 1. Combine results of quantification tools to produce counts matrix for downstream statistical analysis 2. Require circRNAs in matrix to be called by at least *n* quantification tools (consensus filtering) 8. miRNA target prediction 1. [`miRanda`](http://cbio.mskcc.org/miRNA2003/miranda.html) 2. [`TargetScan`](http://www.targetscan.org/cgi-bin/targetscan/data_download.vert72.cgi) 3. Filter results, miRNAs must be called by both tools 9. Differential expression analysis [`DESeq2`](https://bioconductor.org/packages/release/bioc/html/DESeq2.html) 10. MultiQC report [`MultiQC`](http://multiqc.info/) Ouputs given by each step in the pipeline can be viewed in the [output documentation](https://nf-co.re/circrna/dev/output) ## Documentation The nf-core/circrna pipeline comes with documentation about the pipeline: [usage](https://nf-co.re/circrna/usage) and [output](https://nf-co.re/circrna/output). ## Credits `nf-core/circrna` was originally written by Barry Digby ([@BarryDigby](https://github.com/BarryDigby)) from the [National University of Ireland, Galway](http://www.nuigalway.ie/index-internal.html) as a member of Dr. Pilib Ó Broins lab with the financial support of Science Foundation Ireland (Grant number 18/CRT/6214). ## Contributions and Support If you would like to contribute to this pipeline, please see the [contributing guidelines](.github/CONTRIBUTING.md). For further information or help, don't hesitate to get in touch on the [Slack `#circrna` channel](https://nfcore.slack.com/channels/circrna) (you can join with [this invite](https://nf-co.re/join/slack)). ## Citations You can cite the `nf-core` publication as follows: > **The nf-core framework for community-curated bioinformatics pipelines.** > > Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen. > > _Nat Biotechnol._ 2020 Feb 13. doi: [10.1038/s41587-020-0439-x](https://dx.doi.org/10.1038/s41587-020-0439-x). In addition, references of tools and data used in this pipeline are as follows: * **BBDUK** Bushnell, B. (Unpublished). Download: [https://sourceforge.net/projects/bbmap/](https://sourceforge.net/projects/bbmap/) * **bedtools** Quinlan, A.R. & Hall, I.M., (2010). BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics , 26(6), pp.841–842. Available at: [http://dx.doi.org/10.1093/bioinformatics/btq033](http://dx.doi.org/10.1093/bioinformatics/btq033). Download: [https://github.com/arq5x/bedtools2/releases](https://github.com/arq5x/bedtools2/) * **Bowite** Langmead, B., Trapnell, C., Pop, M. et al., (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25. Availabe at: [https://doi.org/10.1186/gb-2009-10-3-r25](https://doi.org/10.1186/gb-2009-10-3-r25). Download: [https://sourceforge.net/projects/bowtie-bio/](https://sourceforge.net/projects/bowtie-bio/) * **Bowtie2** Langmead, B. & Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nature methods, 9(4), p. 357–359. Available at: [10.1038/nmeth.1923](https:/dx.doi.org/10.1038/nmeth.1923). Download: [http://bowtie-bio.sourceforge.net/bowtie2/index.shtml](http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) * **bwa** Li, H., & Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics , 25(14), 1754–1760. Available at: [https://doi.org/10.1093/bioinformatics/btp324](https://doi.org/10.1093/bioinformatics/btp324). Download: [http://bio-bwa.sourceforge.net/bwa.shtml](http://bio-bwa.sourceforge.net/bwa.shtml). * **CIRCexplorer2** Zhang XO, Dong R, Zhang Y, Zhang JL, Luo Z, Zhang J, Chen LL, Yang L. (2016). Diverse alternative back-splicing and alternative splicing landscape of circular RNAs. Genome Res. 2016 Sep;26(9):1277-87. Available at: [https://doi.org/10.1101/gr.202895.115](https://doi.org/10.1101/gr.202895.115). Download: [https://circexplorer2.readthedocs.io/en/latest/tutorial/setup/](https://circexplorer2.readthedocs.io/en/latest/tutorial/setup/) * **circRNA finder** Westholm, J.O., Lai, E.C., et al. (2016). Genome-wide Analysis of Drosophila Circular RNAs Reveals Their Structural and Sequence Properties and Age-Dependent Neural Accumulation Westholm et al. Cell Reports. Available at: [https://doi.org/10.1016/j.celrep.2014.10.062](https://doi.org/10.1016/j.celrep.2014.10.062). Download: [https://github.com/orzechoj/circRNA_finder](https://github.com/orzechoj/circRNA_finder) * **CIRIquant** Zhang, J., Chen, S., Yang, J. et al. (2020). Accurate quantification of circular RNAs identifies extensive circular isoform switching events. Nat Commun 11, 90. Available at: [https://doi.org/10.1038/s41467-019-13840-9](https://doi.org/10.1038/s41467-019-13840-9). Download: [https://github.com/bioinfo-biols/CIRIquant](https://github.com/bioinfo-biols/CIRIquant) * **DCC** Jun Cheng, Franziska Metge, Christoph Dieterich, (2016). Specific identification and quantification of circular RNAs from sequencing data, Bioinformatics, 32(7), 1094–1096. Available at: [https://doi.org/10.1093/bioinformatics/btv656](https://doi.org/10.1093/bioinformatics/btv656). Download: [https://github.com/dieterich-lab/DCC](https://github.com/dieterich-lab/DCC) * **find circ** Memczak, S., Jens, M., Elefsinioti, A., Torti, F., Krueger, J., Rybak, A., Maier, L., Mackowiak, S. D., Gregersen, L. H., Munschauer, M., Loewer, A., Ziebold, U., Landthaler, M., Kocks, C., le Noble, F., & Rajewsky, N. (2013). Circular RNAs are a large class of animal RNAs with regulatory potency. Nature, 495(7441), 333–338. Available at: [https://doi.org/10.1038/nature11928](https://doi.org/10.1038/nature11928). Download: [https://github.com/marvin-jens/find_circ](https://github.com/marvin-jens/find_circ) * **HISAT2** Kim, D., Paggi, J.M., Park, C. et al. (2019). Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37, 907–915 (2019). Available at: [https://doi.org/10.1038/s41587-019-0201-4](https://doi.org/10.1038/s41587-019-0201-4). Download: [http://daehwankimlab.github.io/hisat2/download/](http://daehwankimlab.github.io/hisat2/download/) * **MapSplice2** Wang, K., Liu J., et al. (2010) MapSplice: Accurate mapping of RNA-seq reads for splice junction discovery, Nucleic Acids Research, 38(18), 178. Avaialable at: [https://doi.org/10.1093/nar/gkq622](https://doi.org/10.1093/nar/gkq622). Download: [http://www.netlab.uky.edu/p/bioinfo/MapSplice2Download](http://www.netlab.uky.edu/p/bioinfo/MapSplice2Download) * **miRanda** Enright, A.J., John, B., Gaul, U. et al. (2003). MicroRNA targets in Drosophila. Genome Biol 5, R1. Available at: [https://doi.org/10.1186/gb-2003-5-1-r1](https://doi.org/10.1186/gb-2003-5-1-r1). Download: [http://cbio.mskcc.org/miRNA2003/miranda.html](http://cbio.mskcc.org/miRNA2003/miranda.html). * **Picard** Broad Institute (Unpublished). Download: [http://broadinstitute.github.io/picard/](http://broadinstitute.github.io/picard/) * **R**: R Core Team (2020). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Download: [https://www.R-project.org/](https://www.R-project.org/). * **biomaRt** Durinck S, Spellman PT, Birney E, Huber W. (2009). Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat Protoc. 4(8):1184-91. Available at: [https://doi.org/10.1038/nprot.2009.97](https://doi.org/10.1038/nprot.2009.97). Download: [https://bioconductor.org/packages/release/bioc/html/biomaRt.html](https://bioconductor.org/packages/release/bioc/html/biomaRt.html) * **circlize** Zuguang Gu, Lei Gu, Roland Eils, Matthias Schlesner, Benedikt Brors (2014). circlize implements and enhances circular visualization in R , Bioinformatics, 30,(19) 2811–2812. Available at: [https://doi.org/10.1093/bioinformatics/btu393](https://doi.org/10.1093/bioinformatics/btu393). 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ISBN 978-3-319-24277-4, Download: [https://ggplot2.tidyverse.org](https://ggplot2.tidyverse.org). * **ggpubr** Kassambara A. (2020). ggpubr: 'ggplot2' Based Publication Ready Plots. Download: [https://rpkgs.datanovia.com/ggpubr/](https://rpkgs.datanovia.com/ggpubr/) * **ihw** Ignatiadis, N., Klaus, B., Zaugg, J. et al. (2016). Data-driven hypothesis weighting increases detection power in genome-scale multiple testing. Nat Methods 13, 577–580. Available at: [https://doi.org/10.1038/nmeth.3885](https://doi.org/10.1038/nmeth.3885). Download: [https://bioconductor.org/packages/release/bioc/html/IHW.html](https://bioconductor.org/packages/release/bioc/html/IHW.html) * **PCAtools** Blighe K, Lun A (2020). PCAtools: PCAtools: Everything Principal Components Analysis. Download: [https://bioconductor.org/packages/release/bioc/html/PCAtools.html](https://bioconductor.org/packages/release/bioc/html/PCAtools.html) * **pheatmap** Kolde, R. (2019) Pretty Heatmaps. Download: [https://cran.r-project.org/package=pheatmap](https://cran.r-project.org/package=pheatmap) * **pvclust** Suzuki R., Shimodaira H., (2006). Pvclust: an R package for assessing the uncertainty in hierarchical clustering, Bioinformatics, 22(12), 1540–1542. Available at: [https://doi.org/10.1093/bioinformatics/btl117](https://doi.org/10.1093/bioinformatics/btl117). Download: [https://cran.r-project.org/web/packages/pvclust/index.html](https://cran.r-project.org/web/packages/pvclust/index.html) * **tximport** Soneson C, Love MI, Robinson MD (2015). Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research, 4. Avaialable at: [https://doi.org/10.12688/f1000research.7563.1](https://doi.org/10.12688/f1000research.7563.1). Download: [http://bioconductor.org/packages/release/bioc/html/tximport.html](http://bioconductor.org/packages/release/bioc/html/tximport.html) * **SAMtools** Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., … 1000 Genome Project Data Processing Subgroup. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics , 25(16), 2078–2079. [https://doi.org/10.1093/bioinformatics/btp352](https://doi.org/10.1093/bioinformatics/btp352). Download: [http://www.htslib.org/](http://www.htslib.org/) * **Segemehl** Hoffmann S, Otto C, Kurtz S, Sharma CM, Khaitovich P, Vogel J, Stadler PF, Hackermueller J: "Fast mapping of short sequences with mismatches, insertions and deletions using index structures", PLoS Comput Biol (2009) vol. 5 (9) pp. e1000502. Available at: [https://doi.org/10.1371/journal.pcbi.1000502](https://doi.org/10.1371/journal.pcbi.1000502). Download: [https://www.bioinf.uni-leipzig.de/Software/segemehl/](https://www.bioinf.uni-leipzig.de/Software/segemehl/) * **STAR** Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., & Gingeras, T. R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics (Oxford, England), 29(1), 15–21. Available at: [https://doi.org/10.1093/bioinformatics/bts635](https://doi.org/10.1093/bioinformatics/bts635). Download: [https://github.com/alexdobin/STAR](https://github.com/alexdobin/STAR) * **StringTie** Pertea, M., Pertea, G., Antonescu, C. et al. (2015). StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33, 290–295. Available at: [https://doi.org/10.1038/nbt.3122](https://doi.org/10.1038/nbt.3122). Download: [https://ccb.jhu.edu/software/stringtie/](https://ccb.jhu.edu/software/stringtie/) * **TargetScan** Agarwal V, Bell GW, Nam JW, Bartel DP. (2015). Predicting effective microRNA target sites in mammalian mRNAs. Elife, 4:e05005. Available at: [https://doi.org/10.7554/elife.05005](https://doi.org/10.7554/elife.05005). Download: [http://www.targetscan.org/cgi-bin/targetscan/data_download.vert72.cgi](http://www.targetscan.org/cgi-bin/targetscan/data_download.vert72.cgi) * **ViennaRNA** Lorenz, R., Bernhart, S.H., Höner zu Siederdissen, C. et al. (2011). ViennaRNA Package 2.0. Algorithms Mol Biol 6, 26. Available at: [https://doi.org/10.1186/1748-7188-6-26](https://doi.org/10.1186/1748-7188-6-26). Download: [https://www.tbi.univie.ac.at/RNA/#download](https://www.tbi.univie.ac.at/RNA/#download) ## Test data References Dong Cao (2021). An autoregulation loop in fust-1 for circular RNA regulation in Caenorhabditis elegans. Biorxiv. Available at: [https://doi.org/10.1101/2021.03.22.436400](https://doi.org/10.1101/2021.03.22.436400).