State-of-the-Art Circular RNA Analytics Using the Circtools Software Suite

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2024 Feb 21

PMID 38381332

Authors

Tobias Jakobi

Affiliations

Soon will be listed here.

Abstract

Circular RNAs (circRNAs) are types of RNA molecules that have been discovered relatively recently and have been found to be widely expressed in eukaryotic cells. Unlike canonical linear RNA molecules, circRNAs form a covalently closed continuous loop structure without a 5' or 3' end. They are generated by a process called back-splicing, in which a downstream splice donor site is joined to an upstream splice acceptor site. CircRNAs have been found to play important roles in various biological processes, including gene regulation, alternative splicing, and protein translation. They can act as sponges for microRNAs or RNA-binding proteins and can also encode peptides or proteins. Additionally, circRNAs have been implicated in several diseases, including cancer, neurological disorders, and cardiovascular diseases.This protocol provides all necessary steps to detect and analyze circRNAs in silico from RNA sequencing data using the circtools circRNA analytics software suite. The protocol starts from raw sequencing data with circRNA detection via back-splice events and includes statistical testing of circRNAs as well as primer design for follow-up wet lab experiments.

References

Hoffmann S, Otto C, Doose G, Tanzer A, Langenberger D, Christ S . A multi-split mapping algorithm for circular RNA, splicing, trans-splicing and fusion detection. Genome Biol. 2014; 15(2):R34. PMC: 4056463. DOI: 10.1186/gb-2014-15-2-r34. View

Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A . Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013; 495(7441):333-8. DOI: 10.1038/nature11928. View

Chuang T, Wu C, Chen C, Hung L, Chiang T, Yang M . NCLscan: accurate identification of non-co-linear transcripts (fusion, trans-splicing and circular RNA) with a good balance between sensitivity and precision. Nucleic Acids Res. 2015; 44(3):e29. PMC: 4756807. DOI: 10.1093/nar/gkv1013. View

Westholm J, Miura P, Olson S, Shenker S, Joseph B, Sanfilippo P . Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation. Cell Rep. 2014; 9(5):1966-1980. PMC: 4279448. DOI: 10.1016/j.celrep.2014.10.062. View

Zhang X, Wang H, Zhang Y, Lu X, Chen L, Yang L . Complementary sequence-mediated exon circularization. Cell. 2014; 159(1):134-147. DOI: 10.1016/j.cell.2014.09.001. View

Cheng J, Metge F, Dieterich C . Specific identification and quantification of circular RNAs from sequencing data. Bioinformatics. 2015; 32(7):1094-6. DOI: 10.1093/bioinformatics/btv656. View

Szabo L, Morey R, Palpant N, Wang P, Afari N, Jiang C . Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development. Genome Biol. 2015; 16:126. PMC: 4506483. DOI: 10.1186/s13059-015-0690-5. View

Dobin A, Davis C, Schlesinger F, Drenkow J, Zaleski C, Jha S . STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2012; 29(1):15-21. PMC: 3530905. DOI: 10.1093/bioinformatics/bts635. View

Langmead B, Salzberg S . Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4):357-9. PMC: 3322381. DOI: 10.1038/nmeth.1923. View

10.

Jeck W, Sorrentino J, Wang K, Slevin M, Burd C, Liu J . Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA. 2012; 19(2):141-57. PMC: 3543092. DOI: 10.1261/rna.035667.112. View

11.

Panda A, De S, Grammatikakis I, Munk R, Yang X, Piao Y . High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs. Nucleic Acids Res. 2017; 45(12):e116. PMC: 5499592. DOI: 10.1093/nar/gkx297. View

12.

Xiao M, Wilusz J . An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing G-quadruplexes or structured 3' ends. Nucleic Acids Res. 2019; 47(16):8755-8769. PMC: 6895279. DOI: 10.1093/nar/gkz576. View

13.

Gruning B, Dale R, Sjodin A, Chapman B, Rowe J, Tomkins-Tinch C . Bioconda: sustainable and comprehensive software distribution for the life sciences. Nat Methods. 2018; 15(7):475-476. PMC: 11070151. DOI: 10.1038/s41592-018-0046-7. View

14.

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N . The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16):2078-9. PMC: 2723002. DOI: 10.1093/bioinformatics/btp352. View

15.

Jakobi T, Uvarovskii A, Dieterich C . circtools-a one-stop software solution for circular RNA research. Bioinformatics. 2018; 35(13):2326-2328. PMC: 6596886. DOI: 10.1093/bioinformatics/bty948. View

16.

Roehr J, Dieterich C, Reinert K . Flexbar 3.0 - SIMD and multicore parallelization. Bioinformatics. 2017; 33(18):2941-2942. DOI: 10.1093/bioinformatics/btx330. View

17.

Jakobi T, Czaja-Hasse L, Reinhardt R, Dieterich C . Profiling and Validation of the Circular RNA Repertoire in Adult Murine Hearts. Genomics Proteomics Bioinformatics. 2016; 14(4):216-23. PMC: 4996846. DOI: 10.1016/j.gpb.2016.02.003. View

18.

Hansen T, Veno M, Damgaard C, Kjems J . Comparison of circular RNA prediction tools. Nucleic Acids Res. 2015; 44(6):e58. PMC: 4824091. DOI: 10.1093/nar/gkv1458. View

19.

Altschul S, Gish W, Miller W, Myers E, Lipman D . Basic local alignment search tool. J Mol Biol. 1990; 215(3):403-10. DOI: 10.1016/S0022-2836(05)80360-2. View