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This article focuses on "how to use CIRCexplorer2 to identify ring RNA", interested friends may wish to take a look. The method introduced in this paper is simple, fast and practical. Let's let the editor take you to learn how to use CIRCexplorer2 to identify ring RNA.

CIRCexplorer is a ring-shaped RNA forecasting software designed to predict exonic circRNA at the following URL

Https://github.com/YangLab/CIRCexplorer2

The identification of annular RNA includes two steps: sequence alignment and annular RNA prediction. The software has been updated to v2 version, and the usage has changed greatly compared with v1 version. In v1 version, only tophat-fusion and STAR are supported for sequence alignment to identify junction reads. In v2 version, it is extended to the following five kinds of software

Tophat-Fusion

STAR

BWA

MapSplice

Segemehl

All the commands in v1 version are encapsulated in one script, and v2 version is also improved. At the same time, it provides two ways of single script one-click operation and sub-module operation, which ensures the simplicity and flexibility of the software.

Relatively speaking, the installation of this software is a little complicated, because it depends on a lot of software. Here I will directly post my installation command in docker for your reference.

Docker run-it centosyum install-y epel-releaseyum install-y gcc gcc-c++ make zlib zlib-devel bzip2 bzip2-devel python2 python2-pip python-devel xz xz-devel unzip which ncurses-devel ncurses# CIRCexplorer2pip install circexplorer2# tophat & tophat-fusionwget http://ccb.jhu.edu/software/tophat/downloads/tophat-2.1.1.Linux_x86_64.tar.gztar xvzf tophat-2.1.1.Linux_x86_64.tar.gzcd tophat-2.1.1.Linux_x86_64cp b* C * f * g* j* long_spanning_reads map2gtf prep_reads sam* segment_juncs sra_to_solid tophat* / usr/local/bin/# cufflinkswget http://cole-trapnell-lab.github.io/cufflinks/assets/downloads/cufflinks-2.2.1.Linux_x86_64.tar.gztar xzvf cufflinks-2.2.1.Linux_x86_64.tar.gzcp * / usr/local/bin/# bedtoolswget https://github.com/arq5x/bedtools2/releases/download / v2.25.0/bedtools-2.25.0.tar.gztar-zxvf bedtools-2.25.0.tar.gzcd bedtools2makecd bincp * / usr/local/bin/# UCSCwget http://hgdownload.soe.ucsc.edu/admin/exe/linux.x86_64/genePredToGtfwget http://hgdownload.soe.ucsc.edu/admin/exe/linux.x86_64/gtfToGenePredwget http://hgdownload.soe.ucsc.edu/admin/exe/linux.x86_64/bedGraphToBigWigwget http://hgdownload.soe .ucsc.edu / admin/exe/linux.x86_64/bedToBigBedchmod + x bedGraphToBigWig bedToBigBed genePredToGtf gtfToGenePredmv bedGraphToBigWig bedToBigBed genePredToGtf gtfToGenePred / usr/local/bin/# starwget https://github.com/alexdobin/STAR/archive/2.7.0d.tar.gztar xzvf 2.7.0d.tar.gzcd STAR-2.7.0d/bincd Linux_x86_64_staticcp * / usr/local/bin/# bwawget https://sourceforge.net/projects/bio-bwa/files/bwa-0.7.17.tar. Bz2tar xjvf bwa-0.7.17.tar.bz2cd bwa-0.7.17makecp bwa / usr/local/bin/# mapsplicewget http://protocols.netlab.uky.edu/~zeng/MapSplice-v2.1.7.zipunzip MapSplice-v2.1.7.zipcd MapSplice-v2.1.7make# segemehlwget https://github.com/samtools/htslib/releases/download/1.9/htslib-1.9.tar.bz2tar xjvf htslib-1.9.tar.bz2cd htslib-1.9./configuremakemake installwget http : / / www.bioinf.uni-leipzig.de/Software/segemehl/downloads/segemehl-0.3.4.tar.gztar xzvf segemehl-0.3.4.tar.gzcd segemehl-0.3.4export PKG_CONFIG_PATH=/usr/local/lib/pkgconfig/:$PKG_CONFIG_PATHmakecp segemehl.x / usr/local/bin/

Compared with installation, the use process of the software is much simpler, and the software is divided into the following five functional modules

Align

Parse

Annotate

Assemble

Denovo

Align is used to align sequences to reference genomes; Parse is used to select junction reads;Annotate from the alignment results to predict the transcripts of circular RNA;Assemble used to assemble circular RNA; and Denovo identifies new circular RNA and analyzes variable cutting events on circular RNA according to the results of sequence assembly. The specific usage is as follows

1. Align

Although a variety of sequence alignment software is supported, because the results of tophat are more convenient for subsequent cufflinks software to analyze, it is officially recommended to use tophat for comparison. For single-ended sequence alignment, the code is as follows

CIRCexplorer2 align\-G hg19.gtf\-I bowtie1_index\-j bowtie2_index\-f RNA_seq.fastq\ > CIRCexplorer2_align.log

It is worth noting that the align module only provides the function of using tophat for comparison of single-ended sequences. If you are the result of double-ended sequencing or want to use other software, you can only compare it by hand. STAR software is recommended here, which is faster, but its disadvantage is that it consumes a lot of memory.

2. Parse

Parse is used to parse the results of sequence alignment and supports a variety of software. Take the commonly used STAR as an example, the code is as follows

CIRCexplorer2 parse\-t STAR\ Chimeric.out.junction\ > CIRCexplorer2_parse.log

For the use of other software, please refer to the official documentation. No matter what the comparison software is, the command will eventually generate the following files

Back_spliced_junction.bed

3. Annotation

This step is to identify the circular RNA based on the known linear transcript information, so we need to provide the corresponding annotation file for the reference genome, and the official script is also provided to help us download it.

Fetch_ucsc.py hg19 ref hg19_ref.txt

The code to predict the ring RNA is as follows

CIRCexplorer2 annotate\-r hg19_ref.txt\-g hg19.fa\-b back_spliced_junction.bed\-o circularRNA_known.txt\ > CIRCexplorer2_annotate.log

The-o parameter is the output, and the content is as follows

The meaning of each column is as follows

Since the next two modules can only handle the results of tophat, I'm testing with STAR, so I won't describe its usage here.

If you just want to use this software to predict ring RNA, then there are many sequence alignment software to choose from, but if you want to use full functionality, you must use tophat for comparison.

At this point, I believe you have a deeper understanding of "how to use CIRCexplorer2 to identify annular RNA". You might as well do it in practice. Here is the website, more related content can enter the relevant channels to inquire, follow us, continue to learn!

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