CircAST

1. Introduction

CircAST v1.0.0 (CircRNAs with Alternative Spliced Transcripts) is a computational tool for circular RNA full-length assembly and quantification using RNA-Seq data with the back-spliced events detected by upstream circRNA identification tools, such as UROBORUS, CIRI2, or CIRCexplorer2. Original codes and detailed manuals are included in the downloading URL: https://github.com/xiaofengsong/CircAST/archive/v1.0.2.tar.gz.

Authors: Jing Wu (wujing@njmu.edu.cn), Xiaofeng Song (xfsong@nuaa.edu.cn)
Maintainer: Jing Wu (wujing@njmu.edu.cn)

2. Prerequisites

Software Prerequisites:

CircAST is implemented in Python2 under Linux system. Since several packages are required for using CircAST, we suggest that users install corresponding version of Anaconda(Anaconda 2.5.0 +, Python 2.7 version).

Input files:

CircAST works with three input files. A GTF annotation file, a sorted Sequence Alignment/Map (SAM) of pair-end reads generated by tophat2 and samtools, and a circRNA junction list containing the information of the back-spliced junctions which is generated by upstream identification tools such as CIRCexplorer2/CIRI2/UROBORUS or other similar tools.

GTF annotataion files can be downloaded from https://hgdownload.soe.ucsc.edu/downloads.html (UCSC source is recommanded, such as hg19 and mm10).

SAM file sotsorted by name can be obtained by the following commands:

tophat -o results -p 12 -G /home//genes_homo.gtf /home//index/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome reads_1.fastq reads_2.fastq
samtools sort -n -o accepted_hits.sorted.bam accepted_hits.bam
samtools view accepted_hits.sorted.bam > accepted_hits.sorted.sam

CircRNA lists containing back-spliced junctions can be obtained by CIRCexplorer2/CIRI2/UROBORUS, circ_out/denovo/annotated_circ.txt, result.txt, or circRNA_list.txt. CircRNA lists transformed into such formats using other circRNA back-spliced site detection software are also also feasible.

3. Usage

Command:
    python CircAST.py -L length -T thresh -G GTF -F accepted_hits.sorted.sam -J junction.txt
Options:
-L, --length
      reads length
-T, --threshold
      set threshold for back-splicing junction supporting reads(optional, default: 10)  
-G, --gtf
      gene annotation file (*.gtf file)
-F, --file
      SAM alignment of pair-end reads generated by tophat2 and should be sorted by name
-J, --junction
      circRNA junction list generated by CIRCexplorer2, CIRI, UROBORUS or other tools in similar format
-H, --help
          show this help information

4. Note

The accepted_hits.bam file generated by tophat2 should be sorted by name first, then be converted to accepted_hits.sorted.sam file.

CircAST is compatible with popular upstream circRNA detection tools (CIRCexplorer2, CIRI2, UROBORUS). Other circRNA junction detection tools can also be used in CircAST if their output file format is transformed to CircAST input format files.

CircAST use the GTF annotation file with UCSC compatable GTF format.

The GTF annotation file should be the same one when running CircAST and its upstream softwares.

5. Example

python CircAST.py -L 100 -T 5 -G /home/***/circRNA/Gene/genes.gtf -F accepted_hits.sorted.sam -J circRNA_list.txt

6. Output file format

The first 3 columns are the same with bed file format.
1) Chromosome
2) Start of circular RNA
3) End of circular RNA
4) Name of Circular transcript
5) Parental gene name
6) Strand
7) Number of exons
8) Exon sizes
9) Exon offsets
10) Read counts (junction reads)
11) Expression level (FPKM)
12) Expression level (TPM)
13) Read counts (assigned reads)
Columns of output file are split by tabs ("\t" in python).

7. Additional tool

CircFullSeq.py
    It is designed for extracting the full sequence of circular transctipts in the CircAST result.  
Command:
    python CircFullSeq.py -F /home/**/Chromosomes_homo -I CircAST_result.txt
Options:
    -F, --fafolder
          fa file folder
    -I, --input
          input file
        -O,     --output
              output file
Note that the fa file folder should include all the decompressed file of chromFa.tar.gz, which can be downloaded from UCSC.

8. Reference

Wu J, Li Y, Wang C, et al. CircAST: Full-length Assembly and Quantification of Alternatively Spliced Isoforms in Circular RNAs. Genomics, Proteomics & Bioinformatics, 2020.

9. Contact

Please contact Jing Wu (wujing@njmu.edu.cn) for questions and comments.

xiaofengsong / CircAST

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