FAQ
Q: Which types of sequencing data are supported by CIRCexplorer2? Single or paired-end reads?
A: CIRCexplorer2 will not take advantage of information derived from paired-end reads now. So in practice, paired-end reads could be converted to single reads, and then used in CIRCexplorer2.
Q: Which treatments are required for RNA-seq?
A: If you only use the annotating pipeline, the poly(A)−/ribo− RNA-seq is recommended. If you want to enrich circular RNAs, RNase R treatment could be performed. RNA-seq with only rRNA depletion is acceptable, but it is not the best choice. If you want to use the characterization pipeline, only poly(A)−/ribo− w/o RNase R RNA-seq is acceptable. In addition, poly(A)+ RNA-seq is also required.
Q: What is the criterion to define high-expressed/high-confidence circular RNAs.
A: There is no common rule to define which circular RNAs belong to high-expressed/high-confidence circular RNAs. In practice, we use circular RNA fusion junction cutoff (RPM≥0.1, RPM: Reads Per Million mapped reads) to define them. This cutoff was used in our previous Cell paper, and it works well for our current research.
Q: If I have aligned fastq with TopHat2 in advance and don't want to align them again with CIRCexplorer2 align
, what should I do?
A: You should first convert unmapped.bam
in TopHat2 output folder into fastq format (e.g. unmapped.fq
through tools like bedtools bamtofastq). Then, you could set the --skip-tophat
option and use unmapped.fq
as <fastq>
in CIRCexplorer2 align
command.
Q: Why is there the TopHat2 alignment step in CIRCexplorer2 align
? Is this step necessary?
A: We first align reads onto genome and transcriptome using TopHat2 to reduce false positive reads aligned in the TopHat-Fusion alignment step. It is optional to skip the TopHat2 alignment step simply through set the --skip-tophat
option in in CIRCexplorer2 align
command.