circRNA SequencingNext-generation Sequencing
Service OverviewCircular RNA(circRNA) is a type of non-coding RNA, which are recently found to play vital role in regulatory networks involved in developing, environmental resistance, etc. Distinct from linear RNA molecules, e.g. mRNA, lncRNA, 3' and 5' ends of circRNA are joined together to form a circular structure, which save them from digestion of exonuclease and are more stable than most of linear RNA. CircRNA have been found to have diverse functions in regulating gene expression. CircRNA can perform as ceRNA, which binds miRNA competitively, known as miRNA sponge.
Library construction and sequencing
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CircRNA library construction:
circBase database contains circRNAs of human, mouse, Caenorhabditis.elegans, Chalumnae and Menadoensis. CircRNA is predicteds based on the GU-AG splicing signals. CircRNAs,host genes of which have same start and end sites on the same chromosome, are regarded as same circRNA. Union list of all samples is applied in down-stream analysis.
Distribution and expression level of circRNA are presented on circos diagram.
Differential expression analysis on circRNA
Differential expression analysis is processed on circRNA, which is presented in volcano plot. The differential circRNAs with statistical significancy are coloured in the plot.
Hierarchical clustering of differentially expressed circRNA
Hierarchical cluster analysis enables clustering of circRNAs which share same pattern of expression across different experimental conditions. The genes within the same cluster are more likely to have similar biological functions and be involved in the same functional pathway. Therefore, it facilitates in-depth functional analysis on differentially expressed genes. In this analysis, differentially expressed circRNA will be clustered based on their expression pattern across different groups (experimental conditions).
Differentially expressed circRNA host gene functional analysis
Generation of circRNA from a transcript leads to lost of coding capacity, which further result in distinct phenotype. In order to identify potential biological functions of the differentially expressed circRNA, their host genes are identified and processed for functional analysis including annotation and enrichment analysis.
1Can I do circRNA sequencing without reference genome?
In circRNA prediction, clean reads will be need to mapped against reference genome in order to identify break point of circle. CircRNA prediction can be much less reliable because of mis-assembly in unigenes (generated from non-reference transcriptome sequencing). Therefore, reference genome is required.
2What is the circular mechanism of circRNA?
Answer: There are 5 mechanisms in generating circRNA, including Lariat-driven circularization, RNA binding protein (RBP)-driven circularization, base-pairing driven circularization, GU-riched and C-riched formation of ciRNA, splicing enzyme-driven.
3What's the difference in library construction between circRNA and other RNA?
Answer: There are two strategies in circRNA library construction: 1. Linear RNA removal, which contains only circRNA. 2. Without removing linear RNA. This library containing circRNA, mRNA and lncRNA, which is more suitable for RNA interactions.
4What's the difference between circRNA sequencing and mircroarray?
Answer: CircRNA microarray is designed based on known circRNAs, which is very limited in terms of number and annotations. NGS based circRNA prediction is based on sequence features, which enables identification of known circRNA and prediction of novel circRNA.