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Biclustering of transcriptome sequencing data reveals human tissue-specific circular RNAs
© The Author(s). 2018
Published: 19 January 2018
Emerging evidence has been experimentally confirmed the tissue-specific expression of circRNAs (circRNAs). Global identification of human tissue-specific circRNAs is crucial for the functionality study, which facilitates the discovery of circRNAs for potential diagnostic biomarkers.
In this study, circRNA back-splicing junctions were identified from 465 publicly available transcriptome sequencing samples. The number of reads aligned to these identified junctions was normalized with the read length and sequence depth for each sample. We generated 66 models representing enriched circRNAs among human tissue transcriptome through biclustering algorithm. The result provides thousands of newly identified human tissue-specific circRNAs.
This result suggests that expression of circRNAs is not prompted by random splicing error but serving molecular functional roles. We also identified circRNAs enriched within circulating system, which, along with identified tissue-specific circRNAs, can serve as potential diagnostic biomarkers.
Circular RNAs (circRNAs) are a type of long non-coding RNAs, whose 3′ and 5′ ends joined into a single strand circular form. Although the existence of human circRNAs has been discovered and proven with electron microscopy for more than 30 years , it was only until 2012 with the advance of high throughput sequencing technology the ubiquitous expression of circRNA in mammals was found and proven . Emerging evidence indicates the tissue-specific circRNAs play crucial roles in post-transcriptional level. Several cases of human circRNAs were found to serve as natural microRNA sponges [3, 4]. Biogenesis of circRNAs was found competing with the mRNAs of the host gene. In recent years, cell-free circRNAs were found in salvia and blood plasma [5, 6]. CircRNAs can potentially serve as diagnostic biomarkers for the undercover correlation to the pathogenesis of diseases and human physiological functions, as well as the stable circular forms. Global identification of human tissue-specific circRNAs is crucial for the study of circRNAs functionality.
The junctions between the 3′ and 5′ ends of the circRNAs have been referred as back-splicing junctions. The existence of circRNA within transcriptome sequencing data can be detected through identification of reads spanning these junctions. In previous studies , threshold applied to identify certain junctions as circRNA was that at least two unique reads spanning a head-tail junction. To discover human tissue-specific circRNAs, we collected 465 human transcriptome sequencing runs and applied the established pipeline. Expression level of circRNAs was estimated using the normalized counts of reads spanning the back-splicing junctions . Biclustering  was conducted to detect circRNA expression patterns across different types of human tissues. From the result 66 bicluster models, we found a huge portion of circRNAs express only in the specific tissue type. This result suggests that expression of circRNAs is not prompted by random splicing error but serving molecular functional roles. We also identified circRNAs enriched within circulating system, which, along with identified tissue-specific circRNAs, can serve as potential diagnostic biomarkers.
CircRNAs can be identified using poly A enriched RNA-Seq data
It had been assumed that since the exon originated circRNAs does not go through polyadenylation process after transcribed and spliced, they cannot be identified in the ploy-A enriched RNA-Seq data. However in several recent studies [2, 10, 11] circRNAs were identified in poly-A enriched RNA-Seq data sets, this could be due to the fact that some circular isoforms of the gene are adenine-rich. In this study, we discovered 24,589 unique back-splicing junctions from the 376 selected poly-A enriched RNA-Seq runs. One of the pivotal circRNA cdr1as [3, 4], which was proven to be nature miRNA miR-7 sponge, was found in 107 of our selected runs. Among these runs 71 are poly-A enriched.
Novel back-spicing junctions
Compared with human circRNAs reported in 22 recent studies [4, 6, 7, 12–29], we found 5680 identified circRNAs back-splicing junctions has been reported in other studies. The remaining 92,015 unique back-splicing junctions are considered as novel circRNA candidates. Isoform annotation and the expression profiling can be found in the data base CircNet .
As illustrated in Fig. 1, the biclustering result provides thousands of tissue-specific expressed circRNAs. The nodes containing lower than 10 circRNAs, or connects to more than 3 types of tissues were hidden in the graph. The network demonstrates that circRNA co-expression profile following specific patterns similar to human genes . Some groups of circRNAs express in multiple types of tissues with close correlated function. For example, the 332 circRNAs grouped with bowel, colon and large intestine might have potential physiological roles in the digest system, while the 243 circRNAs enriched in prostate and thyroid might correlate with male reproducing or development. The large amount of circRNA enriched in blood or blood cell samples suggests the ubiquity of circulating circRNAs, which makes circRNAs ideal diagnostic biomarkers. The tissue-specific circRNAs is available in (Additional file 3).
Brain-specific circRNA host genes are enriched with synaptic GO terms
Summary of the putative biomarkers
GO:0031175|neuron projection development
Potential diagnostic biomarkers revealed from the results of the biclustering
Summary of the putative biomarkers
In this study, we identified the potential tissue specific circRNA through conducting biclustering on expression profiles of circRNA across multiple human tissue samples. Despite the promising results, several limitations are worth-mentioning.
First of all, RNA-Seq data set collected in this study are retroactive sourced. Potential Batch effect was inevitable. Expression profiles within poly-A enriched samples were also biased. On the other hand, the expression profile was based on the normalized count of back spliced junction site spanning reads. Without accurate annotation of the full-length sequence of circRNA, this kind of measurement can be limited. Finally, the gene set enrichment conducted in this study was based on the genes locus that intersect with back spliced junction sites. This analysis was based on the assumption that functions of circRNAs correlate with the functions of back spliced junction sites overlapped genes. Weather tissue specific genes correlate with the biogenesis of tissue specific circRNA Is also an ongoing research subject. Further analysis of the issue specific circRNA locus correlation with known tissue specific genes should be conducted in the near future.
From the result 66 bicluster models, we found a huge portion of circRNAs express only in the specific tissue type. This result suggests that expression of circRNAs is not prompted by random splicing error but serving molecular functional roles. We also identified circRNAs enriched within circulating system, which, along with identified tissue-specific circRNAs, can serve as potential diagnostic biomarkers after sufficient experiment verification as well as population studies.
The junction sites with standard deviation of SRPBM among the 465 runs larger than 10 were further selected for biclustering analysis. For searching the coherent expression profiles. The R package ‘isa2’  was used for Iterative Signature Algorithm analysis. Coherent expression pro-files of selected 30000 junctions among the 465 runs was acquired from the bicluster models generated from Iterative Signature Algorithm . A network analysis was conducted on the grouped junctions, and the network was illustrated through Cytoscape . Gene sets enrichment of the circRNA host genes was conducted through DAVID . Back spliced junction sites clustered into models containing only one types of tissue were considered as tissue-specific circRNAs originated.
The authors would like to thank the Ministry of Science and Technology, the National Chiao Tung University and Ministry of Education, Taiwan, and University System of Taiwan (VGHUST), for financially supporting this research.
Publication charges for this work were supported by the Ministry of Science and Technology, Taiwan [MOST103-2628-B-009-001-MY3, MOST105-2627-M-009-007-, MOST105-2319-B-400-002-, MOST104-2911-I-009-509 and MOST 106-2633-B-009-001]. The research reported in this paper was mainly supported by “Aiming for the Top University Program” of the National Chiao Tung University and Ministry of Education, Taiwan, R.O.C.
Availability of data and materials
About this supplement
This article has been published as part of BMC Genomics Volume 19 Supplement 1, 2018: 16th International Conference on Bioinformatics (InCoB 2017): Genomics. The full contents of the supplement are available online at https://bmcgenomics.biomedcentral.com/articles/supplements/volume-19-supplement-1.
This study was proposed by YCL, under the supervision of HDH and CCL. YCL performed the circRNA detection and data collection. The biclustering was conducted by YJC, JRL and CHS. All authors participated in the audition and revision of the manuscript. All authors read and approved the final manuscript.
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