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Additional file 12. Figure S7. Correlation of expression between 24 the most abundant circRNAs and their host genes. (A) CircRNAs with expression significantly correlated with the expression of host genes (Pearson correlation, p < 0.05). (B) CircRNAs with expression independent of their host genes (Pearson correlation, p > 0.05).

Additional file 1. Figure S1. Flowchart for de novo identification of circRNAs in human heart.

Additional file 9. Figure S5. Comparison of circRNAs with 2 unique back-spliced reads observed only once in this study with human circRNAs reported by 3 previous studies and 4 circRNA databases. (A) Four-set venn diagram showing the number of over-lapping circRNAs between the 12,099 circRNAs with 2 unique back-spliced reads observed only in one sample in this study, and human cardiac circRNAs reported by 3 previous studies. (B) Five-set venn diagram showing the number of over-lapping circRNAs between the 12,099 circRNAs with 2 unique back-spliced reads observed only in one sample in this study, and human circRNAs reported by 4 circRNA databases. (C) Three-set venn diagram showing the number of over-lapping circRNAs between the 12,099 circRNAs with 2 unique back-spliced reads observed only in one sample in this study, and 24,256 and 263,738 non-redundant circRNAs obtained by merging circRNAs reported by study of Werfel et al. (n = 16,427), Tan et al. (n = 15,303), and Van et al. (n = 8878), as well as 4 circRNA datasets including circBase (n = 91,986), circBank (n = 140,331), circRNADB (n = 32,883), and CIRCpedia (n = 183,943).

Additional file 11. Figure S6. Comparison of the 2461 high-abundance circRNAs with reported human, mouse and rat circRNAs. (A) Three-set venn diagram showing the number of over-lapping circRNAs between the 2461 high-abundance circRNAs identified in this study, and 24,256 and 263,738 non-redundant circRNAs obtained by merging circRNAs reported by study of Werfel et al. (n = 16,427), Tan et al. (n = 15,303), and Van et al. (n = 8,878), as well as 4 circRNA datasets including circBase (n = 91,986), circBank (n = 140,331), circRNADB (n = 32,883), and CIRCpedia (n = 183,943). (B) Three-set venn diagram showing the number of over-lapping circRNAs between the 2461 high-abundance circRNAs identified in this study, and 61,174 and 20,450 reported mouse and rat circRNAs. The 61,174 non-redundant mouse circRNAs were obtained by merging circRNAs reported by study of Werfel et al. (n = 9474), Jakobi et al. (n = 561), and Tan et al. (n = 2632), as well as 2 circRNA databases including circBase (n = 1756) and CIRCpedia V2 (n = 54,274). The 20,450 non-redundant rat circRNAs were obtained by merging circRNAs reported by study of Werfel et al. (n = 12,256) and CIRCpedia V2 (n = 17,083).

Additional file 4. Figure S2. Validation of the public RNA-seq dataset. (A) Principal Component Analysis (PCA) of the 10 samples (5 normal and 5 DCM hearts) using expression of genes with raw count > 10 in at least 5 samples. (B) Expression of NPPA in normal and DCM hearts from the RNA-seq dataset. PFKM: Fragments Per Kilobase of exon per Million fragments mapped. *p < 0.05 (edgeR).

Additional file 8. Figure S4. Relationship between numbers of circRNAs and exons of their host genes. (A) Schematic diagram of all the circRNAs originated from TTN and (B) RYR2 gene. Arrows point to the transcriptional directions of TTN and RYR2 genes. (C) The numbers of circRNAs increased proportionally with the numbers of exons per gene (binned to 10). (D) Distribution of exon numbers to produce circRNAs.

Additional file 15. Figure S8. Original uncropped PCR gel picture (related to Fig. 5a)

Additional file 7. Figure S3. Analysis of artifactual circRNAs from MYH6 and MYH7 gene. (A) Schematic diagram showing a number of rt-circRNAs originate from MYH6 and MYH7 genes in human heart identified in this study, study of Tan et al. [27] and Werfel et al. [29], and in (B) mouse heart identified in this study and study of Werfel et al. [29], as well as in (C) rat heart revealed in the study of Werfel et al. [29]. Arrows point to the transcriptional directions of MYH6 and MYH7 genes. (D) Heatmap illustrates the rank of the average expression across all studied samples for circRNAs produced from MYH6 and MYH7 genes in this study, and in human, mouse and rat hearts as identified by Tan et al. [27] and Werfel et al. [29]. The numbers in the colored boxes indicate the rank of the circRNAs within the indicated species. (E) Representative example of circMYH6_e35:MYH7_e37 that is likely artifact. The upper schematic diagram illustrating the formation of circMYH6_e35:MYH7_e37 due to read misalignment. The exon 35 of MYH6 (red, the donor exon) and exon 36 of MYH7 (yellow, upstream of the acceptor exon) are homologous. The exon 36 of MYH6 (green, downstream of the donor exon) and exon 37 of MYH7 (blue, the acceptor exon) are highly homologous. Considering a chimeric sequencing read that is supposedly to be aligned back to exon 35 (red line without arrow) and 36 (green line with arrow) of MYH6, due to the high sequence similarity, the down-stream part of the read (green line with arrow) may be misaligned to exon 37 of MYH7 (blue line with arrow), leading to the artifactual identification of back-spliced junction, and versa vice. Dashed lines indicate splicing junctions. The bottom panel shows the sequence alignment of linear MYH6_e35:MYH6_e36, MYH7_e36:MYH7_e37, and the predicted sequence of the circular MYH6_e35:MYH7_e37.