raw sequencing reads  (Biotechnology Information)

Journal: Science Advances

Article Title: Transcriptional readthrough precedes alternative splicing programs triggered in CML cells by imatinib

doi: 10.1126/sciadv.aea2475

Figure Lengend Snippet: ( A ) Change in SPI values from short-read sequencing of mRNA in IM 1h (top) or 18 h (bottom) relative to CTRL. Introns ( n = ~35,300) detected by at least 500 reads and with significant changes in SPI (adjusted P value < 0.05) are marked in blue (20 in IM 1h and 193 in IM 18h). Adjusted P values were determined using chi-square tests for pairs of replicates and the Benjamini-Hochberg procedure. ( B ) Significant changes in alternative splicing in cells treated with JTE or IM 18h. A3SS or A5SS, alternative 3′ or 5′ splice site; MXE, mutually exclusive events. Number of significant events [false discovery rate (FDR) < 0.05] detected by at least 20 reads and with absolute inclusion level difference to CTRL > 0.1. ( C ) Comparison of genes with RTI ≥ 0.2 (RTI) and genes with significant changes in RI and CE at the level of nuclear RNA in IM 18h. Only single intersections with RTI are presented. ( D ) Comparison of genes with significant changes in RI (left) or CE (right) in IM or JTE 18h. ( E ) Sashimi plot for EIF4H (left) and quantification of isoform usage (right) in CTRL versus JTE or IM 18h. Isoforms 1 and 2 represent the skipping and inclusion, respectively, of exon 5 (beige highlight). Percentage of transcripts belonging to each isoform in three replicates (means ± SEM). Significance based on two-tailed Student’s t test (* P < 0.05; *** P < 0.001).

Article Snippet: Long- and short-read sequencing raw data are available through the National Center for Biotechnology Information (NCBI) under GEO accession numbers: GSE283849 , GSE283813 , and GSE310243 .

Techniques: Sequencing, Alternative Splicing, Comparison, Two Tailed Test

Journal: Science Advances

Article Title: Transcriptional readthrough precedes alternative splicing programs triggered in CML cells by imatinib

doi: 10.1126/sciadv.aea2475

Figure Lengend Snippet: ( A ) Examples of reads aligned to RBM14 and RBM4 from long- and short-read sequencing of IM-treated cells. Chimeric reads marked in blue. ( B ) Sashimi plot for RBM14 and RBM4 (chromosome 11: 66616624 to 66644972) based on a single representative CTRL replicate. Only splicing events detected more than five times are shown. Black represents read coverage density in RPKM, gray lines represent splicing events within gene bodies, and the red line represents chimeric splicing. ( C ) Scheme of primer design for real-time PCR semiquantification (qPCR) of chimera expression. ( D ) Expression of selected chimeras [red primers in (C)] in CD34 + -enriched cells from three healthy donors (HD) and two patients with CML (CML), normalized to spike-in controls and presented relative to the expression level of the last exon in the upstream gene [beige primers in (C)]. ( E ) Comparison of readthrough chimeras identified by SOAPfuse analysis of mRNA-seq for CTRL or IM-treated K562 cells. ( F ) Changes in chimera transcript expression normalized to spike-in control referenced to the last exon in the upstream gene and expressed as fold change upon IM treatment. Level in CTRL as 1.0, marked with the dashed line. Significance based on two-way ANOVA test (* P < 0.05; ** P < 0.005; *** P < 0.001).

Article Snippet: Long- and short-read sequencing raw data are available through the National Center for Biotechnology Information (NCBI) under GEO accession numbers: GSE283849 , GSE283813 , and GSE310243 .

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Expressing, Comparison, Control