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Introduction

RNA-stability analysis

It has been suggested that, while exonic read counts in RNA-seq data correspond to steady-state mRNA abundance, changes in the abundance of intronic reads can be used to estimate the change in transcription rate. Through this concept, a change in exonic reads without a corresponding change in intronic reads is diagnostic of differential RNA stability, while concurrent changes in both exonic and intronic reads suggest altered transcription. DiffRAC is a framework that converts unspliced/spliced relationships into a generalized linear model whose parameters can then be inferred from sequencing count data.

Analysis


Initializing DiffRAC framework...

Estimating size factors and dispersions...

Optimizing the bias constant...
0.381966011250105 : 443084.805542457
0.618033988749895 : 451660.927947527
0.76393202250021 : 447436.811077347
0.606281593377457 : 451755.93420737
0.581622715887764 : 451809.600721294
0.587245246809309 : 451815.294125421
0.587578588893278 : 451815.288818795
0.586911904725339 : 451815.259613898
0.587245246809309 : 451815.294125421
The bias constant is 0.587245246809309

Re-estimating dispersion...

Fitting model parameters...

Differentially stabilised genes

UPF3B

Version Author Date
bff9b8f unawaz1996 2023-05-12

Comparison with DEG results

Version Author Date
bff9b8f unawaz1996 2023-05-12
Distribution

Distribution

Version Author Date
bff9b8f unawaz1996 2023-05-12
Distribution of log fold changes of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3B to Controls comparison and their distribution was plotted.

Distribution of log fold changes of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3B to Controls comparison and their distribution was plotted.

Version Author Date
bff9b8f unawaz1996 2023-05-12
Distribution of pvalue of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3B to Controls comparison and their pvalues were plotted to ensure significance of results

Distribution of pvalue of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3B to Controls comparison and their pvalues were plotted to ensure significance of results

Version Author Date
bff9b8f unawaz1996 2023-05-12

UPF3A

Version Author Date
bff9b8f unawaz1996 2023-05-12
01ff011 unawaz1996 2023-05-08

Comparison with DEGs

Version Author Date
bff9b8f unawaz1996 2023-05-12

Version Author Date
bff9b8f unawaz1996 2023-05-12
Distribution of log fold changes of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3A to Controls comparison and their distribution was plotted.

Distribution of log fold changes of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3A to Controls comparison and their distribution was plotted.

Version Author Date
bff9b8f unawaz1996 2023-05-12
Distribution of pvalue of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3A to Controls comparison and their pvalues were plotted to ensure significance of results

Distribution of pvalue of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3A to Controls comparison and their pvalues were plotted to ensure significance of results

Version Author Date
bff9b8f unawaz1996 2023-05-12

UPF3 dKD

Version Author Date
bff9b8f unawaz1996 2023-05-12

Comparison with DEGs

Version Author Date
bff9b8f unawaz1996 2023-05-12

Version Author Date
bff9b8f unawaz1996 2023-05-12
Distribution of log fold changes of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3 dKD to Controls comparison and their distribution was plotted.

Distribution of log fold changes of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3 dKD to Controls comparison and their distribution was plotted.

Version Author Date
bff9b8f unawaz1996 2023-05-12
Distribution of pvalue of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3 dKD to Controls comparison and their pvalues were plotted to ensure significance of results

Distribution of pvalue of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3 dKD to Controls comparison and their pvalues were plotted to ensure significance of results

Version Author Date
bff9b8f unawaz1996 2023-05-12

UPF3A OE

Version Author Date
bff9b8f unawaz1996 2023-05-12

UPF3A OE UPF3B KD

Version Author Date
bff9b8f unawaz1996 2023-05-12

Comparison with DEGs

Version Author Date
bff9b8f unawaz1996 2023-05-12

Version Author Date
bff9b8f unawaz1996 2023-05-12
Distribution of log fold changes of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3 dKD to Controls comparison and their distribution was plotted.

Distribution of log fold changes of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3 dKD to Controls comparison and their distribution was plotted.

Version Author Date
bff9b8f unawaz1996 2023-05-12
Distribution of pvalue of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3 dKD to Controls comparison and their pvalues were plotted to ensure significance of results

Distribution of pvalue of RNA-stability results. The genes that were significantly destabilised/stabilised were overlaapped with DEGs from UPF3 dKD to Controls comparison and their pvalues were plotted to ensure significance of results

Version Author Date
bff9b8f unawaz1996 2023-05-12

Gene set enrichment analysis

Based on the small overlap of DEGs and DSG, it might be that the genes that are differentially stabilised/destabilised are impacted as a secondary effect to NMD inhibition. It would be interesting to see what that overall data represents in terms of pathway enrichment analyses. As we are interested in seeing what gene sets are up-regulated and downregulated, we are conducting a gene set enrichment analysis.


R version 4.2.2 Patched (2022-11-10 r83330)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 22.04.2 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.10.0
LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0

locale:
 [1] LC_CTYPE=en_AU.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_AU.UTF-8        LC_COLLATE=en_AU.UTF-8    
 [5] LC_MONETARY=en_AU.UTF-8    LC_MESSAGES=en_AU.UTF-8   
 [7] LC_PAPER=en_AU.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_AU.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
 [1] grid      stats4    tools     stats     graphics  grDevices utils    
 [8] datasets  methods   base     

other attached packages:
 [1] plyr_1.8.8                    gplots_3.1.3                 
 [3] coin_1.4-2                    survival_3.5-5               
 [5] IsoformSwitchAnalyzeR_2.01.04 pfamAnalyzeR_0.99.0          
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[11] satuRn_1.6.0                  DEXSeq_1.44.0                
[13] BiocParallel_1.32.6           ggrepel_0.9.3                
[15] pander_0.6.5                  msigdbr_7.5.1                
[17] cowplot_1.1.1                 ngsReports_2.0.3             
[19] patchwork_1.1.2               VennDiagram_1.7.3            
[21] futile.logger_1.4.3           UpSetR_1.4.0                 
[23] fgsea_1.24.0                  GOplot_1.0.2                 
[25] RColorBrewer_1.1-3            gridExtra_2.3                
[27] ggdendro_0.1.23               AnnotationHub_3.6.0          
[29] BiocFileCache_2.6.1           dbplyr_2.3.2                 
[31] openxlsx_4.2.5.2              ggiraph_0.8.7                
[33] wasabi_1.0.1                  sleuth_0.30.1                
[35] DT_0.27                       VennDetail_1.14.0            
[37] msigdb_1.6.0                  GSEABase_1.60.0              
[39] graph_1.76.0                  annotate_1.76.0              
[41] XML_3.99-0.14                 pheatmap_1.0.12              
[43] ggvenn_0.1.10                 MetBrewer_0.2.0              
[45] ggpubr_0.6.0                  venn_1.11                    
[47] viridis_0.6.2                 viridisLite_0.4.1            
[49] tximeta_1.16.1                tximport_1.26.1              
[51] goseq_1.50.0                  geneLenDataBase_1.34.0       
[53] BiasedUrn_2.0.9               org.Mm.eg.db_3.16.0          
[55] EnsDb.Mmusculus.v79_2.99.0    ensembldb_2.22.0             
[57] AnnotationFilter_1.22.0       GenomicFeatures_1.50.4       
[59] AnnotationDbi_1.60.2          biomaRt_2.54.1               
[61] edgeR_3.40.2                  limma_3.54.2                 
[63] DESeq2_1.38.3                 SummarizedExperiment_1.28.0  
[65] Biobase_2.58.0                MatrixGenerics_1.10.0        
[67] matrixStats_0.63.0            GenomicRanges_1.50.2         
[69] GenomeInfoDb_1.34.9           IRanges_2.32.0               
[71] S4Vectors_0.36.2              BiocGenerics_0.44.0          
[73] corrplot_0.92                 lubridate_1.9.2              
[75] forcats_1.0.0                 purrr_1.0.1                  
[77] readr_2.1.4                   tidyverse_2.0.0              
[79] stringr_1.5.0                 tidyr_1.3.0                  
[81] scales_1.2.1                  data.table_1.14.8            
[83] readxl_1.4.2                  tibble_3.2.1                 
[85] magrittr_2.0.3                reshape2_1.4.4               
[87] ggplot2_3.4.2                 dplyr_1.1.1                  
[89] eisaR_1.10.0                  workflowr_1.7.0              

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 [61] processx_3.8.0                png_0.1-8                    
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[115] R6_2.5.1                      pillar_1.9.0                 
[117] htmltools_0.5.5               mime_0.12                    
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[123] mvtnorm_1.1-3                 utf8_1.2.3                   
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[127] curl_5.0.0                    gtools_3.9.4                 
[129] zip_2.2.2                     GO.db_3.16.0                 
[131] admisc_0.31                   rmarkdown_2.21               
[133] munsell_0.5.0                 rhdf5_2.42.0                 
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