SpliceWiz: about res_edgeR result only contain events from chromosome 1
Entering edit mode
yyu109 • 0
Last seen 5 days ago
United States


I am following the vignette (https://bioconductor.org/packages/devel/bioc/vignettes/SpliceWiz/inst/doc/SW_QuickStart.html).

The reference files I used are "Homo_sapiens.GRCh38.111.gtf" and "Homo_sapiens.GRCh38.dna.primary_assembly.fa" from Ensembl. They contain information for all the chromosomes.

In the pb_output folder, I can see that there are reads and hits in every chromsome.

However, after the differential analysis, I can only see events detected in chromosome 1.

se_2 <- makeSE(nxtse_path_2)

# Assigning annotations to samples
colData(se_2)$condition <- rep(c("Control", Treated"), each = 3)
colData(se_2)$batch <- rep(c("1", "2", "3"), 2)

# Filtering high-confidence events
se_2.filtered <- se_2[applyFilters(se_2),]

# Performing differential analysis
res_edgeR_2 <- ASE_edgeR(
    se = se_2.filtered,
    test_factor = "condition",
    test_nom = "Treated",
    test_denom = "Control"

For example:

# Check event region
[1] "1:151346607-151347210/-" "1:161043361-161045857/-" "1:154602627-154627953/-" "1:154602627-154627854/-" "1:12580667-12617153/-"

All the EventRegions are started by 1:.

I would like to ask where seems to be the problem? Is there a way to check into se_2 or other dataset to see if the differential analysis was properly performed?

Thank you so much for any help!



R version 4.3.2 (2023-10-31)
Platform: aarch64-apple-darwin20 (64-bit)
Running under: macOS Sonoma 14.3

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.11.0

[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: America/New_York
tzcode source: internal

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] edgeR_4.0.9      limma_3.58.1     fstcore_0.9.18   SpliceWiz_1.4.1  NxtIRFdata_1.8.0

loaded via a namespace (and not attached):
  [1] splines_4.3.2                 later_1.3.2                   BiocIO_1.12.0                 bitops_1.0-7                 
  [5] filelock_1.0.3                tibble_3.2.1                  R.oo_1.25.0                   XML_3.99-0.16                
  [9] lifecycle_1.0.4               lattice_0.22-5                dendextend_1.17.1             magrittr_2.0.3               
 [13] plotly_4.10.4                 sass_0.4.8                    rmarkdown_2.25                jquerylib_0.1.4              
 [17] yaml_2.3.8                    httpuv_1.6.13                 DBI_1.2.0                     RColorBrewer_1.1-3           
 [21] abind_1.4-5                   zlibbioc_1.48.0               rvest_1.0.3                   GenomicRanges_1.54.1         
 [25] purrr_1.0.2                   R.utils_2.12.3                BiocGenerics_0.48.1           RCurl_1.98-1.14              
 [29] rappdirs_0.3.3                seriation_1.5.4               GenomeInfoDbData_1.2.11       IRanges_2.36.0               
 [33] S4Vectors_0.40.2              genefilter_1.84.0             pheatmap_1.0.12               annotate_1.80.0              
 [37] DelayedMatrixStats_1.24.0     codetools_0.2-19              DelayedArray_0.28.0           DT_0.31                      
 [41] xml2_1.3.6                    tidyselect_1.2.0              rhandsontable_0.3.8           viridis_0.6.4                
 [45] TSP_1.2-4                     shinyWidgets_0.8.1            matrixStats_1.2.0             stats4_4.3.2                 
 [49] BiocFileCache_2.10.1          webshot_0.5.5                 GenomicAlignments_1.38.1      jsonlite_1.8.8               
 [53] fst_0.9.8                     ellipsis_0.3.2                survival_3.5-7                iterators_1.0.14             
 [57] foreach_1.5.2                 tools_4.3.2                   progress_1.2.3                Rcpp_1.0.12                  
 [61] glue_1.7.0                    gridExtra_2.3                 SparseArray_1.2.3             xfun_0.41                    
 [65] MatrixGenerics_1.14.0         GenomeInfoDb_1.38.5           dplyr_1.1.4                   ca_0.71.1                    
 [69] HDF5Array_1.30.0              shinydashboard_0.7.2          withr_3.0.0                   BiocManager_1.30.22          
 [73] fastmap_1.1.1                 rhdf5filters_1.14.1           fansi_1.0.6                   digest_0.6.34                
 [77] R6_2.5.1                      mime_0.12                     colorspace_2.1-0              GO.db_3.18.0                 
 [81] RSQLite_2.3.4                 R.methodsS3_1.8.2             utf8_1.2.4                    tidyr_1.3.0                  
 [85] generics_0.1.3                data.table_1.14.10            rtracklayer_1.62.0            prettyunits_1.2.0            
 [89] httr_1.4.7                    htmlwidgets_1.6.4             S4Arrays_1.2.0                pkgconfig_2.0.3              
 [93] gtable_0.3.4                  blob_1.2.4                    registry_0.5-1                XVector_0.42.0               
 [97] htmltools_0.5.7               scales_1.3.0                  Biobase_2.62.0                ompBAM_1.6.0                 
[101] Rsubread_2.16.0               png_0.1-8                     knitr_1.45                    rstudioapi_0.15.0            
[105] rjson_0.2.21                  curl_5.2.0                    cachem_1.0.8                  rhdf5_2.46.1                 
[109] BiocVersion_3.18.1            parallel_4.3.2                AnnotationDbi_1.64.1          restfulr_0.0.15              
[113] pillar_1.9.0                  grid_4.3.2                    vctrs_0.6.5                   promises_1.2.1               
[117] shinyFiles_0.9.3              dbplyr_2.4.0                  xtable_1.8-4                  evaluate_0.23                
[121] cli_3.6.2                     locfit_1.5-9.8                compiler_4.3.2                Rsamtools_2.18.0             
[125] rlang_1.1.3                   crayon_1.5.2                  heatmaply_1.5.0               fs_1.6.3                     
[129] stringi_1.8.3                 viridisLite_0.4.2             BiocParallel_1.36.0           assertthat_0.2.1             
[133] munsell_0.5.0                 Biostrings_2.70.1             lazyeval_0.2.2                Matrix_1.6-5                 
[137] BSgenome_1.70.1               hms_1.1.3                     patchwork_1.2.0               sparseMatrixStats_1.14.0     
[141] bit64_4.0.5                   ggplot2_3.4.4                 Rhdf5lib_1.24.1               KEGGREST_1.42.0              
[145] statmod_1.5.0                 shiny_1.8.0                   SummarizedExperiment_1.32.0   interactiveDisplayBase_1.40.0
[149] AnnotationHub_3.10.0          memoise_2.0.1                 bslib_0.6.1                   bit_4.0.5
SpliceWiz • 223 views
Entering edit mode
a.wong ▴ 10
Last seen 27 days ago

Although not fully documented, one can view the reference that accompanies the NxtSE object by calling the following:

embedded_ref <- ref(se_2)

This returns a list containing a seqInfo object and a number of data tables that summarizes the reference that is used to build the NxtSE object

[1] "seqInfo"     "geneList"    "elements"    "transcripts" "spliceList"  "IRList"     
[7] "ontology"

To verify the chromosomes covered, simply view the seqInfo object:


The fact that only chomosome 1 appears in differential analysis highly suggests that the source reference only contains annotations for chromosome 1, and has nothing to do with the differential analysis steps.

One can confirm this by viewing the genes in the source reference:

geneList <- viewGenes("/path/to/Reference")

Hope this helps!

Entering edit mode

Thank you so much for your help! It turns out that the reference fasta file downloaded from ensembl is somehow truncated. Problem solved when I used the link instead of downloading it to the local environment.


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