Hi all, appreciate any assistance with using Bioconductor's ReportingTools, I'm running into a couple of errors and my Google skills have failed me.
I'm passing in a DESeqResults object into ReportingTools::publish() after first having populated the results object with SYMBOL and ENTREZID from the accompanying ENSEMBL ids using the AnnotationDb::mapIds function.
Unfortunately, I'm getting the following error when I attempt to publish the DESeqResults object and assign annotations:
Error: Ids do not appear to be the correct key for this annotation database.
My results code:
resAmi <- results(dds, contrast = c("Drug","Amitriptyline","DMSO"),alpha=alphaValue, lfcThreshold = 0.58)
resAmi <- subset(resAmi, padj < 0.1
Although I understand the next step is not essential or a hinderance when it comes to generating the HTML reports, I've included it for the sake of completeness: Assigning entrez and symbol IDs using the Ensembl IDs:
ens.str <- substr(rownames(resAmi), 1, 15)
resAmi$symbol <- mapIds(org.Hs.eg.db, keys=ens.str, column="SYMBOL", keytype="ENSEMBL", multiVals="first")
resAmi$entrez <- mapIds(org.Hs.eg.db, keys=ens.str, column="ENTREZID", keytype="ENSEMBL", multiVals="first")
This results in the following DESeqResults object:
baseMean log2FoldChange lfcSE stat pvalue padj symbol
<numeric> <numeric> <numeric> <numeric> <numeric> <numeric> <character>
ENSG00000100053 1.851747 -17.2457 3.50063 -4.76078 1.92847e-06 8.94392e-03 CRYBB3
ENSG00000112473 371.466157 23.8466 5.56999 4.17713 2.95207e-05 8.45537e-02 SLC39A7
ENSG00000127362 0.876239 -19.3906 2.71788 -6.92106 4.48269e-12 1.15555e-07 TAS2R3
ENSG00000205238 1.808115 16.5194 3.35897 4.74532 2.08176e-06 8.94392e-03 SPDYE2
ENSG00000234651 119.459775 18.6629 3.55231 5.09046 3.57190e-07 2.30191e-03 BAG6
ENSG00000284548 24.447361 19.3357 4.10352 4.57065 4.86220e-06 1.56672e-02 METTL9
ENSG00000285238 38.491655 -28.3539 4.34045 -6.39884 1.56558e-10 2.01787e-06 NA
ENSG00000285332 10.242617 16.2567 2.54002 6.17189 6.74790e-10 5.79824e-06 NA
ENSG00000286139 1.425090 -16.6164 3.44713 -4.65212 3.28545e-06 1.20989e-02 ARHGAP11B
entrez
<character>
ENSG00000100053 1417
ENSG00000112473 7922
ENSG00000127362 50831
ENSG00000205238 441273
ENSG00000234651 7917
ENSG00000284548 51108
ENSG00000285238 NA
ENSG00000285332 NA
ENSG00000286139 89839
Notice the two NAs in the entrez ID column, I'm not sure what to do with those!
I then go on to publish, ignoring plots and annotation:
des2Report <- HTMLReport(shortName="RNAseq_analysis_with_DESeq2", title="RNA-seq analysis of differential expression using DESeq2 of human iPSC cells exposed to Amitriptyline.", reportDirectory = "./reports/amitriptyline/")
publish(resAmiBak, des2Report, DataSet=dds)
URL <- finish(des2Report)
browseURL(URL)
This works fine. However, I would like URL links using gene annotations and I would like the stripplots showing the expression values for each transcript from the DataSet (dds).
Firstly when I just call:
publish(resAmi, des2Report, lfc=0.58, make.plots=TRUE, DataSet=dds)
I see a table report but there are no expression stripplots. If I then try to include annotation terms:
publish(resAmi, des2Report, annotation.db="org.Hs.eg.db", lfc=0.58, make.plots=TRUE, DataSet=dds)
I'm faced with the error: Ids do not appear to be the correct key for this annotation database.
I'm not 100% certain what publish() uses for the annotation matches, so have also tried manually retrieved the entrez IDs from annotation.db and using them as row names in the DESeqResult object. I took out all instances where the entrez ID was NA. This still throws the same error.
My sessionInfo():
R version 4.0.2 (2020-06-22)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19041)
Matrix products: default
locale:
[1] LC_COLLATE=English_United Kingdom.1252 LC_CTYPE=English_United Kingdom.1252
[3] LC_MONETARY=English_United Kingdom.1252 LC_NUMERIC=C
[5] LC_TIME=English_United Kingdom.1252
attached base packages:
[1] parallel stats4 stats graphics grDevices utils datasets methods base
other attached packages:
[1] ReportingTools_2.29.0 knitr_1.29 org.Hs.eg.db_3.11.4
[4] AnnotationDbi_1.51.3 RColorBrewer_1.1-2 pheatmap_1.0.12
[7] genefilter_1.71.0 magrittr_1.5 dplyr_1.0.1
[10] GGally_2.0.0 tidyr_1.1.1 broom_0.7.0
[13] ggplot2_3.3.2 vsn_3.57.0 DESeq2_1.29.8
[16] SummarizedExperiment_1.19.6 DelayedArray_0.15.4 matrixStats_0.56.0
[19] Matrix_1.2-18 Biobase_2.49.0 GenomicRanges_1.41.5
[22] GenomeInfoDb_1.25.10 IRanges_2.23.10 S4Vectors_0.27.12
[25] BiocGenerics_0.35.4 tximeta_1.7.7
loaded via a namespace (and not attached):
[1] backports_1.1.8 GOstats_2.55.0 Hmisc_4.4-0
[4] AnnotationHub_2.21.2 BiocFileCache_1.13.1 plyr_1.8.6
[7] lazyeval_0.2.2 GSEABase_1.51.1 splines_4.0.2
[10] BiocParallel_1.23.2 digest_0.6.25 ensembldb_2.13.1
[13] htmltools_0.5.0 GO.db_3.11.4 fansi_0.4.1
[16] checkmate_2.0.0 memoise_1.1.0 BSgenome_1.57.5
[19] cluster_2.1.0 limma_3.45.10 Biostrings_2.57.2
[22] annotate_1.67.0 R.utils_2.9.2 ggbio_1.37.0
[25] askpass_1.1 bdsmatrix_1.3-4 prettyunits_1.1.1
[28] jpeg_0.1-8.1 colorspace_1.4-1 blob_1.2.1
[31] rappdirs_0.3.1 apeglm_1.11.1 xfun_0.16
[34] crayon_1.3.4 RCurl_1.98-1.2 jsonlite_1.7.0
[37] tximport_1.17.3 graph_1.67.1 VariantAnnotation_1.35.3
[40] survival_3.2-3 glue_1.4.1 gtable_0.3.0
[43] zlibbioc_1.35.0 XVector_0.29.3 Rgraphviz_2.33.0
[46] scales_1.1.1 mvtnorm_1.1-1 DBI_1.1.0
[49] edgeR_3.31.4 Rcpp_1.0.5 htmlTable_2.0.1
[52] xtable_1.8-4 progress_1.2.2 emdbook_1.3.12
[55] foreign_0.8-80 bit_4.0.4 OrganismDbi_1.31.0
[58] preprocessCore_1.51.0 Formula_1.2-3 AnnotationForge_1.31.2
[61] htmlwidgets_1.5.1 httr_1.4.2 acepack_1.4.1
[64] ellipsis_0.3.1 R.methodsS3_1.8.0 pkgconfig_2.0.3
[67] reshape_0.8.8 XML_3.99-0.5 farver_2.0.3
[70] nnet_7.3-14 dbplyr_1.4.4 locfit_1.5-9.4
[73] tidyselect_1.1.0 labeling_0.3 rlang_0.4.7
[76] reshape2_1.4.4 later_1.1.0.1 munsell_0.5.0
[79] BiocVersion_3.12.0 tools_4.0.2 cli_2.0.2
[82] generics_0.0.2 RSQLite_2.2.0 evaluate_0.14
[85] stringr_1.4.0 fastmap_1.0.1 yaml_2.2.1
[88] bit64_4.0.2 purrr_0.3.4 AnnotationFilter_1.13.0
[91] RBGL_1.65.0 mime_0.9 R.oo_1.23.0
[94] biomaRt_2.45.2 compiler_4.0.2 rstudioapi_0.11
[97] png_0.1-7 curl_4.3 interactiveDisplayBase_1.27.5
[100] affyio_1.59.0 PFAM.db_3.11.4 tibble_3.0.3
[103] geneplotter_1.67.0 stringi_1.4.6 highr_0.8
[106] GenomicFeatures_1.41.2 lattice_0.20-41 ProtGenerics_1.21.0
[109] vctrs_0.3.2 pillar_1.4.6 lifecycle_0.2.0
[112] BiocManager_1.30.10 data.table_1.13.0 bitops_1.0-6
[115] httpuv_1.5.4 rtracklayer_1.49.5 hwriter_1.3.2
[118] latticeExtra_0.6-29 R6_2.4.1 affy_1.67.0
[121] promises_1.1.1 gridExtra_2.3 dichromat_2.0-0
[124] MASS_7.3-51.6 assertthat_0.2.1 openssl_1.4.2
[127] Category_2.55.0 withr_2.2.0 GenomicAlignments_1.25.3
[130] Rsamtools_2.5.3 GenomeInfoDbData_1.2.3 hms_0.5.3
[133] rpart_4.1-15 grid_4.0.2 coda_0.19-3
[136] rmarkdown_2.3 biovizBase_1.37.0 bbmle_1.0.23.1
[139] base64enc_0.1-3 numDeriv_2016.8-1.1 shiny_1.5.0
