Entering edit mode
I used to run DESeq2/edgeR a lot a few years back (as late as last year), but I have been working on other projects and haven't had need of them of late. I am just starting on a project and was wanting to do some differential expression analysis, so opened up my old scripts and started running them. However, I encounter an error when trying to specify the design of the experiment.
dds <- DESeqDataSet(RSE, design =~ type)
Error in DESeqDataSet(RSE, design = ~type) :
design has a single variable, with all samples having the same value.
use instead a design of '~ 1'. estimateSizeFactors, rlog and the VST can then be used
This is the column data for RSE. I am trying to use type, which has three different groupings, but I get the same error regardless of what I try and use.
DataFrame with 6 rows and 6 columns
fwd rev name class type
<character> <character> <data.frame> <data.frame> <data.frame>
0h-rep1 /Users/matthew/mount.. /Users/matthew/mount.. 0h-rep1 Ct Ct
0h-rep2 /Users/matthew/mount.. /Users/matthew/mount.. 0h-rep2 Ct Ct
24h-rep1 /Users/matthew/mount.. /Users/matthew/mount.. 24h-rep1 Exp Exp1
24h-rep2 /Users/matthew/mount.. /Users/matthew/mount.. 24h-rep2 Exp Exp1
96h-rep1 /Users/matthew/mount.. /Users/matthew/mount.. 96h-rep1 Exp Exp2
96h-rep2 /Users/matthew/mount.. /Users/matthew/mount.. 96h-rep2 Exp Exp2
rep
<data.frame>
0h-rep1 rep1
0h-rep2 rep2
24h-rep1 rep1
24h-rep2 rep2
96h-rep1 rep1
96h-rep2 rep2
I tried this on my old laptop where I originally ran the scripts, as I thought maybe it was a problem with using the latest version of R, but I am for some reason getting the same error there. If you have any tips/advice for where I might be going wrong that would be great!
sessionInfo( )
R version 4.1.2 (2021-11-01)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS Big Sur 11.5.2
Matrix products: default
LAPACK: /Library/Frameworks/R.framework/Versions/4.1/Resources/lib/libRlapack.dylib
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
attached base packages:
[1] grid stats4 stats graphics grDevices utils datasets
[8] methods base
other attached packages:
[1] ggrepel_0.9.1
[2] org.Hs.eg.db_3.14.0
[3] TxDb.Hsapiens.UCSC.hg38.knownGene_3.14.0
[4] BSgenome.Hsapiens.UCSC.hg38_1.4.4
[5] BSgenome_1.62.0
[6] Biostrings_2.62.0
[7] XVector_0.34.0
[8] JASPAR2016_1.22.0
[9] sva_3.42.0
[10] genefilter_1.76.0
[11] mgcv_1.8-38
[12] nlme_3.1-153
[13] edgeR_3.36.0
[14] limma_3.50.0
[15] DESeq2_1.34.0
[16] Gviz_1.38.0
[17] InteractionSet_1.22.0
[18] BiocParallel_1.28.3
[19] GenomicFeatures_1.46.1
[20] AnnotationDbi_1.56.2
[21] reshape2_1.4.4
[22] BiasedUrn_1.07
[23] statmod_1.4.36
[24] forcats_0.5.1
[25] stringr_1.4.0
[26] dplyr_1.0.7
[27] purrr_0.3.4
[28] readr_2.1.1
[29] tidyr_1.1.4
[30] tibble_3.1.6
[31] tidyverse_1.3.1
[32] ggthemes_4.2.4
[33] ggforce_0.3.3
[34] ggplot2_3.3.5
[35] magrittr_2.0.1
[36] viridis_0.6.2
[37] viridisLite_0.4.0
[38] ggseqlogo_0.1
[39] pheatmap_1.0.12
[40] kableExtra_1.3.4
[41] knitr_1.36
[42] CAGEfightR_1.14.0
[43] SummarizedExperiment_1.24.0
[44] Biobase_2.54.0
[45] MatrixGenerics_1.6.0
[46] matrixStats_0.61.0
[47] rtracklayer_1.54.0
[48] GenomicRanges_1.46.1
[49] GenomeInfoDb_1.30.0
[50] IRanges_2.28.0
[51] S4Vectors_0.32.3
[52] BiocGenerics_0.40.0
[53] session_1.0.3
loaded via a namespace (and not attached):
[1] utf8_1.2.2 tidyselect_1.1.1
[3] RSQLite_2.2.9 htmlwidgets_1.5.4
[5] munsell_0.5.0 codetools_0.2-18
[7] withr_2.4.3 colorspace_2.0-2
[9] filelock_1.0.2 highr_0.9
[11] rstudioapi_0.13 labeling_0.4.2
[13] GenomeInfoDbData_1.2.7 polyclip_1.10-0
[15] bit64_4.0.5 farver_2.1.0
[17] vctrs_0.3.8 generics_0.1.1
[19] xfun_0.28 biovizBase_1.42.0
[21] BiocFileCache_2.2.0 R6_2.5.1
[23] locfit_1.5-9.4 AnnotationFilter_1.18.0
[25] bitops_1.0-7 cachem_1.0.6
[27] DelayedArray_0.20.0 assertthat_0.2.1
[29] BiocIO_1.4.0 scales_1.1.1
[31] nnet_7.3-16 gtable_0.3.0
[33] ensembldb_2.18.2 rlang_0.4.12
[35] systemfonts_1.0.2 splines_4.1.2
[37] lazyeval_0.2.2 dichromat_2.0-0
[39] broom_0.7.10 checkmate_2.0.0
[41] BiocManager_1.30.16 yaml_2.2.1
[43] modelr_0.1.8 backports_1.4.0
[45] Hmisc_4.6-0 tools_4.1.2
[47] ellipsis_0.3.2 RColorBrewer_1.1-2
[49] Rcpp_1.0.7 plyr_1.8.6
[51] base64enc_0.1-3 progress_1.2.2
[53] zlibbioc_1.40.0 RCurl_1.98-1.5
[55] prettyunits_1.1.1 rpart_4.1-15
[57] GenomicFiles_1.30.0 haven_2.4.3
[59] cluster_2.1.2 fs_1.5.2
[61] data.table_1.14.2 reprex_2.0.1
[63] ProtGenerics_1.26.0 hms_1.1.1
[65] evaluate_0.14 xtable_1.8-4
[67] XML_3.99-0.8 jpeg_0.1-9
[69] readxl_1.3.1 gridExtra_2.3
[71] compiler_4.1.2 biomaRt_2.50.1
[73] crayon_1.4.2 htmltools_0.5.2
[75] tzdb_0.2.0 Formula_1.2-4
[77] geneplotter_1.72.0 lubridate_1.8.0
[79] DBI_1.1.1 lobstr_1.1.1
[81] tweenr_1.0.2 dbplyr_2.1.1
[83] GenomicInteractions_1.28.0 MASS_7.3-54
[85] rappdirs_0.3.3 Matrix_1.4-0
[87] cli_3.1.0 pryr_0.1.5
[89] igraph_1.2.9 parallel_4.1.2
[91] pkgconfig_2.0.3 GenomicAlignments_1.30.0
[93] foreign_0.8-81 xml2_1.3.3
[95] svglite_2.0.0 annotate_1.72.0
[97] webshot_0.5.2 rvest_1.0.2
[99] VariantAnnotation_1.40.0 digest_0.6.29
[101] rmarkdown_2.11 cellranger_1.1.0
[103] htmlTable_2.3.0 restfulr_0.0.13
[105] curl_4.3.2 Rsamtools_2.10.0
[107] rjson_0.2.20 lifecycle_1.0.1
[109] jsonlite_1.7.2 fansi_0.5.0
[111] pillar_1.6.4 lattice_0.20-45
[113] KEGGREST_1.34.0 fastmap_1.1.0
[115] httr_1.4.2 survival_3.2-13
[117] glue_1.5.1 png_0.1-7
[119] bit_4.0.4 stringi_1.7.6
[121] blob_1.2.2 latticeExtra_0.6-29
[123] memoise_2.0.1
Oh yes, I changed how I built the design table this time round and was reading in the metadata from a file rather than explicitly writing it out myself. I guess I needed to convert the type to factor before adding to the design table.
Thanks for the assist!