Hi, I have been using DESeq2 in RStudio for a long time. I started having problems after updating to R 3.6.0. When using the standard function DESeqDataSetFromTximport like so:

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dds <- DESeq2::DESeqDataSetFromTximport( txi, design, ~treatment+genotype+timepoint) using counts and average transcript lengths from tximport Warning message: In DESeqDataSet(se, design = design, ignoreRank) : some variables in design formula are characters, converting to factors

Where: - txi is a Tximport object: ' ' '

str(txi) List of 4 $ abundance : num [1:35764, 1:150] 0 3.03 14.25 0 0 ... ..- attr(, "dimnames")=List of 2 .. ..$ : chr [1:35764] "Bradi0012s00100" "Bradi0012s00201" "Bradi0014s00100" "Bradi0135s00100" ... .. ..$ : chr [1:150] "A1" "D1" "A3" "B3" ... $ counts : num [1:35764, 1:150] 0 23 228 0 0 ... ..- attr(, "dimnames")=List of 2 .. ..$ : chr [1:35764] "Bradi0012s00100" "Bradi0012s00201" "Bradi0014s00100" "Bradi0135s00100" ... .. ..$ : chr [1:150] "A1" "D1" "A3" "B3" ... $ length : num [1:35764, 1:150] 1481 1340 2820 926 791 ... ..- attr(, "dimnames")=List of 2 .. ..$ : chr [1:35764] "Bradi0012s00100" "Bradi0012s00201" "Bradi0014s00100" "Bradi0135s00100" ... .. ..$ : chr [1:150] "A1" "D1" "A3" "B3" ... $ countsFromAbundance: chr "no" - attr(, "class")= chr "S4" ' ' '

• design is a metadata table: genotype timepoint treatment libType A1 Bd21 1 22.5 single-end D1 Bd21 1 22.5 single-end A3 Bd21 1 90 single-end B3 Bd21 1 90 single-end C3 Bd21 1 90 single-end D3 Bd21 1 90 single-end

But: the result object appears as "Object with null pointer"

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str(dds) Formal class 'DESeqDataSet' [package "DESeq2"] with 8 slots ..@ design :Class 'formula' language ~treatment + genotype + timepoint .. .. ..- attr(, ".Environment")=<environment: r_globalenv=""> ..@ dispersionFunction:function ()
..@ rowRanges :Formal class 'CompressedGRangesList' [package "GenomicRanges"] with 5 slots .. .. ..@ unlistData :Formal class 'GRanges' [package "GenomicRanges"] with 7 slots .. .. .. .. ..@ seqnames :Formal class 'Rle' [package "S4Vectors"] with 4 slots .. .. .. .. .. .. ..@ values : Factor w/ 0 levels: .. .. .. .. .. .. ..@ lengths : int(0) .. .. .. .. .. .. ..@ elementMetadata: NULL .. .. .. .. .. .. ..@ metadata : list() .. .. .. .. ..@ ranges :Formal class 'IRanges' [package "IRanges"] with 6 slots .. .. .. .. .. .. ..@ start : int(0) .. .. .. .. .. .. ..@ width : int(0) .. .. .. .. .. .. ..@ NAMES : NULL .. .. .. .. .. .. ..@ elementType : chr "ANY" .. .. .. .. .. .. ..@ elementMetadata: NULL .. .. .. .. .. .. ..@ metadata : list() .. .. .. .. ..@ strand :Formal class 'Rle' [package "S4Vectors"] with 4 slots .. .. .. .. .. .. ..@ values : Factor w/ 3 levels "+","-","": .. .. .. .. .. .. ..@ lengths : int(0) .. .. .. .. .. .. ..@ elementMetadata: NULL .. .. .. .. .. .. ..@ metadata : list() .. .. .. .. ..@ seqinfo :Formal class 'Seqinfo' [package "GenomeInfoDb"] with 4 slots .. .. .. .. .. .. ..@ seqnames : chr(0) .. .. .. .. .. .. ..@ seqlengths : int(0) .. .. .. .. .. .. ..@ iscircular: logi(0) .. .. .. .. .. .. ..@ genome : chr(0) .. .. .. .. ..@ elementMetadata:Formal class 'DataFrame' [package "S4Vectors"] with 6 slots .. .. .. .. .. .. ..@ rownames : NULL .. .. .. .. .. .. ..@ nrows : int 0 .. .. .. .. .. .. ..@ listData : Named list() .. .. .. .. .. .. ..@ elementType : chr "ANY" .. .. .. .. .. .. ..@ elementMetadata: NULL .. .. .. .. .. .. ..@ metadata : list() .. .. .. .. ..@ elementType : chr "ANY" .. .. .. .. ..@ metadata : list() .. .. ..@ elementMetadata:Formal class 'DataFrame' [package "S4Vectors"] with 6 slots .. .. .. .. ..@ rownames : NULL .. .. .. .. ..@ nrows : int 35764 .. .. .. .. ..@ listData : Named list() .. .. .. .. ..@ elementType : chr "ANY" .. .. .. .. ..@ elementMetadata:Formal class 'DataFrame' [package "S4Vectors"] with 6 slots .. .. .. .. .. .. ..@ rownames : NULL .. .. .. .. .. .. ..@ nrows : int 0 .. .. .. .. .. .. ..@ listData :List of 2 .. .. .. .. .. .. .. ..$ type : chr(0) .. .. .. .. .. .. .. ..$ description: chr(0) .. .. .. .. .. .. ..@ elementType : chr "ANY" .. .. .. .. .. .. ..@ elementMetadata: NULL .. .. .. .. .. .. ..@ metadata : list() .. .. .. .. ..@ metadata : list() .. .. ..@ elementType : chr "GRanges" .. .. ..@ metadata : list() .. .. ..@ partitioning :Formal class 'PartitioningByEnd' [package "IRanges"] with 5 slots .. .. .. .. ..@ end : int [1:35764] 0 0 0 0 0 0 0 0 0 0 ... .. .. .. .. ..@ NAMES : chr [1:35764] "Bradi0012s00100" "Bradi0012s00201" "Bradi0014s00100" "Bradi0135s00100" ... .. .. .. .. ..@ elementType : chr "ANY" .. .. .. .. ..@ elementMetadata: NULL .. .. .. .. ..@ metadata : list() ..@ colData :Formal class 'DataFrame' [package "S4Vectors"] with 6 slots .. .. ..@ rownames : chr [1:150] "A1" "D1" "A3" "B3" ... .. .. ..@ nrows : int 150 .. .. ..@ listData :List of 4 .. .. .. ..$ genotype : Factor w/ 2 levels "Bd21","Bd3.1": 1 1 1 1 1 1 2 2 2 2 ... .. .. .. ..$ timepoint: Factor w/ 10 levels "1","2","3","4",..: 1 1 1 1 1 1 1 1 1 1 ... .. .. .. ..$ treatment: Factor w/ 3 levels "22.5","90","Recovery": 1 1 2 2 2 2 1 1 1 2 ... .. .. .. ..$ libType : chr [1:150] "single-end" "single-end" "single-end" "single-end" ... .. .. ..@ elementType : chr "ANY" .. .. ..@ elementMetadata:Formal class 'DataFrame' [package "S4Vectors"] with 6 slots .. .. .. .. ..@ rownames : NULL .. .. .. .. ..@ nrows : int 4 .. .. .. .. ..@ listData :List of 2 .. .. .. .. .. ..$ type : chr [1:4] "input" "input" "input" "input" .. .. .. .. .. ..$ description: chr [1:4] "" "" "" "" .. .. .. .. ..@ elementType : chr "ANY" .. .. .. .. ..@ elementMetadata: NULL .. .. .. .. ..@ metadata : list() .. .. ..@ metadata : list() ..@ assays :Reference class 'ShallowSimpleListAssays' [package "SummarizedExperiment"] with 1 field .. ..$ data: NULL .. ..and 14 methods. ..@ NAMES : NULL ..@ elementMetadata :Formal class 'DataFrame' [package "S4Vectors"] with 6 slots .. .. ..@ rownames : NULL .. .. ..@ nrows : int 35764 .. .. ..@ listData : Named list() .. .. ..@ elementType : chr "ANY" .. .. ..@ elementMetadata: NULL .. .. ..@ metadata : list() ..@ metadata :List of 1 .. ..$ version:Classes 'packageversion', 'numeric_version' hidden list of 1 .. .. ..$ : int [1:3] 1 24 0 ' ' ' Am I doing anything wrong? I use the same script every time. Thanks! Sarit

This is the session info: ' ' '

sessionInfo() R version 3.6.0 (2019-04-26) Platform: x8664-w64-mingw32/x64 (64-bit) Running under: Windows 10 x64 (build 17134) Matrix products: default Random number generation: RNG: Mersenne-Twister Normal: Inversion Sample: Rounding locale: [1] LCCOLLATE=EnglishUnited States.1252 LCCTYPE=EnglishUnited States.1252
[3] LCMONETARY=EnglishUnited States.1252 LCNUMERIC=C
[5] LCTIME=EnglishUnited States.1252
attached base packages: [1] parallel stats4 stats graphics grDevices utils datasets methods base
other attached packages: [1] WGCNA1.68 fastcluster1.1.25 dynamicTreeCut1.63-1
[4] DiffBind2.12.0 DESeq21.24.0 SummarizedExperiment1.14.0 [7] DelayedArray0.10.0 BiocParallel1.17.18 matrixStats0.54.0
[10] Biobase2.44.0 GenomicRanges1.36.0 GenomeInfoDb1.20.0
[13] IRanges2.18.1 S4Vectors0.22.0 BiocGenerics0.30.0
[16] tximport1.12.1 ggplot23.2.0 readr1.3.1
[19] stringr1.4.0 tibble2.1.3 forcats0.4.0
[22] purrr0.3.2 dplyr0.8.1 tidyr0.8.3
[25] BiocManager1.30.4
loaded via a namespace (and not attached): [1] amap0.8-17 colorspace1.4-1 rjson0.2.20
[4] hwriter1.3.2 htmlTable1.13.1 XVector0.24.0
[7] base64enc0.1-3 rstudioapi0.10 ggrepel0.8.1
[10] bit640.9-7 mvtnorm1.0-10 AnnotationDbi1.46.0
[13] codetools0.2-16 splines3.6.0 doParallel1.0.14
[16] robustbase0.93-5 impute1.58.0 geneplotter1.62.0
[19] knitr1.23 Formula1.2-3 Rsamtools2.0.0
[22] annotate1.62.0 cluster2.0.9 GO.db3.8.2
[25] pheatmap1.0.12 graph1.62.0 rrcov1.4-7
[28] compiler3.6.0 httr1.4.0 GOstats2.50.0
[31] backports1.1.4 assertthat0.2.1 Matrix1.2-17
[34] lazyeval0.2.2 limma3.40.2 acepack1.4.1
[37] htmltools0.3.6 prettyunits1.0.2 tools3.6.0
[40] gtable0.3.0 glue1.3.1 GenomeInfoDbData1.2.1
[43] Category2.50.0 systemPipeR1.18.1 batchtools0.9.11
[46] rappdirs0.3.1 ShortRead1.42.0 Rcpp1.0.1
[49] Biostrings2.52.0 preprocessCore1.46.0 gdata2.18.0
[52] rtracklayer1.44.0 iterators1.0.10 xfun0.7
[55] gtools3.8.1 XML3.98-1.20 DEoptimR1.0-8
[58] edgeR3.26.4 MASS7.3-51.4 zlibbioc1.30.0
[61] scales1.0.0 BSgenome1.52.0 VariantAnnotation1.30.1 [64] hms0.4.2 RBGL1.60.0 RColorBrewer1.1-2
[67] yaml2.2.0 memoise1.1.0 gridExtra2.3
[70] biomaRt2.40.0 rpart4.1-15 latticeExtra0.6-28
[73] stringi1.4.3 RSQLite2.1.1 genefilter1.66.0
[76] pcaPP1.9-73 foreach1.4.4 checkmate1.9.3
[79] GenomicFeatures1.36.1 caTools1.17.1.2 rlang0.3.4
[82] pkgconfig2.0.2 bitops1.0-6 lattice0.20-38
[85] GenomicAlignments1.20.0 htmlwidgets1.3 robust0.4-18
[88] bit1.1-14 tidyselect0.2.5 GSEABase1.46.0
[91] AnnotationForge1.26.0 magrittr1.5 R62.4.0
[94] fit.models0.5-14 gplots3.0.1.1 Hmisc4.2-0
[97] base64url1.4 DBI1.0.0 pillar1.4.1
[100] foreign0.8-71 withr2.1.2 survival2.44-1.1
[103] RCurl1.95-4.12 nnet7.3-12 crayon1.3.4
[106] KernSmooth2.23-15 progress1.2.2 locfit1.5-9.1
[109] grid3.6.0 data.table1.12.2 blob1.1.1
[112] Rgraphviz2.28.0 digest0.6.19 xtable1.8-4
[115] brew1.0-6 munsell_0.5.0

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Updated answer:

https://support.bioconductor.org/p/122184/