Question

DESeq help for comparing contrasts

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muc345 • 0

@muc345-20390

Last seen 4.5 years ago

Hello there,

My issue is that DESeq no longer works and does not give me the correct contrast options. This was working before I updated to DESeq2 1.22.2

Here is my code:

> line.ds <- phyloseq_to_deseq2(ps.phyla, ~ Line)
> diagdds <- DESeq(line.ds, test="Wald", fitType="parametric")
using pre-existing size factors
estimating dispersions
found already estimated dispersions, replacing these
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing

> resultsNames(diagdds)
[1] "Intercept" "Line1"     "Line2"     "Line3"     "Line4"

I'll also show some of the data included in my DESeq object

> line.ds@design
~Line
> line.ds@colData
DataFrame with 40 rows and 9 columns
            X  Genotype     Block     Line Wavelength     Time
     <factor> <integer> <integer> <factor>  <numeric> <factor>
1           1         4         1     N321      0.169    After
10         10         2         2     N326      0.114    After
11         11         2         3     N326      0.137    After
12         12         2         4     N326      0.092    After
13         13         5         1     N336      0.423    After

Now, what I would see before I updated was something like this: N331, N326, N337 etc. that matched the 5 factor levels in "Line".

I can perform the above analysis using a continuous variable with no trouble but do have issues when I group the samples into categories.

platform       x86_64-apple-darwin15.6.0   
arch           x86_64                      
os             darwin15.6.0                
system         x86_64, darwin15.6.0        
status                                     
major          3                           
minor          5.1                         
year           2018                        
month          07                          
day            02                          
svn rev        74947                       
language       R                           
version.string R version 3.5.1 (2018-07-02)
nickname       Feather Spray

I have found that other people have posted similar questions regarding a change to the most up-to-date DESeq package but I have not been able to fix my problem based on the responses to others posts. Hence, why I am posting here.

Any help would be greatly appreciated! Thanks!

deseq2 • 658 views

ADD COMMENT • link updated 5.0 years ago by Michael Love 41k • written 5.0 years ago by muc345 • 0

score 0 · Answer 1 · 2019-04-02

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Michael Love 41k

@mikelove

Last seen 1 hour ago

United States

From your posted code, I'd guess that when you get the resultsNames(dds) of Line1, Line2, ..., that dds$Line will have levels 0,1,2 ... DESeq2 doesn't do any magical renaming of factor levels, it's just pulling straight from dds$Line.

Can you run your code again, and check resultsNames(dds) and dds$Line in the same session?

ADD COMMENT • link 5.0 years ago Michael Love 41k

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Hi Michael,

Thanks for the response.

Here is what I'm seeing:

diagdds <- DESeq(line.ds, test="Wald", fitType="parametric") estimating size factors estimating dispersions gene-wise dispersion estimates mean-dispersion relationship final dispersion estimates fitting model and testing resultsNames(diagdds) [1] "Intercept" "Line1" "Line2" "Line3" "Line4"
diagdds$Line [1] N321 N326 N326 N326 N336 N336 N336 N336 N337 N337 N337 N321 [13] N337 N321 N321 N334 N334 N334 N334 N326 N337 N326 N334 N321 [25] N336 N337 N326 N334 N321 N336 N337 N326 N334 N321 N336 N337 [37] N326 N334 N321 N336 Levels: N321 N326 N334 N336 N337

Thanks!

ADD REPLY • link 5.0 years ago muc345 • 0

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Can you update all packages on your machine and then run BiocManager::valid()

ADD REPLY • link 5.0 years ago Michael Love 41k

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Here is my output:

BiocManager::valid()

sessionInfo()

R version 3.5.1 (2018-07-02) Platform: x86_64-apple-darwin15.6.0 (64-bit) Running under: macOS High Sierra 10.13.6

Matrix products: default BLAS: /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib LAPACK: /Library/Frameworks/R.framework/Versions/3.5/Resources/lib/libRlapack.dylib

locale: [1] enUS.UTF-8/enUS.UTF-8/enUS.UTF-8/C/enUS.UTF-8/en_US.UTF-8

attached base packages: [1] parallel stats4 stats graphics grDevices utils datasets methods base

other attached packages: [1] metagMisc0.0.4 emmeans1.3.3 FSA0.8.22
[4] dunn.test1.3.5 factoextra1.0.5.999 FactoMineR1.41
[7] usethis1.4.0 devtools2.0.1 ggfortify0.4.6
[10] visNetwork2.0.6 igraph1.2.4 microbiomeSeq0.1
[13] naniar0.4.2 tidyr0.8.3 bindrcpp0.2.2
[16] vegan2.5-4 lattice0.20-38 permute0.9-5
[19] RVAideMemoire0.9-73 cowplot0.9.4 DESeq21.22.2
[22] SummarizedExperiment1.12.0 DelayedArray0.8.0 BiocParallel1.16.5
[25] matrixStats0.54.0 Biobase2.42.0 GenomicRanges1.34.0
[28] GenomeInfoDb1.18.1 IRanges2.16.0 S4Vectors0.20.1
[31] BiocGenerics0.28.0 adespatial0.3-4 lmerTest3.1-0
[34] lme41.1-21 Matrix1.2-15 microbiome1.4.2
[37] ggplot23.1.0 phyloseq1.26.1 tibble_2.1.1

loaded via a [1] tidyselect0.2.5 [4] AnnotationDbi1.44.0 [7] RNeXML2.3.0 [10] preprocessCore1.44.0 [13] knitr1.22 [16] rstudioapi0.10 [19] GenomeInfoDbData1.2.0 [22] rprojroot1.3-2 [25] LearnBayes2.15.1 [28] adegenet2.1.1 [31] R62.4.0 [34] bitops1.0-6 [37] scales1.0.0 [40] gtable0.3.0 [43] processx3.3.0 [46] rlang0.3.3 [49] splines3.5.1 [52] impute1.56.0 [55] yaml2.2.0 [58] httpuv1.5.0 [61] spData0.3.0 [64] dynamicTreeCut1.63-1 [67] plyr1.8.4 [70] zlibbioc1.28.0 [73] ps1.3.0 [76] rpart4.1-13 [79] ggrepel0.8.0 ... namespace (and not attached): robust0.4-18 RSQLite2.1.1
htmlwidgets1.3 grid3.5.1
munsell0.5.0 codetools0.2-16
withr2.1.2 colorspace1.4-1
uuid0.1-2 leaps3.0
robustbase0.93-4 labeling0.3
bit640.9-7 rhdf52.26.2
TH.data1.0-10 coda0.19-2
xfun0.6 ggthemes4.1.0
fastcluster1.1.25 adephylo1.1-11
doParallel1.0.14 locfit1.5-9.1
assertthat0.2.1 promises1.0.1
multcomp1.4-10 nnet7.3-12
phylobase0.8.6 KMDA1.0
WGCNA1.66 sandwich2.5-0
genefilter1.64.0 scatterplot3d0.3-41
lazyeval0.2.2 acepack1.4.1
checkmate1.9.1 BiocManager1.30.4
reshape21.4.3 backports1.1.3
Hmisc4.2-0 tools3.5.1
biomformat1.10.1 RColorBrewer1.1-2
sessioninfo1.1.1 Rcpp1.0.1
base64enc0.1-3 progress1.2.0
purrr0.3.2 RCurl1.95-4.12
prettyunits1.0.2 ggpubr0.2
deldir0.1-16 zoo1.8-5
cluster2.0.7-1 fs_1.2.7

ADD REPLY • link 5.0 years ago muc345 • 0

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[82] magrittr1.5 data.table1.12.0 gmodels2.18.1
[85] mvtnorm1.0-10 pkgload1.0.2 hms0.4.2
[88] mime0.6 xtable1.8-3 pbkrtest0.4-7
[91] XML3.98-1.19 gridExtra2.3 compiler3.5.1
[94] KernSmooth2.23-15 crayon1.3.4 minqa1.2.4
[97] htmltools0.3.6 mgcv1.8-26 pcaPP1.9-73
[100] later0.8.0 spdep1.0-2 Formula1.2-3
[103] geneplotter1.60.0 visdat0.5.3 rrcov1.4-7
[106] expm0.999-3 DBI1.0.0 MASS7.3-51.3
[109] boot1.3-20 ade41.7-13 cli1.1.0
[112] adegraphics1.0-15 gdata2.18.0 bindr0.1.1
[115] pkgconfig2.0.2 fit.models0.5-14 flashClust1.01-2
[118] rncl0.8.3 numDeriv2016.8-1 foreign0.8-71
[121] sp1.3-1 xml21.2.0 foreach1.4.4
[124] annotate1.60.0 multtest2.38.0 XVector0.22.0
[127] estimability1.3 stringr1.4.0 callr3.2.0
[130] digest0.6.18 Biostrings2.50.2 htmlTable1.13.1
[133] curl3.3 shiny1.2.0 gtools3.8.1
[136] nloptr1.2.1 nlme3.1-137 jsonlite1.6
[139] Rhdf5lib1.4.2 seqinr3.4-5 desc1.2.0
[142] pillar1.3.1 httr1.4.0 DEoptimR1.0-8
[145] pkgbuild1.0.3 survival2.44-1.1 GO.db3.7.0
[148] glue1.3.1 remotes2.0.2 iterators1.0.10
[151] bit1.1-14 stringi1.4.3 blob1.1.1
[154] latticeExtra0.6-28 memoise1.1.0 dplyr0.8.0.1
[157] ape5.3

Bioconductor version '3.8'

10 packages out-of-date
2 packages too new

create a valid installation with

BiocManager::install(c( "annotate", "BiocParallel", "dada2", "DECIPHER", "expm", "factoextra", "GenomeInfoDb", "mixOmics", "phangorn", "RcppArmadillo", "Rhdf5lib", "Rsamtools" ), update = TRUE, ask = FALSE)

more details: BiocManager::valid()$toonew, BiocManager::valid()$outof_date

Warning message: 10 packages out-of-date; 2 packages too new

ADD REPLY • link 5.0 years ago muc345 • 0

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This could be the problem, can you address this first until valid() doesn’t complain.

ADD REPLY • link 5.0 years ago Michael Love 41k

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Michael,

I'm working on this now. I keep running into this issue: BiocManager::install(c( "expm", "phangorn", "RcppArmadillo" ), update = TRUE, ask = FALSE)

more details: BiocManager::valid()$toonew, BiocManager::valid()$outof_date

Warning message: 3 packages out-of-date; 0 packages too new

So I'm trying to trouble shoot. Be in touch shortly. Thanks!

ADD REPLY • link 5.0 years ago muc345 • 0

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I wonder if at this point, if you re-run the script, where you generate DESeq2 objects from scratch, if you still have the Line1, ... error above.

ADD REPLY • link 5.0 years ago Michael Love 41k

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I was hoping that would work, too. But I still get the Line1/2/3 issue. :(

ADD REPLY • link 5.0 years ago muc345 • 0

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Michael,

I finally got around the fixing all of my packages. Once I had updated versions and the output from BiocManager::valid() was "TRUE", the code worked!

Thank you for your help!

ADD REPLY • link 5.0 years ago muc345 • 0