DESeq2 results change dramatically after scaling + centering of numeric variable
0
Entering edit mode
@matmu
Last seen 4 weeks ago
Germany

To adjust for the data quality, I added the numerical variable percent_mapped to my model:

design = ~ variable_of_interest + percent_mapped
dds = DESeq2::DESeqDataSet(gse, design)

During building of the DESeqDataSet object I got the following warning:

the design formula contains one or more numeric variables that have mean or standard deviation larger than 5 (an arbitrary threshold to trigger this message). it is generally a good idea to center and scale numeric variables in the design to improve GLM convergence.

Since variable_of_interest is categorical, this warning came up because of percent_mapped. The mean of percent_mapped is 91.44303 (standard deviation = 1.369892).

I performed the differential expression analysis (a) without and (b) with scaling + centering and (c) excluding the percent_mapped variable. Code I used for centering and scaling:

colData(gse)$percent_mapped = scale(colData(gse)$percent_mapped, center=TRUE)

Differential expression analysis:

register(MulticoreParam(5))
dds = DESeq(dds, parallel = TRUE)
res = results(dds, contrast = c(variable_of_interest , group1, group2), alpha = 0.05, parallel = TRUE)
res["gene_of_interest",]

For my gene of interest, I got results which are greatly differing between (a) without scaling and (b) + (c) with scaling/centering or by exluding percent_mapped:

(a) baseMean: 1058.808 log2FoldChange: 1.550588 lfcSE: 3.630939 stat: 0.4270487 pvalue: 0.6693438 padj: 0.8398124

(b) baseMean: 1058.808 log2FoldChange: 2.579453 lfcSE: 0.4652964 stat: 5.543676 pvalue: 2.961866e-08 padj: 1.332327e-05

(c) baseMean: 1058.808 log2FoldChange: 2.800934 lfcSE: 0.4835646 stat: 5.792265 pvalue: 6.944332e-09 padj: 4.909011e-06

How can this be? I it is unclear to me, how scaling and centering can effect the DESeq2 results. Can anyone explain what happened? I was assuming that scaling + centering will speed up the fitting of the model and don't affect the results.

Is it recommended to scale + center numeric variables in general?

sessionInfo( )

R version 4.0.1 (2020-06-06)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)

Matrix products: default
BLAS:   /pstore/apps/R/4.0.1-intel-2018b/lib64/R/lib/libR.so
LAPACK: /pstore/apps/R/4.0.1-intel-2018b/lib64/R/modules/lapack.so

locale:
 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C               LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8     LC_MONETARY=en_US.UTF-8   
 [6] LC_MESSAGES=en_US.UTF-8    LC_PAPER=en_US.UTF-8       LC_NAME=C                  LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

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

other attached packages:
 [1] rtracklayer_1.50.0          BiocParallel_1.24.1         DESeq2_1.30.0               SummarizedExperiment_1.20.0 Biobase_2.50.0             
 [6] MatrixGenerics_1.2.0        matrixStats_0.57.0          GenomicRanges_1.42.0        GenomeInfoDb_1.26.2         IRanges_2.24.1             
[11] S4Vectors_0.28.1            BiocGenerics_0.36.0         ggvenn_0.1.7                ggplot2_3.3.3               UpSetR_1.4.0               
[16] tidyr_1.1.2                 msigdbr_7.2.1               biomaRt_2.46.1              ArvadosR_0.0.7              dplyr_1.0.2                

loaded via a namespace (and not attached):
 [1] httr_1.4.2               jsonlite_1.7.2           bit64_4.0.5              splines_4.0.1            assertthat_0.2.1         askpass_1.1             
 [7] BiocManager_1.30.10      BiocFileCache_1.14.0     blob_1.2.1               Rsamtools_2.6.0          GenomeInfoDbData_1.2.4   progress_1.2.2          
[13] pillar_1.4.7             RSQLite_2.2.2            lattice_0.20-41          glue_1.4.2               digest_0.6.27            RColorBrewer_1.1-2      
[19] XVector_0.30.0           colorspace_2.0-0         plyr_1.8.6               Matrix_1.2-18            XML_3.99-0.5             pkgconfig_2.0.3         
[25] genefilter_1.72.0        zlibbioc_1.36.0          purrr_0.3.4              xtable_1.8-6             scales_1.1.1             tibble_3.0.4            
[31] openssl_1.4.3            annotate_1.68.0          generics_0.1.0           ellipsis_0.3.1           withr_2.3.0              cli_2.2.0               
[37] survival_3.1-12          magrittr_2.0.1           crayon_1.3.4             memoise_1.1.0            fansi_0.4.1              xml2_1.3.2              
[43] tools_4.0.1              prettyunits_1.1.1        hms_0.5.3                lifecycle_0.2.0          stringr_1.4.0            munsell_0.5.0           
[49] locfit_1.5-9.4           DelayedArray_0.16.0      Biostrings_2.58.0        AnnotationDbi_1.52.0     compiler_4.0.1           rlang_0.4.10            
[55] RCurl_1.98-1.2           rstudioapi_0.13          rappdirs_0.3.1           bitops_1.0-6             gtable_0.3.0             DBI_1.1.0               
[61] curl_4.3                 R6_2.5.0                 GenomicAlignments_1.26.0 gridExtra_2.3            bit_4.0.4                stringi_1.5.3           
[67] Rcpp_1.0.5               vctrs_0.3.6              geneplotter_1.68.0       dbplyr_2.0.0             tidyselect_1.1.0
DESeq2 • 181 views
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Hi, I will wait for Mike to respond; however, I see no need to include percent_mapped in your design formula. To adjust for quality, some people include, e.g., RIN (RNA Integrity Number). The alignment % (percent_mapped) is not strictly a measure of DNA quality and relates to other things.

Irrespective, by scaling the variable, you are completely altering its distribution. By default, percent_mapped is measured from 0-100% and is assuredly heavily right-skewed when plot via histogram. When you scale it, you are centering it at 0 and essentially transforming it to Z-scores; so, its distribution changes dramatically. Plot it via histogram (before / after) to see this.

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I wrote data quality. Scaling and centering are not new to me.

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You don't need to include percent_mapped in order to adjust for quality. By including it, even as scaled, you are likely introducing bias into the statistical inferences.

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@mikelove
Last seen 2 minutes ago
United States

When you add a covariate with a large mean and small SD, you are creating a near collinearity with the intercept. This is similar to a nearly confounded design: the coefficients can trade off and give the same likelihood, so you are impairing the estimation of the coefficients. Collinearity is part of a typical linear models course.

It is simple to resolve: don't include covariates with a large mean value, just center and scale the continuous-valued covariates before adding to the design. Yes it is recommended.

This is why DESeq2 prints the message to center and scale covariates when the mean is large.

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