Outliers on DESEq2 Results
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Entering edit mode
@Bruno Rodrigo-24702
Last seen 2 days ago
Brazil

I have an RNAseq dataset, where one of the genes I intend to analyze has hundreds of counts ranging from 10 to 12, with a few counts > 9000. I process this data in Deseq2 and get that the gene is differentially expressed across several samples of interest. What can justify these strange counts? Are the results reliable?

library("DESeq2")

        counts2=C
        dds <- DESeqDataSetFromMatrix(countData=counts2, 
                                      colData=expdesign_fucos, 
                                      design=~subtype, tidy = TRUE)


# Rodando a função DESeq
dds <- DESeq(dds)
#normalized.counts = counts(dds, normalized=T, replaced=TRUE)

res = results(dds, alpha = 0.01, lfcThreshold =1, 
                          contr = c("subtype","Her2","LumB"))

summary(res)
out of 15 with nonzero total read count

adjusted p-value < 0.01

LFC > 1.00 (up)    : 0, 0%

LFC < -1.00 (down) : 1, 6.7%

outliers [1]       : 0, 0%

low counts [2]     : 0, 0%

(mean count < 12)

[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

with(colData(dds), table(subtype))

> with(colData(dds), table(subtype))
subtype
Basal  Her2  LumA  LumB 
  187    82   563   215 

mcols(dds)$maxCooks

> mcols(dds)$maxCooks
 [1] NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

The results don't seem to be correct for me as this gene (POFUT1) usually has very low values ​​(between 10-12), some samples with large counts (>9000), and looking at the counts table doesn't show significant differences between #groups.

>subset(res, res$lfcSE < 2 & res$padj < 0.01)

log2 fold change (MLE): subtype Her2 vs LumB 
Wald test p-value: subtype Her2 vs LumB 
DataFrame with 1 row and 6 columns
        baseMean log2FoldChange     lfcSE      stat      pvalue        padj
       <numeric>      <numeric> <numeric> <numeric>   <numeric>   <numeric>

POFUT1   12.2556       -2.15519  0.171235  -6.74621 1.51756e-11 2.27633e-10

sessionInfo( )

R version 4.0.4 (2021-02-15)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19042)

Matrix products: default

locale:
[1] LC_COLLATE=Portuguese_Brazil.1252  LC_CTYPE=Portuguese_Brazil.1252   
[3] LC_MONETARY=Portuguese_Brazil.1252 LC_NUMERIC=C                      
[5] LC_TIME=Portuguese_Brazil.1252    

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

other attached packages:
 [1] EnvStats_2.4.0              genefilter_1.72.1           DESeq2_1.30.1              
 [4] SummarizedExperiment_1.20.0 Biobase_2.50.0              MatrixGenerics_1.2.1       
 [7] matrixStats_0.58.0          GenomicRanges_1.42.0        GenomeInfoDb_1.26.7        
[10] IRanges_2.24.1              S4Vectors_0.28.1            BiocGenerics_0.36.1        

loaded via a namespace (and not attached):
 [1] rgl_0.107.10           Rcpp_1.0.6             locfit_1.5-9.4        
 [4] lattice_0.20-41        digest_0.6.27          assertthat_0.2.1      
 [7] utf8_1.2.1             R6_2.5.0               RSQLite_2.2.7         
[10] httr_1.4.2             ggplot2_3.3.3          pillar_1.6.1          
[13] zlibbioc_1.36.0        rlang_0.4.11           annotate_1.68.0       
[16] blob_1.2.1             Matrix_1.3-2           splines_4.0.4         
[19] BiocParallel_1.24.1    geneplotter_1.68.0     htmlwidgets_1.5.3     
[22] RCurl_1.98-1.3         bit_4.0.4              munsell_0.5.0         
[25] DelayedArray_0.16.3    xfun_0.23              compiler_4.0.4        
[28] pkgconfig_2.0.3        htmltools_0.5.1.1      tidyselect_1.1.1      
[31] tibble_3.1.1           GenomeInfoDbData_1.2.4 XML_3.99-0.6          
[34] fansi_0.4.2            crayon_1.4.1           dplyr_1.0.6           
[37] MASS_7.3-53.1          bitops_1.0-7           grid_4.0.4            
[40] jsonlite_1.7.2         xtable_1.8-4           gtable_0.3.0          
[43] lifecycle_1.0.0        DBI_1.1.1              magrittr_2.0.1        
[46] scales_1.1.1           cachem_1.0.5           XVector_0.30.0        
[49] robustbase_0.93-8      qpcR_1.4-1             ellipsis_0.3.2        
[52] vctrs_0.3.8            generics_0.1.0         RColorBrewer_1.1-2    
[55] tools_4.0.4            bit64_4.0.5            glue_1.4.2            
[58] DEoptimR_1.0-9         purrr_0.3.4            crosstalk_1.1.1       
[61] fastmap_1.1.0          survival_3.2-10        AnnotationDbi_1.52.0  
[64] colorspace_2.0-1       minpack.lm_1.2-1       memoise_2.0.0         
[67] knitr_1.33
RNASeqR DifferentialExpression DESeq2 • 137 views
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0
Entering edit mode
@mikelove
Last seen 7 hours ago
United States

The gene probably has more and higher counts in Her2 compared to LumB. If you don't want such a gene to pop up, you can filter out these genes with something like:

keep <- rowSums(counts(dds) >= 20) >= X
dds <- dds[keep,]
# before DESeq()

Where X is a minimal number of sample to have a count greater than 20.

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