Edge R Error in quantile.default(zresid, probs = prob) : missing values and NaN's not allowed if 'na.rm' is FALSE
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0
Entering edit mode
@3d81e3dc
Last seen 12 weeks ago
Netherlands

Hello there,

I have some problem when I was doing alternative splicing analysis with my RNAseq data via edgeR package.

I have six RNAseq samples(3 control, 3 treated) and I want to analyze the alternative splicing events by R. Here is the Syntax I used.

library(edgeR)
fc <- featureCounts(files=c("file control1.bam",
"file control2.bam",
"file control3.bam",
"file treated1.bam",
"file treated2.bam",
"file treated3.bam"),
annot.ext="file.gtf",
isGTFAnnotationFile=TRUE, 
GTF.featureType = "exon", 
GTF.attrType = "gene_id", 
useMetaFeatures = FALSE, 
allowMultiOverlap = TRUE,
isPairedEnd=TRUE)
fccounts <- fc$counts
metadata <- data.frame(sample_id = colnames(fccounts))
sample <- rep(c("con","treat"),each=3)
metadata$sample <- relevel(factor(sample),"con")
y.all <- DGEList(counts = fc$counts, genes = fc$annotation)
dim(y.all)
head(y.all$genes)
y <- y.all 
y$samples
head(y$genes)
TREAT <- factor(metadata$sample, levels = c("con","treat"))
keep <- filterByExpr(y, group = TREAT)
table(keep)
y <- y[keep, keep.lib.sizes = FALSE]
y <- calcNormFactors(y)
y$samples
plotMDS(y)
Batch <- factor(c(1,2,3,1,2,3))
TREAT <- factor(metadata$sample, levels = c("con","treat"))
design <- model.matrix(~ Batch + TREAT)
design
y <- estimateDisp(y, design, robust = TRUE)
plotBCV(y)
fit <- glmQLFit(y, design, robust = TRUE)
plotQLDisp(fit)
qlf <- glmQLFTest(fit, coef = 4)
topTags(qlf)
is.de <- decideTests(qlf, p.value = 0.05)
summary(is.de)
sp <- diffSpliceDGE(fit, coef = 4, geneid = "GeneID", exonid = "Start")

But at this step the R returned like this:

Error in quantile.default(zresid, probs = prob) : 
missing values and NaN's not allowed if 'na.rm' is FALSE

I have checked the NA values by sum(is.na(fit)) but no NA value was found. So I would like to ask how can I solve this "missing values and NaN's" problems?

Thank you very much for your kindly help!

Qiao

sessionInfo()
R version 4.1.0 (2021-05-18)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19041)

Matrix products: default

locale:
[1] LC_COLLATE=English_Netherlands.936  LC_CTYPE=English_Netherlands.936   
[3] LC_MONETARY=English_Netherlands.936 LC_NUMERIC=C                       
[5] LC_TIME=English_Netherlands.936    

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

other attached packages:
 [1] edgeR_3.32.1                DESeq2_1.30.1               SummarizedExperiment_1.20.0
 [4] Biobase_2.50.0              MatrixGenerics_1.2.1        matrixStats_0.59.0         
 [7] GenomicRanges_1.42.0        GenomeInfoDb_1.26.7         IRanges_2.24.1             
[10] S4Vectors_0.28.1            BiocGenerics_0.36.1         limma_3.46.0               
[13] Rsubread_2.4.3             

loaded via a namespace (and not attached):
 [1] genefilter_1.72.1      statmod_1.4.36         locfit_1.5-9.4        
 [4] tidyselect_1.1.1       purrr_0.3.4            splines_4.1.0         
 [7] lattice_0.20-44        generics_0.1.0         colorspace_2.0-1      
[10] vctrs_0.3.8            utf8_1.2.1             blob_1.2.1            
[13] XML_3.99-0.6           survival_3.2-11        rlang_0.4.11          
[16] pillar_1.6.1           glue_1.4.2             DBI_1.1.1             
[19] BiocParallel_1.24.1    bit64_4.0.5            RColorBrewer_1.1-2    
[22] GenomeInfoDbData_1.2.4 lifecycle_1.0.0        zlibbioc_1.36.0       
[25] munsell_0.5.0          gtable_0.3.0           memoise_2.0.0         
[28] geneplotter_1.68.0     fastmap_1.1.0          AnnotationDbi_1.52.0  
[31] fansi_0.5.0            Rcpp_1.0.6             xtable_1.8-4          
[34] scales_1.1.1           cachem_1.0.5           DelayedArray_0.16.3   
[37] annotate_1.68.0        XVector_0.30.0         bit_4.0.4             
[40] ggplot2_3.3.3          dplyr_1.0.6            grid_4.1.0            
[43] tools_4.1.0            bitops_1.0-7           magrittr_2.0.1        
[46] RCurl_1.98-1.3         RSQLite_2.2.7          tibble_3.1.2          
[49] pkgconfig_2.0.3        crayon_1.4.1           ellipsis_0.3.2        
[52] Matrix_1.3-4           httr_1.4.2             R6_2.5.0              
[55] compiler_4.1.0
diffSpliceDGE RNASeq DifferentialSplicingWorkflow AlternativeSplicing edgeR • 179 views
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0
Entering edit mode
@gordon-smyth
Last seen 3 hours ago
WEHI, Melbourne, Australia

We have not seen this problem ourselves and we can't reproduce it.

Please rerun your data using the current version of Bioconductor and edgeR. I would also always clean up the GTF file using Rsubread::flattenGTF before using diffSpliceDGE.

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