Question

DESeq2; loading htseq-count output; sample condition is described in a different csv file

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mzillur • 0

@mzillur-20118

Last seen 4.8 years ago

Hello there, I have 32 samples without any string 'treated' or something like this. File names are the sample names. In another csv file I have information about those count files (case/control). How can I load these files for differential expression analysis. In the vignettes, they used 'grep' and 'sub' for htseq output, I am not sure how can I adapt this to my dataset. 'https://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#htseq'. I am new in rnaseq analysis. Any help? Best Regards Zillur

deseq2 • 1.8k views

ADD COMMENT • link updated 5.1 years ago by Michael Love 41k • written 5.1 years ago by mzillur • 0

score 0 · Answer 1 · 2019-03-06

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Michael Love 41k

@mikelove

Last seen 9 hours ago

United States

See the match function in R: ?match. This will give you a numeric vector that you can use to put one vector or table in the order of another vector or table. If you're just starting with R, you might try getting some help from someone in your institute who can look over your shoulder.

ADD COMMENT • link 5.1 years ago Michael Love 41k

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Thank you. I have managed to load the dataset. Now facing another problem.

DESeq(dds)
 estimating size factors Error in estimateSizeFactorsForMatrix(counts(object), locfunc = locfunc,  :   
every gene contains at least one zero, cannot compute log geometric means

I have tried dds=dds[rowSums(counts(dds))>32,] But the error persisits. the object is:

class: DESeqDataSet 
dim: 61471 32 
metadata(1): version
assays(1): counts
rownames(61471): 10904 12954 ... gSpikein_ERCC-00171 gSpikein_phiX174
rowData names(0):
colnames(32): A1001 A1006 ... HI021 HI023
colData names(1): condition

Thanks again.

ADD REPLY • link 5.1 years ago mzillur • 0

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This is strange that every gene would contain a zero. What kind of data do you have? What are colSums(counts(dds))?

ADD REPLY • link 5.1 years ago Michael Love 41k

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Thank you. It turns out that one of my samples has zero for all genes. After removing that particular sample, its ok. Now I am having another problem in downstream analysis aka plotting.

> plotCounts(d4,gene=which.min(d4$padj),intgroup = 'condition')
Error in (function (classes, fdef, mtable)  : 
  unable to find an inherited method for function ‘colData’ for signature ‘"DESeqResults"’

I have updated geneplotter after facing the problem. But it didn't solve the error. I have updated it in the same session though. Thanks again and sorry to take your valuable time. Best Regards Zillur

sessionInfo() R version 3.5.2 (2018-12-20) Platform: x86_64-apple-darwin15.6.0 (64-bit) Running under: macOS Sierra 10.12.6

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] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

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

other attached packages:
 [1] geneplotter_1.60.0          annotate_1.60.0             XML_3.98-1.18              
 [4] AnnotationDbi_1.44.0        lattice_0.20-38             tximport_1.10.1            
 [7] readr_1.3.1                 BiocInstaller_1.32.1        DESeq2_1.22.2              
[10] SummarizedExperiment_1.12.0 DelayedArray_0.8.0          BiocParallel_1.16.6        
[13] matrixStats_0.54.0          Biobase_2.42.0              GenomicRanges_1.34.0       
[16] GenomeInfoDb_1.18.2         IRanges_2.16.0              S4Vectors_0.20.1           
[19] BiocGenerics_0.28.0         phytools_0.6-60             maps_3.3.0                 
[22] ape_5.2                    

loaded via a namespace (and not attached):
 [1] nlme_3.1-137            bitops_1.0-6            bit64_0.9-7            
 [4] RColorBrewer_1.1-2      numDeriv_2016.8-1       tools_3.5.2            
 [7] backports_1.1.3         R6_2.4.0                rpart_4.1-13           
[10] Hmisc_4.2-0             DBI_1.0.0               lazyeval_0.2.1         
[13] colorspace_1.4-0        nnet_7.3-12             gridExtra_2.3          
[16] mnormt_1.5-5            phangorn_2.4.0          bit_1.1-14             
[19] compiler_3.5.2          htmlTable_1.13.1        animation_2.6          
[22] expm_0.999-3            scales_1.0.0            checkmate_1.9.1        
[25] genefilter_1.64.0       quadprog_1.5-5          stringr_1.4.0          
[28] digest_0.6.18           foreign_0.8-71          XVector_0.22.0         
[31] base64enc_0.1-3         pkgconfig_2.0.2         htmltools_0.3.6        
[34] plotrix_3.7-4           htmlwidgets_1.3         rlang_0.3.1            
[37] rstudioapi_0.9.0        RSQLite_2.1.1           combinat_0.0-8         
[40] acepack_1.4.1           RCurl_1.95-4.12         magrittr_1.5           
[43] GenomeInfoDbData_1.2.0  Formula_1.2-3           Matrix_1.2-15          
[46] Rcpp_1.0.0              munsell_0.5.0           scatterplot3d_0.3-41   
[49] stringi_1.3.1           clusterGeneration_1.3.4 MASS_7.3-51.1          
[52] zlibbioc_1.28.0         plyr_1.8.4              grid_3.5.2             
[55] blob_1.1.1              crayon_1.3.4            splines_3.5.2          
[58] hms_0.4.2               locfit_1.5-9.1          knitr_1.21             
[61] pillar_1.3.1            igraph_1.2.4            fastmatch_1.1-0        
[64] latticeExtra_0.6-28     data.table_1.12.0       BiocManager_1.30.4     
[67] gtable_0.2.0            ggplot2_3.1.0           xfun_0.5               
[70] xtable_1.8-3            coda_0.19-2             survival_2.43-3        
[73] tibble_2.0.1            memoise_1.1.0           cluster_2.0.7-1

ADD REPLY • link 5.1 years ago mzillur • 0

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If you encounter an error with a function, the best place to check is the help file:

?plotCounts

Take a look at the arguments, and make sure that you are providing the right ones.

Here R is giving you a clue, that you gave it a DESeqResults object and it that's not what it was expecting. What is the expected class of the first argument of plotCounts?