Hi,

I want to clean my RNA-seq data with the SVA package, but the sva() function gives me error:

*Error in density.default(x, adjust = adj) : 'x' contains missing values
In addition: Warning message:
In pf(fstats, df1 = (df1 - df0), df2 = (n - df1)) : NaNs produced*

I attach the code I used since count table preparation and gene filtering:

*dds.txi <- DESeqDataSetFromTximport(txi = txi.kallisto, colData = sample_info, design = ~ Sequencing.lane+Disease)*

*sum(rowSums(counts(dds.txi))==0)
keep <- rowSums(counts(dds.txi)) >= 10
dds.txi <- dds.txi[keep,]*

*dds<-DESeq(dds.txi)    
dds
design<-design(dds)*

*rld<-rlog(dds, blind=T)                   
rld_df<-as.data.frame(assay(rld))*

*mod<-model.matrix(~ Age+Sequencing.lane+Disease, sample_info)
mod0<-model.matrix(~  Age+Sequencing.lane, sample_info)*

*dat<-counts(dds,normalized=T)
n.sv=num.sv(dat,mod,method='leek')
svseq <- sva(assay(rld), mod, mod0, n.sv = n.sv)*

... and my *SessionInfo()*:

*R version 3.6.1 (2019-07-05)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows >= 8 x64 (build 9200)
Matrix products: default
locale:
[1] LC_COLLATE=English_United Kingdom.1252  LC_CTYPE=English_United Kingdom.1252    LC_MONETARY=English_United Kingdom.1252
[4] LC_NUMERIC=C                            LC_TIME=English_United Kingdom.1252    
attached base packages:
 [1] parallel  stats4    grid      stats     graphics  grDevices utils     datasets  methods   base     
other attached packages:
 [1] org.Hs.eg.db_3.8.2           apeglm_1.6.0                 mixOmics_6.8.4               DOSE_3.10.2                 
 [5] pcaGoPromoter.Mm.mm9_1.20.0  pcaGoPromoter.Hs.hg19_1.20.0 pcaGoPromoter_1.28.0         Biostrings_2.52.0           
 [9] XVector_0.24.0               ellipse_0.4.1                ReactomePA_1.28.0            biomaRt_2.40.4              
[13] geneplotter_1.62.0           lattice_0.20-38              limma_3.40.6                 tximport_1.12.3             
[17] sva_3.32.1                   genefilter_1.66.0            mgcv_1.8-28                  nlme_3.1-140                
[21] GSEABase_1.46.0              graph_1.62.0                 annotate_1.62.0              XML_3.98-1.20               
[25] AnnotationDbi_1.46.1         clusterProfiler_3.12.0       IHW_1.12.0                   DESeq2_1.24.0               
[29] SummarizedExperiment_1.14.1  DelayedArray_0.10.0          BiocParallel_1.18.1          Biobase_2.44.0              
[33] GenomicRanges_1.36.1         GenomeInfoDb_1.20.0          IRanges_2.18.2               S4Vectors_0.22.1            
[37] BiocGenerics_0.30.0          rhdf5_2.28.0                 ggrepel_0.8.1                gtools_3.8.1                
[41] stringi_1.4.3                ggforce_0.3.1                MASS_7.3-51.4                rlang_0.4.0                 
[45] survival_2.44-1.1            gridBase_0.4-7               matrixStats_0.55.0           devtools_2.2.1              
[49] usethis_1.5.1                Rcpp_1.0.2                   RSQLite_2.1.2                data.table_1.12.4           
[53] bit_1.1-14                   magrittr_1.5                 scales_1.0.0                 ggbeeswarm_0.6.0            
[57] VennDiagram_1.6.20           futile.logger_1.4.3          colourlovers_0.3.5           pheatmap_1.0.12             
[61] reshape2_1.4.3               forcats_0.4.0                stringr_1.4.0                dplyr_0.8.3                 
[65] purrr_0.3.2                  readr_1.3.1                  tidyr_1.0.0                  tibble_2.1.3                
[69] ggplot2_3.2.1                tidyverse_1.2.1             
loaded via a namespace (and not attached):
  [1] rappdirs_0.3.1         coda_0.19-3            acepack_1.4.1          bit64_0.9-7            knitr_1.25            
  [6] rpart_4.1-15           RCurl_1.95-4.12        generics_0.0.2         callr_3.3.2            cowplot_1.0.0         
 [11] lambda.r_1.2.4         europepmc_0.3          enrichplot_1.4.0       xml2_1.2.2             lubridate_1.7.4       
 [16] assertthat_0.2.1       viridis_0.5.1          xfun_0.10              hms_0.5.1              progress_1.2.2        
 [21] readxl_1.3.1           igraph_1.2.4.1         DBI_1.0.0              htmlwidgets_1.3        rARPACK_0.11-0        
 [26] ellipsis_0.3.0         RSpectra_0.15-0        backports_1.1.5        vctrs_0.2.0            remotes_2.1.0         
 [31] withr_2.1.2            triebeard_0.3.0        checkmate_1.9.4        fdrtool_1.2.15         prettyunits_1.0.2     
 [36] cluster_2.1.0          lazyeval_0.2.2         crayon_1.3.4           pkgconfig_2.0.3        slam_0.1-45           
 [41] labeling_0.3           tweenr_1.0.1           vipor_0.4.5            pkgload_1.0.2          nnet_7.3-12           
 [46] lifecycle_0.1.0        modelr_0.1.5           cellranger_1.1.0       rprojroot_1.3-2        polyclip_1.10-0       
 [51] Matrix_1.2-17          urltools_1.7.3         lpsymphony_1.12.0      Rhdf5lib_1.6.1         base64enc_0.1-3       
 [56] beeswarm_0.2.3         ggridges_0.5.1         processx_3.4.1         png_0.1-7              viridisLite_0.3.0     
 [61] bitops_1.0-6           blob_1.2.0             qvalue_2.16.0          gridGraphics_0.4-1     reactome.db_1.68.0    
 [66] memoise_1.1.0          graphite_1.30.0        plyr_1.8.4             zlibbioc_1.30.0        compiler_3.6.1        
 [71] bbmle_1.0.20           RColorBrewer_1.1-2     cli_1.1.0              ps_1.3.0               htmlTable_1.13.2      
 [76] formatR_1.7            Formula_1.2-3          tidyselect_0.2.5       emdbook_1.3.11         yaml_2.2.0            
 [81] GOSemSim_2.10.0        locfit_1.5-9.1         latticeExtra_0.6-28    fastmatch_1.1-0        tools_3.6.1           
 [86] rstudioapi_0.10        foreign_0.8-71         gridExtra_2.3          farver_1.1.0           ggraph_2.0.0          
 [91] digest_0.6.21          rvcheck_0.1.5          BiocManager_1.30.4     broom_0.5.2            httr_1.4.1            
 [96] colorspace_1.4-1       rvest_0.3.4            fs_1.3.1               splines_3.6.1          graphlayouts_0.5.0    
[101] ggplotify_0.0.4        sessioninfo_1.1.1      xtable_1.8-4           jsonlite_1.6           futile.options_1.0.1  
[106] tidygraph_1.1.2        corpcor_1.6.9          UpSetR_1.4.0           zeallot_0.1.0          testthat_2.2.1        
[111] R6_2.4.0               Hmisc_4.2-0            pillar_1.4.2           htmltools_0.3.6        glue_1.3.1            
[116] fgsea_1.10.1           pkgbuild_1.0.5         numDeriv_2016.8-1.1    GO.db_3.8.2            desc_1.2.0            
[121] munsell_0.5.0          DO.db_2.9              GenomeInfoDbData_1.2.1 haven_2.1.1            gtable_0.3.0* 

Any help would be greatly appreciated!

Theo

First off, note that sva is intended for continuous data that are sort of normal distributed, which is not true of count data. The svaseq function is intended for count data.

Second, when you type in your question, there is a preview pane right below where you are typing, that shows what your post will look like. Bracketing things with an asterisk makes things italicized, which doesn't help with code. Instead you want to either bracket with a triple back-tick (top left key on a QWERTY keyboard) or highlight and click the code button (with the10101010). If your preview doesn't look like what I fixed for you, above, then you need to adjust until it does.

So try using svaseq and post again if it still doesn't work.