Hello All

I have 4 samples M1 , M2 , W1, W2 . I have done alignment with Ecoli ref genome usng hisat2 on this sample i ran stringtie for abundance estimation  Now i am trying to run ball gown on this samples of now i tried this commands:

data_directory <- "/Hisat_stringtie_/string_out/"
bg = ballgown(dataDir=data_directory, samplePattern="string_out_", meas='all')

transcript_fpkm = texpr(bg, 'FPKM')
transcript_cov = texpr(bg, 'cov')
whole_tx_table = texpr(bg, 'all')
exon_mcov = eexpr(bg, 'mcov')
junction_rcount = iexpr(bg)
whole_intron_table = iexpr(bg, 'all')
gene_expression = gexpr(bg)

pData(bg) = data.frame(id=sampleNames(bg), group=rep(c(1,0), each=2))

pheno_data <- pData(bg)

head(pheno_data)

head(bg@dirs)

bg_2 = subset(bg,"rowVars(texpr(bg))>1", genomesubset = TRUE )

stat_results = stattest(bg_2, feature='transcript' ,meas='FPKM', covariate='group')

After running stattest i am getting error:

Error in solve.default(t(mod) %*% mod) :
Lapack routine dgesv: system is exactly singular: U[3,3] = 0

please help me i am not able to understand the error .

Where i am getting wrong

sessionInfo()

R version 3.2.2 (2015-08-14)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 15.10

locale:
 [1] LC_CTYPE=en_IN.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_IN.UTF-8        LC_COLLATE=en_IN.UTF-8    
 [5] LC_MONETARY=en_IN.UTF-8    LC_MESSAGES=en_IN.UTF-8   
 [7] LC_PAPER=en_IN.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_IN.UTF-8 LC_IDENTIFICATION=C       

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

other attached packages:
 [1] dplyr_0.5.0                genefilter_1.52.1         
 [3] BiocStyle_1.8.0            ballgown_2.2.0            
 [5] Rqc_1.4.2                  ggplot2_2.2.0             
 [7] ShortRead_1.28.0           GenomicAlignments_1.6.3   
 [9] SummarizedExperiment_1.0.2 Biobase_2.30.0            
[11] Rsamtools_1.22.0           GenomicRanges_1.22.4      
[13] GenomeInfoDb_1.6.3         Biostrings_2.38.4         
[15] XVector_0.10.0             IRanges_2.4.8             
[17] S4Vectors_0.8.11           BiocGenerics_0.16.1       
[19] BiocParallel_1.4.3        

loaded via a namespace (and not attached):
 [1] splines_3.2.2            GenomicFiles_1.6.2      
 [3] Formula_1.2-1            shiny_0.14.2            
 [5] assertthat_0.1           highr_0.6               
 [7] latticeExtra_0.6-28      BSgenome_1.38.0         
 [9] RSQLite_1.1              lattice_0.20-34         
[11] biovizBase_1.18.0        limma_3.26.9            
[13] digest_0.6.10            RColorBrewer_1.1-2      
[15] colorspace_1.3-1         htmltools_0.3.5         
[17] httpuv_1.3.3             Matrix_1.2-7.1          
[19] plyr_1.8.4               XML_3.98-1.5            
[21] biomaRt_2.26.1           zlibbioc_1.16.0         
[23] xtable_1.8-2             scales_0.4.1            
[25] htmlTable_1.7            tibble_1.2              
[27] annotate_1.48.0          mgcv_1.8-16             
[29] GenomicFeatures_1.22.13  nnet_7.3-12             
[31] lazyeval_0.2.0           survival_2.40-1         
[33] magrittr_1.5             mime_0.5                
[35] evaluate_0.10            memoise_1.0.0           
[37] nlme_3.1-128             hwriter_1.3.2           
[39] foreign_0.8-67           tools_3.2.2             
[41] data.table_1.10.0        stringr_1.1.0           
[43] munsell_0.4.3            cluster_2.0.5           
[45] AnnotationDbi_1.32.3     lambda.r_1.1.9          
[47] futile.logger_1.4.3      grid_3.2.2              
[49] RCurl_1.95-4.8           dichromat_2.0-0         
[51] VariantAnnotation_1.16.4 labeling_0.3            
[53] bitops_1.0-6             gtable_0.2.0            
[55] DBI_0.5-1                markdown_0.7.7          
[57] reshape2_1.4.2           R6_2.2.0                
[59] gridExtra_2.2.1          knitr_1.15.1            
[61] rtracklayer_1.30.4       Hmisc_4.0-0             
[63] futile.options_1.0.0     stringi_1.1.2           
[65] sva_3.18.0               Rcpp_0.12.8             
[67] rpart_4.1-10             acepack_1.4.1  

Thanks

Hello

The problem could be because you only have one sample you are testing for each group. Ballgown can not compute a test statistic for comparing single samples because it can not estimate the variance. You could simply calculate the fold change and then use that as your ranking system, but it is not usually possible to get a measure of statistical significance if you only have one sample in each group. 

Best

Jeff