Question: Model problem using ballgown
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gravatar for Yong Li
20 months ago by
Yong Li50
Yong Li50 wrote:

Hello,

I am having problems to analyzing an RNA-seq dataset using Ballgown. The RNA are from 3 patients and 3 gender and age matched controls. For each individual there are 3 technical replicates, except for one patient with 6 technical replicates. So there are 21 samples in total. I have processed the data following the Nature Protocol paper using HISAT, StringTie and load the data as Ballgown object into R without trouble.

I wanted to find differentially expressed genes between patients and controls, taking into account the information that each of the 3 patients is paired with a control. The phenotype data is as below:

sampleID    case_control    pair    replicate
S10    patient    pair2    p2
S11    patient    pair2    p2
S12    patient    pair2    p2
S13    control    pair3    c3
S14    control    pair3    c3
S15    control    pair3    c3
S16    patient    pair3    p3
S17    patient    pair3    p3
S18    patient    pair3    p3
S19    patient    pair3    p3
S1    control    pair1    c1
S20    patient    pair3    p3
S21    patient    pair3    p3
S2    control    pair1    c1
S3    control    pair1    c1
S4    patient    pair1    p1
S5    patient    pair1    p1
S6    patient    pair1    p1
S7    control    pair2    c2
S8    control    pair2    c2
S9    control    pair2    c2

For that I tried the following command but got an error:

> results_t <- stattest(bg_filt, feature="transcript", covariate="case_control", adjustvars = c("pair", "replicate"), getFC=TRUE, meas="FPKM")

Coefficients not estimable: replicatep1 replicatep2 replicatep3
Error in solve.default(t(mod) %*% mod) :
  system is computationally singular: reciprocal condition number = 4.81307e-27
In addition: Warning message:
Partial NA coefficients for 26438 probe(s)

I can run the above command with adjustvars = c("pair") successfully, but this way I am worrying that the technical replicate information is not used. I have read the other posts dealing with the same ballgown error but still not clear what to do in this case.

Many thanks for your help.

Yong Li

> sessionInfo()
R version 3.2.3 (2015-12-10)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Debian GNU/Linux 8 (jessie)

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

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

other attached packages:
[1] devtools_1.13.5    dplyr_0.7.4        genefilter_1.52.1  RSkittleBrewer_1.1
[5] ballgown_2.2.0    

loaded via a namespace (and not attached):
 [1] Rcpp_0.12.16               pillar_1.2.1              
 [3] bindr_0.1.1                RColorBrewer_1.1-2        
 [5] futile.logger_1.4.3        GenomeInfoDb_1.6.3        
 [7] XVector_0.10.0             bitops_1.0-6              
 [9] futile.options_1.0.0       tools_3.2.3               
[11] zlibbioc_1.16.0            digest_0.6.15             
[13] bit_1.1-12                 tibble_1.4.2              
[15] annotate_1.48.0            RSQLite_2.1.0             
[17] memoise_1.1.0              nlme_3.1-124              
[19] lattice_0.20-35            mgcv_1.8-23               
[21] pkgconfig_2.0.1            rlang_0.2.0               
[23] Matrix_1.2-13              DBI_0.8                   
[25] parallel_3.2.3             bindrcpp_0.2.2            
[27] withr_2.1.2                rtracklayer_1.30.4        
[29] Biostrings_2.38.4          S4Vectors_0.8.11          
[31] IRanges_2.4.8              stats4_3.2.3              
[33] bit64_0.9-7                grid_3.2.3                
[35] glue_1.2.0                 Biobase_2.30.0            
[37] R6_2.2.2                   AnnotationDbi_1.32.3      
[39] survival_2.41-3            XML_3.98-1.10             
[41] BiocParallel_1.4.3         limma_3.26.9              
[43] sva_3.18.0                 magrittr_1.5              
[45] lambda.r_1.2               blob_1.1.1                
[47] Rsamtools_1.22.0           GenomicAlignments_1.6.3   
[49] splines_3.2.3              BiocGenerics_0.16.1       
[51] GenomicRanges_1.22.4       assertthat_0.2.0          
[53] SummarizedExperiment_1.0.2 xtable_1.8-2              
[55] RCurl_1.95-4.10           
ballgown linear models • 292 views
ADD COMMENTlink written 20 months ago by Yong Li50
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