DESeq2 ddsHTSeq error
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Entering edit mode
@matthew-mccormack-2021
Last seen 5 weeks ago
United States

I am running DESeq2 with 100 HTSeq files. My sampleTable is:

sampleTable

      sampleName                     fileName  condition
1      ate1-1_ah    HTseq_ate1-1_ah_HTSeq.txt        ate
2      ate1-2_ah    HTseq_ate1-2_ah_HTSeq.txt        ate
3      azf1-1_ah    HTseq_azf1-1_ah_HTSeq.txt        azf
4      azf1-2_ah    HTseq_azf1-2_ah_HTSeq.txt        azf
5       con_0h_1     HTseq_con_0h_1_HTSeq.txt     con_0h
6       con_0h_2     HTseq_con_0h_2_HTSeq.txt     con_0h
7     con_0p5h_1   HTseq_con_0p5h_1_HTSeq.txt   con_0p5h
8     con_0p5h_2   HTseq_con_0p5h_2_HTSeq.txt   con_0p5h
9      con_12h_1    HTseq_con_12h_1_HTSeq.txt    con_12h
10     con_12h_2    HTseq_con_12h_2_HTSeq.txt    con_12h
11     con_15h_1    HTseq_con_15h_1_HTSeq.txt    con_15h
12     con_15h_2    HTseq_con_15h_2_HTSeq.txt    con_15h
13     con_18h_1    HTseq_con_18h_1_HTSeq.txt    con_18h
14     con_18h_2    HTseq_con_18h_2_HTSeq.txt    con_18h
15      con_1h_1     HTseq_con_1h_1_HTSeq.txt     con_1h
16      con_1h_2     HTseq_con_1h_2_HTSeq.txt     con_1h
17     con_21h_1    HTseq_con_21h_1_HTSeq.txt    con_21h
18     con_21h_2    HTseq_con_21h_2_HTSeq.txt    con_21h
19     con_24h_1    HTseq_con_24h_1_HTSeq.txt     con24h
20     con_24h_2    HTseq_con_24h_2_HTSeq.txt     con24h
21     con_27h_1    HTseq_con_27h_1_HTSeq.txt    con_27h
22     con_27h_2    HTseq_con_27h_2_HTSeq.txt    con_27h
23     con_30h_1    HTseq_con_30h_1_HTSeq.txt    con_30h
24     con_30h_2    HTseq_con_30h_2_HTSeq.txt    con_30h
25      con_3h_1     HTseq_con_3h_1_HTSeq.txt     con_3h
26      con_3h_2     HTseq_con_3h_2_HTSeq.txt     con_3h
27      con_6h_1     HTseq_con_6h_1_HTSeq.txt     con_6h
28      con_6h_2     HTseq_con_6h_2_HTSeq.txt     con_6h
29      con_9h_1     HTseq_con_9h_1_HTSeq.txt     con_9h
30      con_9h_2     HTseq_con_9h_2_HTSeq.txt     con_9h
31       e1-1_ah      HTseq_e1-1_ah_HTSeq.txt         e1
32       e1-2_ah      HTseq_e1-2_ah_HTSeq.txt         e1
33     e104-1_ah    HTseq_e104-1_ah_HTSeq.txt       e104
34     e104-2_ah    HTseq_e104-2_ah_HTSeq.txt       e104
35     e105-1_ah    HTseq_e105-1_ah_HTSeq.txt       e105
36     e105-2_ah    HTseq_e105-2_ah_HTSeq.txt       e105
37     e112-1_ah    HTseq_e112-1_ah_HTSeq.txt       e112
38     e112-2_ah    HTseq_e112-2_ah_HTSeq.txt       e112
39       e4-1_ah      HTseq_e4-1_ah_HTSeq.txt         e4
40       e4-2_ah      HTseq_e4-2_ah_HTSeq.txt         e4
41       e5-1_ah      HTseq_e5-1_ah_HTSeq.txt         e5
42       e5-2_ah      HTseq_e5-2_ah_HTSeq.txt         e5
43       e6-1_ah      HTseq_e6-1_ah_HTSeq.txt         e6
44       e6-2_ah      HTseq_e6-2_ah_HTSeq.txt         e6
45      e98-1_ah     HTseq_e98-1_ah_HTSeq.txt        e98
46      e98-2_ah     HTseq_e98-2_ah_HTSeq.txt        e98
47   flg22_10m_1  HTseq_flg22_10m_1_HTSeq.txt  flg22_10m
48   flg22_10m_2  HTseq_flg22_10m_2_HTSeq.txt  flg22_10m
49   flg22_12h_1  HTseq_flg22_12h_1_HTSeq.txt  flg22_12h
50   flg22_12h_2  HTseq_flg22_12h_2_HTSeq.txt  flg22_12h
51   flg22_15h_1  HTseq_flg22_15h_1_HTSeq.txt  flg22_15h
52   flg22_15h_2  HTseq_flg22_15h_2_HTSeq.txt  flg22_15h
53   flg22_15m_1  HTseq_flg22_15m_1_HTSeq.txt  flg22_15m
54   flg22_15m_2  HTseq_flg22_15m_2_HTSeq.txt  flg22_15m
55   flg22_18h_1  HTseq_flg22_18h_1_HTSeq.txt  flg22_18h
56   flg22_18h_2  HTseq_flg22_18h_2_HTSeq.txt  flg22_18h
57    flg22_1h_1   HTseq_flg22_1h_1_HTSeq.txt   flg22_1h
58    flg22_1h_2   HTseq_flg22_1h_2_HTSeq.txt   flg22_1h
59   flg22_20m_1  HTseq_flg22_20m_1_HTSeq.txt  flg22_20m
60   flg22_20m_2  HTseq_flg22_20m_2_HTSeq.txt  flg22_20m
61   flg22_21h_1  HTseq_flg22_21h_1_HTSeq.txt  flg22_21h
62   flg22_21h_2  HTseq_flg22_21h_2_HTSeq.txt  flg22_21h
63   flg22_24h_1  HTseq_flg22_24h_1_HTSeq.txt  flg22_24h
64   flg22_24h_2  HTseq_flg22_24h_2_HTSeq.txt  flg22_24h
65   flg22_27h_1  HTseq_flg22_27h_1_HTSeq.txt  flg22_27h
66   flg22_27h_2  HTseq_flg22_27h_2_HTSeq.txt  flg22_27h
67  flg22_2p5m_1 HTseq_flg22_2p5m_1_HTSeq.txt flg22_2p5m
68  flg22_2p5m_2 HTseq_flg22_2p5m_2_HTSeq.txt flg22_2p5m
69   flg22_30h_1  HTseq_flg22_30h_1_HTSeq.txt  flg22_30h
70   flg22_30h_2  HTseq_flg22_30h_2_HTSeq.txt  flg22_30h
71   flg22_30m_1  HTseq_flg22_30m_1_HTSeq.txt  flg22_30m
72   flg22_30m_2  HTseq_flg22_30m_2_HTSeq.txt  flg22_30m
73    flg22_3h_1   HTseq_flg22_3h_1_HTSeq.txt   flg22_3h
74    flg22_3h_2   HTseq_flg22_3h_2_HTSeq.txt   flg22_3h
75    flg22_5m_1   HTseq_flg22_5m_1_HTSeq.txt   flg22_5m
76    flg22_5m_2   HTseq_flg22_5m_2_HTSeq.txt   flg22_5m
77    flg22_6h_1   HTseq_flg22_6h_1_HTSeq.txt   flg22_6h
78    flg22_6h_2   HTseq_flg22_6h_2_HTSeq.txt   flg22_6h
79    flg22_9h_1   HTseq_flg22_9h_1_HTSeq.txt   flg22_9h
80    flg22_9h_2   HTseq_flg22_9h_2_HTSeq.txt   flg22_9h
81      m7-1a_ah     HTseq_m7-1a_ah_HTSeq.txt        m7a
82      m7-1b_ah     HTseq_m7-1b_ah_HTSeq.txt        m7b
83      m7-2a_ah     HTseq_m7-2a_ah_HTSeq.txt        m7a
84      m7-2b_ah     HTseq_m7-2b_ah_HTSeq.txt        m7b
85    r2_12-1_ah   HTseq_r2_12-1_ah_HTSeq.txt         r2
86    r2_12-2_ah   HTseq_r2_12-2_ah_HTSeq.txt         r2
87      w15-1_ah     HTseq_w15-1_ah_HTSeq.txt        w15
88      w15-2_ah     HTseq_w15-2_ah_HTSeq.txt        w15
89      w28-1_ah     HTseq_w28-1_ah_HTSeq.txt        w28
90      w28-2_ah     HTseq_w28-2_ah_HTSeq.txt        w28
91      w30-1_ah     HTseq_w30-1_ah_HTSeq.txt        w30
92      w30-2_ah     HTseq_w30-2_ah_HTSeq.txt        w30
93      w40-1_ah     HTseq_w40-1_ah_HTSeq.txt        w40
94      w40-2_ah     HTseq_w40-2_ah_HTSeq.txt        w40
95      z10-1_ah     HTseq_z10-1_ah_HTSeq.txt        z10
96      z10-2_ah     HTseq_z10-2_ah_HTSeq.txt        z10
97      z11-1_ah     HTseq_z11-1_ah_HTSeq.txt        z11
98      z11-2_ah     HTseq_z11-2_ah_HTSeq.txt        z11
99      z12-1_ah     HTseq_z12-1_ah_HTSeq.txt        z12
100     z12-2_ah     HTseq_z12-2_ah_HTSeq.txt        z12

ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = ".", design = ~ condition)

Error in Ops.factor(a$V1, l[[1]]$V1) : 
  level sets of factors are different
In addition: Warning message:
In is.na(e1) | is.na(e2) :
  longer object length is not a multiple of shorter object length

I am running R 3.3.1 on  x86_64-redhat-linux-gnu.

I have tried

> table(file.exists(file.path(".", sampleTable[,2])))

TRUE
100 

And, when each of the 100 .txt files are imported into Excel, they all have the same number of rows and columns. (I won't print the whole thing out, but below is representative for all of the 100. I shows that each file goes from cell A1 to B37341 for each of the 100 files. 

 ='HTseq_ate1-1_ah_HTSeq.txt'!$A$1:$B$37341
 ='HTseq_ate1-2_ah_HTSeq.txt'!$A$1:$B$37341
 ='HTseq_azf1-1_ah_HTSeq.txt'!$A$1:$B$37341
 ='HTseq_azf1-2_ah_HTSeq.txt'!$A$1:$B$37341
 ...
 ='HTseq_z12-1_ah_HTSeq.txt'!$A$1:$B$37341

 ='HTseq_z12-2_ah_HTSeq.txt'!$A$1:$B$37341

So, the files all seem to exist and be the exact same size.

> sessionInfo()
R version 3.3.1 (2016-06-21)
Platform: x86_64-redhat-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)

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

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

other attached packages:
 [1] DESeq2_1.10.1              RcppArmadillo_0.7.800.2.0  Rcpp_0.12.11               Rsubread_1.20.6           
 [5] BiocInstaller_1.20.3       ShortRead_1.28.0           GenomicAlignments_1.6.3    SummarizedExperiment_1.0.2
 [9] Biobase_2.30.0             Rsamtools_1.22.0           GenomicRanges_1.22.4       GenomeInfoDb_1.6.3        
[13] Biostrings_2.38.4          XVector_0.10.0             IRanges_2.4.8              S4Vectors_0.8.11          
[17] BiocParallel_1.4.3         BiocGenerics_0.16.1       

loaded via a namespace (and not attached):
 [1] genefilter_1.52.1    locfit_1.5-9.1       splines_3.3.1        lattice_0.20-35      colorspace_1.3-2     htmltools_0.3.6     
 [7] base64enc_0.1-3      XML_3.98-1.7         survival_2.41-3      rlang_0.1.1          DBI_0.6-1            foreign_0.8-68      
[13] RColorBrewer_1.1-2   lambda.r_1.1.9       plyr_1.8.4           stringr_1.2.0        zlibbioc_1.16.0      munsell_0.4.3       
[19] gtable_0.2.0         futile.logger_1.4.3  hwriter_1.3.2        htmlwidgets_0.8      memoise_1.1.0        latticeExtra_0.6-28 
[25] knitr_1.16           geneplotter_1.48.0   AnnotationDbi_1.32.3 htmlTable_1.9        acepack_1.4.1        xtable_1.8-2        
[31] scales_0.4.1         backports_1.1.0      checkmate_1.8.2      Hmisc_4.0-3          annotate_1.48.0      gridExtra_2.2.1     
[37] ggplot2_2.2.1        digest_0.6.12        stringi_1.1.5        grid_3.3.1           tools_3.3.1          bitops_1.0-6        
[43] magrittr_1.5         RSQLite_1.1-2        lazyeval_0.2.0       tibble_1.3.1         Formula_1.2-1        cluster_2.0.6       
[49] futile.options_1.0.0 Matrix_1.2-10        data.table_1.10.4    rpart_4.1-11         nnet_7.3-12 

 

Matthew

deseq2 • 1.3k views
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Entering edit mode
@mikelove
Last seen 10 hours ago
United States

The test that's failing is one which checks that the first column is identical for all files. So can you do some more spot testing on your end?

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Entering edit mode
Thank you, Michael. This will give me a place to start checking. The first column is the gene names. I will check to see if they are equivalent in all files. Matthew On 5/25/2017 6:30 PM, Michael Love [bioc] wrote: > Activity on a post you are following on support.bioconductor.org > <https: support.bioconductor.org=""> > > User Michael Love <https: support.bioconductor.org="" u="" 5822=""/> wrote > Answer: DESeq2 ddsHTSeq error > <https: support.bioconductor.org="" p="" 96302="" #96308="">: > > The test that's failing is one which checks that the first column is > identical for all files. So can you do some more spot testing on your end? > > ------------------------------------------------------------------------ > > Post tags: deseq2 > > You may reply via email or visit > A: DESeq2 ddsHTSeq error >
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