Error message when trying to use the 'Align' function of the 'Rsubread' package
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@thomasdobrenel-9981
Last seen 5.4 years ago

Macintosh; Intel Mac OS X 10_10_5

Rstudio Version 0.99.903

R version 3.3.1 (2016-06-21) -- "Bug in Your Hair"

Dear colleagues,

I tried to use the 'Rsubread' package for doing an alignment between my RNAseq data and the index of the genome I just created and I have an error message (see below). I first thought that there was something wrong with the index I created but it seems that the error is most likely caused by the reading of the files. I double-checked and the names of the files are exactly identical to the names in the table and are located in the appropriate folder and I also tried to change the name of the FASTQ documents both in the .fastq and in the .txt but this didn't help. Could somebody help me to figure out what can be the cause of this problem and how to solve it? I thank you in advance.

> targets=read.delim("run_info2.txt",header=T)
> targets
FileName CellType Status Type
1 P4355_125_TCTCGCGC-TATAGCCT_L001_R1_001.fastq       PM   DMSO Ctrl
2 P4355_126_TCTCGCGC-ATAGAGGC_L001_R1_001.fastq       PM    AZD Down
3 P4355_127_TCTCGCGC-CCTATCCT_L001_R1_001.fastq    White   DMSO Ctrl
4 P4355_128_TCTCGCGC-GGCTCTGA_L001_R1_001.fastq    White    AZD Down
5 P4355_129_GAATTCGT-AGGCGAAG_L001_R1_001.fastq    White   EtOH Ctrl
6 P4355_130_CTGAAGCT-AGGCGAAG_L001_R1_001.fastq    White   Estr Ctrl
7 P4355_131_TAATGCGC-AGGCGAAG_L001_R1_001.fastq    TOROE   EtOH Ctrl
8 P4355_132_CGGCTATG-AGGCGAAG_L001_R1_001.fastq    TOROE   Estr   Up
> output.files=sub('.fastq','.bam',targetsFileName) > align('mm10',readfile1=targetsFileName,phredOffset=33,input_format='FASTQ',output_file = output.files)
Error in file.exists(readfile1) : invalid 'file' argument
rsubread rnaseq alignment • 854 views
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@gordon-smyth
Last seen 13 hours ago
WEHI, Melbourne, Australia

As Axel has guessed, the problem is that read.delim() has converted your filenames into a factor, and file.exists() will not accept a factor argument. Try this instead:

targets <- read.delim("run_info2.txt", stringsAsFactors=FALSE)
output.files <- sub('.fastq','.bam',targetsFileName) align('mm10',readfile1=targetsFileName,phredOffset=33,input_format='FASTQ',output_file = output.files)
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Hello,

Indeed, you were right, targets$FileName was a factor and not a character and, as such, it couldn't run properly. I then changed the code you gave me and it worked (well, actually, Rstudio finally crashed but at least, it initiated something that I didn't have before). Thank you very much to both of you Tom ADD REPLY 0 Entering edit mode It may help to run it in an ordinary R session instead of in Rstudio. ADD REPLY 1 Entering edit mode Axel Klenk ▴ 980 @axel-klenk-3224 Last seen 13 hours ago Switzerland Dear Thomas, hmm, just an idea -- are you sure that targets$FileName is a character, not a factor?

As the error message claims the problem is with file.exists -- what's the result of

file.exists(targets\$FileName)

?

Cheers,

- axel