Just to keep the group in the loop, there ended up being two problems. The error message posted here was due to an apparent typo in one of the count files. Fixing that solved that problem. There was an additional issue due to a header line having been added (I don't know if this was done by htseq-count or not, I should have asked). Removing that allowed for the creation of a proper DESeqDataSet object.
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Originally posted by pm2012 View PostThanks a lot for help. It was indeed a problem with my count files. I didn't realize I had to redirect the output of HTseq into a different file. I was using file generated with -o option as an input.
I reran the script & was able to generate the correct file (also filtered the last few lines starting with __). The rest of code seems to be working well now.
I also got rid of last colum in sampleTable. It was just one of the many things I was trying to solve my issue.
I am having the same problem that you had back then.
I also just used the file produced by -o option and got the same error message.
How exactly did you redirect your output file to make it compatible with DESeq2?
Thanks
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DESeqDataSet creation error
Hi, I'm a new to learning DESeq,
I am having a similar problem that has been talked about here. This is the error:
Code: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
Code:library("DESeq2") files = c("merged_sample_2.bam_htseq_out.txt","merged_sample_11.bam_htseq_out.txt","merged_sample_20.bam_htseq_out.txt","merged_sample_3.bam_htseq_out.txt","merged_sample_12.bam_htseq_out.txt","merged_sample_21.bam_htseq_out.txt") cond = c("GFP","GFP","GFP","DBM","DBM","DBM") sTable = data.frame(sampleName = files, fileName = files, condition = cond) dds <-DESeqDataSetFromHTSeqCount(sampleTable=sTable, directory = "/Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/DESeq", design = ~condition)
Code:cut -f merged_sample_2.bam_htseq_out.txt | sort | uniq -c
Code:cut: [-cf] list: illegal list value
Last edited by ronaldrcutler; 05-09-2016, 07:35 PM.
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The input files were generated using HT-Seq with this specific command line argument:
Code:htseq-count -f bam -s reverse -i Name -o {0}_htseq_out.txt {1} /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/9.1_Reference_Files/XENLA_UTAmayball_cdna_longest_CHRS2.gff3'.format(files, files)
Code:XF:Z:__alignment_not_unique XF:Z:__alignment_not_unique XF:Z:__alignment_not_unique XF:Z:__alignment_not_unique XF:Z:__alignment_not_unique XF:Z:__alignment_not_unique XF:Z:__alignment_not_unique XF:Z:__alignment_not_unique XF:Z:__alignment_not_unique XF:Z:__no_feature XF:Z:__alignment_not_unique XF:Z:__alignment_not_unique
Code:AAGAB|c.Audic201207_X025945|JGIv7b.000058049_4975593-4992662-__chr3L 27 AAGAB|c.Park201106_X000169|JGIv7a.000035880_844976-861439-__chr3L 0 AAGAB|c.Taira201203egg_X008072|NIGv2.S00000107_247362-264101-__chr3L 0 AAGAB|c.Ueno201210kidney_X002041|NIGv2.S00001669_498925-515626-__chr3L 0 AAGAB|c.UniGene_Xl_S20337254|JGIv7b.000036401_3602075-3619253+__chr3S 424
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Hi, I think you are having the same problem that I originally had when I started using htseq-count: namely using the wrong input to run DESeq.
htseq-count writes the count table directly into STDOUT, while the "-o" option creates an additional sam file, which in most cases you won't need.
If you want htseq-count to write a separate text file for your counts, you can use "> yourfilename.txt".
So you should change "-o" to ">" in your script.
Let me know if it worked.
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I am receiving the same error message when attempting to create my dds from HTSeqCounts: Error in Ops.factor(a$V1, l[[1]]$V1) :
level sets of factors are different.
However, I am absolutely sure the first column of features of my htseq-count files are identical and I have deleted any headers. My count files are identical in format to other former lab members who have run successful DESeq and they are all the same length. I am at a loss for why I would still receive this error!
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