Tweet
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.
-
-
thank you Devon. good to know.Comment
-
Hello pm2012,Originally posted by pm2012 View Post
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?
ThanksComment
-
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:
This is the script I am using: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
I also tried running this code from the command line as mentioned above: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)
But got this error:Code:cut -f merged_sample_2.bam_htseq_out.txt | sort | uniq -c
Any help would be appreciated. Thanks!Code:cut: [-cf] list: illegal list value
Last edited by ronaldrcutler; 05-09-2016, 07:35 PM.Comment
-
I think the error you describe may be due to mismatch between the count tables that you provided (e.g. different number of rows, non-unique rows, typos).
How did you generate your input files? How do your count txt files look?Comment
-
The input files were generated using HT-Seq with this specific command line argument:
The txt file look weird compared to some of my colleagues previous runs. Mine is around 600mb and looks some thing like: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)
While theirs is around 3 mb and looks like (what I know I should be getting):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
This leads me to believe it may be more of a problem with HT-Seq...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
Comment
-
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.Comment
-
Good news, I tried it and started getting the right output files! Will let you know if anything else comes up when running DESeq.Comment
-
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!Comment
-
I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
Comment