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-   -   Error Running DESeq2 (http://seqanswers.com/forums/showthread.php?t=42461)

ronaldrcutler 05-11-2016 02:58 PM

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)
The txt file look weird compared to some of my colleagues previous runs. Mine is around 600mb and looks some thing like:
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

While theirs is around 3 mb and looks like (what I know I should be getting):
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

This leads me to believe it may be more of a problem with HT-Seq...

antoshka 05-11-2016 04:02 PM

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.

ronaldrcutler 05-11-2016 04:05 PM

Good news, I tried it and started getting the right output files! Will let you know if anything else comes up when running DESeq.


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