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Old 05-11-2016, 02:58 PM   #21
ronaldrcutler
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Location: Virginia

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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)
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...
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Old 05-11-2016, 04:02 PM   #22
antoshka
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Location: Irvine, CA

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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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Old 05-11-2016, 04:05 PM   #23
ronaldrcutler
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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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