DEXSeq: Problem running more than 7 pairs of samples

[har]

Hi all,
I have been trying to use DEXSeq for alternative splicing analysis. We have RNA-Seq data for 14 pairs of treated/untreated samples and I'm doing paired analysis following example in the DEXSeq manual.

When I try to do paired analysis for all 14, I get the following error:

ecs = read.HTSeqCounts(countfiles = file.path(inDir, rownames(samples)), design = samples, flattenedfile = annotationfile)

Error in `rownames<-`(`*tmp*`, value = c("ENSG00000000003:001", "ENSG00000000003:002", :
attempt to set rownames on object with no dimensions

This error goes away when I run 7 pairs. I have the latest version of DEXSeq installed.

Thank you

[Simon Anders]

I doubt that this has much to do with the number of samples. Can you post the content of the "samples" data frame (including rownames), and the output of "file.path(inDir, rownames(samples))"? Maybe something is strange with one of the count files?

[har]

Here's the content for "samples".

condition
Alcoholic1a.txt Alcoholic1
Alcoholic1b.txt Alcoholic1
Alcoholic1c.txt Alcoholic1
Alcoholic1d.txt Alcoholic1
Alcoholic1e.txt Alcoholic1
Alcoholic1f.txt Alcoholic1
Alcoholic1g.txt Alcoholic1
Alcoholic1h.txt Alcoholic1
Alcoholic1i.txt Alcoholic1
Alcoholic1j.txt Alcoholic1
Alcoholic1k.txt Alcoholic1
Alcoholic1l.txt Alcoholic1
Alcoholic1m.txt Alcoholic1
Alcoholic1n.txt Alcoholic1
Control1a.txt Control1
Control1b.txt Control1
Control1c.txt Control1
Control1d.txt Control1
Control1e.txt Control1
Control1f.txt Control1
Control1g.txt Control1
Control1h.txt Control1
Control1i.txt Control1
Control1j.txt Control1
Control1k.txt Control1
Control1l.txt Control1
Control1m.txt Control1
Control1n.txt Control1

Unfortunately, I dont have the output for the file.path(inDir, rownames(samples))

Thank you

[Simon Anders]

Unfortunately, I dont have the output for the file.path(inDir, rownames(samples))

Sorry but I guess we'll need this.

[har]

Hi Simon,

I re ran the code and here are the outputs you had asked for.

samples
condition
Alcoholica.txt Alcoholic
Alcoholicb.txt Alcoholic
Alcoholicc.txt Alcoholic
Alcoholicd.txt Alcoholic
Alcoholice.txt Alcoholic
Alcoholicf.txt Alcoholic
Alcoholicg.txt Alcoholic
Alcoholich.txt Alcoholic
Alcoholici.txt Alcoholic
Alcoholicj.txt Alcoholic
Alcoholick.txt Alcoholic
Alcoholicl.txt Alcoholic
Alcoholicm.txt Alcoholic
Alcoholicn.txt Alcoholic
Controla.txt Control
Controlb.txt Control
Controlc.txt Control
Controld.txt Control
Controle.txt Control
Controlf.txt Control
Controlg.txt Control
Controlh.txt Control
Controli.txt Control
Controlj.txt Control
Controlk.txt Control
Controll.txt Control
Controlm.txt Control
Controln.txt Control

d> file.path(inDir, rownames(samples))
[1] "/Users/mayfield/R-dev/package5/data/Alcoholica.txt"
[2] "/Users/mayfield/R-dev/package5/data/Alcoholicb.txt"
[3] "/Users/mayfield/R-dev/package5/data/Alcoholicc.txt"
[4] "/Users/mayfield/R-dev/package5/data/Alcoholicd.txt"
[5] "/Users/mayfield/R-dev/package5/data/Alcoholice.txt"
[6] "/Users/mayfield/R-dev/package5/data/Alcoholicf.txt"
[7] "/Users/mayfield/R-dev/package5/data/Alcoholicg.txt"
[8] "/Users/mayfield/R-dev/package5/data/Alcoholich.txt"
[9] "/Users/mayfield/R-dev/package5/data/Alcoholici.txt"
[10] "/Users/mayfield/R-dev/package5/data/Alcoholicj.txt"
[11] "/Users/mayfield/R-dev/package5/data/Alcoholick.txt"
[12] "/Users/mayfield/R-dev/package5/data/Alcoholicl.txt"
[13] "/Users/mayfield/R-dev/package5/data/Alcoholicm.txt"
[14] "/Users/mayfield/R-dev/package5/data/Alcoholicn.txt"
[15] "/Users/mayfield/R-dev/package5/data/Controla.txt"
[16] "/Users/mayfield/R-dev/package5/data/Controlb.txt"
[17] "/Users/mayfield/R-dev/package5/data/Controlc.txt"
[18] "/Users/mayfield/R-dev/package5/data/Controld.txt"
[19] "/Users/mayfield/R-dev/package5/data/Controle.txt"
[20] "/Users/mayfield/R-dev/package5/data/Controlf.txt"
[21] "/Users/mayfield/R-dev/package5/data/Controlg.txt"
[22] "/Users/mayfield/R-dev/package5/data/Controlh.txt"
[23] "/Users/mayfield/R-dev/package5/data/Controli.txt"
[24] "/Users/mayfield/R-dev/package5/data/Controlj.txt"
[25] "/Users/mayfield/R-dev/package5/data/Controlk.txt"
[26] "/Users/mayfield/R-dev/package5/data/Controll.txt"
[27] "/Users/mayfield/R-dev/package5/data/Controlm.txt"
[28] "/Users/mayfield/R-dev/package5/data/Controln.txt"

Thanks

[areyes]

Could you post the first 10 lines of any of your count files, also from the annotation file?

Alejandro

[har]

Here are the first 10 lines of the annotation file:

1 Homo_sapiens.GRCh37.70.gtf aggregate_gene 11869 14412 . + . gene_id "ENSG00000223972"
1 Homo_sapiens.GRCh37.70.gtf exonic_part 11869 11871 . + . transcripts "ENST00000456328"; exonic_part_number "001"; gene_id "ENSG00000223972"
1 Homo_sapiens.GRCh37.70.gtf exonic_part 11872 11873 . + . transcripts "ENST00000456328+ENST00000515242"; exonic_part_number "002"; gene_id "ENSG00000223972"
1 Homo_sapiens.GRCh37.70.gtf exonic_part 11874 12009 . + . transcripts "ENST00000456328+ENST00000515242+ENST00000518655"; exonic_part_number "003"; gene_id "ENSG00000223972"
1 Homo_sapiens.GRCh37.70.gtf exonic_part 12010 12057 . + . transcripts "ENST00000456328+ENST00000515242+ENST00000450305+ENST00000518655"; exonic_part_number "004"; gene_id "ENSG00000223972"
1 Homo_sapiens.GRCh37.70.gtf exonic_part 12058 12178 . + . transcripts "ENST00000456328+ENST00000515242+ENST00000518655"; exonic_part_number "005"; gene_id "ENSG00000223972"
1 Homo_sapiens.GRCh37.70.gtf exonic_part 12179 12227 . + . transcripts "ENST00000456328+ENST00000515242+ENST00000450305+ENST00000518655"; exonic_part_number "006"; gene_id "ENSG00000223972"
1 Homo_sapiens.GRCh37.70.gtf exonic_part 12595 12612 . + . transcripts "ENST00000518655"; exonic_part_number "007"; gene_id "ENSG00000223972"
1 Homo_sapiens.GRCh37.70.gtf exonic_part 12613 12697 . + . transcripts "ENST00000456328+ENST00000515242+ENST00000450305+ENST00000518655"; exonic_part_number "008"; gene_id "ENSG00000223972"
1 Homo_sapiens.GRCh37.70.gtf exonic_part 12698 12721 . + . transcripts "ENST00000456328+ENST00000515242+ENST00000518655"; exonic_part_number "009"; gene_id "ENSG00000223972"

10 lines of the count file:

ENSG00000000003:001 11
ENSG00000000003:002 5
ENSG00000000003:003 2
ENSG00000000003:004 2
ENSG00000000003:005 3
ENSG00000000003:006 13
ENSG00000000003:007 15
ENSG00000000003:008 3
ENSG00000000003:009 7
ENSG00000000003:010 0


Thank you

[areyes]

Wierd, they look as expected.

Maybe you could try to run the code that is inside the function. It will for sure take you closer to the problem.

Did you make additional moditication to these files after running the python scripts? Do you have something like the lines below at the end of each of your count files?

_ambiguous 51736
_empty 14040641
_lowaqual 0
_notaligned 0

Alejandro

[har]

Thank you all for responding to my question. There was a problem with one of the count files.