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Question: Rsubread stopping half-way through align
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gravatar for Campbell.1933
16 months ago by
Campbell.19330 wrote:

Hi

I've been using Rsubread with a range of new and old FASTQ files and it has been working fine. The latest batch seems to be having some problems and for some of the files I get the following error after it crashes

Traceback:

 1: .C("R_align_wrapper", as.integer(n), as.character(cmd), PACKAGE = "Rsubread")

 2: align(index = "reference_index", minFragLength = 35, nthreads = 30,     readfile1 = "RS-02239140_N3_DMSO_GFP_R1.fastq.gz", output_file = "RWPE.GFP.DMSO.3.BAM")

I think i saw an earlier post about 10 months ago with a similar issue but couldn't see a resolution.

I'm using Rsubread_1.22.2

Anyone got any thoughts?

thanks

 

ADD COMMENTlink modified 15 months ago • written 16 months ago by Campbell.19330

Your post shows a function call but doesn't show any error message. What error message did you get? What you mean by "it crashes"? 

AFAIK there are no unresolved issues with Rsubread unexpectedly stopping.

ADD REPLYlink modified 16 months ago • written 16 months ago by Gordon Smyth32k

That was the error message and then it gives the options 1-4 for how to quit out of R. By crashes then i mean it doesn't do anything else. Maybe that's not the right word?

ADD REPLYlink written 16 months ago by Campbell.19330

The output from traceback is not an error message, so you are keeping us completely in the dark as to what may have gone wrong for you.

ADD REPLYlink written 16 months ago by Gordon Smyth32k

Could you provide your commands and also the full screen output?

ADD REPLYlink modified 16 months ago • written 16 months ago by Wei Shi2.7k

OK will do. I'm traveling so this will take a couple of days. thanks

ADD REPLYlink written 16 months ago by Campbell.19330

The samples divided and run on Hiseq2500 v3 chemistry, high output flowcell, 100SE. The FASTQ files were then catenated.

R version 3.3.0 (2016-05-03) -- "Supposedly Educational" being run on a computer cluster.

In Rsubread

>align(index="reference_index", minFragLength=35, nthreads=30, readfile1="RS-02239142_N4_DMSO_input_R1.fastq.gz", output_file="RWPE.Inp.DMSO.1.BAM")

||                       Threads : 30                                         ||

||                  Phred offset : 33                                         ||

||                     Min votes : 3 / 10                                     ||

||    Maximum allowed mismatches : 3                                          ||

||   Maximum allowed indel bases : 5                                          ||

|| # of best alignments reported : 1                                          ||

||                Unique mapping : yes                                        ||

||                                                                            ||

\\===================== http://subread.sourceforge.net/ ======================//

 

|| The input file contains base space reads.                                  ||

|| The range of Phred scores observed in the data is [2,41] 

 

and then

 

|| The range of Phred scores observed in the data is [2,41]                   ||

....it runs for a while.....

 *** caught segfault ***

address 0x7f3a5ee09fd0, cause 'invalid permissions'

 

Traceback:

 1: .C("R_align_wrapper", as.integer(n), as.character(cmd), PACKAGE = "Rsubread")

 2: align(index = "reference_index", minFragLength = 35, nthreads = 30,     readfile1 = "RS-02239142_N4_DMSO_input_R1.fastq.gz", output_file = "RWPE.Inp.DMSO.1.BAM")

 

 

ADD REPLYlink written 16 months ago by Campbell.19330

I suspect the problem might be related to the concatenation of your fastq files. Can you try to run align() on one of the fastq files before you merge them?

ADD REPLYlink written 16 months ago by Wei Shi2.7k

Yes, that was our thought too, but the non-concatentated file also failed

 

ADD REPLYlink written 16 months ago by Campbell.19330

Could you provide us one of your fastq files in question so we can take a close look? Which reference genome did you use in your mapping?

ADD REPLYlink written 16 months ago by Wei Shi2.7k
1
gravatar for Wei Shi
15 months ago by
Wei Shi2.7k
Australia
Wei Shi2.7k wrote:

Thanks for sending through the data. But we could not reproduce the problem you encountered. Both fastq files were successfully aligned on a linux computer.

Have you checked if you have enough disk space? Could you also provide your session info?

 

ADD COMMENTlink modified 15 months ago • written 15 months ago by Wei Shi2.7k
0
gravatar for Campbell.1933
15 months ago by
Campbell.19330 wrote:

 

We mapped to Hg19

What's the best way to share a fastq file with you?

ADD COMMENTlink written 15 months ago by Campbell.19330
0
gravatar for Wei Shi
15 months ago by
Wei Shi2.7k
Australia
Wei Shi2.7k wrote:

Could you transfer the data via CloudStor which is fast and free to academic users?

https://cloudstor.aarnet.edu.au/sender/

ADD COMMENTlink written 15 months ago by Wei Shi2.7k
0
gravatar for Campbell.1933
15 months ago by
Campbell.19330 wrote:

send you a link to a google drive folder with two fastq files in it. One worked and one didn't. There's also a txt file of the readout from Rsubread for both files.

 

ADD COMMENTlink written 15 months ago by Campbell.19330
0
gravatar for Campbell.1933
15 months ago by
Campbell.19330 wrote:

Hi - did you get the link to share the folder through google drive?

 

ADD COMMENTlink written 15 months ago by Campbell.19330
0
gravatar for Campbell.1933
15 months ago by
Campbell.19330 wrote:

ok 

yes, we think it's something to do with disk space too! 

Ok, i'll get session info

ADD COMMENTlink written 15 months ago by Campbell.19330
0
gravatar for Campbell.1933
15 months ago by
Campbell.19330 wrote:

So, to date, we've been doing as a fisbatch job and then tried modifying the memory allocation

fisbatch --nodes=1 --ntasks-per-node=16 --constraint=CPU-E5-2660

But still seem to be running into the same issue.  

I'm guessing fisbatch won't work always (because of memory allocation issue) and so it will have to  be a slurm job?

 

ADD COMMENTlink written 15 months ago by Campbell.19330

I think you'd better seek help from your system admin.
 

ADD REPLYlink written 15 months ago by Wei Shi2.7k

ok - thanks - yes it sounds like a system admin issue

 

ADD REPLYlink written 15 months ago by Campbell.19330
0
gravatar for Campbell.1933
15 months ago by
Campbell.19330 wrote:

But, one final question - which version of R were you using?

tx

ADD COMMENTlink written 15 months ago by Campbell.19330

R 3.3.0 - the same version as you use.

ADD REPLYlink written 15 months ago by Wei Shi2.7k
0
gravatar for Campbell.1933
15 months ago by
Campbell.19330 wrote:

thanks.

 

ADD COMMENTlink written 15 months ago by Campbell.19330
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