Question: RSEM calculate expression troubleshoot
1
gravatar for prabin.dm
7 months ago by
prabin.dm30
prabin.dm30 wrote:

Hi, I am trying to use RSEM following this [tutorial]

This is the error code I am getting.

readline() on closed filehandle INPUT at /share/pkg/RSEM/1.3.0/bin/rsem-calculate-expression line 350.
Use of uninitialized value $line in scalar chomp at /share/pkg/RSEM/1.3.0/bin/rsem-calculate-expression line 350.
Use of uninitialized value $line in split at /share/pkg/RSEM/1.3.0/bin/rsem-calculate-expression line 352.
Use of uninitialized value $type in numeric ne (!=) at /share/pkg/RSEM/1.3.0/bin/rsem-calculate-expression line 353.

Can anyone explain what this means?

Here is the command I used. I should also mention that I have not used the same datasets in the tutorial I downloaded a different dataset and trying to use it. dataset1

The fastq files were QC-ed with FastQC and trimmed with trimmomatric

# LSBATCH: User input
#!/bin/bash

#BSUB -q long
#BSUB -W 2:0 
#BSUB -n 8 
#BSUB -R "rusage[mem=2048] span[ptile=2]"
#BSUB -J rsem_SRR836120
#BSUB -o out.%J
#BSUB -e err.%J

module load RSEM/1.3.0
module load bowtie2/2.3.2

rsem-calculate-expression -p 8  \
                --bowtie2 --bowtie2-path /share/pkg/bowtie2/2.3.2 \
                --estimate-rspd \
                --append-names \
                --output-genome-bam \
                SRR836120_1.fastq_trim.fastq \
               /ref/mouse_ref \
              /SRR836120_Rsem

module unload RSEM/1.3.0
module unload bowtie2/2.3.2
rsem rna-seq • 430 views
ADD COMMENTlink modified 7 months ago • written 7 months ago by prabin.dm30
2

Snippet from rsem-calculate-expression perl scipt.

348 if ($genGenomeBamF) { 349 open(INPUT, "$refName.ti"); 350 my $line = <input>; chomp($line); 351 close(INPUT); 352 my ($M, $type) = split(/ /, $line); 353 pod2usage(-msg => "No genome information provided, so genome bam file cannot be generated!\n", -exitval => 2, -verbose => 2) if ($type != 0); 354 }

On the line 350 of rsem-calculate-expression script is indicating that reading some content from INPUT file handler.

The usual error readline() on closed filehandle is raise because PERL is unable to open the input file and it subsequently fails to read the contents from it.

No genome information provided, so genome bam file cannot be generated, I think there is some problem with your reference file.

Does your program interrupt?

ADD REPLYlink written 7 months ago by Nitin Narwade380

Thanks Nitin,

I went back and checked there genome reference. And used a prebuild reference. And it worked fine.

ADD REPLYlink written 7 months ago by prabin.dm30

What is the command-line you used? What tutorial are you following, the link didn't get posted.

ADD REPLYlink written 7 months ago by h.mon23k

I updated the post with details. Thank you for your response.

ADD REPLYlink written 7 months ago by prabin.dm30

Did you try running the command interactively (without submitting it as a job)?

ADD REPLYlink written 7 months ago by igor7.3k

Was able to solve it by using a prebuilt reference. Thanks for your response

ADD REPLYlink written 7 months ago by prabin.dm30
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