#8 FASTQ quality value problem

[floflooo]

From Anestis Gkanogiannis:

Greetings Dr. Angly,

I am writing you about a problem we are facing in our lab while using your sequence simulating software, http://sourceforge.net/projects/biogrinder/files/biogrinder/

Specifically, we are trying to generate quality enabled reads dataset (fastq).

The procedure we are following is (as non root user following the instructions for installing local perl lib on a Linux machine):

1) Download and extract code from the link above.

2) perl Makefile.PL

3) make

4) make install

The same procedure was performed on a Unix machine with root privileges.

We execute grinder as follows

grinder -rf reference.fna -tr 100 -md poly4 3e-3 3.3e-8 -fq 1 -ql 30 10

Reference genomes were obtained from ftp://ftp.ncbi.nih.gov/refseq/release/microbial

We observe the following problems on 3 different machines

A)first the output .fastq file contains quality values, but they are all "?", which is 30 based on Sanger quality encoding.

B)second the output .fastq file does not contain any quality values at all

Machine setup was as follow

Machine 1 and 2 are Linux 2.6.18 64bit. Non root user with local perl lib installed. Problems A and B where noticed.

Machine 3 is an Intel MacBook Pro with root privileges. Problem A was noticed.

We would appreciate any advice or help on those problems.

Thank you very much for your time and your valuable work.

Regards,

Anestis Gkanogiannis

[floflooo]

Dear Anestis,

Thank you very much for providing a short, reproducible example. Unfortunately, I am not able to reproduce your issue. This suggests to me that the specific Perl module versions you have are responsible for your issue.

Could you please run the following commands and report which versions you have?

$ perl -v
$ perl -e 'use Bio::Perl; print $Bio::Perl::VERSION."\n";'
1.006902
$ perl -e 'use Getopt::Euclid; print $Getopt::Euclid::VERSION."\n";'
0.4.0
$ perl -e 'use Math::Random::MT; print $Math::Random::MT::VERSION."\n";'
1.16

Notably, if you have an older version of Getopt::Euclid, upgrade it using:
$ sudo cpan Getopt::Euclid

Please let me know if this helps,

Florent

[Anestis Gkanogiannis]

Dear Florent,

The output of the versions on our machines is as follows

$ perl -v
This is perl, v5.8.8 built for x86_64-linux-thread-multi
$ perl -e 'use Bio::Perl; print $Bio::Perl::VERSION."\n";'
1.5
$ perl -e 'use Getopt::Euclid; print $Getopt::Euclid::VERSION."\n";'
0.4.0
$ perl -e 'use Math::Random::MT; print $Math::Random::MT::VERSION."\n";'
1.16

Versions seem current, but unfortunately our problem remains, output fastq file does not contain quality values.

Thanks,
Anestis

[floflooo]

Thanks you for the information, Anestis. Actually, the Bioperl modules you have are quite old. Could you try to upgrade them? On your MacBook Pro with root privileges, this means running:

sudo cpan Bio::Perl

Please let me know if this fixes the issue you have.

[floflooo]

Yes?

[Anonymous]

Hello, I am still waiting systems administrator to reply, since I don't have root privileges to do it myself.

[floflooo]

Anestis, sorry, I thought you had sudo access on this computer.

I can confirm that you need a current version of Bioperl. I tried version 1.4 of Bioperl and noticed the problem you reported (no quality scores in the FASTQ file). Actually, looking at the Grinder code and instructions, version 1.6.901 is required. If the regular Grinder installation procedure is followed, then it should not complete successfully with an older version of Bioperl. The bottom line is that I am quite confident that upgrading Bioperl on machine 3 will solve your problem.

On machine 1 and 2, can you please also report version numbers to confirm that the minimum requirements are met?

Thanks,

Florent