bcl2fastq run error

wintergreen36 · 07-29-2014, 07:19 AM

vanessa@vanessa:~$ /usr/local/bin/configureBclToFastq.pl --input-dir /media/vanessa/UUI/ngs/Data/Intensities/BaseCalls --output-dir /media/vanessa/UUI Unaligned --sample-sheet /media/vanessa/UUI/ngs/Data/Intensities/BaseCalls/SampleSheet.csv
"my" variable $value masks earlier declaration in same statement at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 760.
syntax error at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 747, near "$variable qw(ELAND_FASTQ_FILES_PER_PROCESS)"
Global symbol "$variable" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 749.
syntax error at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 751, near "$directory qw(ELAND_GENOME)"
Global symbol "$self" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$directory" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$project" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$sample" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$lane" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$barcode" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753.
Global symbol "$reference" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753.
syntax error at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 761, near "}"
/usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm has too many errors.
Compilation failed in require at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment.pm line 61.
BEGIN failed--compilation aborted at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment.pm line 61.
Compilation failed in require at /usr/local/bin/configureBclToFastq.pl line 239.
BEGIN failed--compilation aborted at /usr/local/bin/configureBclToFastq.pl line 239.

can any body solve this errors

GenoMax · 07-29-2014, 07:23 AM

Is there a space in the --output-dir declaration "/vanessa/UUI Unaligned"?

wintergreen36 · 07-30-2014, 12:58 AM

Yes ther is space ,

vanessa@vanessa:~$ /usr/local/bin/configureBclToFastq.pl --input-dir /media/vanessa/UUI/ngs/Data/Intensities/BaseCalls --output-dir /media/vanessa/UUI/ Unaligned --sample-sheet /media/vanessa/UUI/ngs/Data/Intensities/BaseCalls/SampleSheet.csv

Please watch the screen shot of usr/local/bin

is there any other requirements other than existed ,like simple XML executables , to begin ?

GenoMax · 07-30-2014, 04:01 AM

When you specify an --output-dir location the "Unaligned" directory is automatically created by the configureBclToFastq.pl script in that directory. You do not need to specify it on the command line.

Omit the "Unaligned" from your command line and the program should work.

You may want to consider adding "--fastq-cluster-count 0" (without quotes) to your command line if you want the sequence output in a single file as opposed to 4 millon fastq sequences each in more than one file.

wintergreen36 · 07-30-2014, 05:41 AM

I solved it , Thanks buddy

wintergreen36 · 08-05-2014, 01:20 AM

@ Genomax

Thanks for the above guidelines
Finally I could able to run the Bcl2fastq , i tried to demultiplex 2X100 pe nextra exome data with 8 bp indices ,unfortunately all fastq files were moved to undetermined_Indices folder , in which sample_lane1 , sample_lane2 has no data except sample sheet ,rest of the folders lane 3 to lane 8 goy sample sheet + fasq.gz files

the command I uses is /usr/local/bin/configureBclToFastq.pl --input-dir
<BaseCalls_dir> --output-dir <Unaligned> --sample-sheet
<BaseCalls_dir>/SampleSheet.csv --use-bases-mask y100n,I8n,Y100N

GenoMax · 08-05-2014, 04:05 AM

If lanes 3-8 worked fine then the format of your sample sheet as well as the command line is fine. Did you look at the Demultiplex_Stats.htm file to ascertain that the demultiplexing had worked as expected (i.e. more reads assigned to samples than are in the "undetermined" pool)? Is the situation reverse for lanes 1 and2 (i.e more reads in undetermined row for lanes 1/2)?

Start looking at the fastq ID headers of the "lane1_undetermined*" files to see if there are any problems with the tag reads (i.e. N's etc).

wintergreen36 · 08-11-2014, 10:09 PM

Hello GEnomax thanks for ur support I SUCCESFULY INSTALLED bcl2fastq BUt while running a project i got the follwing error ,

[2014-08-12 13:23:41] [vanessa] [Temp/L006_R1_demux_summary.xml] Processing BCL files for lane 6, tile 2316, read 1
[2014-08-12 13:24:14] [vanessa] [Temp/L006_R1_demux_summary.xml] runtime error: boost::filesystem::rename: No such file or directory: "/media/vanessa/Sandor_1/Unaligned/Undetermined_indices/Sample_lane6/lane6_Undetermined_L006_R1_001.fastq.tmp.gz", "/media/vanessa/Sandor_1/Unaligned/Undetermined_indices/Sample_lane6/lane6_Undetermined_L006_R1_001.fastq.gz"
make: *** [Temp/L006_R1_demux_summary.xml] Error 2

Could you please guide me how to over come this error

thanks

GenoMax · 08-12-2014, 04:05 AM

Unfortunately not enough information (at least for me) in that error snippet. Have you looked further up in the log file to see if there was any other errors (missing BCL/stat files etc)? Were there any notes from the facility that ran the flowcell about lane 6 experiencing problems during run (no clusters may be a possibility if the error above is real)?

wintergreen36 · 08-12-2014, 05:05 AM

I ran this there was no errors during the run

GenoMax · 08-12-2014, 05:22 AM

Have you tried removing entries for lane 6 samples from your samplesheet and checking to see if the remaining lanes demultiplex?

wintergreen36 · 08-12-2014, 05:31 AM

I haven't , There was no boost libs installed in my system , I just updated libboost all ; started run again

wintergreen36 · 08-12-2014, 05:32 AM

What was the impact of these boost lib on Bcl2fastq run?

wintergreen36 · 08-12-2014, 05:35 AM

There was no boost lib in my system , what's the impact of boost lib on bcl2fastq run ? I just installed lib boost and started the bcl2fastq run again.

GenoMax · 08-12-2014, 05:43 AM

That is odd. You have been able to run bcl2fastq before and I would have though that Boost libraries are required for the software to function. Let us see what happens with the new run.

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07-29-2014, 07:19 AM	#1
wintergreen36 Member Location: india Join Date: Jul 2014 Posts: 19	bcl2fastq run error vanessa@vanessa:~$ /usr/local/bin/configureBclToFastq.pl --input-dir /media/vanessa/UUI/ngs/Data/Intensities/BaseCalls --output-dir /media/vanessa/UUI Unaligned --sample-sheet /media/vanessa/UUI/ngs/Data/Intensities/BaseCalls/SampleSheet.csv "my" variable $value masks earlier declaration in same statement at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 760. syntax error at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 747, near "$variable qw(ELAND_FASTQ_FILES_PER_PROCESS)" Global symbol "$variable" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 749. syntax error at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 751, near "$directory qw(ELAND_GENOME)" Global symbol "$self" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753. Global symbol "$directory" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753. Global symbol "$project" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753. Global symbol "$sample" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753. Global symbol "$lane" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753. Global symbol "$barcode" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753. Global symbol "$reference" requires explicit package name at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 753. syntax error at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm line 761, near "}" /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment/Config.pm has too many errors. Compilation failed in require at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment.pm line 61. BEGIN failed--compilation aborted at /usr/local/lib/bcl2fastq-1.8.3/perl/Casava/Alignment.pm line 61. Compilation failed in require at /usr/local/bin/configureBclToFastq.pl line 239. BEGIN failed--compilation aborted at /usr/local/bin/configureBclToFastq.pl line 239. can any body solve this errors

07-30-2014, 05:41 AM	#5
wintergreen36 Member Location: india Join Date: Jul 2014 Posts: 19	solved I solved it , Thanks buddy

08-05-2014, 01:20 AM	#6
wintergreen36 Member Location: india Join Date: Jul 2014 Posts: 19	@ Genomax Thanks for the above guidelines Finally I could able to run the Bcl2fastq , i tried to demultiplex 2X100 pe nextra exome data with 8 bp indices ,unfortunately all fastq files were moved to undetermined_Indices folder , in which sample_lane1 , sample_lane2 has no data except sample sheet ,rest of the folders lane 3 to lane 8 goy sample sheet + fasq.gz files the command I uses is /usr/local/bin/configureBclToFastq.pl --input-dir <BaseCalls_dir> --output-dir <Unaligned> --sample-sheet <BaseCalls_dir>/SampleSheet.csv --use-bases-mask y100n,I8n,Y100N Last edited by wintergreen36; 08-05-2014 at 01:21 AM. Reason: missing content

07-29-2014, 07:23 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,087	Is there a space in the --output-dir declaration "/vanessa/UUI Unaligned"?

07-30-2014, 04:01 AM	#4
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,087	When you specify an --output-dir location the "Unaligned" directory is automatically created by the configureBclToFastq.pl script in that directory. You do not need to specify it on the command line. Omit the "Unaligned" from your command line and the program should work. You may want to consider adding "--fastq-cluster-count 0" (without quotes) to your command line if you want the sequence output in a single file as opposed to 4 millon fastq sequences each in more than one file.

08-05-2014, 04:05 AM	#7
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,087	If lanes 3-8 worked fine then the format of your sample sheet as well as the command line is fine. Did you look at the Demultiplex_Stats.htm file to ascertain that the demultiplexing had worked as expected (i.e. more reads assigned to samples than are in the "undetermined" pool)? Is the situation reverse for lanes 1 and2 (i.e more reads in undetermined row for lanes 1/2)? Start looking at the fastq ID headers of the "lane1_undetermined*" files to see if there are any problems with the tag reads (i.e. N's etc).

08-11-2014, 10:09 PM	#8
wintergreen36 Member Location: india Join Date: Jul 2014 Posts: 19	Hello GEnomax thanks for ur support I SUCCESFULY INSTALLED bcl2fastq BUt while running a project i got the follwing error , [2014-08-12 13:23:41] [vanessa] [Temp/L006_R1_demux_summary.xml] Processing BCL files for lane 6, tile 2316, read 1 [2014-08-12 13:24:14] [vanessa] [Temp/L006_R1_demux_summary.xml] runtime error: boost::filesystem::rename: No such file or directory: "/media/vanessa/Sandor_1/Unaligned/Undetermined_indices/Sample_lane6/lane6_Undetermined_L006_R1_001.fastq.tmp.gz", "/media/vanessa/Sandor_1/Unaligned/Undetermined_indices/Sample_lane6/lane6_Undetermined_L006_R1_001.fastq.gz" make: *** [Temp/L006_R1_demux_summary.xml] Error 2 Could you please guide me how to over come this error thanks

08-12-2014, 04:05 AM	#9
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,087	Unfortunately not enough information (at least for me) in that error snippet. Have you looked further up in the log file to see if there was any other errors (missing BCL/stat files etc)? Were there any notes from the facility that ran the flowcell about lane 6 experiencing problems during run (no clusters may be a possibility if the error above is real)?

08-12-2014, 05:05 AM	#10
wintergreen36 Member Location: india Join Date: Jul 2014 Posts: 19	I ran this there was no errors during the run

08-12-2014, 05:22 AM	#11
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,087	Have you tried removing entries for lane 6 samples from your samplesheet and checking to see if the remaining lanes demultiplex?

08-12-2014, 05:31 AM	#12
wintergreen36 Member Location: india Join Date: Jul 2014 Posts: 19	I haven't , There was no boost libs installed in my system , I just updated libboost all ; started run again

08-12-2014, 05:32 AM	#13
wintergreen36 Member Location: india Join Date: Jul 2014 Posts: 19	What was the impact of these boost lib on Bcl2fastq run?

08-12-2014, 05:35 AM	#14
wintergreen36 Member Location: india Join Date: Jul 2014 Posts: 19	There was no boost lib in my system , what's the impact of boost lib on bcl2fastq run ? I just installed lib boost and started the bcl2fastq run again.

08-12-2014, 05:43 AM	#15
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,087	That is odd. You have been able to run bcl2fastq before and I would have though that Boost libraries are required for the software to function. Let us see what happens with the new run.