Error while demultipleaxing .bcl to .fastq files

Question

[ravi.uhdnis]

Hi,

I am working in NGS data analysis of WGS (Human), using HiSeq 2500 as platform for data generation. I am encountering an error while demultiplexing bcl to fastq files using 'BclToFastq.pl' script. Following is the command that I used:

/usr/local/bcl2fastq-1.8.4/bin/configureBclToFastq.pl --input-dir ./Data/Intensities/BaseCalls/ --output-dir ./Unaligned --sample-sheet ./SampleSheet_Ana.csv
cd Unaligned
make -j 8 | tee Sceen_out

In Screen_out, I grepped 'Error' as shown below:

[2015-09-22 11:53:26]        [master]        [Temp/L006_R2_demux_summary.xml]        Error: 2015-Sep-22 11:53:26: No such file or directory: /tmp/bcl2fastq-1.8.4/src/c++/lib/alignment/BclReader.cpp(87): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
[2015-09-22 11:53:26]        [master]        [Temp/L006_R1_demux_summary.xml]        Error: 2015-Sep-22 11:53:26: No such file or directory: /tmp/bcl2fastq-1.8.4/src/c++/lib/alignment/BclReader.cpp(87): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
[2015-09-22 11:53:26]        [master]        [Temp/L003_R1_demux_summary.xml]        Error: 2015-Sep-22 11:53:26: No such file or directory: /tmp/bcl2fastq-1.8.4/src/c++/lib/alignment/BclReader.cpp(87): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
make: *** [Temp/L006_R2_demux_summary.xml] Error 1
make: *** [Temp/L003_R1_demux_summary.xml] Error 1
[2015-09-22 11:53:26]        [master]        [Temp/L008_R1_demux_summary.xml]        Error: 2015-Sep-22 11:53:26: No such file or directory: /tmp/bcl2fastq-1.8.4/src/c++/lib/alignment/BclReader.cpp(87): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
make: *** [Temp/L006_R1_demux_summary.xml] Error 1
[2015-09-22 11:53:26]        [master]        [Temp/L008_R2_demux_summary.xml]        Error: 2015-Sep-22 11:53:26: No such file or directory: /tmp/bcl2fastq-1.8.4/src/c++/lib/alignment/BclReader.cpp(87): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
make: *** [Temp/L008_R1_demux_summary.xml] Error 1
[2015-09-22 11:53:26]        [master]        [Temp/L003_R2_demux_summary.xml]        Error: 2015-Sep-22 11:53:26: No such file or directory: /tmp/bcl2fastq-1.8.4/src/c++/lib/alignment/BclReader.cpp(87): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
make: *** [Temp/L008_R2_demux_summary.xml] Error 1
make: *** [Temp/L003_R2_demux_summary.xml] Error 1
[2015-09-22 11:53:26]        [master]        [Temp/L004_R1_demux_summary.xml]        Error: 2015-Sep-22 11:53:26: No such file or directory: /tmp/bcl2fastq-1.8.4/src/c++/lib/alignment/BclReader.cpp(87): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
[2015-09-22 11:53:26]        [master]        [Temp/L004_R2_demux_summary.xml]        Error: 2015-Sep-22 11:53:26: No such file or directory: /tmp/bcl2fastq-1.8.4/src/c++/lib/alignment/BclReader.cpp(87): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
make: *** [Temp/L004_R1_demux_summary.xml] Error 1
make: *** [Temp/L004_R2_demux_summary.xml] Error 1

Anybody have idea about such errors (Throw in function unsigned int) and way to solve it. Also, Is there any other software for doing demultiplexing (.bcl to .fastq conversion) other than by BclToFastq.pl program?

[Devon Ryan]

The 2500 supports Bcl2fastq2, which seems to be a bit less annoying (I switched over a couple weeks ago). You might give that a try. For what it's worth, I suspect that you have an issue with your sample sheet (e.g., a unicode character).

[ravi.uhdnis]

Hi, thanks for response. I found this information on Illumina website

The bcl2fastq2 Conversion Software v2.17 can be used to convert BCL files from all Illumina sequencing systems running RTA version 1.18.54 and above, and replaces v2.16. For conversion of data generated on Illumina sequencing systems using versions of RTA earlier than RTA 1.18.54, use bcl2fastq v1.8.4.

Since my data was generated using "Illumina RTA 1.17.21.3" so I think use of bcl2fastq-1.8.4 is ok. Also, didn't find anything wrong within Samplesheet.csv.

[Dan D]

It looks like missing or corrupted BCL files, though I can't be sure from the current content of the error message.

[ravi.uhdnis]

Yes, there are few lines with error messages e.g.

[2015-09-22 16:48:04]    [master]    [Temp/L003_R2_demux_summary.xml]    : File /storage01/HiSeq/WGS/2015/RV217/150827_SN7001137_0115_AC6MWAACXX/Data/Intensities/BaseCalls/L003/C102.1/s_3_1101.bcl.gz does not exist

but I also tried option --ignore-missing-bcl but that didn't solved the problem.

[Dan D]

If the missing bcls are isolated to a specific cycle you can try masking out that cycle. Otherwise, --ignore-missing-bcl is not likely to salvage things.

[ravi.uhdnis]

True, Since missing files weren't isolated to any specific cycle so this usage was not helpful.

[sklages]

But did you get the same eror message? Do you have full (read) to all data files?

You can also use --ignore-missing-stats and --ignore-missing-control ... though I would track down the problem why there are files missing.

[ravi.uhdnis]

Yes, i got the same error message. No, there was an accidental deletions of some files from one of the 8 lanes, i recovered the whole lane from backup drive but now i am sure there must be deletions of some other files/reads too. Going to recover the whole run data. Thanks everyone for important suggestions n comments.