Issues with CASAVA 1.8.1

[Orr Shomroni]

I am having some trouble with the Bcl to FastQ conversion in CASAVA 1.8.1. I imagine it might be a very stupid question (mostly because I couldn't find any trace of it being asked around the web), but how do I use the position files? When I give the command "make" (after making the "SampleSheet" and "make" files), it doesn't return the FastQ files, but instead returns the repeated error "failed to read position from pos file <path to _pos.txt file>". I know CASAVA can also use .locs and .clocs, but those are the _pos.txt appear on my computer already, so I thought of using them in the configureBclToFastq.pl, but for some reason it goes wrong there. Can anyone help me with this?

[GenoMax]

The BCL to fastq converter is supposed to figure out which type of position file it looks for based on the RTA version.

Do you have access to the entire flowcell data folder for this run?

[Orr Shomroni]

So far as I know, yes.
Ah, I think I found the problem: I get an "Error:<Date> <Time>: No such file or directory: /<path>/BclReader.cpp". I'll figure it out by myself, thank you.

[singhp5]

was this issue ever resolved, if so how? - I have the same problem in in v1.8.2

[GenoMax]

Can you be more specific about what problem you are encountering?

Are you getting the same error as the original poster? Do you have access to the *entire* flowcell folder?

Originally Posted by singhp5

was this issue ever resolved, if so how? - I have the same problem in in v1.8.2

[singhp5]

yes, I have access to the entire flowcell folder, but what I'm doing is running the following command:

/usr/local/bin/configureBclToFastq.pl --input-dir /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls --output-dir /seqout/c6 --sample-sheet /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/SampleSheet.csv --use-bases-mask Y101,I8,I8,Y101 --tiles s_1_1101 --force

which runs, but when I go to /seqout/c6 directory and type make -j 6

I get:

[2012-06-07 18:01:46] [eggbeater.local] [Temp/L001_R2_demux_summary.xml] Dynamic exception type: boost::exception_detail::clone_impl<casava::common::IoException>
[2012-06-07 18:01:46] [eggbeater.local] [Temp/L001_R2_demux_summary.xml] std::exception::what: Couldn't open BCL file /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/Ba$
[2012-06-07 18:01:46] [eggbeater.local] [Temp/L001_R2_demux_summary.xml] : Couldn't open BCL file /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/L001/C167.1/s_8_1101.bcl
make: *** [Temp/L001_R2_demux_summary.xml] Error 1
make: *** Waiting for unfinished jobs....
[2012-06-07 18:01:48] [eggbeater.local] [Temp/L001_R1_demux_summary.xml] Error: 2012-Jun-07 18:01:48: Operation not permitted: /home/software/CASAVA_v1.8.2/src/c++/lib/alignment/BclReader.cpp(97): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
[2012-06-07 18:01:48] [eggbeater.local] [Temp/L001_R1_demux_summary.xml] Dynamic exception type: boost::exception_detail::clone_impl<casava::common::IoException>
[2012-06-07 18:01:48] [eggbeater.local] [Temp/L001_R1_demux_summary.xml] std::exception::what: Couldn't open BCL file /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/L001/C101.1/s_8_1102.bcl
[2012-06-07 18:01:48] [eggbeater.local] [Temp/L001_R1_demux_summary.xml] : Couldn't open BCL file /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/L001/C101.1/s_8_1102.bcl
make: *** [Temp/L001_R1_demux_summary.xml] Error 1

however both files /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/L001/C101.1/s_8_1101.bcl
and /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/L001/C101.1/s_8_1102.bcl are present

[GenoMax]

Originally Posted by singhp5

--use-bases-mask Y101,I8,I8,Y101

The format on this command option may be incorrect (unless you are using dual indexing, are you?).

You should use Y101,I7n,Y101 (or Y101,I8,Y101 if you must). Illumina has generally recommended ignoring the last base in the index read (thus I7n).

Also did you make sure that the relevant folders/files are readable by the "user" account that is running the demultiplexing command.

[singhp5]

yes, using dual indexing , so have to keep all 4 components in the line

I'm using the root account, which has full read/write access on all folder/file, haven't setup any users yet, was planning to once I had successful output.

Still have the same error as above when I try a smaller run:

/usr/local/bin/configureBclToFastq.pl
--input-dir /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls
--output-dir /seqout/DengTest
--sample-sheet /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/Deng_3.csv
--use-bases-mask Y101,I8,I8,Y101 --tiles s_3_111[0-3]

any other ideas?

[sklages]

(a) Try to convert without '--tiles' , just for testing. Do you get the same errors?
(b) And you should have a look at the make output; the real error occurs earlier as
posted before. (c) Just retyping make after the conversion crash should "isolate" the
real error from the other stuff.

my 2p, Sven

[singhp5]

I ran without '--tiles', then repeated make, took the output and looked for the first error message, still the same outcome:

[2012-06-11 10:22:40] [localserv] [ecall_Stats_C0RAPACXX/Matrix/s_1_3_2111_matrix.txt] make Cmd: cat /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/Matrix/s_1_3_2111_matrix.txt >Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2111_matrix.txt.tmp && mv Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2111_matrix.txt.tmp Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2111_matrix.txt
[2012-06-11 10:22:40] [localserv] [ecall_Stats_C0RAPACXX/Matrix/s_1_3_2110_matrix.txt] make Cmd: cat /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/Matrix/s_1_3_2110_matrix.txt >Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2110_matrix.txt.tmp && mv Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2110_matrix.txt.tmp Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2110_matrix.txt
[2012-06-11 10:22:40] [localserv] [ecall_Stats_C0RAPACXX/Matrix/s_1_3_2111_matrix.txt] cat: /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/Matrix/s_1_3_2111_matrix.txt: Operation not permitted
Need a job token; we have children
Duplicate the job FD
Live child 0x01fa3530 (Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2111_matrix.txt) PID 4244
Live child 0x01fa5190 (Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2110_matrix.txt) PID 4243
Live child 0x01f44910 (Temp/L003_R2_demux_summary.xml) PID 31067
Live child 0x01f36c70 (Temp/L003_R1_demux_summary.xml) PID 30999
Reaping losing child 0x01fa3530 PID 4244
make: *** [Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2111_matrix.txt] Error 1
Removing child 0x01fa3530 PID 4244 from chain.
Released token for child 0x01fa3530 (Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2111_matrix.txt).
make: *** Waiting for unfinished jobs....
Live child 0x01fa5190 (Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2110_matrix.txt) PID 4243
Live child 0x01f44910 (Temp/L003_R2_demux_summary.xml) PID 31067
Live child 0x01f36c70 (Temp/L003_R1_demux_summary.xml) PID 30999
[2012-06-11 10:22:40] [localserv] [ecall_Stats_C0RAPACXX/Matrix/s_1_3_2110_matrix.txt] cat: /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/Matrix/s_1_3_2110_matrix.txt: Operation not permitted
Reaping losing child 0x01fa5190 PID 4243
make: *** [Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2110_matrix.txt] Error 1
Removing child 0x01fa5190 PID 4243 from chain.
Released token for child 0x01fa5190 (Basecall_Stats_C0RAPACXX/Matrix/s_1_3_2110_matrix.txt).
Live child 0x01f44910 (Temp/L003_R2_demux_summary.xml) PID 31067
Live child 0x01f36c70 (Temp/L003_R1_demux_summary.xml) PID 30999
Live child 0x01f44910 (Temp/L003_R2_demux_summary.xml) PID 31067
Live child 0x01f36c70 (Temp/L003_R1_demux_summary.xml) PID 30999
[2012-06-11 10:22:40] [localserv] [Temp/L003_R2_demux_summary.xml] Error: 2012-Jun-11 10:22:40: Operation not permitted: /software/CASAVA_v1.8.2/src/c++/lib/alignment/BclReader.cpp(97): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
[2012-06-11 10:22:40] [localserv] [Temp/L003_R2_demux_summary.xml] Dynamic exception type: boost::exception_detail::clone_impl<casava::common::IoException>
[2012-06-11 10:22:40] [localserv] [Temp/L003_R2_demux_summary.xml] std::exception::what: Couldn't open BCL file /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/L003/C168.1/s_3_1101.bcl.
[2012-06-11 10:22:40] [localserv] [Temp/L003_R2_demux_summary.xml] : Couldn't open BCL file /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/L003/C168.1/s_3_1101.bcl.
Reaping losing child 0x01f44910 PID 31067
make: *** [Temp/L003_R2_demux_summary.xml] Error 1
Removing child 0x01f44910 PID 31067 from chain.
Released token for child 0x01f44910 (Temp/L003_R2_demux_summary.xml).
Live child 0x01f36c70 (Temp/L003_R1_demux_summary.xml) PID 30999
Live child 0x01f36c70 (Temp/L003_R1_demux_summary.xml) PID 30999
[2012-06-11 10:22:43] [localserv] [Temp/L003_R1_demux_summary.xml] Error: 2012-Jun-11 10:22:43: Operation not permitted: /software/CASAVA_v1.8.2/src/c++/lib/alignment/BclReader.cpp(97): Throw in function unsigned int casava::alignment::BclReader::readClusterCount()
[2012-06-11 10:22:43] [localserv] [Temp/L003_R1_demux_summary.xml] Dynamic exception type: boost::exception_detail::clone_impl<casava::common::IoException>
[2012-06-11 10:22:43] [localserv] [Temp/L003_R1_demux_summary.xml] std::exception::what: Couldn't open BCL file /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/L003/C101.1/s_3_1101.bcl.
[2012-06-11 10:22:43] [localserv] [Temp/L003_R1_demux_summary.xml] : Couldn't open BCL file /output/120101_SNL151_0018_AC0RAPACXX/Data/Intensities/BaseCalls/L003/C101.1/s_3_1101.bcl.
Reaping losing child 0x01f36c70 PID 30999
make: *** [Temp/L003_R1_demux_summary.xml] Error 1
Removing child 0x01f36c70 PID 30999 from chain.

[singhp5]

hey folks,

I think I may have solved it,

I was using sshfs mount to output directory on storage array,

I switched to nfs mount method and it seems to be running without any error during make process thus far *fingers crossed*

[gabyslysa]

[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L006_R1_001.fastq.gz] opening /home/servidor/Documentos/Unaligned/Project_RNA-JOAS/Sample_not_demultiplexed/not_demultiplexed_NoIndex_L006_R1_001.fastq.tmp.gz
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L004_R1_001.fastq.gz]
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L003_R1_001.fastq.gz] opening /home/servidor/Documentos/Unaligned/Project_RNA-JOAS/Sample_not_demultiplexed/not_demultiplexed_NoIndex_L003_R1_001.fastq.tmp.gz
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L006_R1_001.fastq.gz] Processing BCL files for lane 6, tile 1101, read 1
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L004_R1_001.fastq.gz] opening /home/servidor/Documentos/Unaligned/Project_RNA-JOAS/Sample_not_demultiplexed/not_demultiplexed_NoIndex_L004_R1_001.fastq.tmp.gz
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L004_R1_001.fastq.gz] Processing BCL files for lane 4, tile 1101, read 1
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L006_R1_001.fastq.gz] [2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L003_R1_001.fastq.gz] Error: 2013-Nov-21 19:38:06: No such file or directory: /tmp/BclToFastq/src/c++/lib/alignment/BclReader.cpp(391): Throw in function casava::alignment::PositionsReaderCompressed::PositionsReaderCompressed(const boost::filesystem2:ath&, unsigned int)
Processing BCL files for lane 3, tile 1101, read 1
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L006_R1_001.fastq.gz] Dynamic exception type: boost::exception_detail::clone_impl<casava::common::IoException>
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L006_R1_001.fastq.gz] std::exception::what: Failed to read version from clocs file /home/servidor/Documentos/Gabriela/../L006/s_6_1101.clocs
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L004_R1_001.fastq.gz] Error: 2013-Nov-21 19:38:06: No such file or directory: /tmp/BclToFastq/src/c++/lib/alignment/BclReader.cpp(391): Throw in function casava::alignment::PositionsReaderCompressed::PositionsReaderCompressed(const boost::filesystem2:ath&, unsigned int)
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L006_R1_001.fastq.gz] : Failed to read version from clocs file /home/servidor/Documentos/Gabriela/../L006/s_6_1101.clocs
make: ** [Project_RNA-JOAS/Sample_not_demultiplexed/not_demultiplexed_NoIndex_L006_R1_001.fastq.gz] Erro 1
make: ** Esperando que outros processos terminem.
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L004_R1_001.fastq.gz] Dynamic exception type: boost::exception_detail::clone_impl<casava::common::IoException>
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L003_R1_001.fastq.gz] Error: 2013-Nov-21 19:38:06: No such file or directory: /tmp/BclToFastq/src/c++/lib/alignment/BclReader.cpp(391): Throw in function casava::alignment::PositionsReaderCompressed::PositionsReaderCompressed(const boost::filesystem2:ath&, unsigned int)
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L004_R1_001.fastq.gz] std::exception::what: Failed to read version from clocs file /home/servidor/Documentos/Gabriela/../L004/s_4_1101.clocs
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L003_R1_001.fastq.gz] Dynamic exception type: boost::exception_detail::clone_impl<casava::common::IoException>
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L004_R1_001.fastq.gz] : Failed to read version from clocs file /home/servidor/Documentos/Gabriela/../L004/s_4_1101.clocs
make: ** [Project_RNA-JOAS/Sample_not_demultiplexed/not_demultiplexed_NoIndex_L004_R1_001.fastq.gz] Erro 1
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L003_R1_001.fastq.gz] std::exception::what: Failed to read version from clocs file /home/servidor/Documentos/Gabriela/../L003/s_3_1101.clocs
[2013-11-21 19:38:06] [servidor] [xed/not_demultiplexed_NoIndex_L003_R1_001.fastq.gz] : Failed to read version from clocs file /home/servidor/Documentos/Gabriela/../L003/s_3_1101.clocs
make: ** [Project_RNA-JOAS/Sample_not_demultiplexed/not_demultiplexed_NoIndex_L003_R1_001.fastq.gz] Erro 1

I need help here!

[sklages]

What is "the same problem"? What have you done? Which version of CASAVA are you using? Which OS? What is " /home/servidor/", local FS, NFS or? Your output seems not to be complete.

[GenoMax]

As Sven suggested some more information is required. But at a minimum you seem to be missing (or do not have permission to read) several ".clocs" files. Is this a full folder from an illumina run? Have you used this install of CASAVA before? If this is a first time install have you checked it with the included test data?

[gabyslysa]

Hi

I'm having the same errors described by singhp5. I'm using for the first time CASAVA 1.8.2. I checked the test data. All my data were in a external media and I passed all files to a local directory, in my server. I used this commands:

configureBclToFastq.pl --input-dir /home/servidor/Documentos/Gabriela --output-dir /home/servidor/Documentos/Unaligned --use-bases-mask Y*

The first message was:

...
[2013-11-21 21:40:22] [configureBclToFastq.pl] INFO: Read 1: length = 100: mask = Y*
[2013-11-21 21:40:22] [configureBclToFastq.pl] INFO: Read 2: length = 7: mask = n*
[2013-11-21 21:40:22] [configureBclToFastq.pl] INFO: Running self tests: 'make self_test'
[2013-11-21 21:40:24] [configureBclToFastq.pl] INFO: Running self tests on /home/servidor/Documentos/Unaligned completed with no problems

After that, I did the make command in Unaligned folder

Thank you.

[GenoMax]

What exactly are you trying to do?

Based on the file names it appears that your sequence provider has already done the basecalling (e.g. xed/not_demultiplexed_NoIndex_L003_R1_001.fastq.gz).

If you have some kind of internal barcode/index on these reads then CASAVA can't be used for demultiplexing those.

[gabyslysa]

I'll explain what happened. I made all of my sequencing in another laboratory. We ran two samples per lane of mRNA libraries. But few indexed reads were generated, approximately one million per sample (very little indeed). I'm questioning the preparation of these libraries, because I believe that it was not done properly by this laboratory. But I would like to see the reads before making demultiplexing, to see if I can recover some reads. So I want to do it again converting blc to fastq, without demultiplexing. I want to see the reads generated containing all the information, the sequences containing the indexes. What do you suggest? Thank you. I thought about doing the PICARD.

[GenoMax]

In case of illumina multiplex libraries the index read happens as a separate read so you will not be able to see the tags unless you actually provide a samplesheet and de-multiplex the sample.

If as you say the index read was bad (i.e. N's or whatever) most of the reads will end up in the "undetermined" reads file. You can then parse the reads to determine if the library indeed is bad (look for the tags at the end of the read ID's).

Were there other samples on the flowcell that demultiplexed properly? What was the cluster concentration (k/mm^2)? Generally if you overload a sample the regular reads are generally fine but it is the index reads that suffer since the basecaller has a hard time distinguishing the clusters. You will then not be able to demultiplex the reads ruining the run (as you found).

[sklages]

Good Morning :-)

Be aware as GenoMax already mentioned that the index itself is simply a separate read. Without demultiplexing you loose the information about the indices.

Have a look at the undetermined_indices to see if you have quality problems with the index or if you have another index present in the data.
PM me if you want a simple perl script doing the job for you.

[arkilis]

I just met this problem recently and I solved this by checking CASAVA 1.8.2 manual.

Solution for people who needs this:

There might be many reasons lead to such an error.

If the error msg is missing bcf file or stats files. Try to use parameters: --ignore-missing-bcl or --ignore-missing-stats

The reason also might be file/path permission and mounted drive. However, they are all give the same error msg. (It is quite misleading)