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  • #1

    Flowcell with single and dual indexing demultiplexing issue

    Hello,

    I have received from my lab one HiSeq flowcell with 7 lanes single indexed and the 8th is dual indexed.

    Something goes wrong during the computation.

    Since I would like to put the use-base-mask argument, I prepared a truncated sample sheet for the first 7 lanes, and launched with my always working script with the argument --use-bases-mask Y101,I6N,Y101.

    The program complains that it is not what it found in the conf files :

    Code:
    $ grep -i warning run.err
[2015-02-17 16:03:15]	[configureBclToFastq.pl]	WARNING: No sample sheet data for lane '8'. Skipping lane.
[2015-02-17 16:03:15]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 7 for read 2: expected length from config file is 8
[2015-02-17 16:03:15]	[configureBclToFastq.pl]	WARNING: Read 3: current cycle in mask is 109: first cycle in read is 110
[2015-02-17 16:03:15]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 101 for read 3: expected length from config file is 8
[2015-02-17 16:03:15]	[configureBclToFastq.pl]	WARNING: Read 4: current cycle in mask is 210: first cycle in read is 118
[2015-02-17 16:03:15]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 7 for read 2: expected length from config file is 8
[2015-02-17 16:03:15]	[configureBclToFastq.pl]	WARNING: Read 3: current cycle in mask is 109: first cycle in read is 110
[2015-02-17 16:03:15]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 101 for read 3: expected length from config file is 8
[2015-02-17 16:03:15]	[configureBclToFastq.pl]	WARNING: Read 4: current cycle in mask is 210: first cycle in read is 118
    ...and a little later...

    Code:
    $ grep -i error run.err 
[2015-02-17 16:07:56]	[r103-n67]	[Temp/L003_R3_demux_summary.xml]	Error: 2015-Feb-17 16:07:53: 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/L003_R3_demux_summary.xml] Error 1
[2015-02-17 16:07:56]	[r103-n67]	[Temp/L006_R3_demux_summary.xml]	Error: 2015-Feb-17 16:07:53: 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_R3_demux_summary.xml] Error 1
    And the last lines of my log:

    Code:
    tail run.err
[2015-02-17 17:21:29]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L004/C105.1/s_4_2316.bcl.gz
[2015-02-17 17:21:29]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L004/C106.1/s_4_2316.bcl.gz
[2015-02-17 17:21:29]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L004/C107.1/s_4_2316.bcl.gz
[2015-02-17 17:21:31]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	mapClusterBarcodes: barcode/clusters:
[2015-02-17 17:21:31]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	/run/Demultiplexed_2015-02-17/Undetermined_indices/Sample_lane4:Undetermined:277429
[2015-02-17 17:21:31]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	/run/Demultiplexed_2015-02-17/Project_run/Sample_15:ACTTGA:635531
[2015-02-17 17:21:31]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	/run/Demultiplexed_2015-02-17/Project_run/Sample_87:ACAGTG:639074
[2015-02-17 17:21:31]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	/run/Demultiplexed_2015-02-17/Project_run/Sample_55:GCCAAT:1036142
[2015-02-17 17:21:31]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	/run/Demultiplexed_2015-02-17/Project_run/Sample_25:CAGATC:790545
[2015-02-17 17:21:31]	[r103-n67]	[Temp/L004_R2_demux_summary.xml]	Processing BCL files for lane 4, tile 2316, read 2
    ...and it stops right here, with no other explanation.

    For info :

    Code:
    $ grep Read run/RunInfo.xml 
    <Reads>
      <Read Number="1" NumCycles="101" IsIndexedRead="N" />
      <Read Number="2" NumCycles="8" IsIndexedRead="Y" />
      <Read Number="3" NumCycles="8" IsIndexedRead="Y" />
      <Read Number="4" NumCycles="101" IsIndexedRead="N" />
    </Reads>

    Any idea that could help?

    Cheers!
    Tags: bcl2fastq, demultiplex, illumina, indexing
  • #2
    Disclaimer: Following procedure edits original data files. You will want to make/keep backup of the original RunInfo.xml file before you edit it.

    Edit the RunInfo.xml file so it looks like this (see third line). Now run the analysis with your samplesheet for the first 7 lanes.

    Code:
     <Reads>
      <Read Number="1" NumCycles="101" IsIndexedRead="N" />
      <Read Number="2" NumCycles="8" IsIndexedRead="Y" />
      <Read Number="3" NumCycles="8" IsIndexedRead="[COLOR="Red"][B]N[/B][/COLOR]" />
      <Read Number="4" NumCycles="101" IsIndexedRead="N" />
    </Reads>
    Because we are tinkering with the run data file you will get output that will not follow the standard R1/R2 convention.

    Your read 2 will end up in a file that will have R3 identifier (with corresponding /3 in read ID header). You can change those afterwards.

    Comment

    • #3
      You can restore the original RunInfo.xml file and run the analysis for lane 8 separately to get that data.

      Comment

      • #4
        Thanks GenoMax, I am trying it and I get back at you ASAP.

        Comment

        • #5
          So I backed-up RunInfo.xml, and modified it :

          Code:
          $ grep Read run/RunInfo.xml
    <Reads>
      <Read Number="1" NumCycles="101" IsIndexedRead="N" />
      <Read Number="2" NumCycles="8" IsIndexedRead="Y" />
      <Read Number="3" NumCycles="8" IsIndexedRead="N" />
      <Read Number="4" NumCycles="101" IsIndexedRead="N" />
    </Reads>
          But when I launched it :
          Code:
          $ grep -i warning run.err
[2015-02-18 13:25:47]	[configureBclToFastq.pl]	WARNING: No sample sheet data for lane '8'. Skipping lane.
[2015-02-18 13:25:47]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 7 for read 2: expected length from config file is 8
[2015-02-18 13:25:47]	[configureBclToFastq.pl]	WARNING: Read 3: current cycle in mask is 109: first cycle in read is 110
[2015-02-18 13:25:47]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 101 for read 3: expected length from config file is 8
[2015-02-18 13:25:47]	[configureBclToFastq.pl]	WARNING: Read 4: current cycle in mask is 210: first cycle in read is 118
[2015-02-18 13:25:47]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 7 for read 2: expected length from config file is 8
[2015-02-18 13:25:47]	[configureBclToFastq.pl]	WARNING: Read 3: current cycle in mask is 109: first cycle in read is 110
[2015-02-18 13:25:47]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 101 for read 3: expected length from config file is 8
[2015-02-18 13:25:47]	[configureBclToFastq.pl]	WARNING: Read 4: current cycle in mask is 210: first cycle in read is 118
          and when I look for errors :

          Code:
          $ grep -i error run.err
[2015-02-18 13:30:20]	[r110-n43]	[Temp/L003_R3_demux_summary.xml]	Error: 2015-Feb-18 13:30:15: 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/L003_R3_demux_summary.xml] Error 1
[2015-02-18 13:30:20]	[r110-n43]	[Temp/L006_R3_demux_summary.xml]	Error: 2015-Feb-18 13:30:15: 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_R3_demux_summary.xml] Error 1
          It is running right now, but with all those warnings and errors, I am not sure it will be a happy ending...

          Comment

          • #6
            This run was setup as 101x8x8x101. So if you want to use only 6 bases on the index read then you should use --use-bases-mask Y101,I6n,Y101 instead of --use-bases-mask Y101,I6N,Y101. Otherwise use --use-bases-mask Y101,I7n,Y101.
            Last edited by GenoMax; 02-18-2015, 11:45 AM. Reason: correction

            Comment

            • #7
              I modified the use-base-mask with Y101,I6NN,Y101, same warnings pop up :

              Code:
              [2015-02-18 14:05:47]	[configureBclToFastq.pl]	WARNING: No sample sheet data for lane '8'. Skipping lane.
[2015-02-18 14:05:47]	[configureBclToFastq.pl]	INFO: Read 1: length = 101: mask = YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY
[2015-02-18 14:05:47]	[configureBclToFastq.pl]	INFO: Read 2: length = 8: mask = IIIIIINN
[2015-02-18 14:05:47]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 101 for read 3: expected length from config file is 8
[2015-02-18 14:05:47]	[configureBclToFastq.pl]	INFO: Read 3: length = 101: mask = YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY
[2015-02-18 14:05:47]	[configureBclToFastq.pl]	WARNING: Read 4: current cycle in mask is 211: first cycle in read is 118
[2015-02-18 14:05:47]	[configureBclToFastq.pl]	INFO: Read 4: length = 101: mask = yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy
[2015-02-18 14:05:47]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 101 for read 3: expected length from config file is 8
[2015-02-18 14:05:47]	[configureBclToFastq.pl]	WARNING: Read 4: current cycle in mask is 211: first cycle in read is 118
[2015-02-18 14:05:47]	[configureBclToFastq.pl]	INFO: Running self tests: 'make self_test'
[2015-02-18 14:06:08]	[configureBclToFastq.pl]	INFO: Running self tests on run/Demultiplexed_2015-02-18 completed with no problems
              and early errors too :

              Code:
              [2015-02-18 14:10:25]	[r110-n43]	[Temp/L006_R3_demux_summary.xml]	Error: 2015-Feb-18 14:10:19: 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()
[2015-02-18 14:10:25]	[r110-n43]	[Temp/L003_R3_demux_summary.xml]	Error: 2015-Feb-18 14:10:19: 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()

              Comment

              • #8
                I had a look at CASAVA manual. I think bases-mask option should have been specified with a lower case "n". --use-bases-mask Y101,I6n,Y101. I have corrected the post above.

                Comment

                • #9
                  With small case n :

                  Code:
                  [2015-02-18 15:03:33]	[configureBclToFastq.pl]	WARNING: No sample sheet data for lane '8'. Skipping lane.
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	INFO: Read 1: length = 101: mask = YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 7 for read 2: expected length from config file is 8
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	INFO: Read 2: length = 7: mask = IIIIIIn
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	WARNING: Read 3: current cycle in mask is 109: first cycle in read is 110
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 101 for read 3: expected length from config file is 8
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	INFO: Read 3: length = 101: mask = YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	WARNING: Read 4: current cycle in mask is 210: first cycle in read is 118
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	INFO: Read 4: length = 101: mask = yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 7 for read 2: expected length from config file is 8
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	WARNING: Read 3: current cycle in mask is 109: first cycle in read is 110
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	WARNING: use-base-mask length is 101 for read 3: expected length from config file is 8
[2015-02-18 15:03:33]	[configureBclToFastq.pl]	WARNING: Read 4: current cycle in mask is 210: first cycle in read is 118
                  and :

                  Code:
                  $ grep -i error run.err
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L003_R3_demux_summary.xml]	Error: 2015-Feb-18 15:08:14: 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/L003_R3_demux_summary.xml] Error 1
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	Error: 2015-Feb-18 15:08:14: 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_R3_demux_summary.xml] Error 1

                  Comment

                  • #10
                    Instead of grepping just for "error" can you take a look at what file those two errors (No such file or directory) are being thrown for (and post those lines)? My guess is you may be missing some bcl or stats files.

                    Comment

                    • #11
                      Of course. This is what is around errors :

                      Code:
                      ...
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L004_R2_demux_summary.xml]	Processing BCL files for lane 4, tile 1101, read 2
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	/run/Demultiplexed_2015-02-18/Project_run/Sample_23797:CGATGT:531078
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L003_R3_demux_summary.xml]	Error: 2015-Feb-18 15:08:14: 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()
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	/run/Demultiplexed_2015-02-18/Project_run/Sample_23955:TTAGGC:558024
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	/run/Demultiplexed_2015-02-18/Project_run/Sample_23645:ATCACG:532550
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L003_R1_demux_summary.xml]	/run/Demultiplexed_2015-02-18/Project_run/Sample_24004:TGACCA:604942
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L003_R1_demux_summary.xml]	Processing BCL files for lane 3, tile 1101, read 1
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L003_R3_demux_summary.xml]	Dynamic exception type: boost::exception_detail::clone_impl<casava::common::IoException>
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	/run/Demultiplexed_2015-02-18/Project_run/Sample_24012:TGACCA:644390
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L003_R3_demux_summary.xml]	std::exception::what: File /run/Data/Intensities/BaseCalls/L003/C219.1/s_3_1101.bcl.gz does not exist
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L003_R3_demux_summary.xml]	: File /run/Data/Intensities/BaseCalls/L003/C219.1/s_3_1101.bcl.gz does not exist
make: *** [Temp/L003_R3_demux_summary.xml] Error 1
make: *** Waiting for unfinished jobs....
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	Processing BCL files for lane 6, tile 1101, read 3
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	Error: 2015-Feb-18 15:08:14: 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()
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	Dynamic exception type: boost::exception_detail::clone_impl<casava::common::IoException>
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	std::exception::what: File /run/Data/Intensities/BaseCalls/L006/C219.1/s_6_1101.bcl.gz does not exist
[2015-02-18 15:08:23]	[r104-n67]	[Temp/L006_R3_demux_summary.xml]	: File /run/Data/Intensities/BaseCalls/L006/C219.1/s_6_1101.bcl.gz does not exist
make: *** [Temp/L006_R3_demux_summary.xml] Error 1
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R2_demux_summary.xml]	Barcode BCLs:
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R2_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L003/C102.1/s_3_1102.bcl.gz
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R2_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L003/C103.1/s_3_1102.bcl.gz
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R2_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L003/C104.1/s_3_1102.bcl.gz
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R2_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L003/C105.1/s_3_1102.bcl.gz
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R2_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L003/C106.1/s_3_1102.bcl.gz
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R2_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L003/C107.1/s_3_1102.bcl.gz
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R1_demux_summary.xml]	Barcode BCLs:
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R1_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L003/C102.1/s_3_1102.bcl.gz
[2015-02-18 15:08:49]	[r104-n67]	[Temp/L003_R1_demux_summary.xml]	/run/Data/Intensities/BaseCalls/L003/C103.1/s_3_1102.bcl.gz
...
                      However, I have the RTAComplete.txt in my run folder.

                      Comment

                      • #12
                        As I suspected you are missing bcl files (e.g. /run/Data/Intensities/BaseCalls/L003/C219.1/s_3_1101.bcl.gz).

                        Add --ignore-missing-bcl option to your configureBcltoFastq.pl command and re-run.

                        This is normal and does happen at times. These will be treated as no call.
                        Last edited by GenoMax; 02-18-2015, 12:40 PM.

                        Comment

                        • #13
                          Should I change base-mask?

                          Comment

                          • #14
                            Make it --use-bases-mask Y101,I7n,Y101.
                            Last edited by GenoMax; 02-18-2015, 04:35 PM.

                            Comment

                            • #15
                              Based on the recommended edit of RunInfo.xml, I don't think the recommended --use-bases-mask will behave as expected. The former treats R2 as the sole index read, which means that R3 (the second index read) now becomes read 2.

                              I believe that the solution is to use the original (unedited) RunInfo.xml and --use-bases-mask Y101,I6n*,n*, Y101. This should yield 101bp paired-end data with a single 6bp index. (N.B.-You will also need to include the --ignore-missing-bcl flag)

                              Also, deleting the lane eight entry in the sample sheet may produce errors. I usually include a dummy entry with no index for each lane that I want to skip.

                              Comment

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