Casava1.8.2: One Barcode Per Lane Does Not Work?

Question

[Rm]

Samples were run using one barcode per lane: Casava1.8.2 is giving me below error? When I ran it without using the barcode; it went on fine; but I am not confident of using the reads be cause of potential mix-up of barcoded and unbarcoded reads?

Any suggestions will be highly appreciated:

Error Message:

[2012-04-18 14:28:46]   [configureBclToFastq.pl]        ^[[2;32mINFO: Basecalling software: RTA^[[0m
[2012-04-18 14:28:46]   [configureBclToFastq.pl]        ^[[2;32mINFO:              version: 1.13 (build 48)^[[0m
[2012-04-18 14:28:46]   [configureBclToFastq.pl]        ^[[2;32mINFO: Original use-bases mask: undefined^[[0m
[2012-04-18 14:28:46]   [configureBclToFastq.pl]        ^[[2;32mINFO: Guessed use-bases mask: yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy,yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy^[[0m
[2012-04-18 14:28:46]   [configureBclToFastq.pl]        ^[[1;31mERROR: barcode CGATGT for lane 1 has length 6: expected barcode lenth (including delimiters) is 0^[[0m
[2012-04-18 14:28:46]   [configureBclToFastq.pl]        BACKTRACE:  at /packages/CASAVA_v1.8.2/lib/CASAVA-1.8.2/perl/Casava/Demultiplex.pm line 491
        Casava::Demultiplex::loadSampleSheet('Casava::Demultiplex=HASH(0x839d8f0)') called at /packages/CASAVA_v1.8.2/bin/configureBclToFastq.pl line 400
Died at /packages/CASAVA_v1.8.2/lib/CASAVA-1.8.2/perl/Casava/Common/Log.pm line 310.
make: *** No targets specified and no makefile found.  Stop.

Answer 1

[Stefano Berri]

I do the analysis, do not work directly with the sequencer. Those that do, however, tell we have to make at least 4 barcodes so that there is never the same pase for all sequences at any given cicle. The software use the images to normalise the signal and expect a roughly similar level of all bases. When it finds the tag it get confused as it only see one color.

Not sure this is the reason why it doesn't work for you, but it might.

In the rare cases where we only have 2 samples to run, we tag one sample with two different tags (and merge them later) so that we have 4 in total. Tags have to be as different as possible.

However, I think we do not use exaclty the Illumina protocol when tagging, but here I am not 100% sure... I'll ask and edit this

[Pablo Marin-Garcia]

Illumina has two lasers, red and green, to sequence G/T and C/A and in each cicle you should have a proper representation of both pairs. See an explanation here for nextera: http://www.illumina.com/documents/products%5Ctechnotes%5Ctechnote_nextera_low_plex_pooling_guidelines.pdf. For TrueSeq there is also a list of compatible indexes when you are going to have a high cluster density:Pool of 2 samples: Index #6 GCCAAT Index #12 CTTGTA Pool of 3 samples: Index #4 TGACCA Index #6 GCCAAT Index #12 CTTGTA Pool of 6 samples: Index #2 CGATGT Index #4 TGACCA Index #5 ACAGTG Index #6 GCCAAT Index #7 CAGATC Index #12 CTTGTA

[Rm]

Thanks @S_Berri for your inputs: Information I received from the sequencing guys (wetlab side) is that Whole Genome Sequencing library kit has the barcode but they would not index it. But the barcode is passed on to samplesheet; practically they donot do the "indexing" step in sequencing, so removing the barcode and running the casava should be fine.

Answer 2

[kmcarr00]

The two important clues in the error message are the "Guessed use-bases-mask" and "expected barcode length ... is 0"

CASAVA has guessed that the use-bases-mask to use only includes two reads, each (oddly) 104 cycles long, and no index read between them. This is reiterated by the expected length is 0 line. This suggests to me that the sequencing facility configured the run with no index read, assuming the index would not be required since the samples were not multiplexed. Can you confirm with the sequence provider how the run was performed?

Answer 3

[samsara]

If your run has 1 sample per lane, you should not use any barcode. Barcode is used for multiplexed samples in one lane i.e if one lane has more than one sample then need to use barcode. I suppose it is to identify the samples that are in the same lane. So, if there has been single sample per lane - do not use barcode. That is what illumina support said to me.