[Casava] Error: Barcode Cttgta For Lane 1 Has Length 6: Expected Barcode Lenth Is 7

Question

[Pierre Lindenbaum]

That's the first time I'm using Casava to generate my fastq files.

My command line was:

/path/to/CASAVA/bin/configureBclToFastq.pl \
   --input-dir ../120414_SNL149_0002_AC0KR2ACXX/Data/Intensities/BaseCalls \
  --output-dir 20120720.out \
   --sample-sheet ../120414_SNL149_0002_AC0KR2ACXX/custom.csv --force

the content of custom.csv is:

$ cat ../120414_SNL149_0002_AC0KR2ACXX/custom.csv 

FCID,Lane,SampleID,SampleRef,Index,Description,Control,Recipe,Operator,SampleProject
C0KR2ACXX,1,CD05121,,TGACCA,3pM_each,N,,,DefaultProject
C0KR2ACXX,1,CD10390,,GCCAAT,3pM_each,N,,,DefaultProject
C0KR2ACXX,1,CD10911,,CTTGTA,3pM_each,N,,,DefaultProject
C0KR2ACXX,2,CD05121,,TGACCA,3pM_each,N,,,DefaultProject
(...)
C0KR2ACXX,8,CD10911,,CTTGTA,5pM_each,N,,,DefaultProject
#_IEMVERSION_3_TruSeq DNA/RNA,,,,,,,,,

Output & Error:

[2012-07-23 10:55:26]   [configureBclToFastq.pl]        INFO: Basecalling software: RTA
[2012-07-23 10:55:26]   [configureBclToFastq.pl]        INFO:              version: 1.12 (build 4)
[2012-07-23 10:55:27]   [configureBclToFastq.pl]        WARNING: 'LocationFileType' element not found in ../120414_SNL149_0002_AC0KR2ACXX/Data/Intensities/BaseCalls/../RTAConfiguration.xml
[2012-07-23 10:55:27]   [configureBclToFastq.pl]        INFO: Original use-bases mask: undefined
[2012-07-23 10:55:27]   [configureBclToFastq.pl]        INFO: Guessed use-bases mask: yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy,IIIIIIIn,yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy
[2012-07-23 10:55:27]   [configureBclToFastq.pl]        ERROR: barcode CTTGTA for lane 1 has length 6: expected barcode lenth (including delimiters) is 7
[2012-07-23 10:55:27]   [configureBclToFastq.pl]        BACKTRACE:  at /path/to/CASAVA/lib/CASAVA-1.8.2/perl/Casava/Demultiplex.pm line 491
        Casava::Demultiplex::loadSampleSheet('Casava::Demultiplex=HASH(0x1883e460)') called at /path/to/CASAVA/bin/configureBclToFastq.pl line 400
Died at /path/to/CASAVA/lib/CASAVA-1.8.2/perl/Casava/Common/Log.pm line 310.

How can I fix this ?

Thank you

Pierre

Answer 1

[Istvan Albert]

I think something might not be quite right with the run. I think the lenght of the barcode was not entered correctly when setting up the sequencing run, somewhere in the RunInfo.xml This may lead to other problems down the road, I suspect the total lenght of the read needs to add up correctly.

I vaguely recall some situation - if the barcode is not the right length you can set the --use-bases-mask option and set it as I6

That being said see which version do you have exactly, they change these options around from release to release, the manual for your version has more info.

[Peter Briggs]

This answer was really useful when we had a similar problem with some HiSeq data: it looked like CASAVA wasn't reading the data from RunInfo.xml file, resulting in a similar length mismatch error. Using --use-bases-mask option as suggested fixed it for me.

[Pierre Lindenbaum]

thanks that was helpful. I didn't create this file. It was our very first run here. and we got some problems to set it up.

Answer 2

[Rm]

There might be a possibility of mixing of samples indexed with different barcode lengths: (say one indexed with 6mer barcode and another with larger barcode)

Prepare two sample sheets one with 6-mer and one with other (larger barcode): and run the cassava script separately. Also add this option to your BCL converter: --use-bases-mask Y*,I6n,Y* (I6 or I8 accordingly: i.e.: use all bases from read 1 and 2, and use 6 or 8 bases as the index/barcode.

Answer 3

[swbarnes2]

Pad your index with an extra A, and then CASAVA should run fine.