error in running casava

[Viberance]

I am working with data from MiSeq bcl files, I want to convert it to fastq files using the bcl2fastq.

below is sample of my data

[Header]
IEMFileVersion 4
Investigator Name XXXXX
Experiment Name XXXX_plate01_1pool
Date xx/xx/xxxx
Workflow GenerateFASTQ
Application FASTQ Only
Assay TruSeq LT
Description Test
Chemistry Default

Reads
250
250

[Settings]
ReverseComplement 0
Adapter TTTTTTTTTTTTTTT
AdapterRead2 AAAAAAAAAAAA

[Data]
FCID Lane SampleID Sample_Ref index Description Control Recipe Operator Sample_Project
073388Sm XXXXXXXX
073389Sm XXXXXXXX
073390Sm XXXXXXXX
073391Sm XXXXXXXX
073392Sm XXXXXXXX
073393Sm XXXXXXXX
073394Sm XXXXXXXX
cont1 XXXXXXXX


bash-4.1$ /home/Downloads/CASAVA/bin/configureBclToFastq.pl --output-dir /home/Projects/Data/unaligned --input-dir /home/Projects/Data/Intensities/BaseCalls --fastq-cluster-count 0 --sample-sheet /home/Projects/Data/Intensities/BaseCalls/SampleSheet.csv --tiles s_1_* --force --mismatches 1 --ignore-missing-bcl --ignore-missing-stats --use-bases-mask n
could not find ParserDetails.ini in /home/Downloads/localperl/lib/site_perl/5.20.2/XML/SAX
[2015-02-24 17:18:02] [configureBclToFastq.pl] INFO: Basecalling software: RTA
[2015-02-24 17:18:02] [configureBclToFastq.pl] INFO: version: 1.18 (build 54)
[2015-02-24 17:18:02] [configureBclToFastq.pl] WARNING: Couldn't find run info in /home/Projects/Data/Intensities/BaseCalls/../../../RunInfo.xml
[2015-02-24 17:18:02] [configureBclToFastq.pl] WARNING: Couldn't find RunInfo.xml for /home/Projects/Data/Intensities/BaseCalls
[2015-02-24 17:18:02] [configureBclToFastq.pl] INFO: Original use-bases mask: n
[2015-02-24 17:18:02] [configureBclToFastq.pl] INFO: Guessed use-bases mask: n
ERROR: Wrong number of fields in sample sheet (expected: 10, got 8: IEMFileVersion,4,,,,,,)
at /home/Downloads/CASAVA/lib/bcl2fastq-1.8.4/perl/Casava/Demultiplex.pm line 531

I am running casava for the first time so any help will be appreciated

Thank you

[GenoMax]

You can use a simplified samplesheet like the example here: http://seqanswers.com/forums/showpos...4&postcount=14

See the entire thread for additional information.

[Viberance]

error in running casava

Hi

I have seen this thread, it is still not clear to me, by simplified are you saying to remove the run information in top and just keep the values from column FCID and onwards.

Also in FCID column I have ids and then cont1, cont2 and so on would that be the reason for inconsistent flowcell ID.

thank you

[GenoMax]

You can manually create a Samplesheet.csv file (you can name the file anything, it has to be in comma separated value (CSV) format)) that exactly looks like the example I linked above.

That example contains the minimum information you need to convert BCL files to fastq when de-multiplexing your samples. You will need to grab the last part of the flowcell ID from the folder name (e.g. 000000000-ADB2U).

[Viberance]

my FCID look like this
073388Sm
073389Sm
073390Sm
073391Sm
073392Sm
073393Sm
073394Sm
cont1

if this is not correct where should I look for it

Thanks

[GenoMax]

Those must be your sample ID's. The Samplesheet.csv file that is contained in the raw data folder does not have the Flowcell ID in the file.

Did you get the complete raw data folder from your sequence provider? It should have a date stamp as the starting name (http://support.illumina.com/help/Seq...FileNaming.htm).

[Viberance]

This is how I have it now

[Header]
IEMFileVersion 4
Investigator Name
Experiment Name
Date 0/00/2015
Workflow GenerateFASTQ
Application FASTQ Only
Assay TruSeq LT
Description Test
Chemistry Default

[Reads]
250
250

[Settings]
ReverseComplement 0
Adapter
AdapterRead2

[Data]
FCID Lane Sample_ID SampleRef index Description Control Recipe Operator SampleProject
000000000-ADBFK 070008Sm xxxxxxxx
000000000-ADBFK 070009Sm xxxxxxxx
000000000-ADBFK 070010Sm xxxxxxxx
000000000-ADBFK 070011Sm xxxxxxxx
000000000-ADBFK 070012Sm xxxxxxxx
000000000-ADBFK 070013Sm xxxxxxxx
000000000-ADBFK 070014Sm xxxxxxxx
000000000-ADBFK cont1 xxxxxxxx
000000000-ADBFK 070016Sm xxxxxxxx
000000000-ADBFK 070017Sm xxxxxxxx

I am still getting this error

/home/CASAVA/bin/configureBclToFastq.pl --output-dir /home/unaligned --input-dir /home/DevelopmentRun1/Data/Intensities/BaseCalls --fastq-cluster-count 0 --sample-sheet /home/DevelopmentRun1/SampleSheet1.csv --tiles s_1_* --force --mismatches 1 --ignore-missing-bcl --ignore-missing-stats --use-bases-mask n
could not find ParserDetails.ini in /home/localperl/lib/site_perl/5.20.2/XML/SAX
[2015-02-25 10:38:57] [configureBclToFastq.pl] INFO: Basecalling software: RTA
[2015-02-25 10:38:57] [configureBclToFastq.pl] INFO: version: 1.18 (build 54)
[2015-02-25 10:38:57] [configureBclToFastq.pl] INFO: Original use-bases mask: n
[2015-02-25 10:38:57] [configureBclToFastq.pl] INFO: Guessed use-bases mask: n,IIIIIIIn,yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy
ERROR: FlowCell ID is inconsistent across Sample Sheet lines. Expected: 'Casava:emultiplex::SampleSheet::Csv=HASH(0x2f16e80)->flowCellId()', got Investigator Name
at /home/CASAVA/lib/bcl2fastq-1.8.4/perl/Casava/Demultiplex.pm line 531

Thank you for the kind help

[GenoMax]

Is this a 1D or 2D barcode run?

[GenoMax]

Your samplesheet (if this is a 1D run) needs to look like this:

FCID,Lane,Sample_ID,SampleRef,index,Description,Control,Recipe,Operator,SampleProject
000000000-ADBFK,1,073388Sm,no_ref,PUT_TAG_SEQ_HERE,NA,N,NA,NA,
000000000-ADBFK,1,073389Sm,no_ref,PUT_TAG_SEQ_HERE,NA,N,NA,NA,
000000000-ADBFK,1,073390Sm,no_ref,PUT_TAG_SEQ_HERE,NA,N,NA,NA,
000000000-ADBFK,1,073391Sm,no_ref,PUT_TAG_SEQ_HERE,NA,N,NA,NA,
and so on

The samplesheet that you are using is needed if you were using MiSeq reporter to do the analysis.

[Viberance]

its a 2D run and now my sample sheet looks like this:

FCID,Lane,Sample_ID,SampleRef,index,Description,Control,Recipe,Operator,SampleProject
000000000-ADBFK,1,070008Sm,,CGCCCGCC-AAAAAAAA,,N,,,
000000000-ADBFK,1,070009Sm,,CGCCCGCC-AAAAAAAA,,N,,,
000000000-ADBFK,1,070000Sm,,CGCCCGCC-AAAAAAAA,,N,,,
000000000-ADBFK,1,070011Sm,,CGCCCGCC-AAAAAAAA,,N,,,
000000000-ADBFK,1,070012Sm,,CGCCCGCC-AAAAAAAA,,N,,,
000000000-ADBFK,1,070013Sm,,CGCCCGCC-AAAAAAAA,,N,,,

/home/CASAVA/bin/configureBclToFastq.pl --output-dir /home/DevelopmentRun1/unaligned --input-dir /home/DevelopmentRun1/Data/Intensities/BaseCalls --fastq-cluster-count 0 --sample-sheet /home/DevelopmentRun1/SampleSheet1.csv --tiles s_1_* --force --mismatches 1 --ignore-missing-bcl --ignore-missing-stats --use-bases-mask n
could not find ParserDetails.ini in /home/Downloads/localperl/lib/site_perl/5.20.2/XML/SAX
[2015-02-25 15:00:14] [configureBclToFastq.pl] INFO: Basecalling software: RTA
[2015-02-25 15:00:14] [configureBclToFastq.pl] INFO: version: 1.18 (build 54)
[2015-02-25 15:00:14] [configureBclToFastq.pl] INFO: Original use-bases mask: n
[2015-02-25 15:00:14] [configureBclToFastq.pl] INFO: Guessed use-bases mask: n,IIIIIIIn,yyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyy
[2015-02-25 15:00:14] [configureBclToFastq.pl] ERROR: barcode ACGCATGGATGACTGG for lane 1 has length 16: expected barcode lenth (including delimiters) is 7
[2015-02-25 15:00:14] [configureBclToFastq.pl] BACKTRACE: at /home/Downloads/CASAVA/lib/bcl2fastq-1.8.4/perl/Casava/Demultiplex.pm line 553
Casava:emultiplex::loadSampleSheet('Casava:emultiplex=HASH(0x20f1498)') called at /home/Downloads/CASAVA/bin/configureBclToFastq.pl line 427
Died at /home/Downloads/CASAVA/lib/bcl2fastq-1.8.4/perl/Casava/Common/Log.pm line 310

I have tried to change the --use-bases-mask to different I settings as well but none of them seems to work

[GenoMax]

Use this base mask (if you have 8 bp tags).

--use-bases-mask Y*,I8,I8,Y*

or you could completely omit that option and bcl2fastq will guess the correct values from RunInfo.xml file.

I hope you have separate tags for each sample since giving identical tags to all samples is not going to separate any samples.

[Viberance]

It worked but I don't see a Demultiplex_Stats File and DemultiplexedBustardSummary.xml. Looks like it is putting everything in Undetermined _indices.

[Viberance]

I have it working now thank you for all the guidance.

[GenoMax]

Great. All of your output files should be in the "Unaligned/Basecall_Stats*" and "Unaligned/Project_*" directories. Undetermined pile of sequences goes into "Undetermined_indices" directory.

[Viberance]

yes that's where they are thank you!