Bcl to fastq Conversion Problem

Question: Bcl to fastq Conversion Problem

5.3 years ago by

BioRyder • 180

India

BioRyder • 180 wrote:

Hello,

Below are the summary of Bcl to fastq conversion by using bcl2fastq v2.16.0.10 .In Output folder only lane 4 and 5 are having R1 and R2 reads (18 GB ) and remaining all lanes (1,2,3,6,7,8) are having R1 and R2 reads with zero size. If We are looking into lane summary ,last four columns are showing there were data generated for all lanes. Can any one tell me why the data are missing and reports showing data generated... ?

Clusters (Raw)	Clusters(PF)	Yield (MBases)
3,861,446,400	2,333,759,444	704,795

Flowcell Summary

Lane Summary

Lane	Raw data	Filtered data
#	Clusters	% of the lane	% Perfect barcode	% One mismatch barcode	Clusters	Yield (Mbases)	% PF Clusters	% >= Q30 bases	Mean Quality Score
1	482,680,800	100.00	0.00	0.00	315,981,885	95,427	65.46	87.52	37.35
2	482,680,800	100.00	0.00	0.00	294,018,812	88,794	60.91	87.41	37.31
3	482,680,800	100.00	0.00	0.00	306,362,193	92,521	63.47	86.42	37.07
4	482,680,800	100.00	0.00	0.00	312,679,038	94,429	64.78	87.17	37.22
5	482,680,800	100.00	0.00	0.00	269,671,083	81,441	55.87	84.35	36.42
6	482,680,800	100.00	0.00	0.00	302,801,644	91,446	62.73	84.90	36.60
7	482,680,800	100.00	0.00	0.00	218,432,818	65,967	45.25	77.27	34.33
8	482,680,800	100.00	0.00	0.00	313,811,971	94,771	65.01	86.58	37.12

software error • 3.0k views

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modified 5.2 years ago • written 5.3 years ago by BioRyder • 180

Is everything ending up in the Undetermined_*.fastq.gz files? That would explain the results. This would indicate that your sample sheet is incorrect.

ADD REPLY • link written 5.3 years ago by Devon Ryan ♦ 98k

Hello Devon Ryan,

There is no Undetermined_*.fastq.gz files in out put directory , Because I have not mentioned any index in Sampleshee.csv to convert bcl to fastq.

Below is the Index file.

[Header],,,,,,,,
IEMFileVersion,4,,,,,,,
Date,1/12/15,,,,,,,
Workflow,GenerateFASTQ,,,,,,,
Application,HISeq FASTQ Only,,,,,,,
Assay,,,,,,,,
Description,,,,,,,,
Chemistry,Default,,,,,,,
,,,,,,,,
[Reads],,,,,,,,
151,,,,,,,,
151,,,,,,,,
,,,,,,,,
[Settings],,,,,,,,
ReverseComplement,0,,,,,,,
Adapter,AGATCGGAAGAGCACACGTCTGAACTCCAGTCA,,,,,,,
AdapterRead2,AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT,,,,,,,
,,,,,,,,
[Data],,,,,,,,
Lane,Sample_ID,Sample_Name,Sample_Plate,Sample_Well,I7_Index_ID,index,Sample_Project,Description
1,PhiX_Sample_Stock,PhiX_Sample_Stock,,,,,PhiX,
2,Spix_44G,Spix_44G,,,,,Spix_Macaw,
3,Spix_45G,Spix_45G,,,,,Spix_Macaw,
4,PhiX_Sample_Stock,PhiX_Sample_Stock,,,,,PhiX,
5,Spix_73G,Spix_73G,,,,,Spix_Macaw,
6,Spix_74G,Spix_74G,,,,,Spix_Macaw,
7,Spix_95G,Spix_95G,,,,,Spix_Macaw,
8,Spix_109G,Spix_109G,,,,,Spix_Macaw,

ADD REPLY • link modified 14 months ago by _r_am ♦ 32k • written 5.3 years ago by BioRyder • 180

Presumably it's hitting an error, check the log file.

ADD REPLY • link written 5.3 years ago by Devon Ryan ♦ 98k

We haven't switched to v2, but v1 would exhibit this behavior if there were missing .bcl or .stats files. You can add the relevant flags (in v1, I believe it's --ignore-missing-stats --ignore-missing-bcl) and try again.

ADD REPLY • link modified 14 months ago by _r_am ♦ 32k • written 5.3 years ago by harold.smith.tarheel • 4.6k

5.2 years ago by

BioRyder • 180

India

BioRyder • 180 wrote:

Hi All,

We have identified the problem.The above mentioned problem is happened due to File format of linux Server. Bcl2fastq Version2 is working properly in XFS file system. But If we are using gpfs file system in Linux server,bcl2fastq V2 is generating partial out put file, missing R1 or both R1 and R2 . We have contacted illumina and informed the same. They are internally checking the issue of Bcl2fast with gpfs file system.

ADD COMMENT • link modified 14 months ago by _r_am ♦ 32k • written 5.2 years ago by BioRyder • 180

Wow, that's kind of crazy. Thanks for reporting back!

ADD REPLY • link written 5.2 years ago by Devon Ryan ♦ 98k

What kind of hardware are you using gpfs on? Is this on a cluster that uses a job scheduler?

ADD REPLY • link written 5.2 years ago by GenoMax ♦ 95k

It is on a cluster that uses SLURM scheduler.

ADD REPLY • link modified 14 months ago by _r_am ♦ 32k • written 5.2 years ago by BioRyder • 180

GPFS is not our favorite either but we have not had gross issues like missing files in bulk. It sounds like bcl2fastq/SLURM both think that the jobs are completing properly but the files are missing on storage. Is the storage hardware fully patched/has latest firmware? Have you noticed this problem with other software/processes?

ADD REPLY • link written 5.2 years ago by GenoMax ♦ 95k

5.2 years ago by

BioRyder • 180

India

BioRyder • 180 wrote:

Hello All,

Below is the reply from illumina for the above mentioned GPFS file problem.Hope it will helpful for others

As far as I understand GPFS, while supported by CentOS, is not a default FS and the OS needs to be reconfigured to use it. Illumina development teams use CentOS for the development and validation of our Linux based software, though only with standard settings. I will however make some internal enquiries to seen whether the GPFS files system has been tested. Having made these enquiries I can confirm that we have had reports that GPFS does not handle BCL2FASTQ processes very well. This is due the sheer number of small files that need to be loaded and processed.

ADD COMMENT • link modified 14 months ago by _r_am ♦ 32k • written 5.2 years ago by BioRyder • 180

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