Question: disproportinate fastq sizes from bcl2fastq
0
gravatar for das2000sidd
12 days ago by
das2000sidd30
das2000sidd30 wrote:

Hi Everyone

While demultiplexing fastq files and for some reason for one of our sequencing runs, one set of fastq run on 4 different lanes of Illumina nextseq for a particular sample is generating fastq in the range of 2 .5 to 3 gb while the remaining 15 samples have fastq in the range of 100 to 200 Mb. This particular problem is happened in only one of our sequencing runs and the bcl2fastq log seems okay. Can anyone give some suggestions as to what problem this could be? I do not have much experience with how bcl2fastq works and hence was looking for some advice.

Thank you again

rna-seq bcl2fastq next-gen • 91 views
ADD COMMENTlink modified 12 days ago by swbarnes25.3k • written 12 days ago by das2000sidd30
0
gravatar for genomax
12 days ago by
genomax65k
United States
genomax65k wrote:

Have you looked at the demultiplexing report produced by bcl2fastq? You should be able to find that report in FCID/Unaligned/Reports/html directory, if you are using a local install of bcl2fastq to process the data. Look at the index.html file. If you are using BaseSpace then you probably know where to find the report.

  • A simple explanation could be that the pool you are sequencing is unbalanced with some samples constituting a larger fraction, resulting in correspondingly larger files.
  • If that is not the case then check the "undetermined" pool. If you see a large number of reads landing in the "undetermined" pool you will need to start chasing down issues with index sequences. Perhaps there are N's in indexes which is causing the reads to not demultiplex properly.
ADD COMMENTlink modified 12 days ago • written 12 days ago by genomax65k
1

This one liner will show you some of the most common barcodes in your undetermined fastq file. Might help?

zcat undetermined.fastq.gz | grep "^@" | sed "s/.*://g" | head -n 1000000 | sort | uniq -c | sort -n

ADD REPLYlink written 12 days ago by Madelaine Gogol5.0k
0
gravatar for swbarnes2
12 days ago by
swbarnes25.3k
United States
swbarnes25.3k wrote:

The simplest explanation is that bcl2fastq ran fine, but whoever loaded the instrument didn't normalize all the samples equally.

ADD COMMENTlink modified 12 days ago • written 12 days ago by swbarnes25.3k
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