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Hello. We recently met some problems about casava demultiplexing. Since we used some other kit instead of illumina sequencing kit to prepare the samples, the barcode format of the outcome is different from the illumina style. Thus, we couldn't use Casava to demultiplex the samples. It's still OK for us to use the open source demultiplexing tool for this step, however, when it comes to the alignment, we need some files that were generated from CASAVA demultiplexing step(DemultiplexedBustardSummary.xml & DemultiplexedBustardConfig.xml). Otherwise, we might need to make these files manually which is prone to cause mistakes. Is there some way to solve this problem? Thanks so much.
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Senpeng,
It may still be possible to use the CASAVA 1.8 to demultiplex your samples even though the read format is the not the default (TruSeq). Please note that below is just speculation on my part, based on successful experience with the earlier version of the CASAVA/OLB demultiplexing pipeline.
I'm going to assume that your alternate library prep method places the index (barcode) in-line with read 1 so that it comprises the first bases of read 1. You would still construct the SampleSheet.csv exactly as described in the documentation. The only difference is in the format of the --use-bases-mask when running configureBclToFastq.pl. For the purposes of this example I am going to assume an index of 6 bases long at the beginning of read 1 and the total read is 101 cycles. When you run configureBclToFastq.pl you should use the following:
This tells the pipeline to use the first 6 bases of read 1 as the index (with sample indexes listed in the SampleSheet.csv), the next 94 bases are read 1 and the last cycle is discarded. NOTE: NO commas.Code:# configureBclToFastq.pl --use-bases-mask I6Y94N <other parameters>
I'll repeat just to be clear. I have not tried this myself with CASAVA 1.8. I'm just suggesting it based on my experience with previous versions of CASAVA/demultiplex.pl. They could have changed things so that this doesn't work anymore.
But it costs you virtually nothing to give it a try, just a minimal amount of time, and it certainly isn't going to hurt anything.
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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