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Old 02-06-2020, 05:11 AM   #1
Noveline
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Location: Poland

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Default Problems with MiniSeq sequencing

Hello everyone,

I have a problem with sequencing on MiniSeq. My last 3 sequencing runs were unsuccessful.

At the first sight everything looks fine: Q30 value, cluster density and other parameters. When it comes time to analyze the data it looks like no reads are assign to appropriate samples…
After sequencing we can see that there is 3,6 GB reads, cluster density 168K, Q30=95,9%, phiX = 6%. Control sample that has been prepared earlier and assumed to be at 0,5% identified reads after sequencing is at 0,56% level but % of identified reads for tested samples is 0.
Run was set with BSO and I always run sequencing with FASTQ generation workflow.

What have we checked?
- Results of bioanalyzer shows that prepared libraries have expected amplicon size.
- Run parameters settings and library preparation, indexes, sample pooling were ok and everything was checked at least 2-3 times.
- In raw data (focus image) I can see that clustering is uniform.

Have you got any experience with this kind of issue and have idea what could be the potential source of problems? Is this problem with demultiplexing or indexes, maybe something else?

For me it looks like there is something wrong with indexes and samples cannot be correctly demultiplexed.

I would be really appreciate for any help.
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Old 02-06-2020, 06:25 AM   #2
GenoMax
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While I don't have direct experience with MiniSeq it sounds like a problem with demultiplexing. Do you have an install of bcl2fastq available that you could use offline to do the demux again?
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Old 02-06-2020, 06:42 AM   #3
Noveline
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Quote:
Originally Posted by GenoMax View Post
While I don't have direct experience with MiniSeq it sounds like a problem with demultiplexing. Do you have an install of bcl2fastq available that you could use offline to do the demux again?
Unfortunately, we don't have any conversion tool, because until now there was no need for this. I will ask if there is possibility to install this converter in my lab.
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