Question: My run over-clustered - what does this mean for the data?
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gravatar for CC
16 months ago by
CC20
Melbourne, Australia
CC20 wrote:

Hi, I recently sequenced some libraries on a MiSeq, and I must have got the concentration wrong because it over-clustered. The cluster density was 1997k/mm2. The run still completed and I have data, but I was wondering what I can expect from this data/is it usable?

I had a massive amount of reads - 46.33 million with 35 million passing filter. I used a 600V3 kit and I thought the upper limit of this was 25 million reads so I'm a bit concerned.

Thank you for any advice.

rna-seq miseq illumina • 861 views
ADD COMMENTlink modified 16 months ago by genomax63k • written 16 months ago by CC20

Upper limit is 25M PE reads, while it seems you counted both R1 and R2, for which the upper limit would be 2x25M.

ADD REPLYlink written 16 months ago by WouterDeCoster37k
0
gravatar for chen
16 months ago by
chen1.8k
OpenGene
chen1.8k wrote:

The passing filter seems a bit lower than usual, but the data must be still usable.

Check the data quality with some QC/filtering tools, like FASTQC or fastp

ADD COMMENTlink written 16 months ago by chen1.8k
0
gravatar for colindaven
16 months ago by
colindaven1.1k
Hannover Medical School
colindaven1.1k wrote:

Try aligning it and checking the % alignments and visualization. It's probably still pretty decent but I guess there could be a lot of duplicates.You can exclude these bioinformatically depending on the application.

ADD COMMENTlink written 16 months ago by colindaven1.1k
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gravatar for genomax
16 months ago by
genomax63k
United States
genomax63k wrote:

This run is clustered well beyond the normal spec for a MiSeq v.3 run. As others have said, if you are going to align to a good reference (and if this plain genomic DNA) then the data may still be usable. If this is for any kind of de novo work then you should re-run this with significantly reduced concentration.

ADD COMMENTlink written 16 months ago by genomax63k
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