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  • Miniseq run issues

    Hi,
    I am new to sequencing stuff by myself and till date I have ran at least 30 miniseq runs. Some runs were decent, but most of the runs over clustered in spite of quantifying libraries using NEB library quant kit. I make my own libraries using two sets of PCR and custom designed barcodes, purify and run bioanalyzer. Is there a way to troubleshoot what is exactly wrong? I did contact Illumina and they haven't given me a satisfactory answer ever. Any help would be appreciated.

  • #2
    Puh... hard to say... MiniSeq as far as I know uses unpatterned flow cells so over clustering is definitely more of an issue then with patterned flow cells. Did you have a look at what was the difference in the decent runs and the over clustered ones? How do you determine loading concentration and what concentrations did you load? I guess you are aware of Illuminas Denature and Dilute Guide? Whats the average library size? What kind of libraries are you preparing? Do you see a difference with different library types (for example RNAseq vs WGS)?

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    • #3
      Hi tired_of_overclustering

      I've had to do quite a lot of troubleshooting with MiniSeq libraries so I can try to help!

      How are you purifying your libraries? And if you do a melt curve at the end of the qPCR, how does it look?

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