I recently ran a ~200-400bp library prepped with NexteraXT on the MiSeq using reagents kit v3 for a 2x300 run, paired end, dual index reads: 10pM pooled library with 15% 10pM phiX.
I got a high cluster density of 1185 k/mm2 with only 27% clusters PF and >=Q30 14%.
Does anyone have any ideas for troubleshooting? Could this come from old kits or bubbles on the flowcell, would this even be visible? I did a qPCR (KAPA library quantification) before dilution all samples to 4nM. The qPCR worked fine, so there must be an error afterwards or with the kit.
Could it be that there was a DNase destroying the amplicons?
I'm curious about your ideas and answers.
Thanks in advance
Sarah
I got a high cluster density of 1185 k/mm2 with only 27% clusters PF and >=Q30 14%.
Does anyone have any ideas for troubleshooting? Could this come from old kits or bubbles on the flowcell, would this even be visible? I did a qPCR (KAPA library quantification) before dilution all samples to 4nM. The qPCR worked fine, so there must be an error afterwards or with the kit.
Could it be that there was a DNase destroying the amplicons?
I'm curious about your ideas and answers.
Thanks in advance
Sarah
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