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Old 09-06-2017, 01:56 AM   #1
Jellyfish
Junior Member
 
Location: London

Join Date: Sep 2017
Posts: 4
Default Underclustering in MiSeq run

Hi all, this is my first post here.

I set up a MiSeq run on Monday and I'm trying to troubleshoot why I have underclustering.

So I made a silly quantification mistake where I missed a step in my qPCR analysis, which basically resulted in me calculating my normalisation based off values what were half of what they actually were (e.g. I normalised sample 1 from '40nM' to 4nM, when in reality the original concentration was 80nM.)

..It was just a stupid mistake on my part, but this has obviously resulted in my libraries being twice as concentrated (pooled and normalised to 8nM rather than 4nM).

I diluted the '4nM' library to '15pM' and spiked in 1% phiX for my run....

So I apparently accidentally ran a 30pM library on the MiSeq. But the weirdest thing is that I actually have underclustering where I would have expected it to be massively overclustered! My cluster density is 729K/mm^2 and I can't work out why.

For reference: this is whole genome sequencing prepped with the TruSeq Nano LT kit and run with the 2x300 v3 reagents. I believe I should be aiming for a cluster density of 1200-1400K/mm^2.

Previous runs (with the 2x75 v3 reagents) where I used 15pM libraries have clustered at ~1100K/mm^2.

Any insight into this would be much appreciated! I have another run starting tomorrow and a third next week and I'd really like to get to the bottom of this!

Thank you!!
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