An assay that I have run on the miniseq previously is starting to give me problems and I am at a loss on what it could be. My problem on the surface looks like underloading. My cluster density is around 124 k/mm2 and phiX is around 2.5%. However, I ran an old library the previously gave me good numbers (i.e. 240K/mm2, 1% phix) has dropped to the poor numbers I first mentioned. I swapped out lots of the flow cell and the results recovered. However, the following day the numbers dropped again and even worse on the third day. My cluster density decreased from 240 to 174 to 109 k/mm2. I looked at the image of the clusters and looks heavily clustered and does not look like 109 k/mm2. The other metrics like pass filter and Q30 etc are fantastic, above 95%. The problem is that I lose depth at all of my targeted amplicons as a result of the underclustering. I tried new NaOH and TrisHCl to no avail. It seems that my library is somehow binding less efficiently and phix isnt as affected. On a side note, I have noticed some bubbles in the flow cell but I am not sure if this is normal and introduced during the post run wash cycle. Any ideas will be helpful! Thanks!
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