I am having difficulty figuring out why my cluster density is consistently too high for my 16S sequencing runs on the MiSeq (v2 chemistry). I have been getting a cluster density between 1100-1300, and for 16S, my lab aims for about 800-900. I am loading LESS library than my labmates (they load 12.5-13 pM and get the optimal cluster density while I load 12 pm). Our libraries are prepared in the same way (v4 PCR amplification, Ampure XP bead purification, and Qubit quantification followed by qPCR with standards to get an accurate library concentration). We use the same stock of NaOH to prepare our fresh 0.2 N NaOH solution. We use Tris-HCl to neutralize the NaOH prior to diluting the library down to 20 pm, which we then dilute further.

Does anyone have any ideas on how to troubleshoot this? I cannot figure out what is going on for the life of me; I’ve had labmates observe me and they can’t find anything that stands out either. Any thoughts would be greatly appreciated!