We’re using the xGEN DNA Library Prep MC Kit with UDI-uMI adapters from IDT, a workflow we’ve used successfully for years. However, in our last two independent library preps, we’re seeing very low clustering (~400K/mm²) on the MiSeq (v3 kit), despite good-quality reads and expected PhiX levels.
This issue appears specific to these two libraries:
We suspect something in these libraries is inhibiting cluster formation without affecting standard QC.
We also contacted Illumina support, but couldn’t identify the cause.
Has anyone experienced something similar or have suggestions for what to check?
This issue appears specific to these two libraries:
- The same sequencer works fine for other users.
- Reagents (including NaOH) were tested and ruled out by sequencing in a different lab — same low clustering.
- qPCR quantification of the libraies was confirmed independently in another lab.
- Library QC by Bioanalyzer and qPCR (pre- and post-PCR, and after pooling) looks normal.
- Libraries were cleaned with SPRI beads at least once before sequencing.
We suspect something in these libraries is inhibiting cluster formation without affecting standard QC.
We also contacted Illumina support, but couldn’t identify the cause.
Has anyone experienced something similar or have suggestions for what to check?