Recently, we performed targeted gene sequencing by capture on the Miseq sequencer device. The library was quatified on different systems (Qubit, tape station, qPCR), the final concentration was similar between the three methods at 4nM. However, the sequencing process was interrupted at the clustering step. Analyses of iclusters mages showed a confusing appearance of the cluster generations (see attached images).

We tried to sequence this library three times, changing everything during the dilution of the library (NAOH, water...), the Miseq was also checked by illumina, no relevant problems were highlighted.

In my opinion, I had a problem with my library even though all aspects of a good library are present in this library (tapeStation profile and qPCR quatification).

Do you have any ideas about these profiles?