Strange/poor sequencing quality -troubleshooting -help needed

[yangjianhunt]

Hi All,

We've had three runs of ArcherDx's VariantPlex and FusionPlex sequencing runs on Nextseq 500: using 300 cycles, paired end, high output kit.
We've obtained extremely bad results, which is also perplexing -we can't figure out why it's not working.

The flow cell intensity chart looked very strange as attached:
The first 25 cycles had good intensity at above 5000; but there seemed to have no nucleotide diversity. How can this be?
The thing is: this low diversity disappeared after cycle 25. But the intensity dropped to really low less than 2000.

To explain this library constructs further, I have the fusionPlex (from RNA) workflow diagram attached. The Variant Plex is similar (from DNA).

We did a Dragen RNA quantification analysis, and saw results like the in attached picture:

== would really appreciate any help or input on how the results are like this.

By the way, we added 40% phix control to the library pools, since we know we are working gene panels. But it seems phix spike in did not work too well?

Another thing to mention is that cluster density is at 350, even though we diluted the library pool to 1.2pM.

Thanks a lot!
Jian

[adx2021]

What is the recommended PhiX% recommended for your assay? 40% looks a bit high.

See if increasing the final concentration (1.2pM to 1.4pM maybe) will help with cluster density.

Did you work out the optimal loading concentration previously?

In our experience, Archer Fusionplex/Variantplex libraries are very sensitive to loading concentration. Even 0.2pm difference in loading concentration will have huge difference in cluster density.

[katieloojojo]

Did you resolve this issue with the Archer Vairantplex. I have the same issue with low diversity in the first 20 cycles?
I assume I need to optimise the PhiX spike in to solve the problem.