Re-running denatured libraries on MiSeq

[Samarpana]

Two of my libraries failed on HiSeq, due to focussing error of the instrument. The cluster densities were approx. 800-900 K/mm2 but %PF was low (50-60%). Just to be sure that my libraries are not bad, I want to run them on MiSeq.

I do not have any fresh library remaining with me. Can I use the denatured 20PM ones that I stored after setting the HiSeq run?

Has anyone tried something like this? It's been 10 days since the library has been diluted and they were stored at -20C.

[thermophile]

The word that I've gotten from Illumina is to redenature even if the run fails within the first few hours. So I've never tried using old denatured library (I don't even keep the 20pm library)

[Jessica_L]

In theory, I think you might be okay to use the 20pM denatured libraries? 20pM PhiX is good for something like three weeks, but I'd add a caveat about not assuming they'll just behave the same. Like thermophile, I usually go back to my 4nM and repeat the denaturation step just in case that's part of the reason for a run failure.

Based on the information you provided, like the cluster density and the fact that the run failed due to a focusing error, I'd be a bit more confident that re-running the libraries should yield good results, but there's still a degree of risk involved with going back to the denatured libraries. I'd be interested to know how your libraries perform if you do end up re-running them since I don't think there's a lot of information about the stability of 20pM libraries.

[Samarpana]

Jessica_L: Yes that's exactly why I am hoping it should work. Previously whenever I had to re-run, I always repeated the denaturation step. Just that this time, my library concentrations were too low to repeat that step. I hope our sequencing facility doesn't create any issue regarding it. I will update if it works out. Thanks for your time.

In theory, I think you might be okay to use the 20pM denatured libraries? 20pM PhiX is good for something like three weeks, but I'd add a caveat about not assuming they'll just behave the same. Like thermophile, I usually go back to my 4nM and repeat the denaturation step just in case that's part of the reason for a run failure.

Based on the information you provided, like the cluster density and the fact that the run failed due to a focusing error, I'd be a bit more confident that re-running the libraries should yield good results, but there's still a degree of risk involved with going back to the denatured libraries. I'd be interested to know how your libraries perform if you do end up re-running them since I don't think there's a lot of information about the stability of 20pM libraries.

[Samarpana]

Thanks Thermophile.. If I end up running them, it'll be my first time too. Never tried re-running denatured ones before. I wouldn't have considered it, if I had enough fresh library remaining. Just hoping it works out.

The word that I've gotten from Illumina is to redenature even if the run fails within the first few hours. So I've never tried using old denatured library (I don't even keep the 20pm library)

[thermophile]

Please post how it turns out.

[Samarpana]

Just to update @Thermophile and @Jessica_L: re-running the denatured library was a bad idea. The run failed.
So...I will prepare the library again.