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Old 12-18-2015, 02:34 AM   #6
Xray1
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Location: Germany

Join Date: Jun 2015
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When I get uniformity near 50%, it is usually just that one of two primer pools didn't work. You can check whether amplicons with low coverage are all from the same pool. If so, your ampliseq library prep just didn't work.

Mixing libraries generated with long and short amplicon panels in the templating step might add to the problem. However, most "long amplicon" panels also have shorter amplicons and still work just great on the PGM. I am currently running a long amplicon panel with the shortest amplicon somewhere around 130pb and it shows great results. However, for Chef/PGM (and I think also the OT2) templating settings are different for 200bp and 400bp runs. For Proton you probably always use the same templating and that might cause more proplems when you add more short amplicons. Make sure a certain long amplicon panel works fine on the Proton before mixing with other panels.

You will have to try around a bit I guess, since the long panels are not officially supported. Interesting to read that it can work. Getting longer reads on Proton was one of the things people really hoped for (besides new P-chips.....), so the fact that Ion never got anything going (not even on the S5-540) indicated to me that it just didn't work. I guess most people therefore just avoid even trying this......Do you adjust any of your protocols (amount of library you put in or whatever) to get the long amplicons running?
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