Anyone using QIAGEN 16S/ITS Panel with MiSeq?

HelenaSC · 08-02-2019, 05:17 AM

Hi there!

Anyone using QIAGEN 16S/ITS Panel with MiSeq?

We have good libraries with good profiles, quantified by TapeStation, Qubit and qPCR and we load them according to Qiagen instructions (dilute to 2nM and load at 10pM) and it result in very low cluster density...NaOH is working properly with all the other runs, the Miseq is also giving good results with other type of Runs....anyone is in the same situation? Or anyone is having good results with this Panel sequencing on a MiSeq?

Thanks a lot in advance!

kmcarr · 08-02-2019, 08:37 AM

Are you sure that it really is low cluster density? Do you have access to the Thumbnails? When a flow cell is very overloaded it may falsely report a low cluster density. I ask because our experience with 16S/ITS amplicons on the MiSeq is that they need to be loaded at much lower concentrations (3.0pM). But this is with amplicon we make using the Schloss SOP. Amplicons prepared using a different protocol may require very different loading concentration.

HelenaSC · 08-04-2019, 11:25 PM

Hi there,

THanks a lot for your comments. Yes, I'm sure it is low cluster density, I can see it at the thumbnails and comparing to other runs. We only got like 200K clusters...that is really low

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08-02-2019, 05:17 AM	#1
HelenaSC Member Location: Spain Join Date: Jun 2013 Posts: 21	Anyone using QIAGEN 16S/ITS Panel with MiSeq? Hi there! Anyone using QIAGEN 16S/ITS Panel with MiSeq? We have good libraries with good profiles, quantified by TapeStation, Qubit and qPCR and we load them according to Qiagen instructions (dilute to 2nM and load at 10pM) and it result in very low cluster density...NaOH is working properly with all the other runs, the Miseq is also giving good results with other type of Runs....anyone is in the same situation? Or anyone is having good results with this Panel sequencing on a MiSeq? Thanks a lot in advance!

08-02-2019, 08:37 AM	#2
kmcarr Senior Member Location: USA, Midwest Join Date: May 2008 Posts: 1,178	Are you sure that it really is low cluster density? Do you have access to the Thumbnails? When a flow cell is very overloaded it may falsely report a low cluster density. I ask because our experience with 16S/ITS amplicons on the MiSeq is that they need to be loaded at much lower concentrations (3.0pM). But this is with amplicon we make using the Schloss SOP. Amplicons prepared using a different protocol may require very different loading concentration.

08-04-2019, 11:25 PM	#3
HelenaSC Member Location: Spain Join Date: Jun 2013 Posts: 21	Hi there, THanks a lot for your comments. Yes, I'm sure it is low cluster density, I can see it at the thumbnails and comparing to other runs. We only got like 200K clusters...that is really low