low clustering for Sureselect exome v6 on NextSeq

bsvega · 06-10-2016, 02:29 AM

Hi everybody,
We are doing SureSelect all exome v6 on a High Output 300 cycle NextSeq Kit. The libraries were quantitated with QIASeq qPCR (works very weel with other libraries), and although we load the recomended concentration we always got low clustering (around 98 K/mm2). We also try increassing the loading concentration with the same result.
Anyone has the same issue? Any suggestions?.
Thanks for your help

jteeee2 · 06-10-2016, 04:31 AM

Could you please attach Bioanalyzer traces of your pre and post capture libraries? That information may help troubleshoot the issue.

bsvega · 06-10-2016, 06:13 AM

We use QIAxcell instead of bioanalyzer. I am attaching the files.

jteeee2 · 06-10-2016, 06:57 AM

Thanks for attaching these. The only pertinent comment I have about the traces is that the average library size is a bit larger than we commonly see for captures. Was genomic DNA used as the starting material?

The NextSeq can typically handle fragments of that size with no problem, however. You simply see a significant bias towards smaller fragments being able to cluster effectively. One thing we have seen with Agilent capture libraries is the presence of a poly-G tract at the beginning of the read that can really screw up the NextSeq's base calling software. How does the base diversity look on the run? Are you seeing high "G" calls anywhere?

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06-10-2016, 02:29 AM	#1
bsvega Junior Member Location: Madrid Join Date: Jul 2015 Posts: 9	low clustering for Sureselect exome v6 on NextSeq Hi everybody, We are doing SureSelect all exome v6 on a High Output 300 cycle NextSeq Kit. The libraries were quantitated with QIASeq qPCR (works very weel with other libraries), and although we load the recomended concentration we always got low clustering (around 98 K/mm2). We also try increassing the loading concentration with the same result. Anyone has the same issue? Any suggestions?. Thanks for your help

06-10-2016, 04:31 AM	#2
jteeee2 Member Location: Illinois Join Date: Oct 2014 Posts: 37	Could you please attach Bioanalyzer traces of your pre and post capture libraries? That information may help troubleshoot the issue.

06-10-2016, 06:57 AM	#4
jteeee2 Member Location: Illinois Join Date: Oct 2014 Posts: 37	Thanks for attaching these. The only pertinent comment I have about the traces is that the average library size is a bit larger than we commonly see for captures. Was genomic DNA used as the starting material? The NextSeq can typically handle fragments of that size with no problem, however. You simply see a significant bias towards smaller fragments being able to cluster effectively. One thing we have seen with Agilent capture libraries is the presence of a poly-G tract at the beginning of the read that can really screw up the NextSeq's base calling software. How does the base diversity look on the run? Are you seeing high "G" calls anywhere?