Cluster density Illumina

CatVincent · 11-23-2010, 05:37 AM

Hello, I would like to intoduce myself.
We have been preparing and successfully sequencing a number of transcriptome libraries for PE Illumina sequencing.

We obtained unexpectedly high cluster density with (supposedly) only 5nanoM of a transcriptome library loaded on a flow cell.
The library was prepared using a NEB mRNA sample preparation kit and appeared normal when checked on a Bioanalyzer. <
Has anyone else obtained (too) high cluster density with low sample input?

Any comments welcome.

NextGenSeq · 11-23-2010, 07:07 AM

We switched to KAPA Q-PCR to quantify our libraries. This gives more reproducible cluster densitites.

laurena · 11-23-2010, 10:50 AM

Hi CatVincent,

I recently went to an Illumina Sequencing seminar and from what I heard there, a majority of the users are using the Bioanalyzer in combination with another quantification method: PicoGreen (or RiboGreen in your case), NanoDrop, Qubit or qPCR to better predict cluster densities. It seemed universal that no one uses just the BioA beacuse it's not that predictive (I think someone mentioned within 25%).

Our lab has been using PicoGreen in combination with BioAnalyzing and have been hitting our predicted cluster densities spot on! We're also going to start incorporating qPCR.

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11-23-2010, 05:37 AM	#1
CatVincent Junior Member Location: Germany Join Date: Nov 2010 Posts: 2	Cluster density Illumina Hello, I would like to intoduce myself. We have been preparing and successfully sequencing a number of transcriptome libraries for PE Illumina sequencing. We obtained unexpectedly high cluster density with (supposedly) only 5nanoM of a transcriptome library loaded on a flow cell. The library was prepared using a NEB mRNA sample preparation kit and appeared normal when checked on a Bioanalyzer. < Has anyone else obtained (too) high cluster density with low sample input? Any comments welcome.

11-23-2010, 10:50 AM	#3
laurena Junior Member Location: San Diego Join Date: Aug 2010 Posts: 2	Predicting Cluster Densities Hi CatVincent, I recently went to an Illumina Sequencing seminar and from what I heard there, a majority of the users are using the Bioanalyzer in combination with another quantification method: PicoGreen (or RiboGreen in your case), NanoDrop, Qubit or qPCR to better predict cluster densities. It seemed universal that no one uses just the BioA beacuse it's not that predictive (I think someone mentioned within 25%). Our lab has been using PicoGreen in combination with BioAnalyzing and have been hitting our predicted cluster densities spot on! We're also going to start incorporating qPCR.

11-23-2010, 07:07 AM	#2
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	We switched to KAPA Q-PCR to quantify our libraries. This gives more reproducible cluster densitites.