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Old 08-15-2019, 12:48 PM   #1
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Location: CA

Join Date: Aug 2019
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Default Pooling barcoded DNA for NGS Sequencing

Hi all,

Pretty simple question here. I recently did ATAC seq on 10 samples, they have adapters and are all uniquely barcoded. Iím about to pool to prepare for NGS submission. I was told that the ideal concentration to submit was 10-20nM in approx 20uL & that I should make everything equimolar before pooling. The problem is that most of my samples range from 4-8nM, while only one is above 10nM. Will I need to make the final pooled mixture 4nM since this is my least concentrated sample? I am essentially limited by the sample of lowest concentration, correct? Just making sure Iím not missing something here. Thanks
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Old 08-15-2019, 04:13 PM   #2
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Location: US

Join Date: Dec 2010
Posts: 449

It is not necessary to bring all libraries to the same concentration before pooling - although many labs do it that way.
You could also pool equal amounts of each library (e.g. same number of femtomoles).

In any case, you can always concentrate the pool with a bead-cleanup to reach the number requested by your facility (10-20nM seems a very high request, though).
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