Hi there!
We are doing some tests to sequence different panels in a same FlowCell of NovaSeq 6000. We have already successfully loaded different libraries from different panels in the same FLow Cell. However these panels often produce similarly sized libraries (around 350pb).
Now, we need to include another distinct panel. However, in this time, the panel produces smaller libraries (around 270bp).
We know that different libraries with different sizes show distint clustering efficiency, once smaller libraries cluster more efficiently and probably will be overrepresented compared to larger libraries in a same NGS run.
So, do you have experience in mixture diferents libraries with diferent size to sequencing? Do you have any recommendations regarding the proportions to follow (Considering output per sample, library size, others factors)?
Thank you in advance for your attention regarding this matter.
Hi,
It is not ideal to pool different libraries (of different sizes) in one run.
But it can be done with some considerations. As you mentioned that small libraries will get overrepresented compared to large ones. Therefore while pooling the libraries you need to make sure you add different libraries accordingly.
For eg:
If you have two libraries:
Lib1- 10 samples and size of 500bp
Lib2- 10 samples and size of 250 bp
and you need equal number of reads for all 20 samples (both libraries)
In this situation, you can not pool both libraries in equal proportion. You need to load the larger library in high proportion as compared to the smaller one.
The exact proportion can be determined by doing a spike run if that is possible.
But if the size difference is less than 100bp then I do not think it will skew drastically.
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