library with different sizes
I wish to use MinION to sequence 11 PCR products. Most of them are ~ 2 or 3 kb. Another two are 4kb and 9kb respectively. Is it possible to add the adapter and run them together, although they have different sizes? Thank you very much.
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In theory ONT 2D libraries should not be as sensitive to size variations as illumina/454 amplicon sequences used to be, so it should be possible to have a variety of amplicon sizes.
But make sure you use an excess (something like 2-4X) of MOLAR concentration of the larger fragments compared to the small ones, to compensate for lower ligation and loading efficiencies.
And I would still rather avoid mixing anything under 2kb with larger fragments.
The exact size correction coefficients/functions would have to be worked out empirically. -
You may have to adjust the molar input ratio with the adapter sequences, the protocol contains a note about having smaller fragments.
But mixing shouldn't be a problem.Comment
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