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Old 07-04-2018, 01:04 AM   #2
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,199

For PacBio you can use different quantity and pool based on molar concentration (after ligation step) to obtain relatively equal read coverage.

Points to consider:
1- adding twice the required coverage for plasmid as it might be supercoiled and resist shearing
2- depending on sheared DNA fragment size, it might be difficult to size select because plasmid DNA will shear to smaller fragments so you will need to shear gDNA to similar size which will exclude prepping 20kb library
3- There will be sequencing bias toward shorter fragments

I guess similar issues will apply for Nanopore as well
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