Cell free DNA is generated in a highly specific biological process that yields a characteristic fragmentation length profile. Thus, we recommend preparing a library that does not change the size profile in any way: using the "Mechanical Fragmentation Library Prep Kit" without any additional fragmentation treatment. Our library insert sizes look superb when we use that method.

 

We have used Twist Utopia adapters extensively on the cfDNA samples, and they work great for duplex consensus and getting good assessments of the mutation rates.

 

In a pinch, if a Mechanical Frag. library prep kit is not available, the Enzymatic Fragmentation Library Prep kit can be used, and steps can be taken to minimize the activity of the enzymatic fragmentation:

Assemble fragmentation, end repair, and A-tailing reactions on ice.

Skip the 32°C fragmentation incubation in the thermal cycler: go straight to 65°C for A-tailing.

Pre-heat the thermal cycler to 65°C for A-tailing.

Directly transfer the assembled reactions from ice to the pre-heated 65°C thermal cycler.