Ligate Adapters

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This step ligates adapters to the ends of cfDNA fragments. The UMI DNA index anchors (adapters) contain unique molecular identifiers.

• LIGX (DNA Ligase X)
• UMI DIA (Unique Molecular Identifier DNA Index Anchors)
• Microseal 'B' adhesive seals
• UMI DIA:
– Each well of the UMI DIA plate is for single use only.
– When instructed, pierce the foil seal on the plate for only the number of samples being processed.
– When returning the plate to storage, cover the pierced wells with Microseal 'B' to prevent cross-contamination.
1. Prepare the following consumables:
• LIGX—vortex briefly on medium-high speed to mix, and then centrifuge briefly.
• UMI DIA:

Make sure to use the UMI DNA Index Anchor plate and not the index adapter plate (UDPXXXX).

a. Place on a plate shaker at 1800 rpm for a minimum of 10 seconds.
b. Centrifuge briefly.
2. Save the following LIG program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
• Set the reaction volume to 100 μl
• 30°C for 20 minutes
• 4°C for 1 minute
• Hold at 4°C
1. Add 5 μl LIGX to each well of the LP plate.
2. Pierce the foil seal of the UMI DIA plate using a new pipette tip for each well. Pierce only the number of samples being processed.
3. Add 5 μl UMI DIA to each well of the LP plate.
4. Set a 200 μl pipette to 90 μl, and then pipette 10 times to mix.
5. Apply Microseal 'B', and then centrifuge briefly.
6. Place on the preprogrammed thermal cycler and run the LIG program.
7. When the program reaches the 4°C hold, immediately proceed to the next step.