Sequencing Library Preparation from Degraded DNA Protocol

Blunt-Ending DNA Fragments

1. Set up master mix for blunt-end repair. Include per reaction:
(a) 5.0 μL of 5× End Repair Buffer.
(b) 0.25 μL of Blunt Ending Enzyme.
2. Distribute 5.25 μL of the mix in each labelled PCR tube.
3. Add 19.5 μL of DNA extraction in each individual PCR tube (or bring final volume to 25 μL by adding ddH₂O, according with initial template volume).
4. Incubate the mixture in a thermocycler for 20 min at 20 °C, and then heat-inactivate the enzyme by incubating for 20 min at 72 °C.

Adapter Ligation

1. Remove tube with blunt-ended DNA from the thermocycler and keep on ice.
2. Prepare adapter ligation reaction mix, including per reaction:
(a) 2.5 μL of 10× Ligase Buffer.
(b) 1.0 μL of P1 adapter.
(c) 0.5 μL of dNTP Mix.
(d) 0.5 μL of DNA Ligase.
(e) 2.0 μL of Nick Repair Polymerase.
3. Distribute 6.5 μL of the mix into a new PCR tube.
4. Add 1.0 μL of each of the chosen PGM Barcode X into PCR tube of each respective sample.
5. Add 20.5 μL of blunt-ended DNA.
6. Incubate the mixture in a thermocycler for 15 min at 25 °C, followed by 5 min at 72 °C.

Bead Purification

1. Prepare a fresh 70% Ethanol solution, with a final volume of at least 500 μL × number of samples.
2. Add 40 μL of SPRIselect beads to the product from Subheading 3.3, for a final ratio of 1.8× (which should result in a broad range of recovered fragment size and upper limit of approx. 200 bp).
3. Mix thoroughly by pipetting, and let the tubes incubate for 1 min in the magnetic rack.
4.Remove supernatant without disturbing the magnetic beads.
5. Add 500 μL of freshly prepared 70% ethanol to each sample, mix well, and let the tubes incubate until all beads are captured by the magnetic rack.
6. Remove supernatant without disturbing the magnetic beads, and let them air dry, for a maximum of 5 min.
7. Add 20 μL of ddH₂O to elute DNA, mix well, and let the tubes rest for another minute in the magnetic rack.
8. Remove elution and transfer it into a new (labelled) tube, keep on ice until next reaction.

Amplification

1. Dilute primers by adding 10 μL of primer stock solution (at 100 μM) in 90 μL of ddH₂O.
2. Distribute 15 μL in each PCR tube, and add 5.0 μL of the purified product.
3. In the modern DNA, lab set up and run the thermocycler with the following protocol: denaturation for 10 min at 94 °C, followed by 15 cycles of 30 s at 94 °C, 45 s at 60 °C, and 45 s at 72 °C. Final extension occurs at 72 °C for 5 min.
4. Pool all parallel amplifications for the same sample, eluting the sequencing libraries in 20 μL.
5. Visualize library concentration and fragment size distribution.

Reference:

1. Martins R F, Kampmann M L, Förster D W. Sequencing library preparation from degraded samples for non-illumina sequencing platforms. Ancient DNA: Methods and Protocols, 2019: 85-92.