SNP Genotyping by Multiplex PCR Targeted Amplicon Sequencing Protocol

Multiplex PCR

1. Prepare the primer mix by adding all forward and reverse primers. In the resulting solution, each primer should have a final concentration of 1 μM.
2. Prepare the multiplex master mix by adding all reagents except the DNA template. Prepare sufficient master mix for all samples and PCR blanks.
3. Distribute 16 μl of the master mix into PCR strips/plates and close all lids.
4. To avoid cross-contamination, add 4 μl DNA template to each reaction tube individually (open only one tube or row of tubes at a time).
5. After PCR setup, put the tubes/strips/plate in a thermocycler outside the ancient DNA lab, and run the amplification: initial step at 95 °C for 10 min to activate the hot-start polymerase, followed by 30 cycles of denaturation for 10 s 94 °C, primer annealing for 30 s at the required annealing temperature, and elongation for 30 s at 72 °C. The protocol ends with a final extension step at 72 °C for 4 min.
6. After amplification is finished, dilute the reactions 1:10–1:100 with HPLC grade water. This will be the template for the indexing PCR.

Indexing PCR

1. Prepare the master mix by adding all reagents except the diluted multiplex PCR products and barcoded indexing primers. Prepare sufficient master mix for all samples and PCR blanks.
2. Distribute 7 μl of the master mix into PCR strips/plates and close all lids.
3. Add 2 μl of individual barcode primer to each reaction tube.
4. Add 1 μl of diluted PCR product (from step 1) to each reaction tube individually.
5. After PCR setup, run the indexing PCR in a thermocycler: initial step at 95 °C for 10 min to activate the hot-start polymerase, followed by 10 cycles of denaturation for 15 s at 95 °C, primer annealing for 30 s at 60 °C, and elongation for 60 s at 72 °C. The protocol ends with a final extension step at 72 °C for 4 min.

Library Purification and Quantification

1. Prepare the master mix by adding all reagents except the diluted multiplex PCR products and barcoded indexing primers. Prepare sufficient master mix for all samples and PCR blanks.
2. Purify the reactions on a magnetic 96-well plate using the Agencourt AMPure XP Reagent beads according to manufacturer's instructions. Elute and store DNA in 20 μl HPLC grade water.
3. Quantify the purified amplicon libraries with the Quant-iT™ PicoGreen® dsDNA quantification assay according to manufacturer's instructions.
4. Pool all sample libraries using an equal amount of DNA.

Sequencing

1. Measure the DNA concentration of the pooled library on a Qubit fluorometer according to manufacturer's instructions, and dilute to the required concentration.
2. Sequence purified and pooled amplicon libraries on Illumina sequencers using CS1, CS2, and their reverse complements as sequencing primers according to manufacturer's instructions

Reference:

1. Wutke S, Ludwig A. Targeted PCR amplification and multiplex sequencing of ancient DNA for SNP analysis[J]. Ancient DNA: Methods and Protocols, 2019: 141-147.