concatenated and non-concatenated amplicons from multiplex PCR for 20 genomic target regions
Target regions of the genome were first amplified using gene-specific primers and 30 ng of HD701 of genomic DNA using AmpliTaq Gold DNA polymerase. This first round of PCR amplified the target regions together with flanking spacers on both ends of each amplicon. The resulting PCR product was then amplified with two primer pairs that prime off the spacer sequences and incorporate complementary ConcatSeq adapters to both ends in two separate PCR reactions. Resulting PCR products were then cleaned using KAPA Pure Beads at a 2x ratio by adding 100 μl of beads to 50 μl of PCR reaction and eluted into 50 μl of PCR-grade water. DNA concentration was quantified using the Qubit dsDNA BR Assay Kit. 200-300 ng of each of the two PCR products were then mixed and the final volume was brought to 40 μl with PCR-grade water. An equal volume (40 μl) of NEBuilder HiFi DNA Assembly Master Mix was added and incubated for 1 h at 50°C. Gibson Assembly was followed by clean-up step using KAPA Pure beads (0.8x ratio), followed by Qubit quantification (typically the concentration was ~ 10 ng/μl) and size range analysis using Agilent’s DNA7500 assay. Approximately 100 ng of the concatenated pool was used to prepare PacBio sequencing libraries using the KAPA Hyper Prep Kit. A suitable T-tailed hairpin adapter was first created by self-annealing of oligonucleotide Pr375 (20 μM) using Duplex Buffer and heating for 5 min to 80°C followed by a slow ramp-down (0.2°C per second) to 25°C. Double-stranded DNA concatemers were then subjected to end-repair and A-tailing, and ligated to the hairpin adapters (at roughly a 250:1 ratio of adapters to concatemers) for 30 min at 20°C. Unreacted T-tailed hairpin adapters and concatenated DNA molecules were removed by adding exonuclease III and exonuclease VII (1 μl of each) to the sample and incubating for 30 min at 37°C. The resulting library molecules were cleaned-up with KAPA Pure Beads at 0.8x ratio, and then quantified using Qubit dsDNA HS Assay. On average the final concentration of the sequencing libraries was between 0.5 and 2 ng/μl.