concatenated DNA ladder molecules
Two different complementary T-tailed ConcatSeq adapters were generated by annealing Pr185 with Pr186 and Pr187 with Pr188 at 20 μM final concentration. LMW DNA ladder from NEB was diluted to 10 ng/μl and used as input material. The DNA samples were split into two reactions (with 25 μl comprising ~ 250 ng total DNA amount each) and subjected to the KAPA Hyper Prep assay: end-repair, A-tailing, and ligation to the two T-tailed ConcatSeq adapters. The resulting adapter-ligated fragment pools were then PCR-amplified using either primers Pr185 and Pr186 or Pr187 and Pr188, to enrich for the fragments that had successfully ligated adapters on both ends. DNA concentrations were quantified using the Qubit dsDNA BR Assay Kit. 200-300 ng of each of the two PCR products were then mixed and filled up to 40 μl with PCR-grade water. An equal volume of NEBuilder HiFi DNA Assembly Master Mix was added and incubated for 30, 60, 100, and 120 min at 50°C. Gibson Assembly was followed by clean-up step using KAPA Pure beads (0.8x ratio), followed by Qubit quantification and size range analysis of size range using Agilent’s DNA7500 assay.     PacBio library preparation. 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.