Pacbio barcoded adapters

LR amplicons were purified by AMPure PB beads (Pacific Biosciences), using a bead ratio of 1.5x. Library preparation was done according to protocol ‘Procedure and Checklist—Preparing SMRTbell Libraries using PacBio Barcoded Adapters for Multiplex SMRT Sequencing’ (Pacific Biosciences, Part Number 100-538-700-02). In brief, sample concentrations were measured with Qubit 3.0 (ThermoFisher Scientific). Equimolar amounts of amplicons, adding up to a total input of 200 ng to 1 ug DNA, were used to perform one-step end repair and adapter ligation (using barcoded hairpin adapters), followed by equimolar pooling. The pool was purified with AMPure PB beads, followed by DNA damage repair, exonuclease digestion, and an additional two rounds of AMPure PB beads purification.

We prepared SMRTbell libraries for PacBio RS II sequencing following the manufacturer’s protocol for Multiplex SMRT Sequencing with PacBio Barcoded Adapters (Pacific Biosciences, Menlo Park, CA, USA). Prior to library preparation, amplicons of the three (A, B, C) or four (A, B, C, A-like) different loci, respectively, were pooled in equimolar amounts for each replicate of each individual. Each library contained ten such pooled samples with a DNA concentration of 100 ng per sample. Library preparations were performed according to supplier’s instructions except that after the damage repair step, we again added 1 μl of DNA Damage Repair Mix (Pacific Biosciences, Menlo Park, CA, USA) to each library and incubated the mixture for 30 min at 37 °C. This second damage repair step improved the sequencing by preventing early terminations in the sequencing process due to damages in the DNA template. Sequencing was performed on a PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA) using P6-C4 chemistry and 240 min movies.