Overview of the concatenation procedure. The DNA pool to be sequenced was split into five PCR reactions with distinct primer sets, producing library variants 1–5. The PCR reactions also appended the variants with complementary BsaI restriction sites, which contained the four unique scar sequences (A–D) (yellow, purple, green, grey). The complementary overhangs are shown as black rectangles. Sample-specific barcodes were attached to the 5′-end of library variant 1 and the 3′-end of variant 5. The design enabled controlled and directional assembly of the five library variants upon Golden Gate assembly. The different barcoded concatemers were all combined and SMRTbell hairpin adapters were ligated to the barcodes of each amplicon. PacBio CCS sequencing and the SMRTbell hairpin adapters allowed continuous reads of the large concatemer amplicons producing several subreads. The consensus of the subreads produced high fidelity sequence reads.