BacPhase: Long-insert paired-end sequencing for bin marker construction and genome phasing

In genome sequence analysis, phasing, i.e., determining which genetic variants reside on the same chromosome, is essential for assembling genomic sequences into scaffolds. However, phasing remains challenging for plant species with autopolyploid genomes such as potato (Solanum tuberosum L.) due to the high sequence similarity among homologous and homoeologous chromosomes. Bin marker is a DNA tag for a chromosome segment, helping create genetic maps and find trait-related genes. Traditional bacterial artificial chromosome (BAC)-based methods use end-sequencing of BAC clones to provide information on phasing; however, they need for labor-intensive library construction and selection of single colonies limits these methods. Here, we present BacPhase, an innovative sequence-based approach in which constructed BACs are digested with a restriction enzyme and self-ligated to produce small inserts that can be amplified by PCR and sequenced, thus removing the need for selecting single colonies. The restriction enzyme used affects the evenness and spacing of markers and we evaluated 14 restriction enzymes to select optimal restriction enzymes in multiple crop species. Using PacBio HiFi long-read sequencing to span repetitive regions, we generated 39,484 high-confidence, high-resolution bin markers in potato. Unlike Hi-C, which relies on chromatin interactions, BacPhase uses sequence polymorphisms directly, enabling precise haplotype resolution. Indeed, BacPhase anchored 59.58 ​% of scaffolds to the chromosomes in the autotetraploid potato cultivar C88, substantially improving contiguity without requiring physical maps or Hi-C data. The BacPhase method could facilitate trait mapping, genomic selection, and accelerated breeding in polyploid crops such as potato, sugarcane (Saccharum officinarum), and alfalfa (Medicago sativa).