Schematic showing 3D genome assembly of donkey using horse (EquCab2.0) to assist. We initially assume that the donkey genome is identical to EquCab2.0 (leftmost column), and correct this assumption step-by-step, generating intermediate assemblies (columns 2 and 3), and finally a donkey genome assembly with accurate local sequence and chromosome-length scaffolds (column 4). We illustrate using two representative horse sequences, chr8 and chr24. Bottom row: Contact maps showing reads from donkey aligned to genome assemblies from each step. 2nd-to-last row: Chromograms illustrating locus order, with respect to the final (correct) order, which begins with purple and ends with red. 2nd row: Zooming in on DNA sequence for each step. Gray indicates that the current assembly sequence matches the initial, horse, sequence. Mismatches are shown in color: A (green), C (blue), G (yellow) and T (red). The I icon indicates insertions. Top row: Donkey sequence reads aligned to the current draft. Differences shown as in the 3rd row. The procedure for 3D-assisted assembly is as follows. 1st column: DNA-Seq and Hi-C reads from the donkey are aligned to the assisting horse genome assembly, EquCab2.0 (top two rows), making a contact map for the donkey with respect to EquCab2.0 (bottom row). Small-scale differences between aligned reads and EquCab2.0 are apparent (top two rows). 2nd column: Individual read alignments are examined and corresponding changes are introduced, as in traditional DNA resequencing. The result is a locally corrected genome assembly. Subsequent steps focus on constructing accurate chromosome-length scaffolds for donkey by looking for inconsistencies between donkey Hi-C and the candidate donkey genome assembly. In the bottom row, an inconsistency is highlighted with a pair of scissors: contacts are rare between two long sequences, which are adjacent to each other on a single chromosome in EquCab2.0 (see chromogram), indicating positions on different chromosomes in donkey. As such, they should be placed on separate scaffolds, and this is done in the next step. The resulting assembly (3rd column) contains three long scaffolds. The Hi-C map indicates that the first and last of these scaffolds are in frequent 3D contact, indicating that they are adjacent in the donkey genome. This change is made in the final donkey genome assembly (4th column). This strategy requires very little Hi-C data, compatible with PaleoHi-C, and yields a genome assembly that matches the true donkey genome both at single-base and chromosome scales.
