Extended Data Fig. 5: False duplication mechanisms in genome assembly.

a, False heterotype (haplotype) duplications occurs when more divergent sequence reads from each haplotype A (blue) and B (red) (maternal and paternal) form greater divergent paths in the assembly graph (bubbles), while nearly identical homozygous sequences (black) become collapsed. When the assembly graph is properly formed and correctly resolved (green arrow), one of the haplotype-specific paths (red or blue) is chosen for building a ‘primary’ pseudo-haplotype assembly and the other is set apart as an ‘alternate’ assembly. When the graph is not correctly resolved (purple arrow), one of four types of pattern are formed in the contigs and subsequent scaffolds. Depending on the supporting evidence, the scaffolder either keeps these haplotype contigs on separate scaffolds or brings them together on the same scaffold, often separated by gaps: 1. Separate contigs: both contigs are retained in the primary contig set, an error often observed when haplotype-specific sequences are highly diverged. 2. Flanking contigs: the assembly graph is partially formed, connecting the homozygous sequence of the 5′ side to one haplotype (blue) and the 3′ side to the other haplotype (red). 3. Partial flanking contigs: only one haplotype (blue) flanks one side of the homozygous sequence. 4. Failed connecting of contigs: all haplotype sequences fail to properly connect to flanking homozygous sequences. b, False homotype duplications occur where a sequence from the same genomic locus is duplicated, and are of two types: 1. Overlapping sequences at contig boundaries: in current overlap-layout-consensus assemblers, branching sequences in assembly graphs that are not selected as the primary path have a small overlapping sequence (purple), dovetailing to the primary path where it originated a branch. The size of the duplicated sequence is often the length of a corrected read. Subsequent scaffolding results in tandem duplicated sequences with a gap between. 2. Under-collapsed sequences: sequencing errors in reads (red x) randomly or systematically pile up, forming under-collapsed sequences. Subsequent duplication errors in the scaffolding are similar to the heterotype duplications. Purge_haplotigs¹³ align sequences to themselves to find a smaller sequence that aligns fully to a larger contig or scaffold, and removes heterotype duplication types 1, 3, and 4. Purge_dups¹⁴ additionally uses coverage information to detect heterotype duplication type 2 and homotype duplications. We distinguished the two types of duplications by: 1) haplotype-specific variants in reads aligning at half coverage to each heterotype duplication; 2) differing consensus quality that resulted from read coverage fluctuations when aligning reads to homotype duplications; and 3) k-mer copy number anomalies in which homotype duplications were observed in the assembly with more than the expected number of copies.