VGP Trio Pipeline v1.0–v1.6

The trio pipeline is similarly designed to the standard pipeline, except for the use of parental data (Extended Data Fig. ​Fig.3b).3b). When parental genomes are available, the child’s CLR reads are binned to maternal and paternal haplotypes, and assembled separately as haplotype-specific contigs (haplotigs) using TrioCanu²⁰. In brief, parental specific marker k-mers were collected using Meryl²³ from the parental Illumina WGS reads of the parents. These markers were filtered and used to bin the child’s CLR read. A haplotype was assigned given the markers observed, normalized by the total markers in each haplotype. The subsequent purging, scaffolding, and polishing steps were similarly updated with the use of Purge_Dups¹⁴ (v1.6). We extended binning to linked reads and Hi-C reads, by excluding read pairs that had any parental-specific marker. The binned Hi-C reads were used to scaffold its haplotype assembly, and polished with the binned linked reads from the observation of haplotype switching using the standard polishing approach. During curation, one of the haplotype assemblies with the higher QV and/or contiguity was chosen as the representative haplotype. The heterogametic sex chromosome from the unchosen haplotype was added to the representative assembly. However, while curating several trios, we found that in regions of low divergence between shared parental homogametic sex chromosomes (that is, X or Z), a small fraction of offspring CLR data was mis-assigned to the wrong haplotype. This mis-alignment resulted in a duplicate, low-coverage offspring X or Z assembly in the paternal (for mammals) or maternal (for birds) haplotype, respectively, which required removal during curation. We are working on methods to improve the binning accuracy for resolution of this issue going forward.

For the female zebra finch in particular, contigs were generated before the binning was automated in the Canu assembler as TrioCanu1.7, and therefore a manual binning process was applied as described in the original Trio-binning paper²⁰ (Supplementary Methods). Contigs were assembled for each haplotype using the binned reads, excluding unclassified reads. The contigs were polished with two rounds of Arrow polishing using the binned reads, and scaffolded following the v1.0 pipeline with no purging. Additional scaffolding rounds with Bionano (s4) and Hi-C were applied. Scaffolds were renamed according to the primary scaffold assembly of the same individual (s5), with sex chromosomes grouped as Z in the paternal assembly and W in the maternal assembly following synteny to the Z chromosome from the curated male zebra finch VGP assembly. Two rounds of SR polishing were applied using linked reads, by mapping on both haplotypes. After haplotype switches were discovered, additional rounds of polishing were applied using binned linked reads (Supplementary Methods).