HapSolo: an optimization approach for removing secondary haplotigs during diploid genome assembly and scaffolding

Abstract

Background Despite marked recent improvements in long-read sequencing technology, the assembly of diploid genomes remains a difficult task. A major obstacle is distinguishing between alternative contigs that represent highly heterozygous regions. If primary and secondary contigs are not properly identified, the primary assembly will overrepresent both the size and complexity of the genome, which complicates downstream analysis such as scaffolding. Results Here we illustrate a new method, which we call HapSolo, that identifies secondary contigs and defines a primary assembly based on multiple pairwise contig alignment metrics. HapSolo evaluates candidate primary assemblies using BUSCO scores and then distinguishes among candidate assemblies using a cost function. The cost function can be defined by the user but by default considers the number of missing, duplicated and single BUSCO genes within the assembly. HapSolo performs hill climbing to minimize cost over thousands of candidate assemblies. We illustrate the performance of HapSolo on genome data from three species: the Chardonnay grape (Vitis vinifera), with a genome of 490 Mb, a mosquito (Anopheles funestus; 200 Mb) and the Thorny Skate (Amblyraja radiata; 2650 Mb). Conclusions HapSolo rapidly identified candidate assemblies that yield improvements in assembly metrics, including decreased genome size and improved N50 scores. Contig N50 scores improved by 35%, 9% and 9% for Chardonnay, mosquito and the thorny skate, respectively, relative to unreduced primary assemblies. The benefits of HapSolo were amplified by down-stream analyses, which we illustrated by scaffolding with Hi-C data. We found, for example, that prior to the application of HapSolo, only 52% of the Chardonnay genome was captured in the largest 19 scaffolds, corresponding to the number of chromosomes. After the application of HapSolo, this value increased to ~ 84%. The improvements for the mosquito’s largest three scaffolds, representing the number of chromosomes, were from 61 to 86%, and the improvement was even more pronounced for thorny skate. We compared the scaffolding results to assemblies that were based on PurgeDups for identifying secondary contigs, with generally superior results for HapSolo.

Insights into the domestication of avocado and potential genetic contributors to heterodichogamy

“…We generated 81.2 gigabases, equivalent to roughly 90 × coverage, based on the expected 1C genome size of 896 Mb ( Arumuganathan and Earle 1991 ). We assembled PacBio SMRT reads using Canu (v 2.1), producing a genome of 1,456 Mb with 5,122 contigs, and then applied HapSolo ( Solares et al 2021 ) to remove putative secondary contigs (or haplotigs). The Canu + HapSolo (C + H) genome resulted in a primary assembly of 1,032 Mb, a longest contig of 17 Mb, a BUSCO score of 91%, and an N50 of 3.37 Mb ( Supplementary Table 1 ).…”

“…Once assembled, polishing was performed with 2 passes of PacBio GenomicConsensus v2.33, followed by 2 passes with Pilon v1.23 ( Walker et al 2014 ) using default parameters and 19 × coverage of Gwen short-read Illumina sequencing data. HapSolo v0.1 ( Solares et al 2021 ), which identifies and removes alternative haplotypes, was then run on the assembly with default parameters and 50,000 iterations, producing the Canu + Hapsolo (C + H) assembly. Scaffolding was based on a Gwen × Fuerte genetic map ( Ashworth et al 2019 ) by aligning the C + H assembly using NCBI BLAST v2.2.31+ ( Altschul et al 1990 ).…”

Assessing the quality ofde novoparasite genomes assembled using only Oxford Nanopore Technologies MinION data

“…An alternative method would be to sequence multiple individuals from a highly inbred homozygous line. Many species cannot be maintained in a laboratory setting, however, making the establishment of inbred lines for these taxa challenging or impossible (Solares et al, 2021). It is also now well-recognized that inbreeding can significantly reduce genome size (Price, 1976; Fierst et al, 2015; Roessler et al, 2019), making these lines poor representatives of the species as whole.…”

A second unveiling: Haplotig masking of the eastern oyster genome improves population‐level inference

“…A highly contiguous and annotated genome assembly can be an invaluable tool for population genomic inference (Ellegren, 2014) enabling both genome‐scale and reduced representation methods for sequencing populations (Ekblom & Galindo, 2010; Fonseca et al, 2016; Matz, 2017). However, even as the cost of second‐ and third‐generation sequencing continues to drop (van Dijk et al, 2014), many challenges remain for assembling references for non‐model organisms (Roach et al, 2018; Solares et al, 2021). One of the greatest hurdles to accurate genome assembly is heterozygosity (Kajitani et al, 2014; Safonova et al, 2015; Vinson et al, 2005), a hallmark of many wild plant and animal species, including most insects and marine invertebrates.…”