High-quality genome assembly of ancient asexual rotifer reveals homologous chromosomes that frequently recombine.

Background Long-read sequencing is revolutionizing genome assembly: as PacBio and Nanopore technologies become more accessible in technicity and in cost, long-read assemblers flourish and are starting to deliver chromosome-level assemblies. However, these long reads are usually error-prone, making the generation of a haploid reference out of a diploid genome a difficult enterprise. Failure to properly collapse haplotypes results in fragmented and structurally incorrect assemblies and wreaks havoc on orthology inference pipelines, yet this serious issue is rarely acknowledged and dealt with in genomic projects, and an independent, comparative benchmark of the capacity of assemblers and post-processing tools to properly collapse or purge haplotypes is still lacking. Results We tested different assembly strategies on the genome of the rotifer Adineta vaga, a non-model organism for which high coverages of both PacBio and Nanopore reads were available. The assemblers we tested (Canu, Flye, NextDenovo, Ra, Raven, Shasta and wtdbg2) exhibited strikingly different behaviors when dealing with highly heterozygous regions, resulting in variable amounts of uncollapsed haplotypes. Filtering reads generally improved haploid assemblies, and we also benchmarked three post-processing tools aimed at detecting and purging uncollapsed haplotypes in long-read assemblies: HaploMerger2, purge_haplotigs and purge_dups. Conclusions We provide a thorough evaluation of popular assemblers on a non-model eukaryote genome with variable levels of heterozygosity. Our study highlights several strategies using pre and post-processing approaches to generate haploid assemblies with high continuity and completeness. This benchmark will help users to improve haploid assemblies of non-model organisms, and evaluate the quality of their own assemblies.

The several hundreds of species of bdelloid rotifers are notorious because they represent an ancient clade comprising only asexual lineages 1 . Moreover, most bdelloid species have the ability to withstand complete desiccation and high doses of ionizing radiation, being able to repair their DNA after massive genome breakage 2 . To better understand the impact of long-term asexuality and DNA breakage on genome evolution, a telomere-to-tolemere reference genome assembly of a bdelloid species is critical 3, 4 . Here we present the first, high quality chromosome-scale genome assembly for the bdelloid A. vaga validated using three complementary assembly procedures combined with chromosome conformation capture (Hi-C) data. The different assemblies reveal the same genome architecture and using fluorescent in situ hybridization (FISH), we demonstrate that the A. vaga genome is composed of six pairs of homologous chromosomes, compatible with meiosis. Moreover, the synteny between homoeologous (or ohnologous) chromosomes is also preserved, confirming their paleotetraploidy. The diploid genome structure of A. vaga and the presence of very long homozygous tracts show that recombination between homologous chromosomes occurs in this ancient asexual scandal, either during DSB repair or during meiotic pairing. These homozygosity tracts are mainly observed towards the chromosome ends in the clonal A. vaga suggesting signatures of a parthenogenetic mode of reproduction equivalent to central fusion automixis, in which homologous chromosomes are not segregated during the meiotic division.Bdelloid rotifers are a notorious clade of ancient asexual animals. However, both its longevity (>60 My) and diversity 1 contradicts the expectation that obligatory parthenogenetic animal lineages are evolutionary dead-ends. Historical observations 2 (or lack thereof) have produced a consensus that bdelloid rotifers are strictly parthenogenetic without any meiosis (e.g. no 3 males or hermaphrodites 1 , ameiotic genome structure in the model species Adineta vaga 3 , apomictic oogenesis 5, 6 ). However, 4 recent studies brought doubt regarding the supposed absence of meiotic recombination in these microscopic animals. These 5 include a drop of linkage disequilibrium with increasing distance between loci in A. vaga 7 , signatures of gene conversion 3, 8 , 6 heterozygosity levels falling within the range observed for sexual metazoans 3, 4, 9 and reports of allele sharing between bdelloid 7 individuals 7, 10-12 . 8In addition to its asexual evolution, the bdelloid rotifer A. vaga also became a model species for its extreme resistance to 9 desiccation and radiation, with implications for space research. Both prolonged desiccation and radiation induce oxidative 10 1 stress and massive genome breakage that A. vaga seem to handle well, maintaining high survival and fecundity rates while 11 efficiently repairing its DNA double strand breaks (DSBs) 2 . The exact nature of their DSB repair mechanism remains unknown, 12 but homologous recombination (HR) should ...

Third-generation sequencing, also called long-read sequencing, has revolutionized genome assembly: as PacBio and Nanopore technologies have become more accessible in technicity and in cost (with decreasing error rates and increasing read lengths), longread assemblers have flourished and are starting to deliver chromosome-level assemblies. However, an independent, comparative assessment of the performance of these programs on a common, real-life dataset is still lacking. To fill this gap, we tested the efficiency of long-read assemblers on the genome of the rotifer Adineta vaga, a non-model organism for which both PacBio and Nanopore reads were available. Although all the assemblers included in our benchmark aimed to produce a haploid genome assembly with collapsed haplotypes, we observed strikingly different behaviors of these assemblers on highly heterozygous regions: allelic regions that were most divergent were sometimes not merged, resulting in variable amounts of duplicated regions. We identified three strategies to alleviate this problem: setting a read-length threshold to filter out shorter reads; choosing an assembler less prone to retaining uncollapsed haplotypes; and post-processing the assembled set of contigs using a downstream tool to remove uncollapsed haplotypes. These three strategies are not mutually exclusive and, when combined, generate haploid assemblies with genome sizes, coverage distributions, and k-mer completeness matching expectations.

Until recently, obligate asexuality was often considered an evolutionary dead end. However, recent advances suggest that conventional sexual reproduction, defined as the alternation of meiosis and fertilization, is not the only sustainable eukaryotic lifestyle. Moreover, different modes of asexual reproduction are observed in nature, raising the question of the diverse mechanisms responsible for the long-term survival and adaptation of strict asexuals. One possible way to study the molecular-genetic consequences of the loss of meiotic recombination is to scrutinize the genomes of asexuals of ancient and more recent origins. The first genome draft of an ancient asexual species, the bdelloid rotifer Adineta vaga, was recently made available, revealing a peculiar genomic structure in which allelic regions were massively rearranged and sometimes found on the same chromosome. Such genome organization devoid of homologous chromosomes appears incompatible with meiotic pairing and segregation, and represents therefore a compelling genomic signature of asexuality. Besides, the genome of A. vaga contains around 8 % of genes of apparent nonmetazoan origin, a percentage much higher than observed in most eukaryotes. Interestingly, a similar percentage of genes of nonmetazoan origin was independently inferred from a large-scale transcriptome analysis of the bdelloid rotifer Adineta ricciae. In this chapter, we conducted a comparative study between these two closely related species using reciprocal best blast hits, followed by functional annotation using the GOANNA pipeline. Around 10 % of all the orthologs identified between the two species were putatively acquired by horizontal gene transfer and lots of them were associated to hydrolases (18 %) and oxidoreductases (16 %) functions. We hypothesize that these acquisitions may have helped bdelloids to adapt to multiple food sources and