Semi-Supervised Pipeline for Autonomous Annotation of SARS-CoV-2 Genomes

SARS-CoV-2 genomic sequencing efforts have scaled dramatically to address the current global pandemic and aid public health. However, autonomous genome annotation of SARS-CoV-2 genes, proteins, and domains is not readily accomplished by existing methods and results in missing or incorrect sequences. To overcome this limitation, we developed a novel semi-supervised pipeline for automated gene, protein, and functional domain annotation of SARS-CoV-2 genomes that differentiates itself by not relying on the use of a single reference genome and by overcoming atypical genomic traits that challenge traditional bioinformatic methods. We analyzed an initial corpus of 66,000 SARS-CoV-2 genome sequences collected from labs across the world using our method and identified the comprehensive set of known proteins with 98.5% set membership accuracy and 99.1% accuracy in length prediction, compared to proteome references, including Replicase polyprotein 1ab (with its transcriptional slippage site). Compared to other published tools, such as Prokka (base) and VAPiD, we yielded a 6.4- and 1.8-fold increase in protein annotations. Our method generated 13,000,000 gene, protein, and domain sequences—some conserved across time and geography and others representing emerging variants. We observed 3362 non-redundant sequences per protein on average within this corpus and described key D614G and N501Y variants spatiotemporally in the initial genome corpus. For spike glycoprotein domains, we achieved greater than 97.9% sequence identity to references and characterized receptor binding domain variants. We further demonstrated the robustness and extensibility of our method on an additional 4000 variant diverse genomes containing all named variants of concern and interest as of August 2021. In this cohort, we successfully identified all keystone spike glycoprotein mutations in our predicted protein sequences with greater than 99% accuracy as well as demonstrating high accuracy of the protein and domain annotations. This work comprehensively presents the molecular targets to refine biomedical interventions for SARS-CoV-2 with a scalable, high-accuracy method to analyze newly sequenced infections as they arise.

Tetraploidy accelerates adaption under drug-selection in a fungal pathogen

Baseline ploidy significantly impacts evolutionary trajectories, and in particular, tetraploidy has been associated with higher rates of adaptation compared to haploidy and diploidy. While the majority of experimental evolution studies investigating ploidy use Saccharomyces cerivisiae, the fungal pathogen Candida albicans is a powerful system to investigate ploidy dynamics, particularly in the context of antifungal drug resistance. C. albicans laboratory and clinical strains are predominantly diploid, but have also been isolated as haploid and polyploid. Here, we evolved diploid and tetraploid C. albicans for ∼60 days in the antifungal drug caspofungin. Tetraploid-evolved lines adapted faster than diploid-evolved lines and reached higher levels of caspofungin resistance. While diploid-evolved lines generally maintained their initial genome size, tetraploid-evolved lines rapidly underwent genome-size reductions and did so prior to caspofungin adaption. Furthermore, fitness costs in the absence of drug selection were significantly less in tetraploid-evolved lines compared to the diploid-evolved lines. Taken together, this work supports a model of adaptation in which the tetraploid state is transient but its ability to rapidly transition ploidy states improves adaptative outcomes and may drive drug resistance in fungal pathogens.

Genetic Predictors of Lithium Response

Lithium remains a first-line pharmacological treatment of bipolar disorder (BD). However, treatment response is heterogeneous, with several lines of evidence implicating genetic factors. Unfortunately, neither hypothesis-driven approaches nor initial genome-wide association studies (GWAS) were successful in identifying genetic drivers of response heterogeneity, probably due to low statistical power and different phenotype measurements. Recently, a GWAS of the Consortium of Lithium Genetics (ConLiGen) has identified four single nucleotide polymorphisms (SNPs) mediating response to lithium, located in genes for two long non-coding RNAs. This success was only possible by international collaboration and the use of an established lithium response scale. The findings await further replication.

Adaptive evolution of genomically recodedEscherichia coli

Efforts are underway to construct several recoded genomes anticipated to exhibit multivirus resistance, enhanced nonstandard amino acid (nsAA) incorporation, and capability for synthetic biocontainment. Although our laboratory pioneered the first genomically recoded organism (Escherichia colistrain C321.∆A), its fitness is far lower than that of its nonrecoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for nsAA-linked applications requiring defined media, such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.∆A for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceeded the growth rates of both the ancestral C321.∆A and nonrecoded strains. We used next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstructed individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occurred only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared with application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.∆A strain.

A very recent whole genome duplication in Potamopyrgus antipodarum predates multiple origins of asexuality & associated polyploidy

Potamopyrgus antipodarum, a New Zealand freshwater snail, is a powerful system to study the maintenance of sexual reproduction. Obligate asexual P. antipodarum (herein, Pa) lineages include both triploids and tetraploids that are products of multiple separate transitions from diploid sexual ancestors. Distinct diploid sexual and polyploid asexual lineages coexist and compete; these separate lineages can be considered replicated natural experiments. We have shown that harmful mutations are accumulating at a higher rate in asexual than in sexual Pa, demonstrating the utility of this system as a model for investigating the evolution of sex at the genomic level. In order to better understand the causes and consequences of transitions to asexuality, we have sequenced multiple genomes and transcriptomes of Pa and a close relative, P. estuarinus (herein, Pe) a diploid sexual species. The diploid genome size of Pe is ~0.6X of the genome size of diploid Pa, inspiring us to investigate whether the most recent common ancestor of Pa had experienced a whole-genome duplication (WGD) event prior to the diversification of its many sexual and asexual lineages. In addition to its clear relevance to understanding the evolutionary history of this species, by being so recent, this apparent WGD will also be especially powerful in understanding events immediately following WGD. Our initial genome assembly of a model sexual Pa lineage was consistent with this possibility, indicating high fractions (~35%) of scaffolds containing extended, nearly identical, duplicated regions. This result also partly explains our general difficulty with assembling the genome, despite generating >100X genome coverage using multiple methodologies. Even considering the limitations of our current genome assembly, we used the assembly to test a series of predictions under the hypothesis of recent whole-genome duplication, all of which are consistent with WGD. These tests have shown: 1) a marked excess of duplicated copies of genes in Pa which are maintained in single copy in other animals, 2) implausibly high "heterozygosity" estimates in our model Pa sexual genome, presumably resulting from non-allelic comparisons, 3) higher sequence identity between thousands of Pa-specific paralogous genes, when compared to their Pe orthologs. These and additional lines of evidence will be presented and evaluated. Together, our results suggest that this initial genome-wide duplication event might have played a key role in the subsequent evolutionary trajectory of this species, potentially facilitating its repeated diversification into multiple asexual lineages. We are now generating additional long-range genome scaffolds for Pa using multiple methods, as well as improving the coverage and quality of the Pe genome. We will use these new data to conduct definitive phylogenomic tests of this especially remarkable whole genome duplication.

A very recent whole genome duplication in Potamopyrgus antipodarum predates multiple origins of asexuality & associated polyploidy

Potamopyrgus antipodarum, a New Zealand freshwater snail, is a powerful system to study the maintenance of sexual reproduction. Obligate asexual P. antipodarum (herein, Pa) lineages include both triploids and tetraploids that are products of multiple separate transitions from diploid sexual ancestors. Distinct diploid sexual and polyploid asexual lineages coexist and compete; these separate lineages can be considered replicated natural experiments. We have shown that harmful mutations are accumulating at a higher rate in asexual than in sexual Pa, demonstrating the utility of this system as a model for investigating the evolution of sex at the genomic level. In order to better understand the causes and consequences of transitions to asexuality, we have sequenced multiple genomes and transcriptomes of Pa and a close relative, P. estuarinus (herein, Pe) a diploid sexual species. The diploid genome size of Pe is ~0.6X of the genome size of diploid Pa, inspiring us to investigate whether the most recent common ancestor of Pa had experienced a whole-genome duplication (WGD) event prior to the diversification of its many sexual and asexual lineages. In addition to its clear relevance to understanding the evolutionary history of this species, by being so recent, this apparent WGD will also be especially powerful in understanding events immediately following WGD. Our initial genome assembly of a model sexual Pa lineage was consistent with this possibility, indicating high fractions (~35%) of scaffolds containing extended, nearly identical, duplicated regions. This result also partly explains our general difficulty with assembling the genome, despite generating >100X genome coverage using multiple methodologies. Even considering the limitations of our current genome assembly, we used the assembly to test a series of predictions under the hypothesis of recent whole-genome duplication, all of which are consistent with WGD. These tests have shown: 1) a marked excess of duplicated copies of genes in Pa which are maintained in single copy in other animals, 2) implausibly high "heterozygosity" estimates in our model Pa sexual genome, presumably resulting from non-allelic comparisons, 3) higher sequence identity between thousands of Pa-specific paralogous genes, when compared to their Pe orthologs. These and additional lines of evidence will be presented and evaluated. Together, our results suggest that this initial genome-wide duplication event might have played a key role in the subsequent evolutionary trajectory of this species, potentially facilitating its repeated diversification into multiple asexual lineages. We are now generating additional long-range genome scaffolds for Pa using multiple methods, as well as improving the coverage and quality of the Pe genome. We will use these new data to conduct definitive phylogenomic tests of this especially remarkable whole genome duplication.

Initial genome sequencing of the sugarcane CP 96-1252 complex hybrid

The CP 96-1252 cultivar of sugarcane is a complex hybrid of commercial importance. DNA was extracted from lab-grown leaf tissue and sequenced. The raw Illumina DNA sequencing results provide 101 Gbp of genome sequence reads. The dataset is available from https://www.ncbi.nlm.nih.gov/bioproject/PRJNA345486/.