Genome Sequencing Approaches Research Articles

Genome and transcriptome analysis of the beet armyworm Spodoptera exigua reveals targets for pest control.

The genus Spodoptera (Lepidoptera: Noctuidae) includes some of the most infamous insect pests of cultivated plants including Spodoptera frugiperda, Spodoptera litura, and Spodoptera exigua. To effectively develop targeted pest control strategies for diverse Spodoptera species, genomic resources are highly desired. To this aim, we provide the genome assembly and developmental transcriptome comprising all major life stages of S. exigua, the beet armyworm. Spodoptera exigua is a polyphagous herbivore that can feed on > 130 host plants, including several economically important crops. The 419 Mb beet armyworm genome was sequenced from a female S. exigua pupa. Using a hybrid genome sequencing approach (Nanopore long-read data and Illumina short read), a high-quality genome assembly was achieved (N50 = 1.1 Mb). An official gene set (18,477 transcripts) was generated by automatic annotation and by using transcriptomic RNA-seq datasets of 18 S. exigua samples as supporting evidence. In-depth analyses of developmental stage-specific expression combined with gene tree analyses of identified homologous genes across Lepidoptera genomes revealed four potential genes of interest (three of them Spodoptera-specific) upregulated during first- and third-instar larval stages for targeted pest-outbreak management. The beet armyworm genome sequence and developmental transcriptome covering all major developmental stages provide critical insights into the biology of this devastating polyphagous insect pest species worldwide. In addition, comparative genomic analyses across Lepidoptera significantly advance our knowledge to further control other invasive Spodoptera species and reveals potential lineage-specific target genes for pest control strategies.

Ophiostomatoid fungi associated with pine bark beetles and infested pines in south-eastern Australia, including Graphilbum ipis-grandicollis sp. nov.

The ophiostomatoid fungi are an assemblage of ascomycetes which are arguably best-known for their associations with bark and ambrosia beetles (Curculonidae) and blue stain (sap stain) of many economically important tree species. These fungi are considered a significant threat to coniferous forests, which has resulted in numerous studies characterising the diversity of bark beetles and their ophiostomatoid associates globally. The diversity of ophiostomatoid fungi present in Australian pine plantations, however, remains largely undetermined. The aims of this study were therefore to reconsider the diversity of ophiostomatoid fungi associated with Pinus in Australia, and to establish the baseline of expected taxa found within these plantation ecosystems. To achieve this, we reviewed Australian plant pathogen reference collections, and analysed samples collected during forest health surveillance programs from the major pine growing regions in south-eastern Australia. In total, 135 ophiostomatoid isolates (15 from reference collections and 120 collected during the current study) were assessed using morphological identification and ITS screening which putatively distinguished 15 taxonomic groups. Whole genome sequencing (WGS) of representative isolates from each taxon was performed to obtain high-quality sequence data for multi-locus phylogenetic analysis. Our results revealed a greater than expected diversity, expanding the status of ophiostomatoid fungi associated with Pinus in Australia to include 14 species from six genera in the Ophiostomatales and a single species residing in the Microascales. While most of these were already known to science, our study includes seven first records for Australia and the description of one new species, Graphilbum ipis-grandicollis sp. nov.. This study also provides an early example of whole genome sequencing (WGS) approaches replacing traditional PCR-based methods for taxonomic surveys. This not only allowed for robust multi-locus sequence extraction during taxonomic assessment, but also permitted the rapid establishment of a curated genomic database for ophiostomatoid fungi which will continue to aid in the development of improved diagnostic resources and capabilities for Australian biosecurity.

Genotypic analysis of the female BPH/5 mouse, a model of superimposed preeclampsia.

Animal models that recapitulate human diseases and disorders are widely used to investigate etiology, diagnosis, and treatment of those conditions in people. Disorders during pregnancy are particularly difficult to explore as interventions in pregnant women are not easily performed. Therefore, models that allow for pre-conception investigations are advantageous for elucidating the mechanisms involved in adverse pregnancy outcomes that are responsible for both maternal and fetal morbidity, such as preeclampsia. The Blood Pressure High (BPH)/5 mouse model has been used extensively to study the pathogenesis of preeclampsia. The female BPH/5 mouse is obese with increased adiposity and borderline hypertension, both of which are exacerbated with pregnancy making it a model of superimposed preeclampsia. Thus, the BPH/5 model shares traits with a large majority of women with pre-existing conditions that predisposes them to preeclampsia. We sought to explore the genome of the BPH/5 female mouse and determine the genetic underpinnings that may contribute to preeclampsia-associated phenotypes in this model. Using a whole genome sequencing approach, we are the first to characterize the genetic mutations in BPH/5 female mice that make it unique from the closely related BPH/2 model and the normotensive background strain, C57Bl/6. We found the BPH/5 female mouse to be uniquely different from BPH/2 and C57Bl/6 mice with a genetically complex landscape. The majority of non-synonymous consequences within the coding region of BPH/5 females were missense mutations found most abundant on chromosome X when comparing BPH/5 and BPH/2, and on chromosome 8 when comparing BPH/5 to C57Bl/6. Genetic mutations in BPH/5 females largely belong to immune system-related processes, with overlap between BPH/5 and BPH/2 models. Further studies examining each gene mutation during pregnancy are warranted to determine key contributors to the BPH/5 preeclamptic-like phenotype and to identify genetic similarities to women that develop preeclampsia.

Whole genome sequencing of two human rhinovirus A types (A101 and A15) detected in Kenya, 2016-2018

Background: Virus genome sequencing is increasingly utilized in epidemiological surveillance. Genomic data allows comprehensive evaluation of underlying viral diversity and epidemiology to inform control. For human rhinovirus (HRV), genomic amplification and sequencing is challenging due to numerous types, high genetic diversity and inadequate reference sequences. Methods: We developed a tiled amplicon type-specific protocol for genome amplification and sequencing on the Illumina MiSeq platform of two HRV types, A15 and A101. We then assessed added value in analyzing whole genomes relative to the VP4/2 region only in the investigation of HRV molecular epidemiology within the community in Kilifi, coastal Kenya. Results: We processed 73 samples collected between 2016-2018, and 48 yielded at least 70% HRV genome coverage. These included all A101 samples (n=10) and 38 (60.3%) A15 samples. Phylogenetic analysis revealed that the Kilifi A101 sequences interspersed with global A101 genomes available in GenBank collected between 1999-2016. On the other hand, our A15 sequences formed a monophyletic group separate from the global genomes collected in 2008 and 2019. Improved phylogenetic resolution was observed with the genome phylogenies compared to the VP4/2 phylogenies. Conclusions: We present a type-specific full genome sequencing approach for obtaining HRV genomic data and characterizing infections.

Candidate probiotic Lactiplantibacillus plantarum HNU082 rapidly and convergently evolves within human, mice, and zebrafish gut but differentially influences the resident microbiome

BackgroundImproving probiotic engraftment in the human gut requires a thorough understanding of the in vivo adaptive strategies of probiotics in diverse contexts. However, for most probiotic strains, these in vivo genetic processes are still poorly characterized. Here, we investigated the effects of gut selection pressures from human, mice, and zebrafish on the genetic stability of a candidate probiotic Lactiplantibacillus plantarum HNU082 (Lp082) as well as its ecological and evolutionary impacts on the indigenous gut microbiota using shotgun metagenomic sequencing in combination with isolate resequencing methods.ResultsWe combined both metagenomics and isolate whole genome sequencing approaches to systematically study the gut-adaptive evolution of probiotic L. plantarum and the ecological and evolutionary changes of resident gut microbiomes in response to probiotic ingestion in multiple host species. Independent of host model, Lp082 colonized and adapted to the gut by acquiring highly consistent single-nucleotide mutations, which primarily modulated carbohydrate utilization and acid tolerance. We cultivated the probiotic mutants and validated that these gut-adapted mutations were genetically stable for at least 3 months and improved their fitness in vitro. In turn, resident gut microbial strains, especially competing strains with Lp082 (e.g., Bacteroides spp. and Bifidobacterium spp.), actively responded to Lp082 engraftment by accumulating 10–70 times more evolutionary changes than usual. Human gut microbiota exhibited a higher ecological and genetic stability than that of mice.ConclusionsCollectively, our results suggest a highly convergent adaptation strategy of Lp082 across three different host environments. In contrast, the evolutionary changes within the resident gut microbes in response to Lp082 were more divergent and host-specific; however, these changes were not associated with any adverse outcomes. This work lays a theoretical foundation for leveraging animal models for ex vivo engineering of probiotics to improve engraftment outcomes in humans.1TYRt-mQpEqyYou2DA7bqRVideo abstract

Use of whole genome analysis to identify shared genomic variants across breeds in canine mitral valve disease.

Familial mitral valve prolapse in human beings has been associated with several genetic variants; however, in most cases, a known variant has not been identified. Dogs also have a naturally occurring form of familial mitral valve disease (MMVD) with similarities to the human disease. A shared genetic background and clinical phenotype of this disease in some dog breeds has indicated that the disease may share a common genetic cause. We evaluated DNA from 50 affected dogs from five different dog breeds in a whole genome sequencing approach to identify shared variants across and within breeds that could be associated with MMVD. No single causative genetic mutation was found from the 50 dogs with MMVD. Ten variants were identified in 37/50 dogs around and within the MED13L gene. These variants were no longer associated with MMVD when evaluated with a larger cohort including both affected and unaffected dogs. No high/moderate impact variants were identified in 10/10 miniature poodles, one was identified in 10/10 Yorkshire Terriers and 10/10 dachshunds, respectively, 14 were identified in 10/10 Miniature schnauzers, and 19 in 10/10 CKCS. Only one of these could be associated with the cardiac valve (Chr12:36801705, COL12A1; CKCS) but when evaluated in an additional 100 affected CKCS the variant was only identified in 84/100 affected dogs, perhaps indicating genetic heterogeneity in this disease. Our findings indicate that development of MMVD in the dog may be related to a combination of genetic and environmental factors that impact specific molecular pathways rather than a single shared genetic variant across or within breeds.

Therapeutic targeting of chromatin: status and opportunities.

The molecular characterization of mechanisms underlying transcriptional control and epigenetic inheritance since the 1990s has paved the way for the development of targeted therapies that modulate these pathways. In the past two decades, cancer genome sequencing approaches have uncovered a plethora of mutations in chromatin modifying enzymes across tumor types, and systematic genetic screens have identified many of these proteins as specific vulnerabilities in certain cancers. Now is the time when many of these basic and translational efforts start to bear fruit and more and more chromatin-targeting drugs are entering the clinic. At the same time, novel pharmacological approaches harbor the potential to modulate chromatin in unprecedented fashion, thus generating entirely novel opportunities. Here, we review the current status of chromatin targets in oncology and describe a vision for the epigenome-modulating drugs of the future.

Genome sequencing unveils a regulatory landscape of platelet reactivity

Platelet aggregation at the site of atherosclerotic vascular injury is the underlying pathophysiology of myocardial infarction and stroke. To build upon prior GWAS, here we report on 16 loci identified through a whole genome sequencing (WGS) approach in 3,855 NHLBI Trans-Omics for Precision Medicine (TOPMed) participants deeply phenotyped for platelet aggregation. We identify the RGS18 locus, which encodes a myeloerythroid lineage-specific regulator of G-protein signaling that co-localizes with expression quantitative trait loci (eQTL) signatures for RGS18 expression in platelets. Gene-based approaches implicate the SVEP1 gene, a known contributor of coronary artery disease risk. Sentinel variants at RGS18 and PEAR1 are associated with thrombosis risk and increased gastrointestinal bleeding risk, respectively. Our WGS findings add to previously identified GWAS loci, provide insights regarding the mechanism(s) by which genetics may influence cardiovascular disease risk, and underscore the importance of rare variant and regulatory approaches to identifying loci contributing to complex phenotypes.

SARS-CoV-2 Variants in Lebanon: Evolution and Current Situation.

Simple SummaryThanks to whole genome sequencing approaches, the proteins that compose the SARS-CoV-2 virus that causes COVID-19 are compared, and mutations and differences highlighted. This allows strains to be classified into clades or lineages, according to marker mutations in their proteins as well as relatedness to certain epidemiological events. In this study, 58 SARS-CoV-2 Lebanese strains were analyzed. They were classified into four GISAID clades and 11 Pango lineages. Moreover, the mutational survey revealed several mutations in the encoded proteins, particularly the structural ones, in which 22 uncommon mutations were found, 21 of which were in strains sequenced within this study. The latter also showed an interesting combination of mutations in their spike protein, the key element in the virus’ interaction with cellular receptors. In summary, this study highlights the key features of sequenced Lebanese SARS-CoV-2 genomes, including their classification, phylogenetic relationship, and mutations.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seen a worldwide spread since its emergence in 2019, including to Lebanon, where 534,968 confirmed cases (8% of the population) and 7569 deaths have been reported as of 14 May 2021. With the genome sequencing of strains from various countries, several classification systems were established via genome comparison. For instance, the GISAID clades classification highlights key mutations in the encoded proteins that could potentially affect the virus’ infectivity and transmission rates. In this study, 58 genomes of Lebanese SARS-CoV-2 strains were analyzed, 28 of which were sequenced for this study, and 30 retrieved from the GISAID and GenBank databases. We aimed to classify these strains, establish their phylogenetic relationships, and extract the mutations causing amino acid substitutions within, particularly, the structural proteins. The sequenced Lebanese SARS-COV-2 strains were classified into four GISAID clades and 11 Pango lineages. Moreover, 21 uncommon mutations in the structural proteins were found in the newly sequenced strains, underlining interesting combinations of mutations in the spike proteins. Hence, this study constitutes an observation and description of the current SARS-CoV-2 genetic and clade situation in Lebanon according to the available sequenced strains.

Advances in Cereal Crop Genomics for Resilience under Climate Change.

Adapting to climate change, providing sufficient human food and nutritional needs, and securing sufficient energy supplies will call for a radical transformation from the current conventional adaptation approaches to more broad-based and transformative alternatives. This entails diversifying the agricultural system and boosting productivity of major cereal crops through development of climate-resilient cultivars that can sustainably maintain higher yields under climate change conditions, expanding our focus to crop wild relatives, and better exploitation of underutilized crop species. This is facilitated by the recent developments in plant genomics, such as advances in genome sequencing, assembly, and annotation, as well as gene editing technologies, which have increased the availability of high-quality reference genomes for various model and non-model plant species. This has necessitated genomics-assisted breeding of crops, including underutilized species, consequently broadening genetic variation of the available germplasm; improving the discovery of novel alleles controlling important agronomic traits; and enhancing creation of new crop cultivars with improved tolerance to biotic and abiotic stresses and superior nutritive quality. Here, therefore, we summarize these recent developments in plant genomics and their application, with particular reference to cereal crops (including underutilized species). Particularly, we discuss genome sequencing approaches, quantitative trait loci (QTL) mapping and genome-wide association (GWAS) studies, directed mutagenesis, plant non-coding RNAs, precise gene editing technologies such as CRISPR-Cas9, and complementation of crop genotyping by crop phenotyping. We then conclude by providing an outlook that, as we step into the future, high-throughput phenotyping, pan-genomics, transposable elements analysis, and machine learning hold much promise for crop improvements related to climate resilience and nutritional superiority.

Familial thrombocytopenia: The long and short of it.

In this issue, Wahlster, Verboon, and colleagues (2021. J. Exp. Med.https://doi.org/10.1084/jem.20210444) describe a multigenerational family with inherited thrombocytopenia where the causal variant was not identified using conventional genome sequencing approaches. Long-read sequencing and RNA sequencing revealed a complex structural variant, causing overexpression of a pathogenic gain-of-function WAC-ANKRD26 fusion transcript.

A Nosocomial Outbreak of Invasive Listeriosis in An Italian Hospital: Epidemiological and Genomic Features.

Listeria monocytogenes (L. monocytogenes) is a widespread opportunistic pathogen that causes the listeriosis foodborne disease. This bacterium has become a common contaminant of handled food, and a relevant public health issue. Here we describe a nosocomial outbreak of listeriosis caused by an ST451 strain of L. monocytogenes involving three cancer and one immunocompromised patients hospitalized in different units from the same hospital during September and October 2020. The epidemiological investigation was conducted using traditional microbiological methodology combined with a whole genome sequencing approach. The source of contamination was identified in the kitchen hospital, where a meat slicer used to prepare patients’ meals was tested positive to the same sequence type (ST) of L. monocytogenes. This is the first report of an outbreak of listeriosis caused by ST451 in Italy.

Familial thrombocytopenia due to a complex structural variant resulting in a WAC-ANKRD26 fusion transcript.

Advances in genome sequencing have resulted in the identification of the causes for numerous rare diseases. However, many cases remain unsolved with standard molecular analyses. We describe a family presenting with a phenotype resembling inherited thrombocytopenia 2 (THC2). THC2 is generally caused by single nucleotide variants that prevent silencing of ANKRD26 expression during hematopoietic differentiation. Short-read whole-exome and genome sequencing approaches were unable to identify a causal variant in this family. Using long-read whole-genome sequencing, a large complex structural variant involving a paired-duplication inversion was identified. Through functional studies, we show that this structural variant results in a pathogenic gain-of-function WAC-ANKRD26 fusion transcript. Our findings illustrate how complex structural variants that may be missed by conventional genome sequencing approaches can cause human disease.

Targeted Genome Sequencing (TG-Seq) Approaches to Detect Plant Viruses.

Globally, high-throughput sequencing (HTS) has been used for virus detection in germplasm certification programs. However, sequencing costs have impeded its implementation as a routine diagnostic certification tool. In this study, the targeted genome sequencing (TG-Seq) approach was developed to simultaneously detect multiple (four) viral species of; Pea early browning virus (PEBV), Cucumber mosaic virus (CMV), Bean yellow mosaic virus (BYMV) and Pea seedborne mosaic virus (PSbMV). TG-Seq detected all the expected viral amplicons within multiplex PCR (mPCR) reactions. In contrast, the expected PCR amplicons were not detected by gel electrophoresis (GE). For example, for CMV, GE only detected RNA1 and RNA2 while TG-Seq detected all the three RNA components of CMV. In an mPCR to amplify all four viruses, TG-Seq readily detected each virus with more than 732,277 sequence reads mapping to each amplicon. In addition, TG-Seq also detected all four amplicons within a 10−8 serial dilution that were not detectable by GE. Our current findings reveal that the TG-Seq approach offers significant potential and is a highly sensitive targeted approach for detecting multiple plant viruses within a given biological sample. This is the first study describing direct HTS of plant virus mPCR products. These findings have major implications for grain germplasm healthy certification programs and biosecurity management in relation to pathogen entry into Australia and elsewhere.

Diversity and composition of gut microbiome of cervical cancer patients: Do results of 16S rRNA sequencing and whole genome sequencing approaches align?

BackgroundNext generation sequencing has progressed rapidly, characterizing microbial communities beyond culture-based or biochemical techniques. 16S ribosomal RNA gene sequencing (16S) produces reliable taxonomic classifications and relative abundances, while shotgun metagenome sequencing (WMS) allows higher taxonomic and functional resolution at greater cost. The purpose of this study was to determine if 16S and WMS provide congruent information for our patient population from paired fecal microbiome samples. ResultsComparative indices were highly congruent between 16S and WMS. The most abundant genera for 16S and WMS data did not overlap. Overlap was observed at the Phylum level, as expected. However, relative abundances correlated poorly between the two methodologies (all P-value>0.05). Hierarchical clustering of both sequencing analyses identified overlapping enterotypes. Both approaches were in agreement with regard to demographic variables. ConclusionDiversity, evenness and richness are comparable when using 16S and WMS techniques, however relative abundances of individual genera are not. Clinical associations with diversity and evenness metrics were similarly identified with WMS or 16S.

Profile of Small RNAs, vDNA Forms and Viral Integrations in Late Chikungunya Virus Infection of Aedes albopictus Mosquitoes.

The Asian tiger mosquito Aedes albopictus is contributing to the (re)-emergence of Chikungunya virus (CHIKV). To gain insights into the molecular underpinning of viral persistence, which renders a mosquito a life-long vector, we coupled small RNA and whole genome sequencing approaches on carcasses and ovaries of mosquitoes sampled 14 days post CHIKV infection and investigated the profile of small RNAs and the presence of vDNA fragments. Since Aedes genomes harbor nonretroviral Endogenous Viral Elements (nrEVEs) which confers tolerance to cognate viral infections in ovaries, we also tested whether nrEVEs are formed after CHIKV infection. We show that while small interfering (si)RNAs are evenly distributed along the full viral genome, PIWI-interacting (pi)RNAs mostly arise from a ~1000 bp window, from which a unique vDNA fragment is identified. CHIKV infection does not result in the formation of new nrEVEs, but piRNAs derived from existing nrEVEs correlate with differential expression of an endogenous transcript. These results demonstrate that all three RNAi pathways contribute to the homeostasis during the late stage of CHIKV infection, but in different ways, ranging from directly targeting the viral sequence to regulating the expression of mosquito transcripts and expand the role of nrEVEs beyond immunity against cognate viruses.

Whole genome sequencing in the Middle Eastern Qatari population identifies genetic associations with 45 clinically relevant traits

Clinical laboratory tests play a pivotal role in medical decision making, but little is known about their genetic variability between populations. We report a genome-wide association study with 45 clinically relevant traits from the population of Qatar using a whole genome sequencing approach in a discovery set of 6218 individuals and replication in 7768 subjects. Trait heritability is more similar between Qatari and European populations (r = 0.81) than with Africans (r = 0.44). We identify 281 distinct variant-trait-associations at genome wide significance that replicate known associations. Allele frequencies for replicated loci show higher correlations with European (r = 0.94) than with African (r = 0.85) or Japanese (r = 0.80) populations. We find differences in linkage disequilibrium patterns and in effect sizes of the replicated loci compared to previous reports. We also report 17 novel and Qatari-predominate signals providing insights into the biological pathways regulating these traits. We observe that European-derived polygenic scores (PGS) have reduced predictive performance in the Qatari population which could have implications for the translation of PGS between populations and their future application in precision medicine.

Diverging patterns of introgression from Schistosoma bovis across S. haematobium African lineages

Hybridization is a fascinating evolutionary phenomenon that raises the question of how species maintain their integrity. Inter-species hybridization occurs between certain Schistosoma species that can cause important public health and veterinary issues. In particular hybrids between Schistosoma haematobium and S. bovis associated with humans and animals respectively are frequently identified in Africa. Recent genomic evidence indicates that some S. haematobium populations show signatures of genomic introgression from S. bovis. Here, we conducted a genomic comparative study and investigated the genomic relationships between S. haematobium, S. bovis and their hybrids using 19 isolates originating from a wide geographical range over Africa, including samples initially classified as S. haematobium (n = 11), S. bovis (n = 6) and S. haematobium x S. bovis hybrids (n = 2). Based on a whole genomic sequencing approach, we developed 56,181 SNPs that allowed a clear differentiation of S. bovis isolates from a genomic cluster including all S. haematobium isolates and a natural S. haematobium-bovis hybrid. All the isolates from the S. haematobium cluster except the isolate from Madagascar harbored signatures of genomic introgression from S. bovis. Isolates from Corsica, Mali and Egypt harbored the S. bovis-like Invadolysin gene, an introgressed tract that has been previously detected in some introgressed S. haematobium populations from Niger. Together our results highlight the fact that introgression from S. bovis is widespread across S. haematobium and that the observed introgression is unidirectional.

Molecular Epidemiology of Measles in California, United States—2019

In 2019, the United States (US) experienced the highest number of measles importations and cases in the postelimination era. More than a quarter of imported cases entered the US through California. Measles surveillance efforts in California resulted in the identification of 26 importations, 6 outbreaks, and 72 cases in 2019. Only genotype B3 and D8 measles strains were detected. Genotype-specific differences were noted in the incidence of vaccine failures, hospitalizations, and severe complications among cases. A targeted whole genome sequencing approach provided higher-resolution discrimination between epidemiologically linked and sporadically introduced strains than conventional N450 sequencing. Our report underscores the importance of ensuring appropriate measles vaccination status, especially prior to international travel to measles-endemic regions, and highlights the value of a strong measles surveillance system in minimizing outbreaks and preserving measles elimination status in the US.

A novel canis lupus familiaris reference genome improves variant resolution for use in breed-specific GWAS.

Reference genome fidelity is critically important for genome wide association studies, yet most vary widely from the study population. A typical whole genome sequencing approach implies short-read technologies resulting in fragmented assemblies with regions of ambiguity. Further information is lost by economic necessity when genotyping populations, as lower resolution technologies such as genotyping arrays are commonly used. Here, we present a phased reference genome for Canis lupus familiaris using high molecular weight DNA-sequencing technologies. We tested wet laboratory and bioinformatic approaches to demonstrate a minimum workflow to generate the 2.4 gigabase genome for a Labrador Retriever. The de novo assembly required eight Oxford Nanopore R9.4 flowcells (∼23X depth) and running a 10X Genomics library on the equivalent of one lane of an Illumina NovaSeq S1 flowcell (∼88X depth), bringing the cost of generating a nearly complete reference genome to less than $10K (USD). Mapping of short-read data from 10 Labrador Retrievers against this reference resulted in 1% more aligned reads versus the current reference (CanFam3.1, P < 0.001), and a 15% reduction of variant calls, increasing the chance of identifying true, low-effect size variants in a genome-wide association studies. We believe that by incorporating the cost to produce a full genome assembly into any large-scale genotyping project, an investigator can improve study power, decrease costs, and optimize the overall scientific value of their study.