Reference Genome Assembly Research Articles

Guidelines for Gene and Genome Assembly Nomenclature

Abstract The rapid increase in the number of reference-quality genome assemblies presents significant new opportunities for genomic research. However, the absence of standardized naming conventions for genome assemblies and annotations across datasets creates substantial challenges. Inconsistent naming hinders the identification of correct assemblies, complicates the integration of bioinformatics pipelines, and makes it difficult to link assemblies across multiple resources. To address this, we developed a specification for standardizing the naming of reference genome assemblies, to improve consistency across datasets and facilitate interoperability. This specification was created with FAIR (Findable, Accessible, Interoperable, and Reusable) practices in mind, ensuring that reference assemblies are easier to locate, access, and reuse across research communities. Additionally, it has been designed to comply with primary genomic data repositories, including members of the International Nucleotide Sequence Database Collaboration (INSDC) consortium, ensuring compatibility with widely used databases. While initially tailored to the agricultural genomics community, the specification is adaptable for use across different taxa. Widespread adoption of this standardized nomenclature would streamline assembly management, better enable cross-species analyses, and improve the reproducibility of research. It would also enhance natural language processing applications that depend on consistent reference assembly names in genomic literature, promoting greater integration and automated analysis of genomic data. This is a good time to consider more consistent genomic data nomenclature as many research communities and data resources are now finding themselves juggling multiple datasets from multiple data providers.

Telomere-to-telomere sheep genome assembly identifies variants associated with wool fineness.

Ongoing efforts to improve sheep reference genome assemblies still leave many gaps and incomplete regions, resulting in a few common failures and errors in genomic studies. Here, we report a 2.85-Gb gap-free telomere-to-telomere genome of a ram (T2T-sheep1.0), including all autosomes and the X and Y chromosomes. This genome adds 220.05 Mb of previously unresolved regions and 754 new genes to the most updated reference assembly ARS-UI_Ramb_v3.0; it contains four types of repeat units (SatI, SatII, SatIII and CenY) in centromeric regions. T2T-sheep1.0 has a base accuracy of more than 99.999%, corrects several structural errors in previous reference assemblies and improves structural variant detection in repetitive sequences. Alignment of whole-genome short-read sequences of global domestic and wild sheep against T2T-sheep1.0 identifies 2,664,979 new single-nucleotide polymorphisms in previously unresolved regions, which improves the population genetic analyses and detection of selective signals for domestication (for example, ABCC4) and wool fineness (for example, FOXQ1).

A chromosome-scale reference genome assembly for Triplophysa lixianensis

In this study, we constructed a chromosome-scale reference genome assembly for Lixian plateau loach, Triplophysa lixianensis, by integration of MGI short-read, PacBio HiFi long-read and Hi-C sequencing technologies. A 668-Mb haplotypic genome assembly was obtained for a female T. lixianensis, and 98.91% of the assembled sequences were anchored into 25 chromosomes. This assembly owned a moderate repeat content (35.63%) and an annotation of 23,774 protein-coding genes, among them 94.15% were predicted with functions. The assembled genome of T. lixianensis shared a good syntenic relationship with previously published data of its relative T. dalaica. Taken together, our genome data presented here provide a valuable genetic resource for in-depth evolutionary and functional studies, as well as molecular breeding and conservation of this valuable fish species to elevate its ecological and economical values.

Highlighting the Importance of Correct Sex Identification in Chondrichthyan Genomic Studies, Using the White Shark as an Example

The increased availability of reference genome assemblies of sharks and rays has contributed greatly to our understanding of their biology, including their sex-determination mechanisms. However, several publicly available genome assemblies of sharks and rays appear to be missing information about the sex of the source individuals. This can confound the investigation into genetic sex-determining elements and hinder the discovery of sex-specific patterns. Herein, we highlight the importance of clear and accurate sex identification in sharks and rays for future genome assemblies, using an example of a white shark (Carcharodon carcharias) genome, in which the phenotypically assigned sex conflicts with the genetic information. This genome assembly was reported to be sourced from a juvenile female (BioSample: SAMN01915239). We analyzed the assembly by mapping its available genome sequences to the current white shark reference genome assembly and compared the read coverage to sequences collected from other samples. Evidence suggests that this specimen is genetically male, which contradicts its assignment based on phenotype. Therefore, we urge researchers to provide as much accurate information (e.g., sex, sampling localities, and life history) as possible when publishing genome assemblies for sharks and rays (or for any other organism).

Jan and mini-Jan, a model system for potato functional genomics.

Potato ( Solanum tuberosum ) is the third most important food crop in the world. Although the potato genome has been fully sequenced, functional genomics research of potato lags relative to other major food crops due primarily to the lack of a model experimental potato line. Here, we present a diploid potato line, 'Jan', which possesses all essential characteristics for facile functional genomics studies. Jan has a high level of homozygosity after seven generations of self-pollination. Jan is vigorous and highly fertile with outstanding tuber traits, high regeneration rates, and excellent transformation efficiencies. We generated a chromosome-scale genome assembly for Jan, annotated genes, and identified syntelogs relative to the potato reference genome assembly DMv6.1 to facilitate functional genomics. To miniaturize plant architecture, we developed two "mini-Jan" lines with compact and dwarf plant stature using CRISPR/Cas9-mediated mutagenesis targeting the Dwarf and Erecta genes related to growth. Mini-Jan mutants are fully fertile and will permit higher-throughput studies in limited growth chamber and greenhouse space. Thus, Jan and mini-Jan provide an outstanding model system that can be leveraged for gene editing and functional genomics research in potato.

A high-quality chromosome-level genome assembly of the traditional Chinese medicinal herb Zanthoxylum nitidum

Dried roots of Zanthoxylum nitidum (2n=2x=70, family Rutaceae) was referred as “Liang-Mian-Zhen” in Chinese, acting as a valuable species due to its notable pharmacological activities. Herein, we combined PacBio HiFi data together with Hi-C mapping technology to construct a chromosome-scale reference genome assembly for Z. nitidum. The assembly reached a length of 2.24 Gb, successfully anchoring 99.31% of sequences onto 35 pseudo-chromosomes. Among these, 26 chromosomes achieved telomere-to-telomere assembly, and 11 chromosomes were gap-free. The contigs N50 and scaffolds N50 reached 57.61 Mb and 78.00 Mb, respectively. Transposable elements comprised 81.44% of the Z. nitidum genome, and over 78% of them were long-terminal repeat retrotransposon elements. Furthermore, 32,737 protein-coding genes were identified and 99.38% of all were functionally annotated. The completeness of the genome assembly and final gene sets reached 97.83% and 96.47% based on Benchmarking Universal Single-Copy Orthologs (BUSCO), respectively. Taken together, our results provided a high-quality chromosome-level assembly of Z. nitidum genome and will be a valuable resource that will facilitate breeding varieties with higher alkaloids content.

Masu salmon species complex relationships and sex chromosomes revealed from analyses of the masu salmon (Oncorhynchus masou masou) genome assembly.

Masu salmon (Oncorhynchus masou) are the only Pacific salmon endemic to Asia. Some researchers prefer to categorize these salmon into four subspecies (masu - O. m. masou, amago - O. m. ishikawae, Biwa - O. m. subsp., and Formosan - O. m. formosanus), while others prefer individual species designations. Even though the masu salmon fishery is thousands of years old, classification of the diversity within the masu salmon species complex remains elusive. In this study, a genetic map and reference genome assembly were generated for one species/subspecies (masu) to provide resources for understanding the species complex. In O. m. masou, the sex chromosome was determined to be chromosome 7. Resequenced genomes from two other putative subspecies (amago and Biwa) provided evidence that they do not share the same sex chromosome. Principal component and admixture analyses clustered the amago and Biwa salmon close together. This supported previous findings of a close relationship between amago and Biwa salmon and a more distant relationship to masu salmon for both. Additional analyses of the masu salmon species complex will benefit from using the new reference genome assembly.

A chromosome-level reference genome for the common bed bug, Cimex lectularius, with identification of sex chromosomes.

The common bed bug, Cimex lectularius, is a globally distributed pest insect of medical, veterinary, and economic importance. Previous reference genome assemblies for this species were generated from short read sequencing data, resulting in a ~650Mb composed of thousands of contigs. Here, we present a haplotype-resolved, chromosome-level reference genome, generated from an adult Harlen strain female specimen. Using PacBio long read and Omni-C proximity sequencing, we generated a 540Mb genome with 15 chromosomes (13 autosomes and 2 sex chromosomes - X1X2) with an N50 > 30Mb and BUSCO > 90%. Previous karyotyping efforts indicate an XY sex chromosome system, with 2n=26 and X1X1X2X2 females and X1X2Y males; however significant fragmentation of the X chromosome has also been reported. We further use whole genome resequencing data from males and females to identify the X1 and X2 chromosomes based on sex biases in coverage. This highly contiguous reference genome assembly provides a much-improved resource for identifying chromosomal genome architecture, and for interpreting patterns of urban outbreaks and signatures of selection linked to insecticide resistance.

Extensive longevity and DNA virus-driven adaptation in nearctic Myotis bats.

The genus Myotis is one of the largest clades of bats, and exhibits some of the most extreme variation in lifespans among mammals alongside unique adaptations to viral tolerance and immune defense. To study the evolution of longevity-associated traits and infectious disease, we generated near-complete genome assemblies and cell lines for 8 closely related species of Myotis. Using genome-wide screens of positive selection, analyses of structural variation, and functional experiments in primary cell lines, we identify new patterns of adaptation contributing to longevity, cancer resistance, and viral interactions in bats. We find that Myotis bats have some of the most significant variation in cancer risk across mammals and demonstrate a unique DNA damage response in primary cells of the long-lived M. lucifugus. We also find evidence of abundant adaptation in response to DNA viruses - but not RNA viruses - in Myotis and other bats in sharp contrast with other mammals, potentially contributing to the role of bats as reservoirs of zoonoses. Together, our results demonstrate how genomics and primary cells derived from diverse taxa uncover the molecular bases of extreme adaptations in non-model organisms.

Genetic advancements and future directions in ruminant livestock breeding: from reference genomes to multiomics innovations.

Ruminant livestock provide a rich source of products, such as meat, milk, and wool, and play a critical role in global food security and nutrition. Over the past few decades, genomic studies of ruminant livestock have provided valuable insights into their domestication and the genetic basis of economically important traits, facilitating the breeding of elite varieties. In this review, we summarize the main advancements for domestic ruminants in reference genome assemblies, population genomics, and the identification of functional genes or variants for phenotypic traits. These traits include meat and carcass quality, reproduction, milk production, feed efficiency, wool and cashmere yield, horn development, tail type, coat color, environmental adaptation, and disease resistance. Functional genomic research is entering a new era with the advancements of graphical pangenomics and telomere-to-telomere (T2T) gap-free genome assembly. These advancements promise to improve our understanding of domestication and the molecular mechanisms underlying economically important traits in ruminant livestock. Finally, we provide new perspectives and future directions for genomic research on ruminant genomes. We suggest how ever-increasing multiomics datasets will facilitate future studies and molecular breeding in livestock, including the potential to uncover novel genetic mechanisms underlying phenotypic traits, to enable more accurate genomic prediction models, and to accelerate genetic improvement programs.

Tailoring Genomic Selection for Bos taurus indicus: A Comprehensive Review of SNP Arrays and Reference Genomes.

Advances in SNP arrays and reference genome assemblies have significantly transformed cattle genomics, particularly for Bos taurus indicus (Zebu cattle). Many commercial SNP arrays were originally designed for Bos taurus taurus, leading to ascertainment bias and the exclusion of crucial SNPs specific to Zebu populations. This review assesses progress in SNP array and reference genome development, with a focus on efforts tailored to Zebu populations and their impact on genomic selection and breeding efficiency. We reviewed the relevant literature on the development of SNP arrays, reference genome assemblies, and SNP genotyping techniques used for Zebu cattle. Emphasis was placed on SNP arrays specifically designed for Zebu breeds, evaluating their contributions to genomic evaluations and identifying limitations in prediction accuracy. Recent advancements, such as GeneSeek's low- and high-density SNP panels, have aimed to reduce ascertainment bias and include key SNPs for Zebu populations by providing breed-specific panels. These panels have been instrumental in identifying genomic regions associated with economically important traits in Nellore cattle. Studies show that tailored SNP arrays and breed-specific reference genomes can enhance genetic diversity assessment and improve genomic predictions, supporting more effective breeding programs for Zebu cattle. Improved SNP arrays and breed-specific reference genomes are crucial for accurate genomic selection in Zebu cattle. Future efforts should prioritize expanding de novo genome assemblies, reducing ascertainment bias, and developing cost-effective genotyping solutions tailored to Zebu populations. Targeted genomic tools will ultimately enable more efficient breeding practices and enhance genomic selection for economically important traits in B. t. indicus cattle.

The rice genome annotation project: an updated database for mining the rice genome.

Rice (Oryza sativa L.) is a major cereal crop that provides calories across the world. With a small genome, rice has been used extensively as a model for genetic and genomic studies in the Poaceae. Since the release of the first rice genome sequence in 2002, an improved reference genome assembly, multiple whole genome assemblies, extensive gene expression profiles, and resequencing data from over 3000 rice accessions have been generated. To facilitate access to the rice genome for plant biologists, we updated the Rice Genome Annotation Project database (RGAP; https://rice.uga.edu) with new datasets including 16 whole genome rice assemblies and sequence variants generated from multiple rice pan-genome projects including the 3000 Rice Genomes Project. We updated gene expression abundance data with 80 RNA-sequencing datasets and to facilitate gene function discovery, performed gene coexpression resulting in 39 coexpression modules that capture highly connected sets of co-regulated genes. To facilitate comparative genome analyses, 32335 syntelogs were identified between the Nipponbare reference genome and other rice genomes and 19371 syntelogs were identified between Nipponbare and four other Poaceae genomes. Infrastructure improvements to the RGAP database include an upgraded genome browser and data access portals, enhanced website securityand increased performance of the website.

A high-quality chromosome-level reference genome assembly of Tibetan antelope (Pantholops hodgsonii)

Tibetan antelope (Pantholops hodgsonii), a wild ruminant endemic to the Qinghai-Tibetan Plateau (QTP) in China, has evolved a series of genetic and physiological adaptation strategies to thrive in the harsh plateau environments. However, limited research on the genome of this species exists. Here, we established a high-quality chromosome-level reference genome assembly of the Tibetan antelope using PacBio HiFi, DNBSEQ, and Hi-C sequencing data. The assembly, totaling 3.13 GB, consists of 31 chromosomes (29 + X + partial Y), with a Scaffold N50 length of 92.23 Mb. The quality value (QV) and Benchmarking Universal Single-Copy Ortholog (BUSCO) score were 70.14 and 98.20%, respectively, indicating that our genome sequence is of high quality and completeness. Our genome not only contribute to the genetic conservation of Tibetan antelope but also provides a valuable resource for genetic, ecological, and evolutionary research within the sub-family Caprinae.

Construction and evaluation of a new rat reference genome assembly, GRCr8, from long reads and long-range scaffolding.

We report the construction and analysis of a new reference genome assembly for Rattus norvegicus, the laboratory rat, a widely used experimental animal model organism. The assembly has been adopted as the rat reference assembly by the Genome Reference Consortium and is named GRCr8. The assembly has employed 40× Pacific Biosciences (PacBio) HiFi sequencing coverage and scaffolding using optical mapping and Hi-C. We used genomic DNA from a male BN/NHsdMcwi (BN) rat of the same strain and from the same colony as the prior reference assembly, mRatBN7.2. The assembly is at chromosome level with 98.7% of the sequence assigned to chromosomes. All chromosomes have increased in size compared with the prior assembly and k-mer analysis indicates that the subject animal is fully inbred and that the genome is represented as a single haploid assembly. Notable increases are observed in Chromosomes 3, 11, and 12 in the prospective rDNA regions. In addition, Chr Y has increased threefold in size and is more consistent with the rat karyotype than previous assemblies. Several other chromosomes have grown by the incorporation of sizable discrete new blocks. These contain highly repetitive sequences and encode numerous previously unannotated genes. In addition, centromeric sequences are incorporated in most chromosomes. Genome annotation has been performed by NCBI RefSeq, which confirms improvement in assembly quality and adds more than 1100 new protein coding genes. PacBio Iso-Seq data have been acquired from multiple tissues of the subject animal and are released concurrently with the new assembly to aid further analyses.

The Benefits of Whole-Exome Sequencing in the Differential Diagnosis of Hypophosphatasia.

Hypophosphatasia (HPP) is a rare inherited disorder characterized by the decreased activity of tissue-nonspecific alkaline phosphatase (TNSALP), caused by mutations in the ALPL gene. The aim of this study was to conduct differential diagnostics in HPP patients using whole-exome sequencing (WES). The medical records of HPP patients and the genetic testing of the ALPL gene were reviewed. Seven patients were recruited and underwent WES using the Illumina or MGI sequencing platforms. All of the exome samples were matched onto a GRCh38.p13 reference genome assembly by using the Genome Analysis ToolKit (GATK) and the BWA MEM read aligner. We present the clinical and molecular findings of the seven patients referred for genetic analyses due to a clinical and biochemical suspicion of HPP. In two patients out of three (with identified heterozygous variants in the ALPL gene), we also identified c.682T>A in exon 3 of the WNT10A gene and c.3470del in exon 23 of the SMC1A gene variants for the first time. In four patients, variants in the ALPL gene were not detected, but WES allowed us to identify for the first time rare variants (c.5651A>C in exon 36 of the TRIO gene, c.880T>G in exon 6 of the TRPV4 gene, c.32078-1G>T in intron 159 of the TTN gene, c.47720_47721del in exon 235 of the TTN gene, and c.1946G>A in exon 15 of the SLC5A1 gene) and to conduct differential diagnostics with HPP. Using WES, for the first time, we demonstrate the possibility of early differential diagnostics in HPP patients with other rare genetic diseases.

The Duck 1000 Genomes Project: Achievements and perspectives

AbstractThe duck (Anas platyrhynchos) is not only a vital farm animal but also an excellent model for genetic dissection of economic traits. The integration of multiomics data provides a powerful approach to elucidate the genetic basis of domestication and phenotype variation. Since its inception in 2014, the Duck 1000 Genomes Project has aimed to uncover the genetic foundation of key economic traits in ducks by combining multiomics data including genomic, transcriptomic, and metabolomic from various natural and segregating populations. This paper summarizes the strategies and achievements of the Duck 1000 Genomes Project, highlighting the reference genome assembly, genome evolution analysis, and the identification of genes and causative mutations responsible for key economic traits in ducks. We also discuss perspectives and potential challenges in functional genomic studies that could further accelerate duck molecular breeding.

Pangenome – its Aspect and Prospect

Pangenome is a very new discipline showing a collection of unique and variable genomes of any species in one model. This discipline is a combination of three subjects of Biology like Computer Science and Applied Mathematics. All genes from all species or strains are called Pangenomes. Pangenome was first discovered during the preparation of protein-based vaccine of the G group of Streptococcus sp. which is the main cause of neonatal life-threatening disease by a team of scientists led by Tettelin. Conventional method of Vaccine preparation was not successful due to the existence of five different serotypes in this organism. Then Tettelin with groups of scientists tried to use the genome sequence information for preparing protein- based vaccine in G group of Streptococcus. During their studies, two gap free genomes were found in this G group of bacteria. After sequencing studies of many strains, they found several regions of diversity (genomic diversity) among different isolates of same strain. After sequencing a large number of genomes, they realized that genes belong to isolates present in the species but not present in genomes. Then the Pangenome concept came to their mind and genes were classified into Core genome, Accessory or Dispensable genome and Species-specific genomes. Bacteria genomes have a distinct number of genes but there are differences in the genomes of different strains of a single species that is mainly due to horizontal gene transfer of genes from other bacteria. The Pangenome concept and its discovery was discussed. Plant genomes are dynamic with whole genome duplication, tandem duplication, transposable element duplication, chromosomal rearrangements or structural variations like deletion, inversion, translocation and recombination between species. Several methods have already been tried for the improvement of crop plants like Marker Assisted Selection (MAS), QTL mapping, Single Nucleotide Polymorphisms (SNP), Copy number variations and Genome Wide Association Studies (GWAS) by comparing with Reference Genome Assemblies representing the genome sequence of the genome of an individual or organism. But the Reference genome is not able to locate all genetic variations of a species. To overcome this limitation the concept of Pangenomes may help in the crop improvement which has been discussed. Pangenomic studies have already been done in many crop plants like Rice, Wheat, Maize, Brassica, Soybean etc. Some of all these aspects have been discussed.

Three decades of rat genomics: approaching the finish(ed) line.

The rat, Rattus norvegicus, has provided an important model for investigation of a range of characteristics of biomedical importance. Here we survey the origins of this species, its introduction into laboratory research, and the emergence of genetic and genomic methods that utilize this model organism. Genomic studies have yielded important progress and provided new insight into several biologically important traits. However, some studies have been impeded by the lack of a complete and accurate reference genome for this species. New sequencing and genome assembly methods applied to the rat have resulted in a new reference genome assembly, GRCr8, which is a near telomere-to-telomere assembly of high base-level accuracy that incorporates several elements not captured in prior assemblies. As genome assembly methods continue to advance and production costs become a less significant obstacle, genome assemblies for multiple inbred rat strains are emerging. These assemblies will allow a rat pangenome assembly to be constructed that captures all the genetic variations in strains selected for their utility in research and will overcome reference bias, a limitation associated with reliance on a single reference assembly. By this means, the full utility of this model organism to genomic studies will begin to be revealed.

Long-read de novo genome assembly of Gulf toadfish (Opsanus beta)

BackgroundThe family Batrachoididae are a group of ecologically important teleost fishes with unique life histories, behavior, and physiology that has made them popular model organisms. Batrachoididae remain understudied in the realm of genomics, with only four reference genome assemblies available for the family, with three being highly fragmented and not up to current assembly standards. Among these is the Gulf toadfish, Opsanus beta, a model organism for serotonin physiology which has recently been bred in captivity.ResultsHere we present a new, de novo genome and transcriptome assemblies for the Gulf toadfish using PacBio long read technology. The genome size of the final assembly is 2.1 gigabases, which is among the largest teleost genomes. This new assembly improves significantly upon the currently available reference for Opsanus beta with a final scaffold count of 62, of which 23 are chromosome scale, an N50 of 98,402,768, and a BUSCO completeness score of 97.3%. Annotation with ab initio and transcriptome-based methods generated 41,076 gene models. The genome is highly repetitive, with ~ 70% of the genome composed of simple repeats and transposable elements. Satellite DNA analysis identified potential telomeric and centromeric regions.ConclusionsThis improved assembly represents a valuable resource for future research using this important model organism and to teleost genomics more broadly.

High-quality chromosome-level genomic insights into molecular adaptation to low-temperature stress in Madhuca longifolia in southern subtropical China

BackgroundMadhuca longifolia, the energy-producing and medicinal tropical tree originally from southern India, faces difficulties in adapting to the low temperatures of late autumn and early winter in subtropical southern China, impacting its usability. Therefore, understanding the molecular mechanisms controlling the ability of this species to adapt to environmental challenges is essential for optimising horticulture efforts. Accordingly, this study aimed to elucidate the molecular responses of M. longifolia to low-temperature stress through genomic and transcriptomic analyses to inform strategies for its effective cultivation and utilisation in colder climates.ResultsHerein, the high-quality reference genome and genomic assembly for M. longifolia are presented for the first time. Using Illumina sequencing, Hi-C technology, and PacBio HiFi sequencing, we assembled a chromosome-level genome approximately 737.92 Mb in size, investigated its genomic features, and conducted an evolutionary analysis of the genus Madhuca. Additionally, using transcriptome sequencing, we identified 17,941 differentially expressed genes related to low-temperature response. Through bioinformatics analysis of the WRKY gene family, 15 genes crucial for M. longifolia low-temperature resistance were identified.ConclusionsThis research not only lays the groundwork for the successful ecological adaptation and cultivation of M. longifolia in China’s southern subtropical regions but also offers valuable insights for the genetic enhancement of cold tolerance in tropical species, contributing to their sustainable horticulture and broader industrial, medicinal, and agricultural use.