Biology subjects

Smith, T. P. L.

Publications and source records attributed to Smith, T. P. L..

16 recordsLinked to original sources

bioRxiv2025

Bighorn sheep T2T genome assembly reveals differences in immune genes: a potential cause of high morbidity due to respiratory pathogens

The bighorn sheep (Ovis canadensis), despite its close relation to domestic sheep, suffer higher morbidity and mortality from respiratory disease complexes, likely due to genetic differences in immune responses. Unraveling highly repetitive regions such as immune loci and genetic differences was problematic until now. We generated a bighorn sheep telomere-to-telomere assembly, adding 14.28% of novel sequence compared to the previous reference. This enabled the first complete immune loci annotation revealing the IGL and TR loci are significantly short in bighorn sheep. Importantly, a critical immune gene GBP5 and ZNF501, involved in Golgi-mediated immune response, are lacking in bighorn but present in domestic sheep. Re-analysis of a Mycoplasma ovipneumoniae carriage study, using this assembly, identified the immune gene CAPN2 as a key genetic marker for disease carriage, not observable in the original study. This work provides a critical resource for identifying phenotype-linked genetic variation and exploring evolutionary adaptations of bighorn sheep.

genomics↗

bioRxiv2025

Finishing a complete giraffe genome from telomere to telomere with Verkko-Fillet

High-quality genome assemblies are essential for understanding speciation, evolution, and for building reference genomes and pan-genomes. Despite major advances in long-read sequencing technologies and graph-based assembly algorithms, current assemblies often remain incomplete and require refinement, including correction of haplotype switching and resolution of gaps, particularly within repetitive regions such as ribosomal DNA clusters and recent segmental duplications. These limitations largely stem from challenges in graph curation and are not adequately addressed by conventional polishing methods, which focus primarily on nucleotide-level corrections. To overcome these issues, we developed Verkko-Fillet, a Python-based interactive framework for genome graph inspection, editing, and curation that also provides step-by-step guidelines for downstream polishing. Verkko-Fillet takes as input Verkko output files, including the assembly graph, haplotype paths, Hi-C contacts, and additional ONT reads or alternative assemblies aligned to the graph, and offers tools for visualizing, modifying, and exporting curated assembly graphs. It enables users to track changes, resolve complex structural features, fill gaps, and enhance assembly quality beyond conventional polishing. As a case study, we applied Verkko-Fillet to the giraffe genome (Giraffa tippelskirchi), refining a draft assembly (QV 61.5) to a telomere-to-telomere (T2T) reference genome (QV 73.6) by improving both contiguity and completeness. Along with the T2T diploid giraffe genome, we provide gene annotations on this assembly as a valuable resource. Our results highlight the critical role of graph-based curation in producing high-quality, gapless, T2T assemblies that are ready for downstream biological analyses.

bioinformatics↗

bioRxiv2025

Population structure and domestication history of the Javan banteng (Bos javanicus javanicus)

The domestication of the banteng in Southeast Asia is one of the Worlds least known livestock domestications, yet a vital component of the agricultural system in Indonesia and surrounding countries. Here we generated the first reference genome of the banteng and used it to analyze a set of 78 resequenced wild and domesticated bantengs, including 19 newly generated whole-genome sequenced samples of which three are historical samples. We found low heterozygosity and significant differentiation, the latter primarily driven by recent genetic drift and inbreeding in two populations, and clearly attributable to anthropogenically driven founder events or ex-situ breeding. Population structure when excluding these two populations was limited, and we found that the evolutionary divergence between wild and domestic banteng is moderate (FST = 0.14), relatively young (10,356 years), and with post-divergence gene flow. We found only weak signals of a domestication bottleneck between [~]6100-2900 years ago, and genetic diversity is on average higher in domestic than in wild banteng. Despite the soft domestication history, we found 56 candidate genes under selection during domestication, with the leptin receptor gene (LEPR) of particular interest due to the robust selection signal across methods, and its known association to metabolism, obesity and energy homeostasis. Finally, genetic load estimation revealed that Bali cattle in Australia have high realized load, while Bali cattle from Bali have high masked load. These findings provide the first genomic insights into an understudied bovine that is Critically Endangered in its wild form, and agriculturally important in its domesticated form.

evolutionary biology↗

bioRxiv2024

Most Beefalo cattle have no detectable bison genetic ancestry

Hybridization is common among lineages in the genus Bos, often mediated through human management for the selection of adaptive or desirable traits. A recent example is the American Beefalo cattle breed, which was developed in the 1970s and defined as a hybrid between American bison (Bison bison) and cattle (Bos taurus). The American Beefalo Association (ABA) typically require [3/8] bison ancestry to qualify as Beefalo. Here, we sought to characterize admixed ancestry among Beefalo as a component of a larger project to understand the role of hybridization in shaping present-day diversity in bison and cattle. We generated genomic data from 50 historical and present-day Beefalo and bison hybrids, including several important founding animals, as well as from 10 bison originating from commercial herds that represent potential sources of bison ancestry in Beefalo. We found that most Beefalo did not contain detectable bison ancestry. No individual Beefalo within our data set satisfies the ancestry requirements specified by the ABA, although several Beefalo had smaller proportions of bison ancestry (2-18%). Some beefalo had detectable zebu cattle ancestry (2-38%), suggesting that hybridization of taurine and zebu cattle may contribute to morphological similarity between some Beefalo and bison. Overall, ancestry profiles of Beefalo and bison hybrid genomes are consistent with repeated backcrossing to either parental species rather than the breeding between hybrids themselves, implying significant barriers to gene flow between bison and cattle. Our results call into question the [3/8] bison ancestry targeted by the breed association and demonstrate the value of genomic information in examining claims of interspecies gene flow among Bos species.

evolutionary biology↗

bioRxiv2023

African bush pigs exhibit porous species boundaries and appeared in Madagascar concurrently with human arrival

Several African mammals exhibit a phylogeographic pattern where closely related taxa are split between West/Central and East/Southern Africa, but their evolutionary relationships and histories remain controversial. Bushpigs (Potamochoerus larvatus) and red river hogs (P. porcus) are recognised as separate species due to morphological distinctions, a perceived lack of interbreeding at contact, and putatively old divergence times, but historically, they were considered conspecific. Moreover, the presence of Malagasy bushpigs as the sole large terrestrial mammal shared with the African mainland raises intriguing questions about its origin and arrival in Madagascar. Analyses of 67 whole genomes revealed a genetic continuum between the two species, with putative signatures of historical gene flow, variable FST values, and a recent divergence time (<500,000 years). Thus, our study challenges key arguments for splitting Potamochoerus into two species and suggests their speciation might be incomplete. Our findings also indicate that Malagasy bushpigs diverged from southern African populations and underwent a limited bottleneck 1,000-5,000 years ago, concurrent with human arrival in Madagascar. These results shed new light on the evolutionary history of an iconic and widespread African genus and provide insight into the longstanding biogeographic puzzle surrounding the bushpigs presence in Madagascar.

evolutionary biology↗

bioRxiv2023

A reference assembly for the legume cover crop, hairy vetch (Vicia villosa)

Vicia villosa is an incompletely domesticated annual legume of the Fabaceae family native to Europe and Western Asia. V. villosa is widely used as a cover crop and as a forage due to its ability to withstand harsh winters. A reference-quality genome assembly (Vvill1.0) was prepared from low error rate long sequence reads to improve genetic-based trait selection of this species. The Vvill1.0 assembly includes seven scaffolds corresponding to the seven estimated linkage groups and comprising approximately 68% of the total genome size of 2.03 gigabase pairs (Gbp). This assembly is expected to be a useful resource for genetic improvement of this emerging cover crop species as well as to provide useful insights into plant genome evolution.

genomics↗

bioRxiv2022

Functional genomics of cattle through integration of multi-omics data

Functional annotation of the bovine genome was performed by characterizing the spectrum of RNA transcription using a multi-omics approach, combining long- and short-read transcript sequencing and orthogonal data to identify promoters and enhancers and to determine boundaries of open chromatin. A total number of 171,985 unique transcripts (50% protein-coding) representing 35,150 unique genes (64% protein-coding) were identified across tissues. Among them, 159,033 transcripts (92% of the total) were structurally validated by independent datasets such as PacBio Iso-seq, ONT-seq, de novo assembled transcripts from RNA-seq, or Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive independent data from different technologies such as WTTS-seq, RAMPAGE, ChIP-seq, and ATAC-seq. A large proportion of identified transcripts (69%) were novel, of which 87% were produced by known genes and 13% by novel genes. A median of two 5 untranslated regions was detected per gene, an increase from Ensembl and NCBI annotations (single). Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as non-coding genes in fetal tissues, but as protein coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 known gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available QTL data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. These validated results show significant improvement over current bovine genome annotations.

bioinformatics↗

bioRxiv2022

Chromosome-scale assembly of the highly heterozygous genome of red clover (Trifolium pratense L.), an allogamous forage crop species

Red clover (Trifolium pratense L.) is used as a forage crop due to a variety of favorable traits relative to other crops. Improved varieties have been developed through conventional breeding approaches, but progress could be accelerated and gene discovery facilitated using modern genomic methods. Existing short-read based genome assemblies of the ~420 Megabase (Mb) genome are fragmented into >135,000 contigs with numerous errors in order and orientation within scaffolds, likely due to the biology of the plant which displays gametophytic self-incompatibility resulting in inherent high heterozygosity. A high-quality long-read based assembly of red clover is presented that reduces the number of contigs by more than 500-fold, improves the per-base quality, and increases the contig N50 statistic by three orders of magnitude. The 413.5 Mb assembly is nearly 20% longer than the 350 Mb short read assembly, closer to the predicted genome size. Quality measures are presented and full-length isoform sequence of RNA transcripts reported for use in assessing accuracy and for future annotation of the genome. The assembly accurately represents the seven main linkage groups present in the genome of an allogamous (outcrossing), highly heterozygous plant species.

genomics↗

bioRxiv2021

Bovine pangenome reveals trait-associated structural variation from diverse assembly inputs

Advantages of pangenomes over linear reference assemblies for genome research have recently been established. However, potential effects of sequence platform and assembly approach, or of combining assemblies created by different approaches, on pangenome construction have not been investigated. We generated haplotype-resolved assemblies from the offspring of three bovine trios representing increasing levels of heterozygosity that each demonstrate a substantial improvement in contiguity, completeness, and accuracy over the current Bos taurus reference genome. Diploid coverage as low as 20x for HiFi or 60x for ONT was sufficient to produce two haplotype-resolved assemblies meeting the standards set by the Vertebrate Genome Project. Structural variant-based pangenomes created from the haplotype-resolved assemblies demonstrated significant consensus regardless of sequence platform, assembler algorithm, or coverage. Inspecting pangenome topologies identified 90 thousand structural variants including 931 overlapping with coding sequences; this approach revealed variants affecting QRICH2, PRDM9, HSPA1A, TAS2R46, and GC that have potential to affect phenotype.

genomics↗

bioRxiv2021

Revealing how variations in antibody repertoires correlate with vaccine responses

An important challenge in vaccine development is to figure out why a vaccine succeeds in some individuals and fails in others. Although antibody repertoires hold a key to answering this question, there have been very few personalized immunogenomics studies so far aimed at revealing how variations in immunoglobulin genes affect a vaccine response. We conducted an immunosequencing study of 204 calves vaccinated against bovine respiratory disease (BRD) with the goal to reveal variations in immunoglobulin genes and somatic hypermutations that impact the efficacy of vaccine response. Our study represents the largest longitudinal personalized immunogenomics study reported to date across all species, including humans. To analyze the generated dataset, we developed an algorithm for identifying variations of the immunoglobulin genes (as well as frequent somatic hypermutations) that affect various features of the antibody repertoire and titers of neutralizing antibodies. In contrast to relatively short human antibodies, cattle have a large fraction of ultralong antibodies that have opened new therapeutic opportunities. Our study revealed that ultralong antibodies are a key component of the immune response against the costliest disease of beef cattle in North America. The detected variants of the cattle immunoglobulin genes, which are implicated in the success/failure of the BRD vaccine, have the potential to direct the selection of individual cattle for ongoing breeding programs.

immunology↗

bioRxiv2021

Accurate viral genome reconstruction and host assignment with proximity-ligation sequencing

Viruses play crucial roles in the ecology of microbial communities, yet they remain relatively understudied in their native environments. Despite many advancements in high-throughput whole-genome sequencing (WGS), sequence assembly, and annotation of viruses, the reconstruction of full-length viral genomes directly from metagenomic sequencing is possible only for the most abundant phages and requires long-read sequencing technologies. Additionally, the prediction of their cellular hosts remains difficult from conventional metagenomic sequencing alone. To address these gaps in the field and to accelerate the study of viruses directly in their native microbiomes, we developed an end-to-end bioinformatics platform for viral genome reconstruction and host attribution from metagenomic data using proximity-ligation sequencing (i.e., Hi-C). We demonstrate the capabilities of the platform by recovering and characterizing the metavirome of a variety of metagenomes, including a fecal microbiome that has also been sequenced with accurate long reads, allowing for the assessment and benchmarking of the new methods. The platform can accurately extract numerous near-complete viral genomes even from highly fragmented short-read assemblies and can reliably predict their cellular hosts with minimal false positives. To our knowledge, this is the first software for performing these tasks. Being significantly cheaper than long-read sequencing of comparable depth, the incorporation of proximity-ligation sequencing in microbiome research shows promise to greatly accelerate future advancements in the field.

genomics↗

bioRxiv2020

Global analysis of transcription start sites in the new ovine reference genome (Oar rambouillet v1.0)

The overall aim of the Ovine FAANG project is to provide a comprehensive annotation of the new highly contiguous sheep reference genome sequence (Oar rambouillet v1.0). Mapping of transcription start sites (TSS) is a key first step in understanding transcript regulation and diversity. Using 56 tissue samples collected from the reference ewe Benz2616 we have performed a global analysis of TSS and TSS- Enhancer clusters using Cap Analysis Gene Expression (CAGE) sequencing. CAGE measures RNA expression by 5 cap-trapping and has been specifically designed to allow the characterization of TSS within promoters to single-nucleotide resolution. We have adapted an analysis pipeline that uses TagDust2 for clean-up and trimming, Bowtie2 for mapping, CAGEfightR for clustering and the Integrative Genomics Viewer (IGV) for visualization. Mapping of CAGE tags indicated that the expression levels of CAGE tag clusters varied across tissues. Expression profiles across tissues were validated using corresponding polyA+ mRNA-Seq data from the same samples. After removal of CAGE tags with < 10 read counts, 39.3% of TSS overlapped with 5 ends of 31,113 transcripts that had been previously annotated by NCBI (out of a total of 56,308 from the NCBI annotation). For 25,195 of the transcripts, previously annotated by NCBI, no TSS meeting stringent criteria were identified. A further 14.7% of TSS mapped to within 50bp of annotated promoter regions. Intersecting these predicted TSS regions with annotated promoter regions ({+/-}50bp) revealed 46% of the predicted TSS were novel and previously un-annotated. Using whole genome bisulphite sequencing data from the same tissues we were able to determine that a proportion of these novel TSS were hypo-methylated (32.2%) indicating that they are likely to be reproducible rather than noise. This global analysis of TSS in sheep will significantly enhance the annotation of gene models in the new ovine reference assembly. Our analyses provide one of the highest resolution annotations of transcript regulation and diversity in a livestock species to date.

genomics↗

bioRxiv2019

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

BackgroundAssemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions in their entirety with higher continuity and accuracy than is possible with other methods.\n\nResultsWe used trio binning to assemble reference genomes for two species from a single individual using an interspecies cross of yak (Bos grunniens) and cattle (Bos taurus). The high heterozygosity inherent to interspecies hybrids allowed us to confidently assign >99% of long reads from the F1 offspring to parental bins using unique k-mers from parental short reads. Both the maternal (yak) and paternal (cattle) assemblies contain over one third of the acrocentric chromosomes, including the two largest chromosomes, in single haplotigs.\n\nConclusionsThese haplotigs are the first vertebrate chromosome arms to be assembled gap-free and fully phased, and the first time assemblies for two species have been created from a single individual. Both assemblies are the most continuous currently available for non-model vertebrates.

bioinformatics↗

bioRxiv2018

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation

The characterization of microbial communities by metagenomic approaches has been enhanced by recent improvements in short-read sequencing efficiency and assembly algorithms. We describe the results of adding long-read sequencing to the mix of technologies used to assemble a highly complex cattle rumen microbial community, and compare the assembly to current short read-based methods applied to the same sample. Contigs in the long-read assembly were 7-fold longer on average, and contained 7-fold more complete open reading frames (ORF), than the short read assembly, despite having three-fold lower sequence depth. The linkages between long-read contigs, provided by proximity ligation data, supported identification of 188 novel viral-host associations in the rumen microbial community that suggest cross-species infectivity of specific viral strains. The improved contiguity of the long-read assembly also identified 94 antimicrobial resistance genes, compared to only seven alleles identified in the short-read assembly. Overall, we demonstrate a combination of experimental and computational methods that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

microbiology↗

bioRxiv2018

FALCON-Phase: Integrating PacBio and Hi-C data for phased diploid genomes

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. These assemblies can be created in various ways, such as use of tissues that contain single-haplotype (haploid) genomes, or by co-sequencing of parental genomes, but these approaches can be impractical in many situations. We present FALCON-Phase, which integrates long-read sequencing data and ultra-long-range Hi-C chromatin interaction data of a diploid individual to create high-quality, phased diploid genome assemblies. The method was evaluated by application to three datasets, including human, cattle, and zebra finch, for which high-quality, fully haplotype resolved assemblies were available for benchmarking. Phasing algorithm accuracy was affected by heterozygosity of the individual sequenced, with higher accuracy for cattle and zebra finch (>97%) compared to human (82%). In addition, scaffolding with the same Hi-C chromatin contact data resulted in phased chromosome-scale scaffolds.

genomics↗

bioRxiv2018

Complete assembly of parental haplotypes with trio binning

Reference genome projects have historically selected inbred individuals to minimize heterozygosity and simplify assembly. We challenge this dogma and present a new approach designed specifically for heterozygous genomes. \"Trio binning\" uses short reads from two parental genomes to partition long reads from an offspring into haplotype-specific sets prior to assembly. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction. On a benchmark human trio, this method achieved high accuracy and recovered complex structural variants missed by alternative approaches. To demonstrate its effectiveness on a heterozygous genome, we sequenced an F1 cross between cattle subspecies Bos taurus taurus and Bos taurus indicus, and completely assembled both parental haplotypes with NG50 haplotig sizes >20 Mbp and 99.998% accuracy, surpassing the quality of current cattle reference genomes. We propose trio binning as a new best practice for diploid genome assembly that will enable new studies of haplotype variation and inheritance.

genomics↗