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Background Pacific Biosciences HiFi read technology is currently the industry standard for high accuracy long-read sequencing that has been widely adopted by large sequencing and assembly initiatives for generation of de novo assemblies in non-model organisms. Though adapter contamination filtering is routine in traditional short-read analysis pipelines, it has not been widely adopted for HiFi workflows. Results Analysis of 55 publicly available HiFi datasets revealed that a read-sanitation step to remove sequence artifacts derived from PacBio library preparation from read pools is necessary as adapter sequences can be erroneously integrated into assemblies. Conclusions Here we describe the nature of adapter contaminated reads, their consequences in assembly, and present HiFiAdapterFilt, a simple and memory efficient solution for removing adapter contaminated reads prior to assembly.

Background The Vespa lineage of hornets demonstrate the potential to displace native species and cause significant damage to US apiculture through predation. In spite of introductions in recent years to North America, eradication efforts have prevented the Northern Giant Hornet, Vespa mandarinia , from establishing. Improved genomic resources could offer insight into the traits that define this lineage: large body size variance, adaptability to new environments, and high potential for invasiveness. Results We sequenced and assembled genomes of two lineages of the northern giant hornet, Vespa mandarinia , and one of the European hornet, Vespa crabro , using HiFi long read sequencing technology. We found centromeric and pericentric satellite repeats account for nearly half the total DNA of the hornet genomes and their identities were largely unique across species, indicating active, independent expansion. The intraspecific northern giant hornet genomes exhibit asymmetrical expansion across homologous chromosomal regions localized with Hi-C scaffolding. We leveraged pangenomic alignments of the hornet genomes to identify derived mutations, particularly those that affect repeat content and transposable elements (TEs). We found that TEs do not contribute to the bulk repeat content and show no differential expansion in genic space. Conclusions Large tandemly repetitive DNA account for large structural variations across the Vespa and between V. mandarinia . Localization within the genomes necessitated a suite of tools to support the assembly and identification of those elements. Typical repercussions of long-term reductions in population size – namely, reduced diversity and TE expansion – are not present. The degree to which alternative explanations, such as cell and body size selection or centromeric drive, cause massive, localized repeat amplification will require more extensive sampling.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata , and Zeugodacus cucurbitae , a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp − mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni . The conserved phenotype and independent nature of wp − mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests. The white pupae (wp) phenotype has been used for decades to selectively remove females of tephritid species in genetic sexing, but the determining gene is unknown. Here, the authors show that wp phenotype is produced by parallel mutations in a Major Facilitator Superfamily domain containing gene across multiple species.

Hi-C characterizes three-dimensional chromatin organization, facilitates haplotype phasing, and enables genome-assembly scaffolding, but encounters difficulties across complex regions. By coupling chromosome conformation capture (3 C ) with PacBio H iFi long-read sequencing, here we develop a method (CiFi) that enables analysis of genomic interactions across repetitive regions. Starting with as little as 60,000 cells (sub-microgram DNA), the method produces multi-kilobasepair HiFi reads that contain multiple interacting, concatenated segments (~350 bp to 2 kbp). This multiplicity and increase in segment length versus standard short-read-based Hi-C improves read-mapping efficiency and coverage in repetitive regions and enhances haplotype phasing. CiFi pairwise interactions are largely concordant with Hi-C from a human lymphoblastoid cell line, with gains in assigning topologically associating domains across centromeres, segmental duplications, and human disease-associated genomic hotspots. As CiFi requires less input versus established methods, we apply the approach to characterize single small insects: assaying chromatin interactions across the genome from an Anopheles coluzzii mosquito and producing a chromosome-scale scaffolded assembly from a Ceratitis capitata Mediterranean fruit fly. Together, CiFi enables assessment of chromosome-scale interactions of previously recalcitrant low-complexity loci, low-input samples, and small organisms. Hi-C methods for studying 3D genome structure typically require millions of cells and struggle with repetitive regions. Here, authors develop CiFi, combining 3C with PacBio HiFi sequencing, enabling chromatin analysis from as few as 60,000 cells and chromosome-scale assembly from small samples.

Genetic sexing strains (GSS) used in sterile insect technique (SIT) programs are textbook examples of how classical Mendelian genetics can be directly implemented in the management of agricultural insect pests. Although the foundation of traditionally developed GSS are single locus, autosomal recessive traits, their genetic basis are largely unknown. With the advent of modern genomic techniques, the genetic basis of sexing traits in GSS can now be further investigated. This study is the first of its kind to integrate traditional genetic techniques with emerging genomics to characterize a GSS using the tephritid fruit fly pest Bactrocera cucurbitae as a model. These techniques include whole-genome sequencing, the development of a mapping population and linkage map, and quantitative trait analysis. The experiment designed to map the genetic sexing trait in B. cucurbitae, white pupae (wp), also enabled the generation of a chromosome-scale genome assembly by integrating the linkage map with the assembly. Quantitative trait loci analysis revealed SNP loci near position 42 MB on chromosome 3 to be tightly linked to wp. Gene annotation and synteny analysis show a near perfect relationship between chromosomes in B. cucurbitae and Muller elements A–E in Drosophila melanogaster. This chromosome-scale genome assembly is complete, has high contiguity, was generated using a minimal input DNA, and will be used to further characterize the genetic mechanisms underlying wp. Knowledge of the genetic basis of genetic sexing traits can be used to improve SIT in this species and expand it to other economically important Diptera.

The phylum Arthropoda includes species crucial for ecosystem stability, soil health, crop production, and others that present obstacles to crop and animal agriculture. The United States Department of Agriculture’s Agricultural Research Service initiated the Ag100Pest Initiative to generate reference genome assemblies of arthropods that are (or may become) pests to agricultural production and global food security. We describe the project goals, process, status, and future. The first three years of the project were focused on species selection, specimen collection, and the construction of lab and bioinformatics pipelines for the efficient production of assemblies at scale. Contig-level assemblies of 47 species are presented, all of which were generated from single specimens. Lessons learned and optimizations leading to the current pipeline are discussed. The project name implies a target of 100 species, but the efficiencies gained during the project have supported an expansion of the original goal and a total of 158 species are currently in the pipeline. We anticipate that the processes described in the paper will help other arthropod research groups or other consortia considering genome assembly at scale.

The braconid wasp Fopius arisanus (Sonan) is an important biological control agent of tropical and subtropical pest fruit flies, including two important global pests, the Mediterranean fruit fly (Ceratitis capitata), and the oriental fruit fly (Bactrocera dorsalis). The goal of this study was to develop foundational genomic resources for this species to provide tools that can be used to answer questions exploring the multitrophic interactions between the host and parasitoid in this important research system. Here, we present a whole genome assembly of F. arisanus, derived from a pool of haploid offspring from a single unmated female. The genome is ∼154 Mb in size, with a N50 contig and scaffold size of 51,867 bp and 0.98 Mb, respectively. Utilizing existing RNA-Seq data for this species, as well as publicly available peptide sequences from related Hymenoptera, a high quality gene annotation set, which includes 10,991 protein coding genes, was generated. Prior to this assembly submission, no RefSeq proteins were present for this species. Parasitic wasps play an important role in a diverse ecosystem as well as a role in biological control of agricultural pests. This whole genome assembly and annotation data represents the first genome-scale assembly for this species or any closely related Opiine, and are publicly available in the National Center for Biotechnology Information Genome and RefSeq databases, providing a much needed genomic resource for this hymenopteran group.

The boll weevil, Anthonomus grandis grandis Boheman, is one of the most historically impactful insects due to its near destruction of the US cotton industry in the early 20th century. Contemporary efforts to manage this insect primarily use pheromone baited traps for detection and organophosphate insecticides for control, but this strategy is not sustainable due to financial and environmental costs. We present a high-quality boll weevil genome assembly, consisting of 306 scaffolds with approximately 24,000 annotated genes, as a first step in the identification of gene targets for novel pest control. Gene content and transposable element distribution are similar to those found in other Curculionidae genomes; however, this is the most contiguous and only assembly reported to date for a member in the species-rich genus Anthonomus. Transcriptome profiles across larval, pupal, and adult life stages led to identification of several genes and gene families that could present targets for novel control strategies.

Anthurium amnicola is in the monocot family Araceae, subfamily Pothoideae and is a contributing species in Hawaii floriculture industry hybrids. To support future genetic improvements to this commodity, we sequenced and assembled the A. amnicola genome to chromosome-scale using PacBio HiFi and short-read Hi-C sequencing. A total of 98.51% of the 4.79 Gb genome is anchored into 15 chromosomes, with 99.2% gene completeness and a high LTR assembly index (LAI) score of 21.73, indicative of a complete, high-quality assembly. Annotation reveals the presence of 20,380 protein-coding genes, with 78.52% of the genome composed of repetitive sequences, predominantly long terminal repeat retrotransposons (LTR-RT). Phylogenetic analysis identified evolutionary relationships between A. amnicola and representative species in the Araceae and other plant families. This study provides the first reference genome sequence for the neotropical genus Anthurium and insights into Araceae evolution, benefiting the floriculture industry and evolutionary studies.

The remarkable diversity of insect pigmentation offers a captivating avenue for studying evolution and genetics. In tephritids, understanding the molecular basis of mutant traits is also crucial for applied entomology, enabling the creation of genetic sexing strains through genome editing, thus facilitating sex-sorting before sterile insect releases. Here, we present evidence from classical and modern genetics showing that the black pupae (bp) phenotype in the GUA10 strain of Anastrepha ludens is caused by a large deletion at the ebony locus, removing the gene’s entire coding region. Targeted knockout of ebony induced analogous bp phenotypes across six major tephritid agricultural pests, demonstrating that disruption of Ebony alone is sufficient to produce the mutant trait in distantly related species. This functional characterization further allowed a deeper exploration of Ebony’s role in pigmentation and development across life stages in diverse species. Our findings offer key insights for molecular engineering of sexing strains based on the bp marker and for future evolutionary developmental biology studies in tephritids. This study integrates classical and modern genetics to show that the ebony gene is linked to the black pupae phenotype in tephritids, offering new insights into Ebony’s role in the evolutionary developmental biology of this diverse group of flies