Read Sequencing Technologies Research Articles

Maptcha: an efficient parallel workflow for hybrid genome scaffolding

BackgroundGenome assembly, which involves reconstructing a target genome, relies on scaffolding methods to organize and link partially assembled fragments. The rapid evolution of long read sequencing technologies toward more accurate long reads, coupled with the continued use of short read technologies, has created a unique need for hybrid assembly workflows. The construction of accurate genomic scaffolds in hybrid workflows is complicated due to scale, sequencing technology diversity (e.g., short vs. long reads, contigs or partial assemblies), and repetitive regions within a target genome.ResultsIn this paper, we present a new parallel workflow for hybrid genome scaffolding that would allow combining pre-constructed partial assemblies with newly sequenced long reads toward an improved assembly. More specifically, the workflow, called Maptcha, is aimed at generating long scaffolds of a target genome, from two sets of input sequences—an already constructed partial assembly of contigs, and a set of newly sequenced long reads. Our scaffolding approach internally uses an alignment-free mapping step to build a ⟨\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\langle $$\\end{document}contig,contig⟩\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\rangle $$\\end{document} graph using long reads as linking information. Subsequently, this graph is used to generate scaffolds. We present and evaluate a graph-theoretic “wiring” heuristic to perform this scaffolding step. To enable efficient workload management in a parallel setting, we use a batching technique that partitions the scaffolding tasks so that the more expensive alignment-based assembly step at the end can be efficiently parallelized. This step also allows the use of any standalone assembler for generating the final scaffolds.ConclusionsOur experiments with Maptcha on a variety of input genomes, and comparison against two state-of-the-art hybrid scaffolders demonstrate that Maptcha is able to generate longer and more accurate scaffolds substantially faster. In almost all cases, the scaffolds produced by Maptcha are at least an order of magnitude longer (in some cases two orders) than the scaffolds produced by state-of-the-art tools. Maptcha runs significantly faster too, reducing time-to-solution from hours to minutes for most input cases. We also performed a coverage experiment by varying the sequencing coverage depth for long reads, which demonstrated the potential of Maptcha to generate significantly longer scaffolds in low coverage settings (1×\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$1\ imes $$\\end{document}–10×\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$10\ imes $$\\end{document}).

LongTR: genome-wide profiling of genetic variation at tandem repeats from long reads

Tandem repeats are frequent across the human genome, and variation in repeat length has been linked to a variety of traits. Recent improvements in long read sequencing technologies have the potential to greatly improve tandem repeat analysis, especially for long or complex repeats. Here, we introduce LongTR, which accurately genotypes tandem repeats from high-fidelity long reads available from both PacBio and Oxford Nanopore Technologies. LongTR is freely available at https://github.com/gymrek-lab/longtr and https://zenodo.org/doi/10.5281/zenodo.11403979.

De Novo Assembly and Annotation of the Siganus fuscescens (Houttuyn, 1782) Genome: Marking a Pioneering Advance for the Siganidae Family.

This study presents the first draft genome of Siganus fuscescens, and thereby establishes the first whole-genome sequence for a species in the Siganidae family. Leveraging both long and short read sequencing technologies, i.e., Oxford Nanopore and Illumina sequencing, we successfully assembled a mitogenome spanning 16.494 Kb and a first haploid genome encompassing 498Mb. The assembled genome accounted for a 99.6% of the estimated genome size and was organized into 164 contigs with an N50 of 7.2Mb. This genome assembly showed a GC content of 42.9% and a high Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness score of 99.5% using actinopterygii_odb10 lineage, thereby meeting stringent quality standards. In addition to its structural aspects, our study also examined the functional genomics of this species, including the intricate capacity to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) and secrete venom. Notably, our analyses revealed various repeats elements, which collectively constituted 17.43% of the genome. Moreover, annotation of 28,351 genes uncovered both shared genetic signatures and those that are unique to S. fuscescens. Our assembled genome also displayed a moderate prevalence of gene duplication compared to other fish species, which suggests that this species has a distinctive evolutionary trajectory and potentially unique functional constraints. Taken altogether, this genomic resource establishes a robust foundation for future research on the biology, evolution, and the aquaculture potential of S. fuscescens.

A chromosome-scale genome assembly of the nipa palm hispid beetle Octodonta nipae

Nipa palm hispid beetle (Octodonta nipae) is an insect species that is native to Malaysia but has spread to southern China and beyond, seriously threatening palm production. A lack of high-quality genome resources has hindered understanding of the insect’s invasive characteristics and ecological adaptations. Here, we combined Illumina short read, PacBio long-read, and high-throughput chromosome conformation capture (Hi-C) sequencing technologies to generate a high-quality, chromosome-scale genome assembly of nipa palm hispid beetle. The genome assembly was 1.31 Gb in size, consisting of nine chromosomes. The contig and scaffold N50 values were 1.022 Mb and 148.6 Mb, respectively. The genome assembly completeness was estimated at 99.1%. Annotation revealed 16,305 protein-coding genes and 62.16% repeat sequences. This high-quality genome assembly is a valuable resource that will contribute to understanding of the genetic factors underlying the invasive characteristics of nipa palm hispid beetle, ultimately promoting development of efficient control policies.

A novel F type plasmid encoding mcr-10 in a clinical Enterobacter ludwigii strain from a tertiary hospital in the Czech Republic

ObjectiveHere we describe a novel IncFIA plasmid harboring mcr-10 gene in a clinical Enterobacter ludwigii strain isolated at the University Hospital in Pilsen in the Czech Republic. MethodsThe strain was subjected to antibiotic susceptibility testing. Whole genome sequencing was performed using Illumina for short read sequencing and Oxford Nanopore Technologies for long-read sequencing followed by hybrid assembly. The resulting genome was used to detect species using average nucleotide identity, resistance genes, plasmid replicon and MLST (using center for genomic epidemiology databases; ResFinder, PlasmidFinder and MLST repsectively) and virulence genes using VFDB. ResultsΤhe strain showed susceptibility against tetracycline, cefuroxime and chloramphenicol, and was susceptible to the 2nd and 3rd generation of cephalosporins, carbapenems and colistin. Genome analysis identified the strain as E. ludwigii sequence type ST20 and located the mcr-10 gene on an IncFIA (HI1)/IncFII (Yp) plasmid (pI9455333_MCR10; 129,863 bp). Upon blasting the nucleotide sequence of pI9455333_MCR10 against NCBI database; no similar plasmid sequence was detected implying a novel plasmid structure. Nevertheless, it showed a partial similarity with: pRHBSTW-00123_3 and FDAARGOS 1432 which were detected in Enterobacter cloacae complex (ECC) strains in wastewater samples in 2017 in UK and in 2021 in the USA respectively, and pEC81-mcr which was detected in a clinical Escherichia coli strain in 2020 in China. Moreover, I9455333cz genome carried virulence genes coding for: curli fibers, fimbrial adherence determinants, siderophore aerobactin, iron uptake proteins, and regulators of sigma factor. ConclusionIn conclusion, we identified a novel IncF plasmid harboring mcr-10 gene in a clinical Enterobacter ludwigii strain. To our knowledge, this is the first clinical report of mcr-10 in the Czech Republic.

Abstract 1764: Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions

Abstract Background Acute myeloid leukemia with complex karyotype (CKAML) does frequently show del(5q), deletion of chromosome 5q. Previous work in MDS and AML showed that CKAML form clusters distinguishing del(5q) cases with commonly retained regions (CRRs) in the telomeric (≥5q34) and centromeric (≤5q14.2) ends from cases with loss of the telomeric and/or centromeric regions. Interestingly, del(5q) patients with CRRs were shown to have less genomic lesions and were associated with a better prognosis than other 5q-deleted CKAML cases. Aim: Combining the advantages of two different long read sequencing technologies, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), we aimed to more comprehensively characterize the molecular landscape of CKAML with del(5q). Our integrative workflow enabled an in-depth characterization of copy number variations (CNV), genomic rearrangements, transcriptomic and epigenomic changes. Methods: Low coverage whole genome sequencing (WGS) using ONT was performed on 209 patients with CKAML to identify del(5q) cases for further in-depth analysis. For 10 selected cases with known CRR status, genomic deep sequencing using PacBio Revio and ONT PromethION was performed, as well as PacBio Revio MAS-seq for fusion transcript detection. Results: A mean sequencing depth of 3-fold (range 0.4-17.3-fold) was obtained by low coverage WGS of the 209 CKAML cases. With a resolution of 0.1 Mbp n=134 del(5q) cases were identified in our CKAML cohort (64%) and commonly deleted regions on 5q could be resolved at 5q21.2 to 5q23.3 and 5q33.3 to 5q35 (Fisher test, FDR Benjamini-Hochberg, p<10−30). These regions separated del(5q) in n=52 cases showing CRR retention and n=82 showing partially no CRR, (either the centromeric or telomeric regions, n=74, or of both regions, n=8 involved). Subsequent co-occurrence analysis provided evidence of an association between the loss of telomeric 5q region with a 3p12-25 deletion. In-depth analysis (30-50 fold) of ten distinct del(5q) cases by PacBio and ONT revealed additional insights into the complex clonal composition of del(5q) cases. For example, one case showed two distinct subclones with 5q14.2-5q35.1 deletion and with translocation t(3;5)(p14;q14) respectively. Moreover, we could demonstrate that 5q-aberrations are far more complex than initially thought showing, e.g. inverted terminal duplications that resulted in fusion transcripts involving PDE4D and leading to its deregulated expression. Conclusion: Using our long read deep sequencing approach we could further delineate the structural complexity of del(5q) CKAML. While gene expression profiling, methylation analysis and correlation of findings with clinical outcome are ongoing and will be presented at the meeting, our results help to further pinpoint the biology underlying del(5q) and to further refine this heterogeneous CKAML subgroup. Citation Format: Felix Schick, Ekaterina Slonova, Eric Straeng, Sarah Huet, Maurizio Raso, Marlon Tilgner, Frederik Damm, Pierre Sujobert, Olga Blau, Lars Bullinger, Anna Dolnik. Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1764.

Long read sequencing on its way to the routine diagnostics of genetic diseases.

The clinical application of technological progress in the identification of DNA alterations has always led to improvements of diagnostic yields in genetic medicine. At chromosome side, from cytogenetic techniques evaluating number and gross structural defects to genomic microarrays detecting cryptic copy number variants, and at molecular level, from Sanger method studying the nucleotide sequence of single genes to the high-throughput next-generation sequencing (NGS) technologies, resolution and sensitivity progressively increased expanding considerably the range of detectable DNA anomalies and alongside of Mendelian disorders with known genetic causes. However, particular genomic regions (i.e., repetitive and GC-rich sequences) are inefficiently analyzed by standard genetic tests, still relying on laborious, time-consuming and low-sensitive approaches (i.e., southern-blot for repeat expansion or long-PCR for genes with highly homologous pseudogenes), accounting for at least part of the patients with undiagnosed genetic disorders. Third generation sequencing, generating long reads with improved mappability, is more suitable for the detection of structural alterations and defects in hardly accessible genomic regions. Although recently implemented and not yet clinically available, long read sequencing (LRS) technologies have already shown their potential in genetic medicine research that might greatly impact on diagnostic yield and reporting times, through their translation to clinical settings. The main investigated LRS application concerns the identification of structural variants and repeat expansions, probably because techniques for their detection have not evolved as rapidly as those dedicated to single nucleotide variants (SNV) identification: gold standard analyses are karyotyping and microarrays for balanced and unbalanced chromosome rearrangements, respectively, and southern blot and repeat-primed PCR for the amplification and sizing of expanded alleles, impaired by limited resolution and sensitivity that have not been significantly improved by the advent of NGS. Nevertheless, more recently, with the increased accuracy provided by the latest product releases, LRS has been tested also for SNV detection, especially in genes with highly homologous pseudogenes and for haplotype reconstruction to assess the parental origin of alleles with de novo pathogenic variants. We provide a review of relevant recent scientific papers exploring LRS potential in the diagnosis of genetic diseases and its potential future applications in routine genetic testing.

Long read transcriptome sequencing of a sugarcane hybrid and its progenitors, Saccharum officinarum and S. spontaneum.

Commercial sugarcane hybrids are derivatives from Saccharum officinarum and Saccharum spontaneum hybrids containing the full complement of S. officinarum and a few S. spontaneum chromosomes and recombinants with favorable agronomic characters from both the species. The combination of the two sub-genomes in varying proportions in addition to the recombinants presents a challenge in the study of gene expression and regulation in the hybrid. We now report the transcriptome analysis of the two progenitor species and a modern commercial sugarcane hybrid through long read sequencing technology. Transcripts were profiled in the two progenitor species S. officinarum (Black Cheribon), and S. spontaneum (Coimbatore accession) and a recent high yielding, high sugar variety Co 11015. The composition and contribution of the progenitors to a hybrid with respect to sugar, biomass, and disease resistance were established. Sugar related transcripts originated from S. officinarum while several stress and senescence related transcripts were from S. spontaneum in the hybrid. The hybrid had a higher number of transcripts related to sugar transporters, invertases, transcription factors, trehalose, UDP sugars, and cellulose than the two progenitor species. Both S. officinarum and the hybrid had an abundance of novel genes like sugar phosphate translocator, while S. spontaneum had just one. In general, the hybrid shared a larger number of transcripts with S. officinarum than with S. spontaneum, reflecting the genomic contribution, while the progenitors shared very few transcripts between them. The common isoforms among the three genotypes and unique isoforms specific to each genotype indicate that there is a high scope for improvement of the modern hybrids by utilizing novel gene isoforms from the progenitor species.

Characterization of Proteus mirabilis and associated plasmids isolated from anaerobic dairy cattle manure digesters.

Proteus mirabilis is an opportunistic pathogen associated with a variety of human infections, including urinary tract infections. The prevalence of P. mirabilis in foods of animal origin and in the manure by-products created in animal production is not well documented. Further, the prevalence and persistence of extended-spectrum cephalosporin (ESC) resistant P. mirabilis is largely unknown. In this study, we characterized ESC-resistant P. mirabilis recovered from various stages of dairy manure anaerobic digestion. Isolates were screened by PCR for blaCTX-M, blaCMY and blaSHV, and antimicrobial susceptibility testing was performed. Fifty-six P. mirabilis carrying CTX-M were sequenced with short and long read sequencing technologies, and the assembled chromosomes and plasmids were compared. ESC-resistant Proteus was found in four of the six manure digesters, an indication that not all digesters were colonized with resistant strains. Both CTX-M-1 and CTX-M-15 plasmids were found in P. mirabilis isolates. Transfer of plasmid DNA by conjugation was also explored, with ESC-resistance plasmids able to transfer to Escherichia coli at high frequency. We concluded that P. mirabilis can harbour and transfer ESC-resistance genes and plasmids, and may be an overlooked reservoir of antimicrobial resistance.

Abstract P1008: Characterizing Complex Tandem Repeat Variants And Their Contribution To Congenital Heart Disease

Congenital heart disease (CHD) is a major cause of childhood mortality and lifelong morbidity in children and adults. CHD has a strong genetic basis, with exome and short read whole genome sequencing (srWGS) identifying genetic contributions to CHD risk in around 45% of affected individuals, while the remaining 55% have not had a specific genetic risk identified. Variation in tandem repeats (TRs), defined as repeated sequences of base pairs, has been implicated in several diseases and can affect gene expression. TR polymorphism in coding or regulatory regions of CHD related genes can alter function and expression, thereby modifying risk of CHD in patients. Identifying variants in TRs has been a challenge due to multiple variants at each site, as well as difficulty resolving long expansions with short read sequencing technologies. New bioinformatic tools have enabled identification of high-confidence short-TR (STR) variants in srWGS. Additionally, long read whole genome sequencing (lrWGS), with read lengths of 10-20 kilobases, enables genotyping of longer TRs that are missed by srWGS. We hypothesized that patients with CHD will have a higher burden of de novo and transmitted TRs near known CHD genes. We identified de novo and transmitted STR variants near known CHD genes in srWGS data from 1900 CHD proband and their parents in the Pediatric Cardiac Genomics Consortium (PCGC), and 1611 non-CHD probands and their parents. Transmitted STR variants in CHD probands were enriched in 5’-UTR, introns, and promoters of CHD genes compared to non-CHD probands. We identified large de novo repeats near known CHD genes such as NOTCH2 and RBFOX2 . Additionally we used Pacbio HiFi lrWGS on 150 PCGC probands without an established etiology for CHD. Combining these analyses, we identified 3,243 repeat regions with low read depth in srWGS, and therefore unable to be resolved accurately, near known CHD genes and from lrWGS we assess length polymorphisms using Tandem Repeat Finder. TRs that harbor large variation in length were identified within intronic regions of CHD genes including FLT4 , NFATC1 , and PKD1 . We suggest that polymorphic repeat regions provide new candidate variants or altering gene transcription or splicing and thereby contribute to CHD.

Diversity of genetic platforms harboring the blaPER-2 gene in Enterobacterales and insights into the role of ISPa12 in its mobilization and dissemination

The production of PER-like extended-spectrum β-lactamases has recently been associated with reduced susceptibility to the last resort drugs aztreonam/avibactam and cefiderocol. PER-2 has been mainly confined to Argentina and neighboring countries. Until now, only three plasmids harboring blaPER-2 genes have been characterized but very little is known about the involvement of different plasmid groups in its dissemination. The diversity of genetic platforms associated with blaPER-2 genes from a collection of PER-producing Enterobacterales was analysed by describing the close environment and the plasmid backbones. Full sequences of 11 plasmids were obtained by short read (Illumina) and long read (Oxford Nanopore or PacBio) sequencing technologies. De novo assemblies, annotation and sequence analysis were performed by Unicycler, Prokka and BLAST. Plasmid analysis revealed that the blaPER-2 gene is encoded on plasmids of different incompatibility groups (A, C, FIB, HI1B, N2), indicating that this gene may have been disseminated through a variety of plasmids. Comparison with the few publicly available nucleotide sequences describing the blaPER-2 genetic environment, including those from the environmental species Pararheinheimera spp. (considered as the progenitor of blaPER genes), indicates a role of ISPa12 in blaPER-2 gene mobilization from the chromosome of Pararheinheimera spp. Also, the blaPER-2 gene was carried by a novel ISPa12-composite transposon, Tn7390. In addition, its association with ISKox2-like elements in the close genetic environment in all plasmids analysed suggests a role of these insertion sequence elements in further dissemination of blaPER-2 genes.

Insplico: effective computational tool for studying splicing order of adjacent introns genome-wide with short and long RNA-seq reads.

Although splicing occurs largely co-transcriptionally, the order by which introns are removed does not necessarily follow the order in which they are transcribed. Whereas several genomic features are known to influence whether or not an intron is spliced before its downstream neighbor, multiple questions related to adjacent introns' splicing order (AISO) remain unanswered. Here, we present Insplico, the first standalone software for quantifying AISO that works with both short and long read sequencing technologies. We first demonstrate its applicability and effectiveness using simulated reads and by recapitulating previously reported AISO patterns, which unveiled overlooked biases associated with long read sequencing. We next show that AISO around individual exons is remarkably constant across cell and tissue types and even upon major spliceosomal disruption, and it is evolutionarily conserved between human and mouse brains. We also establish a set of universal features associated with AISO patterns across various animal and plant species. Finally, we used Insplico to investigate AISO in the context of tissue-specific exons, particularly focusing on SRRM4-dependent microexons. We found that the majority of such microexons have non-canonical AISO, in which the downstream intron is spliced first, and we suggest two potential modes of SRRM4 regulation of microexons related to their AISO and various splicing-related features. Insplico is available on gitlab.com/aghr/insplico.

Abstract 260: Long RNA and DNA sequencing reveals HEK293 diversity between commercial sources

Abstract HEK293 cells are used extensively in cancer research, with more than 4800 articles between 2017 and 2022 (PubMed search “cancer[MeSH Major Topic] and hek293”). Studies involving transfection to overexpress, knock down, or modify proteins in these cells are common. While HEK293 cells are relatively easy to grow and maintain, early use of high throughput sequencing Yao-Cheng et. al. identified genomic variability between different sources of cell lines and revealed unexpected details about their developmental lineage (likely adrenal, not kidney)[1]. Genomic or transcriptomic reorganization may complicate interpretation of results from targeted intervention experiments. Here, we evaluate HEK293 cells from various commercial sources using long read sequencing technology. Oxford Nanopore direct cDNA profiling was used to compare isoform usage and examine gene fusions. Long DNA libraries were prepared using a high molecular weight isolation method, sequenced on Oxford Nanopore, and assembled to produce long contiguous fragments. Genome assemblies generated from long read sequencing support the genomic diversity reported by Yao-Cheng et.al, though the specific copy number variants are distinct from those previously reported. Assembled genomes and transcriptomes for these specific lots of HEK293 cells will be available at NCBI. Given the plastic nature of HEK293 cells, we recommend confirmation of genomic and transcriptomic structure of the specific cells in use before and after intervention. [1] Lin, Yao-Cheng, Morgane Boone, Leander Meuris, Irma Lemmens, Nadine Van Roy, Arne Soete, Joke Reumers, et al. “Genome Dynamics of the Human Embryonic Kidney 293 Lineage in Response to Cell Biology Manipulations.” Nature Communications 5, no. 1 (September 3, 2014): 4767. https://doi.org/10.1038/ncomms5767. Citation Format: Laura Blum, Brittany Sexton, Jonathan C. Sanford, Evan Janzen, Luo Sun, Paul A. Koetsier, Keerthana Krishnan, Lynne Apone, Bradley W. Langhorst. Long RNA and DNA sequencing reveals HEK293 diversity between commercial sources [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 260.

Novel Alu-mediated deletions of the SMN1 gene were identified by ultra-long read sequencing technology in patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by biallelic variants of the survival motor neuron 1 (SMN1) gene. In this study, our aim was to make a molecular diagnosis in two patients with SMA carrying only one SMN1 copy number. Using ultra-long read sequencing (Ultra-LRS), 1415 bp deletion and 3348 bp deletion of the SMN1 gene were identified in patient 1 and the father of patient 2, respectively. Ultra-LRS revealed two novel deletions, starting from the SMN1 promoter to intron 1. It also accurately provided the location of the deletion breakpoints in the SMN1 gene: chr5 g.70,924,798–70,926,212 for a 1415 bp deletion; chr5 g.70,922,695–70,926,042 for a 3348 bp deletion. By analyzing the breakpoint junctions, we identified that these genomic sequences were composed of Alu sequences, including AluJb, AluYm1, AluSq, and AluYm1, indicating that Alu-mediated rearrangements are a mechanism of SMN1 deletion events. In addition, full-length SMN1 transcripts and SMN protein in patient 1 were significantly decreased (p < 0.01), suggesting that a 1415 bp deletion that included the transcription and translation initiation sites of the SMN1 gene had severe consequences for SMN expression. Ultra-LRS can easily distinguish highly homozygous genes compared to other detection technologies, which is useful for detecting SMN1 intragenic mutations, to quickly discover structural rearrangements and to precisely present the breakpoint positions.

The genome of the egg parasitoid Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae), a model organism and biocontrol agent of stink bugs

Trissolcus basalis (Wollaston) is a minute parasitic wasp that develops in the eggs of stink bugs. Over the past 30 years, Tr. basalis has become a model organism for studying host finding, patch defense behavior, and chemical ecology. As an entry point to better understand the molecular basis of these factors, in addition to filling a critical gap in the genomic resources available for parasitic Hymenoptera, we sequenced and assembled the genome of Tr. basalis using short (454, Illumina) and long read (Oxford Nanopore) sequencing technologies. The three sequencing methods produced 32 million reads (4.10 Gb; 27.9×), which were assembled into 7,586 scaffolds. The 147 Mb (N50: 42.8 kb) assembly contains complete sequences for 93.1% of the insect BUSCO dataset, and an extensive annotation protocol resulted in 14,158 protein-coding gene models, 12,197 (86%) of which had a blast hit in GenBank. Repetitive elements comprised 13.8% of the genome, and a phylogenomic analysis recovered Tr. basalis as sister to Chalcidoidea, a result in line with other studies. We identified 174 rapidly evolving gene families in Tr. basalis, including olfactory receptors and pheromone/general odorant binding proteins. These genetic elements are an obligatory portion of the parasitoid-host relationship, and the draft genome of Tr. basalis has and will continue to be useful in elucidating these relationships at finer resolution.

Accelerating Minimap2 for Accurate Long Read Alignment on GPUs.

Long read sequencing technology is becoming increasingly popular for Precision Medicine applications like Whole Genome Sequencing (WGS) and microbial abundance estimation. Minimap2 is the state-of-the-art aligner and mapper used by the leading long read sequencing technologies, today. However, Minimap2 on CPUs is very slow for long noisy reads. ~60-70% of the run-time on a CPU comes from the highly sequential chaining step in Minimap2. On the other hand, most Point-of-Care computational workflows in long read sequencing use Graphics Processing Units (GPUs). We present minimap2-accelerated (mm2-ax), a heterogeneous design for sequence mapping and alignment where minimap2's compute intensive chaining step is sped up on the GPU and demonstrate its time and cost benefits. We extract better intra-read parallelism from chaining without losing mapping accuracy by forward transforming Minimap2's chaining algorithm. Moreover, we better utilize the high memory available on modern cloud instances apart from better workload balancing, data locality and minimal branch divergence on the GPU. We show mm2-ax on an NVIDIA A100 GPU improves the chaining step with 5.41 - 2.57X speedup and 4.07 - 1.93X speedup : costup over the fastest version of Minimap2, mm2-fast, benchmarked on a Google Cloud Platform instance of 30 SIMD cores.

Extraction of long k-mers using spaced seeds.

The extraction of k-mers from reads is an important task in many bioinformatics applications, such as all DNA sequence analysis methods based on de Bruijn graphs. These methods tend to be more accurate when the used k-mers are unique in the analyzed DNA, and thus the use of longer k-mers is preferred. When the read lengths of short read sequencing technologies increase, the error rate will become the determining factor for the largest possible value of k. Here we propose LoMeX which uses spaced seeds to extract long k-mers accurately even in the presence of sequencing errors. Our experiments show that LoMeX can extract long k-mers from current Illumina reads with a similar or higher recall than a standard k-mer counting tool. Furthermore, our experiments on simulated data show that when the read length further increases enabling even longer k-mers, the performance of standard k-mer counters declines, whereas LoMeX still extracts long k-mers successfully.

Chromosome-level Genome of the Muskrat (Ondatra zibethicus)

The muskrat (Ondatra zibethicus) is a semi-aquatic rodent species with ecological, economic, and medicinal importance. Here, we present an improved genome assembly, which is the first high-quality chromosome-level genome of the muskrat with high completeness and contiguity assembled using single-tube long fragment read, BGISEQ, and Hi-C sequencing technologies. The genome size of the final assembly was 2.63 Gb with 27 pseudochromosomes. The length of scaffold N50 reached 80.25 Mb with a Benchmarking Universal Single-Copy Ortholog score of 91.3%. We identified a 66.98 Mb X chromosome and a 1.14-Mb Y-linked genome region, and these sex-linked regions were validated by resequencing 32 extra male individuals. We predicted 19,396 protein-coding genes, among which 19,395 (99.99%) were functionally annotated. The expanded gene families in the muskrat genome were found to be enriched in several organic synthesis- and metabolism-related Gene Ontology terms, suggesting the likely genomic basis for the production and secretion of musk. This chromosome-level genome represents a valuable resource for improving our understanding of muskrat ecology and musk secretion.

Ongoing endeavors to detect mobilization of transposable elements

Transposable elements (TEs) are DNA sequences capable of mobilization from one location to another in the genome. Since the discovery of ‘Dissociation (Dc) locus’ by Barbara McClintock in maize (1), mounting evidence in the era of genomics indicates that a significant fraction of most eukaryotic genomes is composed of TE sequences, involving in various aspects of biological processes such as development, physiology, diseases and evolution. Although technical advances in genomics have discovered numerous functional impacts of TE across species, our understanding of TEs is still ongoing process due to challenges resulted from complexity and abundance of TEs in the genome. In this mini-review, we briefly summarize biology of TEs and their impacts on the host genome, emphasizing importance of understanding TE landscape in the genome. Then, we introduce recent endeavors especially in vivo retrotransposition assays and long read sequencing technology for identifying de novo insertions/TE polymorphism, which will broaden our knowledge of extraordinary relationship between genomic cohabitants and their host.

Progress in Plant Genome Sequencing

The genome sequence of any organism is key to understanding the biology and utility of that organism. Plants have diverse, complex and sometimes very large nuclear genomes, mitochondrial genomes and much smaller and more highly conserved chloroplast genomes. Plant genome sequences underpin our understanding of plant biology and serve as a key platform for the genetic selection and improvement of crop plants to achieve food security. The development of technology that can capture large volumes of sequence data at low costs and with high accuracy has driven the acceleration of plant genome sequencing advancements. More recently, the development of long read sequencing technology has been a key advance for supporting the accurate sequencing and assembly of chromosome-level plant genomes. This review explored the progress in the sequencing and assembly of plant genomes and the outcomes of plant genome sequencing to date. The outcomes support the conservation of biodiversity, adaptations to climate change and improvements in the sustainability of agriculture, which support food and nutritional security.