Oxford Nanopore Technologies Reads Research Articles

Phased nanopore assembly with Shasta and modular graph phasing with GFAse.

Reference-free genome phasing is vital for understanding allele inheritance and the impact of single-molecule DNA variation on phenotypes. To achieve thorough phasing across homozygous or repetitive regions of the genome, long-read sequencing technologies are often used to perform phased de novo assembly. As a step toward reducing the cost and complexity of this type of analysis, we describe new methods for accurately phasing Oxford Nanopore Technologies (ONT) sequence data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of ONT PromethION sequencing, including those using proximity ligation, and show that newer, higher accuracy ONT reads substantially improve assembly quality.

Profiling genome-wide methylation in two maples: Fine-scale approaches to detection with nanopore technology.

DNA methylation is critical to the regulation of transposable elements and gene expression and can play an important role in the adaptation of stress response mechanisms in plants. Traditional methods of methylation quantification rely on bisulfite conversion that can compromise accuracy. Recent advances in long-read sequencing technologies allow for methylation detection in real time. The associated algorithms that interpret these modifications have evolved from strictly statistical approaches to Hidden Markov Models and, recently, deep learning approaches. Much of the existing software focuses on methylation in the CG context, but methylation in other contexts is important to quantify, as it is extensively leveraged in plants. Here, we present methylation profiles for two maple species across the full range of 5mC sequence contexts using Oxford Nanopore Technologies (ONT) long-reads. Hybrid and reference-guided assemblies were generated for two new Acer accessions: Acer negundo (box elder; 65x ONT and 111X Illumina) and Acer saccharum (sugar maple; 93x ONT and 148X Illumina). The ONT reads generated for these assemblies were re-basecalled, and methylation detection was conducted in a custom pipeline with the published Acer references (PacBio assemblies) and hybrid assemblies reported herein to generate four epigenomes. Examination of the transposable element landscape revealed the dominance of LTR Copia elements and patterns of methylation associated with different classes of TEs. Methylation distributions were examined at high resolution across gene and repeat density and described within the broader angiosperm context, and more narrowly in the context of gene family dynamics and candidate nutrient stress genes.

Comparative Genomic Analysis of Colletotrichum lini Strains with Different Virulence on Flax.

Colletotrichum lini is a flax fungal pathogen. The genus comprises differently virulent strains, leading to significant yield losses. However, there were no attempts to investigate the molecular mechanisms of C. lini pathogenicity from high-quality genome assemblies until this study. In this work, we sequenced the genomes of three C. lini strains of high (#390-1), medium (#757), and low (#771) virulence. We obtained more than 100× genome coverage with Oxford Nanopore Technologies reads (N50 = 12.1, 6.1, 5.0 kb) and more than 50× genome coverage with Illumina data (150 + 150 bp). Several assembly strategies were tested. The final assemblies were obtained using the Canu-Racon ×2-Medaka-Polca scheme. The assembled genomes had a size of 54.0-55.3 Mb, 26-32 contigs, N50 values > 5 Mb, and BUSCO completeness > 96%. A comparative genomic analysis showed high similarity among mitochondrial and nuclear genomes. However, a rearrangement event and the loss of a 0.7 Mb contig were revealed. After genome annotation with Funannotate, secreting proteins were selected using SignalP, and candidate effectors were predicted among them using EffectorP. The analysis of the InterPro annotations of predicted effectors revealed unique protein categories in each strain. The assembled genomes and the conducted comparative analysis extend the knowledge of the genetic diversity of C. lini and form the basis for establishing the molecular mechanisms of its pathogenicity.

Assembling highly repetitive Xanthomonas TALomes using Oxford Nanopore sequencing

BackgroundMost plant-pathogenic Xanthomonas bacteria harbor transcription activator-like effector (TALE) genes, which function as transcriptional activators of host plant genes and support infection. The entire repertoire of up to 29 TALE genes of a Xanthomonas strain is also referred to as TALome. The DNA-binding domain of TALEs is comprised of highly conserved repeats and TALE genes often occur in gene clusters, which precludes the assembly of TALE-carrying Xanthomonas genomes based on standard sequencing approaches.ResultsHere, we report the successful assembly of the 5 Mbp genomes of five Xanthomonas strains from Oxford Nanopore Technologies (ONT) sequencing data. For one of these strains, Xanthomonas oryzae pv. oryzae (Xoo) PXO35, we illustrate why Illumina short reads and longer PacBio reads are insufficient to fully resolve the genome. While ONT reads are perfectly suited to yield highly contiguous genomes, they suffer from a specific error profile within homopolymers. To still yield complete and correct TALomes from ONT assemblies, we present a computational correction pipeline specifically tailored to TALE genes, which yields at least comparable accuracy as Illumina-based polishing. We further systematically assess the ONT-based pipeline for its multiplexing capacity and find that, combined with computational correction, the complete TALome of Xoo PXO35 could have been reconstructed from less than 20,000 ONT reads.ConclusionsOur results indicate that multiplexed ONT sequencing combined with a computational correction of TALE genes constitutes a highly capable tool for characterizing the TALomes of huge collections of Xanthomonas strains in the future.

Nanopore-only assemblies for genomic surveillance of the global priority drug-resistant pathogen, Klebsiella pneumoniae.

Oxford Nanopore Technologies (ONT) sequencing has rich potential for genomic epidemiology and public health investigations of bacterial pathogens, particularly in low-resource settings and at the point of care, due to its portability and affordability. However, low base-call accuracy has limited the reliability of ONT data for critical tasks such as antimicrobial resistance (AMR) and virulence gene detection and typing, serotype prediction, and cluster identification. Thus, Illumina sequencing remains the standard for genomic surveillance despite higher capital and running costs. We tested the accuracy of ONT-only assemblies for common applied bacterial genomics tasks (genotyping and cluster detection, implemented via Kleborate, Kaptive and Pathogenwatch), using data from 54 unique Klebsiella pneumoniae isolates. ONT reads generated via MinION with R9.4.1 flowcells were basecalled using three alternative models [Fast, High-accuracy (HAC) and Super-accuracy (SUP), available within ONT's Guppy software], assembled with Flye and polished using Medaka. Accuracy of typing using ONT-only assemblies was compared with that of Illumina-only and hybrid ONT+Illumina assemblies, constructed from the same isolates as reference standards. The most resource-intensive ONT-assembly approach (SUP basecalling, with or without Medaka polishing) performed best, yielding reliable capsule (K) type calls for all strains (100 % exact or best matching locus), reliable multi-locus sequence type (MLST) assignment (98.3 % exact match or single-locus variants), and good detection of acquired AMR genes and mutations (88-100 % correct identification across the various drug classes). Distance-based trees generated from SUP+Medaka assemblies accurately reflected overall genetic relationships between isolates. The definition of outbreak clusters from ONT-only assemblies was problematic due to inflation of SNP counts by high base-call errors. However, ONT data could be reliably used to 'rule out' isolates of distinct lineages from suspected transmission clusters. HAC basecalling + Medaka polishing performed similarly to SUP basecalling without polishing. Therefore, we recommend investing compute resources into basecalling (SUP model), wherever compute resources and time allow, and note that polishing is also worthwhile for improved performance. Overall, our results show that MLST, K type and AMR determinants can be reliably identified with ONT-only R9.4.1 flowcell data. However, cluster detection remains challenging with this technology.

Comparison of structural variants detected by PacBio-CLR and ONT sequencing in pear

BackgroundStructural variations (SVs) have recently become a topic of great interest in the area of genetic diversity and trait regulation. As genomic sequencing technologies have rapidly advanced, longer reads have been used to identify SVs at high resolution and with increased accuracy. It is important to choose a suitable sequencing platform and appropriate sequencing depth for SV detection in the pear genome.ResultsIn this study, two types of long reads from sequencing platforms, continuous long reads from Pacific Biosciences (PB-CLR) and long reads from Oxford Nanopore Technologies (ONT), were used to comprehensively analyze and compare SVs in the pear genome. The mapping rate of long reads was higher when the program Minimap2 rather than the other three mapping tools (NGMLR, LRA and Winnowmap2) was used. Three SV detection programs (Sniffles_v2, CuteSV, and Nanovar) were compared, and Nanovar had the highest sensitivity in detecting SVs at low sequencing depth (10–15×). A sequencing depth of 15× was suitable for SV detection in the pear genome using Nanovar. SVs detected by Sniffles_v2 and CuteSV with ONT reads had the high overlap with presence/absence variations (PAVs) in the pear cultivars ‘Bartlett’ and ‘Dangshansuli’, both of them with 38% of insertions and 55% of deletions overlapping with PAVs at sequencing depth of 30×. For the ONT sequencing data, over 37,526 SVs spanning ~ 28 Mb were identified by all three software packages for the ‘Bartlett’ and ‘Dangshansuli’ genomes. Those SVs were annotated and combined with transcriptome profiles derived from ‘Bartlett’ and ‘Dangshansuli’ fruit flesh at 60 days after cross-pollination. Several genes related to levels of sugars, acid, stone cells, and aromatic compounds were identified among the SVs. Transcription factors were then predicted among those genes, and results included bHLH, ERF, and MYB genes.ConclusionSV detection is of great significance in exploring phenotypic differences between pear varieties. Our study provides a framework for assessment of different SV software packages and sequencing platforms that can be applied in other plant genome studies. Based on these analyses, ONT sequencing data was determined to be more suitable than PB-CLR for SV detection in the pear genome. This analysis model will facilitate screening of genes related to agronomic traits in other crops.

ONT-Based Alternative Assemblies Impact on the Annotations of Unique versus Repetitive Features in the Genome of a Romanian Strain of Drosophila melanogaster

To date, different strategies of whole-genome sequencing (WGS) have been developed in order to understand the genome structure and functions. However, the analysis of genomic sequences obtained from natural populations is challenging and the biological interpretation of sequencing data remains the main issue. The MinION device developed by Oxford Nanopore Technologies (ONT) is able to generate long reads with minimal costs and time requirements. These valuable assets qualify it as a suitable method for performing WGS, especially in small laboratories. The long reads resulted using this sequencing approach can cover large structural variants and repetitive sequences commonly present in the genomes of eukaryotes. Using MinION, we performed two WGS assessments of a Romanian local strain of Drosophila melanogaster, referred to as Horezu_LaPeri (Horezu). In total, 1,317,857 reads with a size of 8.9 gigabytes (Gb) were generated. Canu and Flye de novo assembly tools were employed to obtain four distinct assemblies with both unfiltered and filtered reads, achieving maximum reference genome coverages of 94.8% (Canu) and 91.4% (Flye). In order to test the quality of these assemblies, we performed a two-step evaluation. Firstly, we considered the BUSCO scores and inquired for a supplemental set of genes using BLAST. Subsequently, we appraised the total content of natural transposons (NTs) relative to the reference genome (ISO1 strain) and mapped the mdg1 retroelement as a resolution assayer. Our results reveal that filtered data provide only slightly enhanced results when considering genes identification, but the use of unfiltered data had a consistent positive impact on the global evaluation of the NTs content. Our comparative studies also revealed differences between Flye and Canu assemblies regarding the annotation of unique versus repetitive genomic features. In our hands, Flye proved to be moderately better for gene identification, while Canu clearly outperformed Flye for NTs analysis. Data concerning the NTs content were compared to those obtained with ONT for the D. melanogaster ISO1 strain, revealing that our strategy conducted to better results. Additionally, the parameters of our ONT reads and assemblies are similar to those reported for ONT experiments performed on various model organisms, revealing that our assembly data are appropriate for a proficient annotation of the Horezu genome.

The Phylogenetic Position of the Enigmatic, Polypodium hydriforme (Cnidaria, Polypodiozoa): Insights from Mitochondrial Genomes.

Polypodium hydriforme is an enigmatic parasite that belongs to the phylum Cnidaria. Its taxonomic position has been debated: whereas it was previously suggested to be part of Medusozoa, recent phylogenomic analyses based on nuclear genes support the view that P. hydriforme and Myxozoa form a clade called Endocnidozoa. Medusozoans have linear mitochondrial (mt) chromosomes, whereas myxozoans, as most metazoan species, have circular chromosomes. In this work, we determined the structure of the mt genome of P. hydriforme, using Illumina and Oxford Nanopore Technologies reads, and showed that it is circular. This suggests that P. hydriforme is not nested within Medusozoa, as this would entail linearization followed by recirculation. Instead, our results support the view that P. hydriforme is a sister clade to Myxozoa, and mt linearization in the lineage leading to medusozoans occurred after the divergence of Myxozoa + P. hydriforme. Detailed analyses of the assembled P. hydriforme mt genome show that: (1) it is encoded on a single circular chromosome with an estimated size of ∼93,000 base pairs, making it one of the largest metazoan mt genomes; (2) around 78% of the genome encompasses a noncoding region composed of several repeat types; (3) similar to Myxozoa, no mt tRNAs were identified; (4) the codon TGA is a stop codon and does not encode for tryptophan as in other cnidarians; (5) similar to myxozoan mt genomes, it is extremely fast evolving.

Subtyping Evaluation of Salmonella Enteritidis Using Single Nucleotide Polymorphism and Core Genome Multilocus Sequence Typing with Nanopore Reads.

Whole-genome sequencing (WGS) for public health surveillance and epidemiological investigation of foodborne pathogens predominantly relies on sequencing platforms that generate short reads. Continuous improvement of long-read nanopore sequencing, such as Oxford nanopore technologies (ONT), presents a potential for leveraging multiple advantages of the technology in public health and food industry settings, including rapid turnaround and onsite applicability in addition to superior read length. Using an established cohort of Salmonella Enteritidis isolates for subtyping evaluation, we assessed the technical readiness of nanopore long read sequencing for single nucleotide polymorphism (SNP) analysis and core-genome multilocus sequence typing (cgMLST) of a major foodborne pathogen. By multiplexing three isolates per flow cell, we generated sufficient sequencing depths in <7 h of sequencing for robust subtyping. SNP calls by ONT and Illumina reads were highly concordant despite homopolymer errors in ONT reads (R9.4.1 chemistry). In silico correction of such errors allowed accurate allelic calling for cgMLST and allelic difference measurements to facilitate heuristic detection of outbreak isolates. IMPORTANCE Evaluation, standardization, and implementation of the ONT approach to WGS-based, strain-level subtyping is challenging, in part due to its relatively high base-calling error rates and frequent iterations of sequencing chemistry and bioinformatic analytics. Our study established a baseline for the continuously evolving nanopore technology as a viable solution to high-quality subtyping of Salmonella, delivering comparable subtyping performance when used standalone or together with short-read platforms. This study paves the way for evaluating and optimizing the logistics of implementing the ONT approach for foodborne pathogen surveillance in specific settings.

Dressing like a pathogen: Comparative analysis of different Pseudomonas genomospecies wearing different features to infect Corylus avellana

AbstractDuring the last decade, the European hazelnut (Corylus avellana) has been threatened by several bacterial and fungal infections. In particular, at least four different Pseudomonas species have developed the capability to converge into the same host. However, one of these species, Pseudomonas avellanae, may infect other hosts as well. Here, we newly provide the complete genome sequence of two Pseudomonas avellanae strains obtained by a combination of long Oxford Nanopore Technologies reads (ONT) and short Illumina reads. These have been included in a comparative analysis with all the genomes of Pseudomonas strains known to infect Corylus avellana available on the NCBI database, and the genomes of Pseudomonas avellanae strains infecting other host plants, like Prunus, Actinidia and from the river epilithon. The Pseudomonas strains clustered according to their taxonomy, host and geographic origins. The identification of specific traits related to each population cluster could give us insights regarding the host–pathogen interaction and the host range specificity. Indeed, the Pseudomonas avellanae strains infecting hazelnut have a peculiar set of three type III secretion effectors, while the Pseudomonas avellanae infecting Prunus and Actinidia are carrying the genomic WHOP island, necessary for woody‐host infection. The syringae cluster, instead, contains more phytotoxins, the ice nucleation cluster, but less effectors. Thus, here, we confirmed that the convergence into the same host was possible due to different unrelated mechanisms of infection.

Chasing perfection: validation and polishing strategies for telomere-to-telomere genome assemblies.

Advances in long-read sequencing technologies and genome assembly methods have enabled the recent completion of the first telomere-to-telomere human genome assembly, which resolves complex segmental duplications and large tandem repeats, including centromeric satellite arrays in a complete hydatidiform mole (CHM13). Although derived from highly accurate sequences, evaluation revealed evidence of small errors and structural misassemblies in the initial draft assembly. To correct these errors, we designed a new repeat-aware polishing strategy that made accurate assembly corrections in large repeats without overcorrection, ultimately fixing 51% of the existing errors and improving the assembly quality value from 70.2 to 73.9 measured from PacBio high-fidelity and Illumina k-mers. By comparing our results to standard automated polishing tools, we outline common polishing errors and offer practical suggestions for genome projects with limited resources. We also show how sequencing biases in both high-fidelity and Oxford Nanopore Technologies reads cause signature assembly errors that can be corrected with a diverse panel of sequencing technologies.

KOREF_S1: phased, parental trio-binned Korean reference genome using long reads and Hi-C sequencing methods.

BackgroundKOREF is the Korean reference genome, which was constructed with various sequencing technologies including long reads, short reads, and optical mapping methods. It is also the first East Asian multiomic reference genome accompanied by extensive clinical information, time-series and multiomic data, and parental sequencing data. However, it was still not a chromosome-scale reference. Here, we updated the previous KOREF assembly to a new chromosome-level haploid assembly of KOREF, KOREF_S1v2.1. Oxford Nanopore Technologies (ONT) PromethION, Pacific Biosciences HiFi-CCS, and Hi-C technology were used to build the most accurate East Asian reference assembled so far.ResultsWe produced 705 Gb ONT reads and 114 Gb Pacific Biosciences HiFi reads, and corrected ONT reads by Pacific Biosciences reads. The corrected ultra-long reads reached higher accuracy of 1.4% base errors than the previous KOREF_S1v1.0, which was mainly built with short reads. KOREF has parental genome information, and we successfully phased it using a trio-binning method, acquiring a near-complete haploid-assembly. The final assembly resulted in total length of 2.9 Gb with an N50 of 150 Mb, and the longest scaffold covered 97.3% of GRCh38’s chromosome 2. In addition, the final assembly showed high base accuracy, with <0.01% base errors.ConclusionsKOREF_S1v2.1 is the first chromosome-scale haploid assembly of the Korean reference genome with high contiguity and accuracy. Our study provides useful resources of the Korean reference genome and demonstrates a new strategy of hybrid assembly that combines ONT's PromethION and PacBio's HiFi-CCS.

Sequencing of individual barcoded cDNAs using Pacific Biosciences and Oxford Nanopore Technologies reveals platform-specific error patterns.

Long-read transcriptomics require understanding error sources inherent to technologies. Current approaches cannot compare methods for an individual RNA molecule. Here, we present a novel platform-comparison method that combines barcoding strategies and long-read sequencing to sequence cDNA copies representing an individual RNA molecule on both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). We compare these long-read pairs in terms of sequence content and isoform patterns. Although individual read pairs show high similarity, we find differences in (1) aligned length, (2) transcription start site (TSS), (3) polyadenylation site (poly(A)-site) assignment, and (4) exon–intron structures. Overall, 25% of read pairs disagree on either TSS, poly(A)-site, or splice site. Intron-chain disagreement typically arises from alignment errors of microexons and complicated splice sites. Our single-molecule technology comparison reveals that inconsistencies are often caused by sequencing error–induced inaccurate ONT alignments, especially to downstream GUNNGU donor motifs. However, annotation-disagreeing upstream shifts in NAGNAG acceptors in ONT are often confirmed by PacBio and are thus likely real. In both barcoded and nonbarcoded ONT reads, we find that intron number and proximity of GU/AGs better predict inconsistencies with the annotation than read quality alone. We summarize these findings in an annotation-based algorithm for spliced alignment correction that improves subsequent transcript construction with ONT reads.

Accurate profiling of forensic autosomal STRs using the Oxford Nanopore Technologies MinION device

The high variability characteristic of short tandem repeat (STR) markers is harnessed for human identification in forensic genetic analyses. Despite the power and reliability of current typing techniques, sequence-level information both within and around STRs are masked in the length-based profiles generated. Forensic STR typing using next generation sequencing (NGS) has therefore gained attention as an alternative to traditional capillary electrophoresis (CE) approaches. In this proof-of-principle study, we evaluate the forensic applicability of the newest and smallest NGS platform available – the Oxford Nanopore Technologies (ONT) MinION device. Although nanopore sequencing on the handheld MinION offers numerous advantages, including low startup cost and on-site sample processing, the relatively high error rate and lack of forensic-specific analysis software has prevented accurate profiling across STR panels in previous studies. Here we present STRspy, a streamlined method capable of producing length- and sequence-based STR allele designations from noisy, error-prone third generation sequencing reads. To assess the capabilities of STRspy, seven reference samples (female: n = 2; male: n = 5) were amplified at 15 and 30 PCR cycles using the Promega PowerSeq 46GY System and sequenced on the ONT MinION device in triplicate. Basecalled reads were then processed with STRspy using a custom database containing alleles reported in the STRSeq BioProject NIST 1036 dataset. Resultant STR allele designations and flanking region single nucleotide polymorphism (SNP) calls were compared to the manufacturer-validated genotypes for each sample. STRspy generated robust and reliable genotypes across all autosomal STR loci amplified with 30 PCR cycles, achieving 100% concordance based on both length and sequence. Furthermore, we were able to identify flanking region SNPs in the 15-cycle dataset with > 90% accuracy. These results demonstrate that when analyzed with STRspy ONT reads can reveal additional variation in and around STR loci depending on read coverage. As the first and only third generation sequencing platform-specific method to successfully profile the entire panel of autosomal STRs amplified by a commercially available multiplex, STRspy significantly increases the feasibility of nanopore sequencing in forensic applications.

Benchmarking Oxford Nanopore read assemblers for high-quality molluscan genomes.

Choosing the optimum assembly approach is essential to achieving a high-quality genome assembly suitable for comparative and evolutionary genomic investigations. Significant recent progress in long-read sequencing technologies such as PacBio and Oxford Nanopore Technologies (ONT) has also brought about a large variety of assemblers. Although these have been extensively tested on model species such as Homo sapiens and Drosophila melanogaster, such benchmarking has not been done in Mollusca, which lacks widely adopted model species. Molluscan genomes are notoriously rich in repeats and are often highly heterozygous, making their assembly challenging. Here, we benchmarked 10 assemblers based on ONT raw reads from two published molluscan genomes of differing properties, the gastropod Chrysomallon squamiferum (356.6 Mb, 1.59% heterozygosity) and the bivalve Mytilus coruscus (1593 Mb, 1.94% heterozygosity). By optimizing the assembly pipeline, we greatly improved both genomes from previously published versions. Our results suggested that 40-50X of ONT reads are sufficient for high-quality genomes, with Flye being the recommended assembler for compact and less heterozygous genomes exemplified by C. squamiferum, while NextDenovo excelled for more repetitive and heterozygous molluscan genomes exemplified by M. coruscus. A phylogenomic analysis using the two updated genomes with 32 other published high-quality lophotrochozoan genomes resulted in maximum support across all nodes, and we show that improved genome quality also leads to more complete matrices for phylogenomic inferences. Our benchmarking will ensure efficiency in future assemblies for molluscs and perhaps also for other marine phyla with few genomes available. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Comparison of different sequencing strategies for assembling chromosome-level genomes of extremophiles with variable GC content

SummaryIn this study, six bacterial isolates with variable GC, including Escherichia coli as mesophilic reference strain, were selected to compare hybrid assembly strategies based on next-generation sequencing (NGS) of short reads, single-tube long-fragment reads (stLFR) sequencing, and Oxford Nanopore Technologies (ONT) sequencing platforms. We obtained the complete genomes using the hybrid assembler Unicycler based on the NGS and ONT reads; others were de novo assembled using NGS, stLFR, and ONT reads by using different strategies. The contiguity, accuracy, completeness, sequencing costs, and DNA material requirements of the investigated strategies were compared systematically. Although all sequencing data could be assembled into accurate whole-genome sequences, the stLFR sequencing data yield a scaffold with more contiguity with more completeness of gene function than NGS sequencing assemblies. Our research provides a low-cost chromosome-level genome assembly strategy for large-scale sequencing of extremophile genomes with different GC contents.

Complete hybrid genome assembly of clinical multidrug-resistant Bacteroides fragilis isolates enables comprehensive identification of antimicrobial-resistance genes and plasmids.

Bacteroides fragilis constitutes a significant part of the normal human gut microbiota and can also act as an opportunistic pathogen. Antimicrobial resistance (AMR) and the prevalence of AMR genes are increasing, and prediction of antimicrobial susceptibility based on sequence information could support targeted antimicrobial therapy in a clinical setting. Complete identification of insertion sequence (IS) elements carrying promoter sequences upstream of resistance genes is necessary for prediction of AMR. However, de novo assemblies from short reads alone are often fractured due to repeat regions and the presence of multiple copies of identical IS elements. Identification of plasmids in clinical isolates can aid in the surveillance of the dissemination of AMR, and comprehensive sequence databases support microbiome and metagenomic studies. We tested several short-read, hybrid and long-lead assembly pipelines by assembling the type strain B. fragilis CCUG4856T (=ATCC25285=NCTC9343) with Illumina short reads and long reads generated by Oxford Nanopore Technologies (ONT) MinION sequencing. Hybrid assembly with Unicycler, using quality filtered Illumina reads and Filtlong filtered and Canu-corrected ONT reads, produced the assembly of highest quality. This approach was then applied to six clinical multidrug-resistant B. fragilis isolates and, with minimal manual finishing of chromosomal assemblies of three isolates, complete, circular assemblies of all isolates were produced. Eleven circular, putative plasmids were identified in the six assemblies, of which only three corresponded to a known cultured Bacteroides plasmid. Complete IS elements could be identified upstream of AMR genes; however, there was not complete correlation between the absence of IS elements and antimicrobial susceptibility. As our knowledge on factors that increase expression of resistance genes in the absence of IS elements is limited, further research is needed prior to implementing AMR prediction for B. fragilis from whole-genome sequencing.

Comparison of long-read sequencing technologies in the hybrid assembly of complex bacterial genomes.

Illumina sequencing allows rapid, cheap and accurate whole genome bacterial analyses, but short reads (<300 bp) do not usually enable complete genome assembly. Long-read sequencing greatly assists with resolving complex bacterial genomes, particularly when combined with short-read Illumina data (hybrid assembly). However, it is not clear how different long-read sequencing methods affect hybrid assembly accuracy. Relative automation of the assembly process is also crucial to facilitating high-throughput complete bacterial genome reconstruction, avoiding multiple bespoke filtering and data manipulation steps. In this study, we compared hybrid assemblies for 20 bacterial isolates, including two reference strains, using Illumina sequencing and long reads from either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio) sequencing platforms. We chose isolates from the family Enterobacteriaceae, as these frequently have highly plastic, repetitive genetic structures, and complete genome reconstruction for these species is relevant for a precise understanding of the epidemiology of antimicrobial resistance. We de novo assembled genomes using the hybrid assembler Unicycler and compared different read processing strategies, as well as comparing to long-read-only assembly with Flye followed by short-read polishing with Pilon. Hybrid assembly with either PacBio or ONT reads facilitated high-quality genome reconstruction, and was superior to the long-read assembly and polishing approach evaluated with respect to accuracy and completeness. Combining ONT and Illumina reads fully resolved most genomes without additional manual steps, and at a lower consumables cost per isolate in our setting. Automated hybrid assembly is a powerful tool for complete and accurate bacterial genome assembly.

Whole-genome sequencing and analysis of the Chinese herbal plant Gelsemium elegans

BackgroundGelsemium elegans (G. elegans) (2n = 2x = 16) is genus of flowering plants belonging to the Gelsemicaeae family. MethodHere, a high-quality genome assembly using the Oxford Nanopore Technologies (ONT) platform and high-throughput chromosome conformation capture techniques (Hi-C) were used. ResultsA total of 56.11 Gb of raw GridION X5 platform ONT reads (6.23 Gb per cell) were generated. After filtering, 53.45 Gb of clean reads were obtained, giving 160 × coverage depth. The de novo genome assemblies 335.13 Mb, close to the 338 Mb estimated by k-mer analysis, was generated with contig N50 of 10.23 Mb. The vast majority (99.2%) of the G. elegans assembled sequence was anchored onto 8 pseudo-chromosomes. The genome completeness was then evaluated and 1338 of the 1440 conserved genes (92.9%) could be found in the assembly. Genome annotation revealed that 43.16% of the G. elegans genome is composed of repetitive elements and 23.9% is composed of long terminal repeat elements. We predicted 26,768 protein-coding genes, of which 84.56% were functionally annotated. ConclusionThe genomic sequences of G. elegans could be a valuable source for comparative genomic analysis in the Gelsemicaeae family and will be useful for understanding the phylogenetic relationships of the indole alkaloid metabolism.

Completing bacterial genome assemblies with multiplex MinION sequencing.

Illumina sequencing platforms have enabled widespread bacterial whole genome sequencing. While Illumina data is appropriate for many analyses, its short read length limits its ability to resolve genomic structure. This has major implications for tracking the spread of mobile genetic elements, including those which carry antimicrobial resistance determinants. Fully resolving a bacterial genome requires long-read sequencing such as those generated by Oxford Nanopore Technologies (ONT) platforms. Here we describe our use of the ONT MinION to sequence 12 isolates of Klebsiella pneumoniae on a single flow cell. We assembled each genome using a combination of ONT reads and previously available Illumina reads, and little to no manual intervention was needed to achieve fully resolved assemblies using the Unicycler hybrid assembler. Assembling only ONT reads with Canu was less effective, resulting in fewer resolved genomes and higher error rates even following error correction with Nanopolish. We demonstrate that multiplexed ONT sequencing is a valuable tool for high-throughput bacterial genome finishing. Specifically, we advocate the use of Illumina sequencing as a first analysis step, followed by ONT reads as needed to resolve genomic structure.