Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing

PLoS Computational Biology, Journal Year: 2023, Volume and Issue: 19(3), P. e1010905 - e1010905

Published: March 2, 2023

A perfect bacterial genome assembly is one where the assembled sequence an exact match for organism’s genome—each replicon complete and contains no errors. While this has been difficult to achieve in past, improvements long-read sequencing, assemblers, polishers have brought assemblies within reach. Here, we describe our recommended approach assembling a perfection using combination of Oxford Nanopore Technologies long reads Illumina short reads: Trycycler assembly, Medaka polishing, Polypolish short-read followed by other polishing tools manual curation. We also discuss potential pitfalls might encounter when challenging genomes, provide online tutorial with sample data ( github.com/rrwick/perfect-bacterial-genome-tutorial ).

Language: Английский

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Genome assembly in the telomere-to-telomere era

Nature Reviews Genetics, Journal Year: 2024, Volume and Issue: 25(9), P. 658 - 670

Published: April 22, 2024

Language: Английский

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How low can you go? Short-read polishing of Oxford Nanopore bacterial genome assemblies

Microbial Genomics, Journal Year: 2024, Volume and Issue: 10(6)

Published: June 4, 2024

It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing usually required for perfection. However, the effect of depth on performance not well understood. Here, we introduce Pypolca (with default and careful parameters) Polypolish v0.6.0 a new parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default Polypolish-careful commonly false-positive errors at low read depth; (2) most benefit occurs by 25× (3) almost never introduces any (4) Pypolca-careful single effective polisher. Overall, recommend following strategies: alone when very (<5×), (5–25×), sufficient (>25×).

Language: Английский

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Plastid Genome Assembly Using Long‐read data

Molecular Ecology Resources, Journal Year: 2023, Volume and Issue: 23(6), P. 1442 - 1457

Published: March 20, 2023

Although plastid genome (plastome) structure is highly conserved across most seed plants, investigations during the past two decades have revealed several disparately related lineages that experienced substantial rearrangements. Most plastomes contain a large inverted repeat and single-copy regions, few dispersed repeats; however, of some taxa harbour long sequences (>300 bp). These repeats make it challenging to assemble complete using short-read data, leading misassemblies consensus with spurious Single-molecule, long-read sequencing has potential overcome these challenges, yet there no on effective method for accurately assembling data. We generated pipeline, Genome Assembly Using Long-read data (ptGAUL), address problem plastome assembly from Oxford Nanopore Technologies (ONT) or Pacific Biosciences platforms. demonstrated efficacy ptGAUL pipeline 16 published sets. showed quickly produces accurate unbiased assemblies only ~50× coverage Additionally, we deployed four new Juncus (Juncaceae) ONT reads. Our results many rearrangements in compared basal Poales. The available GitHub: https://github.com/Bean061/ptgaul.

Language: Английский

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How low can you go? Short-read polishing of Oxford Nanopore bacterial genome assemblies

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 10, 2024

Abstract It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing still required for perfection. However, the effect of depth on performance not well understood. Here, we introduce Pypolca (with default and careful parameters) Polypolish v0.6.0 a new parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default Polypolish-careful commonly false-positive errors at low depth; (2) most benefit occurs by 25× (3) never introduces any (4) Pypolca-careful single effective polisher. Overall, recommend following strategies: alone when very (<5×), (5–25×), sufficient (>25×). Data Summary open-source freely available Bioconda, PyPI, GitHub ( github.com/gbouras13/pypolca ). Bioconda github.com/rrwick/Polypolish All code data reproduce analyses figures are github.com/gbouras13/depth_vs_polishing_analysis . FASTQ sequencing reads BioProject PRJNA1042815 A detailed list accessions can be found in Table S1.

Language: Английский

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Chromosome-level genome assemblies of Nicotiana tabacum, Nicotiana sylvestris, and Nicotiana tomentosiformis

Scientific Data, Journal Year: 2024, Volume and Issue: 11(1)

Published: Jan. 26, 2024

Abstract The Solanaceae species Nicotiana tabacum , an economically important crop plant cultivated worldwide, is allotetraploid that appeared about 200,000 years ago as the result of hybridization diploid ancestors sylvestris and tomentosiformis . previously published genome assemblies for these three relied primarily on short-reads, obtained pseudochromosomes only partially covered genomes. In this study, we generated annotated de novo chromosome-level genomes N. which contain 3.99 Gb, 2.32 1.74 respectively sequence data, with 97.6%, 99.5%, 95.9% aligned in chromosomes, represent 99.2%, 98.3%, 98.5% near-universal single-copy orthologs genes. completion levels are comparable to other reference genomes, enabling more efficient synteny-based cross-species research.

Language: Английский

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The mitochondrial genome of Carex pseudochinensis H. Lév. & Vaniot, an endemic sedge in Korea

Mitochondrial DNA Part B, Journal Year: 2025, Volume and Issue: 10(1), P. 88 - 93

Published: Jan. 2, 2025

Carex pseudochinensis H. Lév. & Vaniot is an endemic species in Korea and included the clade of section Paludosae recent classification system. We present complete mitochondrial genome sequence C. based on POLAP pipeline with both long- short-read sequences. The 997,628 bp length, containing two large regions 536.94 419.04 kbp, respectively, a pair direct repeat about 20.25 kbp. contains 57 genes, including 31 protein-coding 20 tRNAs, 6 rRNAs. Phylogenetic analysis proteomes, those from ten related taxa, confirmed close phylogenetic relationship between breviculmis pseudochinensis.

Language: Английский

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Comparative Genomics Points to Ecological Drivers of Genomic Divergence Among Intertidal Limpets

Molecular Ecology Resources, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 31, 2025

ABSTRACT Comparative genomic studies of closely related taxa are important for our understanding the causes divergence on a changing Earth. This being said, resources available marine intertidal molluscs limited and currently, there few publicly high‐quality annotated genomes species in general. Here we report transcriptome assemblies six Patellogastropoda genome annotations three these ( Scurria scurra , viridula zebrina ). analysis using suggest that recently diverging lineages (10–20 Mya) have experienced similar amounts contractions expansions but across different gene families. Furthermore, differences among diverged reflected variation amount coding noncoding material genomes, such as repetitive elements lengths transcripts introns exons. Additionally, functional ontologies species‐specific duplicated genes together with demographic inference support finding recent members genus aligns their unique ecological characteristics. Overall, presented here will be valuable future adaptation habitats whole.

Language: Английский

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Discovery of a novel fungal Tc toxin complex and a functional myco- serpin via a unique two-by-two comparative genomics pipeline

Research Square (Research Square), Journal Year: 2025, Volume and Issue: unknown

Published: March 21, 2025

Background Fungi have been a rich source of pharmaceuticals such as antibiotics, immunosuppressants, and cholesterol-lowering drugs; however, their therapeutic potential remains largely untapped due to difficulties in culturing elucidating the genetic basis beneficial traits. contain 'cryptic' genes that are expressed under certain, often obscure, growth conditions can produce complex compounds difficult synthesize economically. Developments genome sequencing DNA-synthesis technologies offer new opportunities using biotechnological techniques, accurately identifying useful novel genes, prerequisite for approaches, challenging. Results We present ‘two-by-two’ comparative genomics pipeline comprehensive gene analysis selected fungal groups, enabling more confident identification unique across analyzed species. The approach compares sets from two strains same species with those different or families within order. Self-clustering orthologs provide higher confidence species-specific proteins help reduce noise low-quality assemblies prediction errors. validated our method on well-studied group fungi, discovering first functional myco-serpin an undescribed Tc toxin complex. Using knockout approach, we implicated both proteins’ roles insect host infection process this entomopathogenic Conclusions Elucidating underlying traits fungi presents significant challenges, relatively aspects lifestyles. Our two-by-two offers broad applications mining bioprospecting exemplified study by discovery myco-serpin. identified high sequence identity serpin other pathogenic strains, including known infect humans. Furthermore, be adapted organisms architectures similar fungi.

Language: Английский

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Kastor: a reference-based comparative approach for assessment and correction of gene-fragmenting errors in long-read assemblies of small genomes

BMC Genomics, Journal Year: 2025, Volume and Issue: 26(1)

Published: April 18, 2025

Abstract Long read sequencing technologies provide an efficient approach to generating highly contiguous and informative assemblies. However, higher relative error rates can introduce frameshifts premature stop codons that pseudogenize genes, hindering downstream analyses. We developed a software tool detects gene-fragmenting errors in draft assemblies of small genomes through comparison with curated set reference genome sequences raw information. In our presented example, detected represent less than 0.05% the genome, but when corrected reduced rate pseudogenes from 23.3 5.6% example long assemblies, comparable short demonstrate this detect assembly generated correct them de-fragment genes.

Language: Английский

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