High-quality metagenome assembly from long accurate reads with metaMDBG

Nature Biotechnology, Journal Year: 2024, Volume and Issue: 42(9), P. 1378 - 1383

Published: Jan. 2, 2024

Abstract We introduce metaMDBG, a metagenomics assembler for PacBio HiFi reads. MetaMDBG combines de Bruijn graph assembly in minimizer space with an iterative over sequences of minimizers to address variations genome coverage depth and abundance-based filtering strategy simplify strain complexity. For complex communities, we obtained up twice as many high-quality circularized prokaryotic metagenome-assembled genomes existing methods had better recovery viruses plasmids.

Language: Английский

---

Antibiotic resistance: A key microbial survival mechanism that threatens public health

Cell Host & Microbe, Journal Year: 2024, Volume and Issue: 32(6), P. 837 - 851

Published: June 1, 2024

Language: Английский

---

Rapid genomic sequencing for genetic disease diagnosis and therapy in intensive care units: a review

npj Genomic Medicine, Journal Year: 2024, Volume and Issue: 9(1)

Published: Feb. 27, 2024

Abstract Single locus (Mendelian) diseases are a leading cause of childhood hospitalization, intensive care unit (ICU) admission, mortality, and healthcare cost. Rapid genome sequencing (RGS), ultra-rapid (URGS), rapid exome (RES) diagnostic tests for genetic ICU patients. In 44 studies children in ICUs with unknown etiology, 37% received diagnosis, 26% had consequent changes management, net costs were reduced by $14,265 per child tested URGS, RGS, or RES. URGS outperformed RGS RES faster time to higher rate diagnosis clinical utility. Diagnostic outcomes will improve as methods evolve, decrease, testing is implemented within precision medicine delivery systems attuned needs. currently performed <5% the ~200,000 likely benefit annually due lack payor coverage, inadequate reimbursement, hospital policies, hospitalist unfamiliarity, under-recognition possible diseases, current formatting rather than system. The gap between actual optimal increasing since expanded use lags growth those through new therapies. There sufficient evidence conclude that should be considered all uncertain etiology at admission. Minimally, ordered early during admissions critically ill infants suspected diseases.

Language: Английский

---

Sequencing accuracy and systematic errors of nanopore direct RNA sequencing

BMC Genomics, Journal Year: 2024, Volume and Issue: 25(1)

Published: May 28, 2024

Abstract Background Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about base modifications and poly-A tail lengths. Although many published studies have been expanding potential of dRNA-seq, its accuracy error patterns remain understudied. Results We present first comprehensive evaluation characterisation systematic errors in dRNA-seq data from diverse organisms synthetic vitro transcribed RNAs. found that for kits SQK-RNA001 SQK-RNA002, median read ranged 87% 92% across species, deletions significantly outnumbered mismatches insertions. Due their high abundance transcriptome, heteropolymers short homopolymers were major contributors overall errors. also observed biases all species at levels single nucleotides motifs. In general, cytosine/uracil-rich regions more likely be erroneous than guanines adenines. By examining raw signal data, we identified underlying signal-level features potentially associated with dependency sequence contexts. While quality scores used approximate rates levels, failure detect DNA adapters may a source loss. comparing distinct basecallers, reason some are attributable insufficiency rather algorithmic (basecalling) artefacts. Lastly, generated using latest SQK-RNA004 kit released end 2023 although increased, largely identical compared previous kits. Conclusions As investigation errors, this study offers overview reproducible datasets, identifies insufficiency, lays foundation correction methods.

Language: Английский

---

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

---

Hybracter: enabling scalable, automated, complete and accurate bacterial genome assemblies

Microbial Genomics, Journal Year: 2024, Volume and Issue: 10(5)

Published: May 8, 2024

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- long-reads) assembly approaches. Complete allow a deeper understanding evolution genomic variation beyond single nucleotide variants. They also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance genes. However, small plasmids missed or misassembled by algorithms. Here, we present Hybracter allows fast, automatic scalable recovery near-perfect first approach. can be run either as assembler only assembler. We compared to existing automated tools diverse panel samples varying levels with manually curated ground truth reference genomes. demonstrate is more accurate faster than gold standard Unicycler. show long-reads most comparable methods accurately recovering plasmids.

Language: Английский

---

Do we still need Illumina sequencing data? Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and the Rapid v14 library prep kit for Gram negative bacteria whole genome assemblies

Canadian Journal of Microbiology, Journal Year: 2024, Volume and Issue: 70(5), P. 178 - 189

Published: Feb. 14, 2024

The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read that can bridge repetitive problematic regions. Oxford Nanopore Technologies (ONT) produce platforms they continually improving their technology to obtain higher quality read is approaching the obtained such as Illumina. As these innovations continue, we evaluated how much ONT coverage produced by Rapid Barcoding Kit v14 (SQK-RBK114) necessary generate high-quality hybrid long-read-only for panel carbapenemase-producing Enterobacterales bacterial isolates. We found 30× sufficient if Illumina available, more (at least 100× recommended assemblies. polishing still single nucleotide variants (SNVs) INDELs in also examined antimicrobial resistance genes could be accurately identified data, Flye regardless detected >96% at 100% identity length. Overall, an optimal strategy (i.e., plasmid characterization AMR detection) but finer-scale analyses SNV) benefit data.

Language: Английский

---

The long and short of it: benchmarking viromics using Illumina, Nanopore and PacBio sequencing technologies

Microbial Genomics, Journal Year: 2024, Volume and Issue: 10(2)

Published: Feb. 20, 2024

Viral metagenomics has fuelled a rapid change in our understanding of global viral diversity and ecology. Long-read sequencing hybrid assembly approaches that combine long- short-read technologies are now being widely implemented bacterial genomics metagenomics. However, the use long-read to investigate communities is still its infancy. While Nanopore PacBio have been applied metagenomics, it not known what extent different will impact reconstruction community. Thus, we constructed mock bacteriophage community previously sequenced phage genomes them using Illumina, tested number approaches. When single technology, Illumina assemblies were best at recovering genomes. Nanopore- PacBio-only performed poorly comparison both genome recovery error rates, which varied with assembler used. The had errors manifested as SNPs INDELs frequencies 41 157 % higher than found only assemblies, respectively. 12 78 Illumina-only Despite high-read coverage, long-read-only recovered maximum one complete from any assembly, unless reads down-sampled prior assembly. Overall approach was by combination reads, reduced rates levels comparable short-read-only assemblies. approach. differences between technology downstream impacts on gene prediction, subsequent estimates within sample. These findings provide starting point for others choice algorithms analysis viromes.

Language: Английский

---

Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing

Microbial Genomics, Journal Year: 2024, Volume and Issue: 10(5)

Published: May 7, 2024

Whole-genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate complete assemblies have largely only historically been achievable using hybrid long- short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved over R9.4.1/kit10 combination, however long-read contained more errors compared to Illumina-ONT assemblies. ONT since released R10.4.1/kit14 upgrade recommended use Bovine Serum Albumin (BSA) during library preparation, both which reportedly increase accuracy yield. They also updated basecallers trained native DNA containing methylation sites intended fix systematic basecalling errors, including common adenosine (A) guanine (G) cytosine (C) thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four reference strains, namely Escherichia coli , Klebsiella pneumoniae Pseudomonas aeruginosa Staphylococcus aureus nine genetically diverse E. bloodstream infection-associated isolates from different phylogroups sequence types, with without BSA. These sequences were de novo assembled against Illumina-corrected genomes. In this small evaluation 13 that nanopore long-read-only R10.4.1/kit 14 methylated produce ≥40×depth, sufficient be cost-effective ONT/Illumina sequencing in our setting.

Language: Английский

---

Nanopore sequencing: flourishing in its teenage years

Journal of genetics and genomics/Journal of Genetics and Genomics, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 1, 2024

Language: Английский

---