Trycycler: consensus long-read assemblies for bacterial genomes.
Ontology highlight
ABSTRACT:
SUBMITTER: Wick RR
PROVIDER: S-EPMC8442456 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Long-read-only bacterial genome assemblies usually contain residual errors, most commonly homopolymer-length errors. Short-read polishing tools can use short reads to fix these errors, but most rely on short-read alignment which is unreliable in repeat regions. Errors in such regions are therefore challenging to fix and often remain after short-read polishing. Here we introduce Polypolish, a new short-read polisher which uses all-per-read alignments to repair errors in repeat sequences that other polishers cannot. Polypolish performed well in benchmarking tests using both simulated and real reads, and it almost never introduced errors during polishing. The best results were achieved by using Polypolish in combination with other short-read polishers.
| S-EPMC8812927 | biostudies-literature
Project description:DNA assembly is a core methodological step in metagenomic pipelines used to study the structure and function within microbial communities. Here we investigate the utility of Pacific Biosciences long and high accuracy circular consensus sequencing (CCS) reads for metagenomic projects. We compared the application and performance of both PacBio CCS and Illumina HiSeq data with assembly and taxonomic binning algorithms using metagenomic samples representing a complex microbial community. Eight SMRT cells produced approximately 94 Mb of CCS reads from a biogas reactor microbiome sample that averaged 1319 nt in length and 99.7% accuracy. CCS data assembly generated a comparative number of large contigs greater than 1 kb, to those assembled from a ~190x larger HiSeq dataset (~18 Gb) produced from the same sample (i.e approximately 62% of total contigs). Hybrid assemblies using PacBio CCS and HiSeq contigs produced improvements in assembly statistics, including an increase in the average contig length and number of large contigs. The incorporation of CCS data produced significant enhancements in taxonomic binning and genome reconstruction of two dominant phylotypes, which assembled and binned poorly using HiSeq data alone. Collectively these results illustrate the value of PacBio CCS reads in certain metagenomics applications.
| S-EPMC4860591 | biostudies-literature
Project description: Not available
| S-EPMC11007910 | biostudies-literature
Project description:BackgroundThe recent introduction of the Pacific Biosciences RS single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. The long read sequence information is promised to enhance the quality of incomplete and inaccurate draft assemblies constructed from Next Generation Sequencing (NGS) data.ResultsHere we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using PacBio RS long read information as a backbone. On a test set comprising six bacterial draft genomes, assembled using either a single Illumina MiSeq or Roche 454 library, we show that even a 50× coverage of uncorrected PacBio RS long reads is sufficient to drastically reduce the number of contigs. Comparisons to the AHA scaffolder indicate our strategy is better capable of producing (nearly) complete bacterial genomes.ConclusionsThe current work describes our SSPACE-LongRead software which is designed to upgrade incomplete draft genomes using single molecule sequences. We conclude that the recent advances of the PacBio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner.
| S-EPMC4076250 | biostudies-literature
Project description:BackgroundLong-read sequencing in metagenomics facilitates the assembly of complete genomes out of complex microbial communities. These genomes include essential biologic information such as the ribosomal genes or the mobile genetic elements, which are usually missed with short-reads. We applied long-read metagenomics with Nanopore sequencing to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a dog fecal sample.ResultsWe used nanopore long-read metagenomics and frameshift aware correction on a canine fecal sample and retrieved eight single-contig HQ MAGs, which were > 90% complete with < 5% contamination, and contained most ribosomal genes and tRNAs. At the technical level, we demonstrated that a high-molecular-weight DNA extraction improved the metagenomics assembly contiguity, the recovery of the rRNA operons, and the retrieval of longer and circular contigs that are potential HQ MAGs. These HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Catenibacterium, Blautia, and Enterococcus genera. Linking our results to previous gastrointestinal microbiome reports (metagenome or 16S rRNA-based), we found that some bacterial species on the gastrointestinal tract seem to be more canid-specific -Succinivibrio, Prevotellamassilia, Phascolarctobacterium, Blautia_A sp900541345-, whereas others are more broadly distributed among animal and human microbiomes -Sutterella, Catenibacterium, Enterococcus, and Blautia sp003287895. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Moreover, we show that long reads are essential to detect mobilome functions, usually missed in short-read MAGs.ConclusionsWe recovered eight single-contig HQ MAGs from canine feces of a healthy dog with nanopore long-reads. We also retrieved relevant biological insights from these specific bacterial species previously missed in public databases, such as complete ribosomal operons and mobilome functions. The high-molecular-weight DNA extraction improved the assembly's contiguity, whereas the high-accuracy basecalling, the raw read error correction, the assembly polishing, and the frameshift correction reduced the insertion and deletion errors. Both experimental and analytical steps ensured the retrieval of complete bacterial genomes.
| S-EPMC8103633 | biostudies-literature
Project description:BackgroundOxford Nanopore provides high throughput sequencing platforms able to reconstruct complete bacterial genomes with 99.95% accuracy. However, even small levels of error can obscure the phylogenetic relationships between closely related isolates. Polishing tools have been developed to correct these errors, but it is uncertain if they obtain the accuracy needed for the high-resolution source tracking of foodborne illness outbreaks.ResultsWe tested 132 combinations of assembly and short- and long-read polishing tools to assess their accuracy for reconstructing the genome sequences of 15 highly similar Salmonella enterica serovar Newport isolates from a 2020 onion outbreak. While long-read polishing alone improved accuracy, near perfect accuracy (99.9999% accuracy or ~ 5 nucleotide errors across the 4.8 Mbp genome, excluding low confidence regions) was only obtained by pipelines that combined both long- and short-read polishing tools. Notably, medaka was a more accurate and efficient long-read polisher than Racon. Among short-read polishers, NextPolish showed the highest accuracy, but Pilon, Polypolish, and POLCA performed similarly. Among the 5 best performing pipelines, polishing with medaka followed by NextPolish was the most common combination. Importantly, the order of polishing tools mattered i.e., using less accurate tools after more accurate ones introduced errors. Indels in homopolymers and repetitive regions, where the short reads could not be uniquely mapped, remained the most challenging errors to correct.ConclusionsShort reads are still needed to correct errors in nanopore sequenced assemblies to obtain the accuracy required for source tracking investigations. Our granular assessment of the performance of the polishing pipelines allowed us to suggest best practices for tool users and areas for improvement for tool developers.
| S-EPMC11232133 | biostudies-literature
Project description:BackgroundLong read technologies have revolutionized de novo genome assembly by generating contigs orders of magnitude longer than that of short read assemblies. Although assembly contiguity has increased, it usually does not reconstruct a full chromosome or an arm of the chromosome, resulting in an unfinished chromosome level assembly. To increase the contiguity of the assembly to the chromosome level, different strategies are used which exploit long range contact information between chromosomes in the genome.MethodsWe develop a scalable and computationally efficient scaffolding method that can boost the assembly contiguity to a large extent using genome-wide chromatin interaction data such as Hi-C.Resultswe demonstrate an algorithm that uses Hi-C data for longer-range scaffolding of de novo long read genome assemblies. We tested our methods on the human and goat genome assemblies. We compare our scaffolds with the scaffolds generated by LACHESIS based on various metrics.ConclusionOur new algorithm SALSA produces more accurate scaffolds compared to the existing state of the art method LACHESIS.
| S-EPMC5508778 | biostudies-literature
Project description:IntroductionGenome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time.MethodsIn this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%.Results and discussionFor a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.
| S-EPMC10225699 | biostudies-literature
Project description:Cassava genomes assembled with single-molecule long reads, optical and Hi-C maps reveal narrow genetic diversity and mono-allelic expression
| PRJEB27129 | ENA
Project description: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.
| S-EPMC6807382 | biostudies-literature