Nanopore Sequencing Is a Credible Alternative to Recover Complete Genomes of Geminiviruses.
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ABSTRACT:
SUBMITTER: Ben Chehida S
PROVIDER: S-EPMC8147096 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Obtaining complete, high-quality reference genomes is essential to the study of any organism. Recent advances in nanopore sequencing, as well as genome assembly and analysis methods, have made it possible to obtain complete bacterial genomes from metagenomic (i.e., multispecies) samples, including those from the human microbiome. In this study, methods are presented to obtain complete bacterial genomes from human saliva using complementary Oxford Nanopore (ONT) and Illumina sequencing. Applied to 3 human saliva samples, these methods resulted in 11 complete bacterial genomes: 3 Saccharibacteria clade G6 (also known as Ca. Nanogingivalaceae HMT-870), 1 Saccharibacteria clade G1 HMT-348, 2 Rothia mucilaginosa, 2 Actinomyces graevenitzii, 1 Mogibacterium diversum, 1 Lachnospiraceae HMT-096, and 1 Lancefieldella parvula; and one circular chromosome of Ruminococcaceae HMT-075 (which likely has at least 2 chromosomes). The 4 Saccharibacteria genomes, as well as the Actinomyces graeventizii genomes, represented the first complete genomes from their respective bacterial taxa. Aside from the complete genomes, the assemblies contained 147 contigs of over 500,000 bp each and thousands of smaller contigs, together representing a myriad of additional draft genomes including many which are likely nearly complete. The complete genomes enabled highly accurate pangenome analysis, which identified unique and missing features of each genome compared to its closest relatives with complete genomes available in public repositories. These features provide clues as to the lifestyle and ecological role of these bacteria within the human oral microbiota, which will be particularly useful in designing future studies of the taxa that have never been isolated or cultivated. IMPORTANCE Obtaining complete and accurate genomes is crucial to the study of any organism. Previously, obtaining complete genomes of bacteria, including those of the human microbiome, frequently required isolation of the organism, as well as low-throughput, manual sequencing methods to resolve repeat regions. Advancements in long-read sequencing technologies, including Oxford Nanopore (ONT), have made it possible to obtain complete, closed bacterial genomes from metagenomic samples. This study reports methods to obtain complete genomes from the human oral microbiome using complementary ONT and Illumina sequencing of saliva samples. Eleven complete genomes were obtained from 3 human saliva samples, with genomes of Saccharibacteria HMT-870, Saccharibacteria HMT-348, and Actinomyces graeventzii being the first complete genomes from their respective taxa. Obtaining complete bacterial genomes in a high-throughput manner will help illuminate the metabolic and ecological roles of important members of the human microbiota, particularly those that have remained recalcitrant to isolation and cultivation.
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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.
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Project description:Whole-genome sequencing (WGS) is becoming widely used in clinical medicine in diagnostic contexts and to inform treatment choice. Here we evaluate the potential of the Oxford Nanopore Technologies (ONT) MinION long-read sequencer for routine WGS by sequencing the reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome and severe immune dysregulation. We develop and apply a novel reference panel-free analytical method to infer and then exploit phase information which improves single-nucleotide variant (SNV) calling performance from otherwise modest levels. In the clinical sample, we identify and directly phase two non-synonymous de novo variants in SAMD9L, (OMIM #159550) inferring that they lie on the same paternal haplotype. Whilst consensus SNV-calling error rates from ONT data remain substantially higher than those from short-read methods, we demonstrate the substantial benefits of analytical innovation. Ongoing improvements to base-calling and SNV-calling methodology must continue for nanopore sequencing to establish itself as a primary method for clinical WGS.
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Project description:Mitochondrial DNA sequence data are often utilized in disease studies, conservation genetics and forensic identification. The current approaches for sequencing the full mtGenome typically require several rounds of PCR enrichment during Sanger or MPS protocols followed by fairly tedious assembly and analysis. Here we describe an efficient approach to sequencing directly from genomic DNA samples without prior enrichment or extensive library preparation steps. A comparison is made between libraries sequenced directly from native DNA and the same samples sequenced from libraries generated with nine overlapping mtDNA amplicons on the Oxford Nanopore MinION™ device. The native and amplicon library preparation methods and alternative base calling strategies were assessed to establish error rates and identify trends of discordance between the two library preparation approaches. For the complete mtGenome, 16 569 nucleotides, an overall error rate of approximately 1.00% was observed. As expected with mtDNA, the majority of error was detected in homopolymeric regions. The use of a modified basecaller that corrects for ambiguous signal in homopolymeric stretches reduced the error rate for both library preparation methods to approximately 0.30%. Our study indicates that direct mtDNA sequencing from native DNA on the MinION™ device provides comparable results to those obtained from common mtDNA sequencing methods and is a reliable alternative to approaches using PCR-enriched libraries.
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Project description:High-throughput sequencing has enabled genome skimming approaches to produce complete mitochondrial genomes (mitogenomes) for species identification and phylogenomics purposes. In particular, the portable sequencing device from Oxford Nanopore Technologies (ONT) has the potential to facilitate hands-on training from sampling to sequencing and interpretation of mitogenomes. In this study, we present the results from sampling and sequencing of six gastropod mitogenomes (Aplysia argus, Cellana orientalis, Cellana toreuma, Conus ebraeus, Conus miles and Tylothais aculeata) from a graduate level biodiversity course. The students were able to produce mitogenomes from sampling to annotation using existing protocols and programs. Approximately 4 Gb of sequence was produced from 16 Flongle and one MinION flow cells, averaging 235 Mb and N50 = 4.4 kb per flow cell. Five of the six 14.1-18 kb mitogenomes were circlised containing all 13 core protein coding genes. Additional Illumina sequencing revealed that the ONT assemblies spanned over highly AT rich sequences in the control region that were otherwise missing in Illumina-assembled mitogenomes, but still contained a base error of one every 70.8-346.7 bp under the fast mode basecalling with the majority occurring at homopolymer regions. Our findings suggest that the portable MinION device can be used to rapidly produce low-cost mitogenomes onsite and tailored to genomics-based training in biodiversity research.
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Project description:Recombinant adeno-associated viruses (rAAVs) are currently the most prominently investigated vector platform for human gene therapy. The rAAV capsid serves as a potent and efficient vehicle for delivering genetic payloads into the host cell, while the vector genome determines the function and effectiveness of these biotherapies. However, current production schemes yield vectors that may consist of heterogeneous populations, compromising their potencies. The development of next-generation sequencing methods within the past few years have helped investigators profile the diversity and relative abundances of heterogenous species in vector preparations. Specifically, long-read sequencing methods, like single molecule real-time (SMRT) sequencing, have been used to uncover truncations, chimeric genomes, and inverted terminal repeat (ITR) mutations in vectors. Unfortunately, these sequencing platforms may be inaccessible to investigators with limited resources, require large amounts of input material, or may require long wait times for sequencing and analyses. Recent advances with nanopore sequencing have helped to bridge the gap for quick and relatively inexpensive long-read sequencing needs. However, their limitations and sample biases are not well-defined for sequencing rAAV. In this study, we explored the capacity for nanopore sequencing to directly interrogate rAAV content to obtain full-length resolution of encapsidated genomes. We found that the nanopore platform can cover the entirety of rAAV genomes from ITR to ITR without the need for pre-fragmentation. However, the accuracy for base calling was low, resulting in a high degree of miscalled bases and false indels. These false indels led to read-length compression; thus, assessing heterogeneity based on read length is not advisable with current nanopore technologies. Nonetheless, nanopore sequencing was able to correctly identify truncation hotspots in single-strand and self-complementary vectors similar to SMRT sequencing. In summary, nanopore sequencing can serve as a rapid and low-cost alternative for proofing AAV vectors.
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Project description:Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, Schistosoma haematobium, and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.
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Project description:Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline-NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications.
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Project description:Nanopore sequencing of five sorghum genomes
| PRJEB39174 | ENA