A comprehensive toolkit to enable MinION sequencing in any laboratory

Schalamun, Miriam; Kainer, David; Beavan, Eleanor; Nagar, Ramawatar; Eccles, David; Rathjen, John P.; Lanfear, Robert; Schwessinger, Benjamin

doi:10.1101/289579

Cited by 6 publications

(5 citation statements)

References 23 publications

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“…Nevertheless, many groups are now working on establishing high-molecular-weight DNA extraction protocols (e.g. [86, 87]), suggesting that the remaining issues are unlikely to be insurmountable for most species.…”

Section: Discussionmentioning

confidence: 99%

Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case

Wang

Schalamun

Suarez

et al. 2018

Preprint

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Section: Discussionmentioning

confidence: 99%

Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case

Wang

Schalamun

Suarez

et al. 2018

Preprint

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“…The nanopore sequencing throughput is affected by many factors, which include the number of active pores remaining in the flow cell, DNA purity and integrity, library concentration, and sequencing time (25). Therefore, the absolute number of the aligned reads does not reflect the absolute abundance of certain bacteria.…”

Section: Relative Abundance Calculationmentioning

confidence: 99%

Nanopore 16S Amplicon Sequencing Enhances the Understanding of Pathogens in Medically Intractable Diabetic Foot Infections

et al. 2021

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Diabetic foot infections (DFIs) cause substantial morbidity and mortality. The mainstay of the treatment is empiric antibiotics and surgical debridement in severe cases. In this study, we performed nanopore 16S rDNA sequencing from the debridement specimens of DFIs. Fifty-four surgical debridement specimens obtained from 45 patients with medically intractable DFI were included. The 16S rDNA PCR was performed on each specimen, and Nanopore sequencing was performed for up to 3 h. The reads were aligned to the BLAST database, and the results were compared with conventional culture studies. The 16S sequencing results revealed that the majority of the DFIs (44 of 54, 81.5%) were polymicrobial infections. All bacteria isolated by conventional culture studies were detected by 16S sequencing. Several anaerobes (Prevotella, Finegoldia, Anaerococcus, Bacteroides) were commonly identified by 16S sequencing but were frequently missed by culture studies. In many cases, certain bacteria only revealed by the 16S sequencing were more abundant than the bacteria isolated by the culture studies. In conclusion, nanopore 16S sequencing was capable of pathogen identification in DFIs and has many advantages over conventional culture studies. Nanopore 16S sequencing enables a comprehensive understanding of the bacteria involved in DFIs.

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“…DNA was extracted from fresh, young leaf material collected from a single plant of the R . idaeus cultivars ‘Autumn Bliss’ and ‘Malling Jewel’ using a high molecular weight genomic DNA extraction protocol [ 13 ]. Long-read sequencing libraries were prepared using the SQK-LSK108 Ligation Sequencing Kit (Oxford Nanopore Technologies) from approximately 1 μg of high molecular weight genomic DNA, following the manufacturer’s protocol.…”

Section: Methodsmentioning

confidence: 99%

Chromosome-scale genome sequence assemblies of the ‘Autumn Bliss’ and ‘Malling Jewel’ cultivars of the highly heterozygous red raspberry (Rubus idaeus L.) derived from long-read Oxford Nanopore sequence data

et al. 2023

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Red raspberry (Rubus idaeus L.) is an economically valuable soft-fruit species with a relatively small (~300 Mb) but highly heterozygous diploid (2n = 2x = 14) genome. Chromosome-scale genome sequences are a vital tool in unravelling the genetic complexity controlling traits of interest in crop plants such as red raspberry, as well as for functional genomics, evolutionary studies, and pan-genomics diversity studies. In this study, we developed genome sequences of a primocane fruiting variety (‘Autumn Bliss’) and a floricane variety (‘Malling Jewel’). The use of long-read Oxford Nanopore Technologies sequencing data yielded long read lengths that permitted well resolved genome sequences for the two cultivars to be assembled. The de novo assemblies of ‘Malling Jewel’ and ‘Autumn Bliss’ contained 79 and 136 contigs respectively, and 263.0 Mb of the ‘Autumn Bliss’ and 265.5 Mb of the ‘Malling Jewel’ assembly could be anchored unambiguously to a previously published red raspberry genome sequence of the cultivar ‘Anitra’. Single copy ortholog analysis (BUSCO) revealed high levels of completeness in both genomes sequenced, with 97.4% of sequences identified in ‘Autumn Bliss’ and 97.7% in ‘Malling Jewel’. The density of repetitive sequence contained in the ‘Autumn Bliss’ and ‘Malling Jewel’ assemblies was significantly higher than in the previously published assembly and centromeric and telomeric regions were identified in both assemblies. A total of 42,823 protein coding regions were identified in the ‘Autumn Bliss’ assembly, whilst 43,027 were identified in the ‘Malling Jewel’ assembly. These chromosome-scale genome sequences represent an excellent genomics resource for red raspberry, particularly around the highly repetitive centromeric and telomeric regions of the genome that are less complete in the previously published ‘Anitra’ genome sequence.

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A comprehensive toolkit to enable MinION sequencing in any laboratory

Cited by 6 publications

References 23 publications

Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case

Assembly of chloroplast genomes with long- and short-read data: a comparison of approaches using Eucalyptus pauciflora as a test case

Nanopore 16S Amplicon Sequencing Enhances the Understanding of Pathogens in Medically Intractable Diabetic Foot Infections

Chromosome-scale genome sequence assemblies of the ‘Autumn Bliss’ and ‘Malling Jewel’ cultivars of the highly heterozygous red raspberry (Rubus idaeus L.) derived from long-read Oxford Nanopore sequence data

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