Sequencing of human genomes with nanopore technology

Bowden, Rory; Davies, R. W.; Heger, Andreas; Pagnamenta, Alistair T.; Cesare, Mariateresa de; Oikkonen, Laura; Parkes, Duncan; Freeman, Colin; Dhalla, Fatima; Patel, Smita Y.; Popitsch, Niko; Clc., Ip; Roberts, Hannah; Salatino, Silvia; Lockstone, Helen; Lunter, Gerton; Taylor, Jenny C.; Buck, David; Simpson, Michael A.; Donnelly, Peter

doi:10.1038/s41467-019-09637-5

Cited by 153 publications

(124 citation statements)

References 43 publications

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“…To test the proposed BOSS-RUNS strategy and investigate its potential in a range of of µ is dependent on the efficiency of basecalling and mapping and µ = 500 is consistent 287 with our experiences with real time analysis using GPU basecalling [7]. Genetic This results in a realistic [14] average fragment length of about λ = 6,300 bp. In 293 practical applications, a decision strategy is not required for fragments shorter than µ.…”

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confidence: 62%

“…• 'sequencing bias' scenario: we simulate variation in the proportions and 300 orientations of acquired fragments from different locations across the genome. We 301 modelled realistic 10-fold variation in sequencing bias (realized coverage for naive 302 sequencing) between the regions with highest and lowest sequencing bias [14], with 303 10 sequencing bias peaks and troughs, by setting F This scenario resembles the case in which one is interested in a multi-locus 310 sequence typing of a bacterial sample [15].…”

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confidence: 99%

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Dynamic, adaptive sampling during nanopore sequencing using Bayesian experimental design

Maio

Manser

Munro

et al. 2020

Preprint

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Real-time selective sequencing of individual DNA fragments, or 'Read Until', allows the focusing of Oxford Nanopore Technology sequencing on pre-selected genomic regions.This can lead to large improvements in DNA sequencing performance in many scenarios where only part of the DNA content of a sample is of interest. This approach is based on the idea of deciding whether to sequence a fragment completely after having sequenced only a small initial part of it. If, based on this small part, the fragment is not deemed of (sufficient) interest it is rejected and sequencing is continued on a new fragment. To date, only simple decision strategies based on location within a genome have been proposed to determine what fragments are of interest. We present a new mathematical model and algorithm for the real-time assessment of the value of prospective fragments.Our decision framework is based not only on which genomic regions are a priori interesting, but also on which fragments have so far been sequenced, and so on the current information available regarding the genome being sequenced. As such, our strategy can adapt dynamically during each run, focusing sequencing efforts in areas of highest uncertainty (typically areas currently low coverage). We show that our approach 101 For each position i of a reference genome of length N , we denote π i (g) the 102 location-specific prior on genotypes g ∈ G before any data have been observed. In all 103 applications below, when considering a haploid genome, we define the prior of reference 104 February 7, 2020 5/31 nucleotide b R at position i as π i (b R ) = 1 − θ, with θ the genetic diversity of the 105 considered population. Conversely, π i (g) = θ/3 if g = b R . 106When considering diploid sequenced genomes, we still assume a haploid reference 107 genome, with reference nucleotide at a given position denoted b R . In the case of a 108 diploid unphased genome being sequenced, we define π i ({b R , b R }) = 1 − θ, and 109 π i ({g, g}) = p homo θ/3 if g = b R , with p homo being the proportion of site differences from 110 a reference that are expected to be homozygous, and π i ({g, b R }) = (1 − p homo )θ/3 for 111 g = b R . We ignore the possibility of a heterozygous genome being sequenced with both 112 alleles different from the reference genome. These prior probability definitions also 113 ignore differences in mutation rates across nucleotides and genome positions and do not 114 use prior knowledge on SNP locations derived from the population; when available, 115 these aspects could however easily be included in the definition of π i (g). 116

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confidence: 62%

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confidence: 99%

Dynamic, adaptive sampling during nanopore sequencing using Bayesian experimental design

Maio

Manser

Munro

et al. 2020

Preprint

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“…For the Nanopore (hereafter, long-read) data, we initially ran SNV calling using FreeBayes 38 , as described in 39 , on reads aligning to reference chromosome 17. This chromosome was chosen because it was known to harbour mutations of interest, as described above.…”

Section: Methods and Supplementarymentioning

confidence: 99%

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Roberts

Lopopolo

Pagnamenta

et al. 2020

Preprint

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Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies (ONT) PromethION platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables genome-wide detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from the long-read data achieved good specificity and sensitivity.However, results of somatic SNV calling highlight the need for the development of specialized joint calling algorithms. We find the comparative performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.

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“…During past decades, high throughput DNA sequencing technologies had been advanced significantly, including Illumina short reads sequencing, Pacific biosciences and Oxford nanopore long reads sequencing [9,10]. It had been illustrated that nanopore long reads sequencing technology can be used to assemble genomes from different species, including bacterial [11], human [12,13], and rice [14]. It had been proved that chromosome-scale assemblies of human and mouse genomes can be generated by integrating short-reads DNA sequencing and Hi-C chromatin interaction mate-pair sequencing [15].…”

Section: Introductionmentioning

confidence: 99%

De novo assembly of trachidermus fasciatus genome by nanopore sequencing

Xie

Zhang

et al. 2020

Preprint

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Trachidermus fasciatus is a roughskin sculpin fish widely located at the coastal areas of East Asia. Due to the environmental destruction and overfishing, the populations of this species have been under threat. It is important to have a reference genome to study the population genetics, domestic farming, and genetic resource protection. However, currently, there is no reference genome for Trachidermus fasciatus, which has greatly hurdled the studies on this species. In this study, we proposed to integrate nanopore long reads sequencing, Illumina short reads sequencing and Hi-C methods to thoroughly de novo assemble the genome of Trachidermus fasciatus. Our results provided a chromosome-level high quality genome assembly with a total length of about 543 Mb, and with N50 of 23 Mb. Based on de novo gene prediction and RNA sequencing information, a total of 38728 genes were found, including 23191 protein coding genes, 2149 small RNAs, 5572 rRNAs, and 7816 tRNAs. Besides, about 23% of the genome area is covered by the repetitive elements. Furthermore, The BUSCO evaluation of the completeness of the assembled genome is more than 96%, and the single base accuracy is 99.997%. Our study provided the first whole genome reference for the species of Trachidermus fasciatus, which might greatly facilitate the future studies on this species.

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Sequencing of human genomes with nanopore technology

Cited by 153 publications

References 43 publications

Dynamic, adaptive sampling during nanopore sequencing using Bayesian experimental design

Dynamic, adaptive sampling during nanopore sequencing using Bayesian experimental design

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

De novo assembly of trachidermus fasciatus genome by nanopore sequencing

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