scholarly journals Barcode aware adaptive sampling for Oxford Nanopore sequencers.

2021 ◽  
Author(s):  
Alexander Payne ◽  
Rory Munro ◽  
Nadine Holmes ◽  
Christopher Moore ◽  
Matthew Carlile ◽  
...  
Keyword(s):  
Copy Number ◽  
Adaptive Sampling ◽  
Structural Variation ◽  
Flow Cell ◽  
Unique Property ◽  
Nanopore Sequencing ◽  
Human Genomes ◽  
Oxford Nanopore ◽  
Selection Of

Adaptive sampling enables selection of individual molecules from sequencing libraries, a unique property of nanopore sequencing. Here we develop our adaptive sampling tool readfish to become "barcode-aware" enabling selection of different targets within barcoded samples or filtering out individual barcodes. We show that multiple human genomes can be assessed for copy number and structural variation on a single sequencing flow cell using sample specific customised target panels.

scholarly journals Nanopore Sequencing of a Forensic STR Multiplex Reveals Loci Suitable for Single-Contributor STR Profiling

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 381 ◽  
Author(s):  
Olivier Tytgat ◽  
Yannick Gansemans ◽  
Jana Weymaere ◽  
Kaat Rubben ◽  
Dieter Deforce ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Massively Parallel Sequencing ◽  
Flow Cell ◽  
Nanopore Sequencing ◽  
Flow Cells ◽  
Pattern Complexity ◽  
Str Loci ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies ◽  
Short Tandem

Nanopore sequencing for forensic short tandem repeats (STR) genotyping comes with the advantages associated with massively parallel sequencing (MPS) without the need for a high up-front device cost, but genotyping is inaccurate, partially due to the occurrence of homopolymers in STR loci. The goal of this study was to apply the latest progress in nanopore sequencing by Oxford Nanopore Technologies in the field of STR genotyping. The experiments were performed using the state of the art R9.4 flow cell and the most recent R10 flow cell, which was specifically designed to improve consensus accuracy of homopolymers. Two single-contributor samples and one mixture sample were genotyped using Illumina sequencing, Nanopore R9.4 sequencing, and Nanopore R10 sequencing. The accuracy of genotyping was comparable for both types of flow cells, although the R10 flow cell provided improved data quality for loci characterized by the presence of homopolymers. We identify locus-dependent characteristics hindering accurate STR genotyping, providing insights for the design of a panel of STR loci suited for nanopore sequencing. Repeat number, the number of different reference alleles for the locus, repeat pattern complexity, flanking region complexity, and the presence of homopolymers are identified as unfavorable locus characteristics. For single-contributor samples and for a limited set of the commonly used STR loci, nanopore sequencing could be applied. However, the technology is not mature enough yet for implementation in routine forensic workflows.

scholarly journals Detection of single nucleotide and copy number variants in the Fabry disease-associated GLA gene using nanopore sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Albina Nowak ◽  
Omer Murik ◽  
Tzvia Mann ◽  
David A. Zeevi ◽  
Gheona Altarescu
Keyword(s):  
Fabry Disease ◽  
Copy Number ◽  
New Technology ◽  
Copy Number Variants ◽  
Cost Effective ◽  
Nanopore Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sanger Sequence ◽  
Gla Gene

AbstractMore than 900 variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence. We aimed to design and validate a method for sequencing the GLA gene using long-read Oxford Nanopore sequencing technology. Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long-read sequencing of a 13 kb PCR amplicon. We used minimap2 to align the long-read data and Nanopolish and Sniffles to call variants. All the variants detected by Sanger (including a deep intronic variant) were also detected by long-read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology. Our long-read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.

scholarly journals Detection of Single Nucleotide and Copy Number Variants in the Fabry Disease-associated GLA Gene Using Nanopore Sequencing

2021 ◽  
Author(s):  
Albina Nowak ◽  
Omer Murik ◽  
Tzvia Mann ◽  
David A. Zeevi ◽  
Gheona Altarescu
Keyword(s):  
Fabry Disease ◽  
Copy Number ◽  
New Technology ◽  
Copy Number Variants ◽  
Cost Effective ◽  
Nanopore Sequencing ◽  
Oxford Nanopore ◽  
Long Read ◽  
Sanger Sequence ◽  
Gla Gene

Abstract Introduction: More than one thousand variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence.Aims: We aimed to design and validate a method for sequencing the GLA gene using long read Oxford Nanopore sequencing technology.Methods: Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long read sequencing of a 13kb PCR amplicon. We used minimap2 to align the long read data and Nanopolish and Sniffles to call variants.Results: All the variants detected by Sanger (including a deep intronic variant) were also detected by long read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology.Conclusions: Our long read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.

scholarly journals Cas9 targeted enrichment of mobile elements using nanopore sequencing

2021 ◽  
Author(s):  
Torrin L. McDonald ◽  
Weichen Zhou ◽  
Christopher Castro ◽  
Camille Mumm ◽  
Jessica A. Switzenberg ◽  
...  
Keyword(s):  
Genetic Disorders ◽  
Mobile Element ◽  
Flow Cell ◽  
Read Length ◽  
Nanopore Sequencing ◽  
Short Read Sequencing ◽  
Human Genomes ◽  
Long Read ◽  
Genomic Regions ◽  
Targeted Enrichment

AbstractMobile element insertions (MEIs) are highly repetitive genomic sequences that contribute to inter- and intra-individual genetic variation and can lead to genetic disorders. Targeted and whole-genome approaches using short-read sequencing have been developed to identify reference and non-reference MEIs; however, the read length hampers detection of these elements in complex genomic regions. Here, we pair Cas9 targeted nanopore sequencing with computational methodologies to capture active MEIs in human genomes. We demonstrate parallel enrichment for distinct classes of MEIs, averaging 44% of reads on targeted signals. We show an individual flow cell can recover a remarkable fraction of MEIs (97% L1Hs, 93% AluYb, 51% AluYa, 99% SVA_F, and 65% SVA_E). We identify twenty-one non-reference MEIs in GM12878 overlooked by modern, long-read analysis pipelines, primarily in repetitive genomic regions. This work introduces the utility of nanopore sequencing for MEI enrichment and lays the foundation for rapid discovery of elusive, repetitive genetic elements.

scholarly journals SENSV: Detecting Structural Variations with Precise Breakpoints using Low-Depth WGS Data from a Single Oxford Nanopore MinION Flowcell

2021 ◽  
Author(s):  
Henry CM Leung ◽  
Huijing Yu ◽  
Yifan Zhang ◽  
Wing Sze Leung ◽  
Ivan FM Lo ◽  
...  
Keyword(s):  
Structural Variation ◽  
Genetic Disorders ◽  
Genetic Disorder ◽  
Simulated Data ◽  
High Sensitivity ◽  
Flow Cell ◽  
Unbalanced Translocation ◽  
Structural Variations ◽  
Oxford Nanopore ◽  
New Algorithms

AbstractStructural variation (SV) is a major cause of genetic disorders. In this paper, we show that low-depth (specifically, 4x) whole-genome sequencing using a single Oxford Nanopore MinION flow cell suffices to support sensitive detection of SV, in particular, pathogenic SV for supporting clinical diagnosis. Existing SV calling software, when using 4x ONT WGS data, often fails to detect pathogenic SV especially in the form of long deletion, terminal deletion, duplication, and unbalanced translocation. Our new SV calling software SENSV is able to achieve high sensitivity for all types of SV and a breakpoint precision typically ±100 bp, both features are important for clinical concerns. The improvement achieved by SENSV stems from several new algorithms. We evaluated SENSV and other software using both real and simulated data. The former was based on 24 patient samples, each diagnosed with a genetic disorder. SENSV found the pathogenic SV in 22 out of 24 cases (all heterozygous, size from hundreds of Kbp to a few Mbp), reporting breakpoints within 100 bp of the true answers. No existing software can detect the pathogenic SV in more than 10 out of 24 cases, even when the breakpoint requirement is relaxed to ±2,000 bp.

scholarly journals Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1811
Author(s):  
Liina Kinkar ◽  
Robin Gasser ◽  
Bonnie Webster ◽  
David Rollinson ◽  
D. Littlewood ◽  
...  
Keyword(s):  
Structural Variation ◽  
Mitochondrial Genomes ◽  
Nanopore Sequencing ◽  
Coding Region ◽  
High Coverage ◽  
Protein Coding ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genomic Dnas ◽  
And Function

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.

scholarly journals Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Marcos Valenzuela-Ortega ◽  
Christopher French
Keyword(s):  
Copy Number ◽  
Life Science ◽  
Insertion Site ◽  
Golden Gate ◽  
Common Elements ◽  
Modern Life ◽  
Specific Host ◽  
Modular Cloning ◽  
Golden Gate Cloning ◽  
Selection Of

Abstract Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.

scholarly journals Genome and transcriptome of a pathogenic yeast, Candida nivariensis

2021 ◽  
Author(s):  
Yunfan Fan ◽  
Andrew N Gale ◽  
Anna Bailey ◽  
Kali Barnes ◽  
Kiersten Colotti ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Nanopore Sequencing ◽  
Cdna Sequencing ◽  
Pathogenic Yeast ◽  
Mitochondrial Sequence ◽  
Dna And Rna ◽  
Oxford Nanopore ◽  
Candida Nivariensis ◽  
Oxford Nanopore Technologies

Abstract We present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies (ONT) and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.

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