scholarly journals Localization of balanced chromosome translocation breakpoints by long-read sequencing on the Oxford Nanopore platform

2018 ◽  
Author(s):  
Liang Hu ◽  
Fan Liang ◽  
Dehua Cheng ◽  
Zhiyuan Zhang ◽  
Guoliang Yu ◽  
...  
Keyword(s):  
Genetic Diagnosis ◽  
Chromosome Translocation ◽  
Therapeutic Monitoring ◽  
Whole Genome ◽  
Precise Localization ◽  
Single Base ◽  
Oxford Nanopore ◽  
Long Read ◽  
Translocation Breakpoints ◽  
Balanced Translocations

AbstractStructural variants (SVs) in genomes, including translocations, inversions, insertions, deletions and duplications, remain difficult to be detected reliably by traditional genomic technologies. In particular, balanced translocations and inversions cannot be detected by microarrays since they do not alter chromosome copy numbers; they cannot be reliably detected by short-read sequencing either, since many breakpoints are located within repetitive regions of the genome that are unmappable by short reads. However, the detection and the precise localization of breakpoints at the nucleotide level are important to study the genetic causes in patients carrying balanced translocations or inversions. Long-read sequencing techniques, such as the Oxford Nanopore Technology (ONT), may detect these SVs in a more direct, efficient and accurate manner. In this study, we applied whole-genome long-read sequencing on the Oxford Nanopore GridION sequencer to detect the breakpoints from 6 carriers of balanced translocations and one carrier of inversion, where SVs had initially been detected by karyotyping at the chromosome level. The results showed that all the balanced translocations were detected with ∼10X coverage and were consistent with the karyotyping results. PCR and Sanger sequencing confirmed 8 of the 14 breakpoints to single base resolution, yet other breakpoints cannot be refined to single-base due to their localization at highly repetitive regions or pericentromeric regions, or due to the possible presence of local deletions/duplications. Our results indicate that low-coverage whole-genome sequencing is an ideal tool for the precise localization of most translocation breakpoints and may provide haplotype information on the breakpoint-linked SNPs, which may be widely applied in SV detection, therapeutic monitoring, assisted reproduction technology (ART) and preimplantation genetic diagnosis (PGD).

scholarly journals Location of Balanced Chromosome-Translocation Breakpoints by Long-Read Sequencing on the Oxford Nanopore Platform

2020 ◽  
Vol 10 ◽  
Author(s):  
Liang Hu ◽  
Fan Liang ◽  
Dehua Cheng ◽  
Zhiyuan Zhang ◽  
Guoliang Yu ◽  
...  
Keyword(s):  
Chromosome Translocation ◽  
Oxford Nanopore ◽  
Long Read ◽  
Translocation Breakpoints

scholarly journals Whole-Genome Sequencing of a Human Clinical Isolate of the Novel Species Klebsiella quasivariicola sp. nov

2017 ◽  
Vol 5 (42) ◽  
Author(s):  
S. Wesley Long ◽  
Sarah E. Linson ◽  
Matthew Ojeda Saavedra ◽  
Concepcion Cantu ◽  
James J. Davis ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Novel Species ◽  
Whole Genome ◽  
The Novel ◽  
Short Read ◽  
Oxford Nanopore ◽  
Long Read

ABSTRACT In a study of 1,777 Klebsiella strains, we discovered KPN1705, which was distinct from all recognized Klebsiella spp. We closed the genome of strain KPN1705 using a hybrid of Illumina short-read and Oxford Nanopore long-read technologies. For this novel species, we propose the name Klebsiella quasivariicola sp. nov.

scholarly journals Mobile Colistin Resistance Genetic Determinants of Non-Typhoid Salmonella enterica Isolates from Russia

2021 ◽  
Vol 9 (12) ◽  
pp. 2515
Author(s):  
Konstantin V. Kuleshov ◽  
Anastasia S. Pavlova ◽  
Elizaveta D. Shedko ◽  
Yulia V. Mikhaylova ◽  
Gabriele Margos ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enterica ◽  
Diarrheal Disease ◽  
Whole Genome ◽  
Genetic Determinants ◽  
Colistin Resistance ◽  
Oxford Nanopore ◽  
Susceptible Isolate ◽  
Long Read

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently poses a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carrying mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n = 586). mcr-1 genes were detected using a previously developed qPCR assay, and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates, including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans, carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene-carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological backgrounds.

Amplicon and metagenomic analysis of MERS-CoV and the microbiome in patients with severe Middle East respiratory syndrome (MERS)

2020 ◽  
Author(s):  
Waleed Aljabr ◽  
Muhannad Alruwaili ◽  
Rebekah Penrice-Randal ◽  
Abdulrahman Alrezaihi ◽  
Abbie Jasmine Harrison ◽  
...  
Keyword(s):  
Middle East ◽  
Middle East Respiratory Syndrome ◽  
Metagenomic Analysis ◽  
Read Length ◽  
Clinical Samples ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Genotype Information ◽  
Long Read ◽  
Health Complications

AbstractMiddle East Respiratory Syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional spill overs into humans. The severity of infection in humans is influenced by numerous factors and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are co-incident in the Middle East and a rapid way is required of sequencing MERS-CoV to derive genotype information for molecular epidemiology. Additionally, complicating factors in MERS-CoV infections are co-infections that require clinical management. The ability to rapidly characterise these infections would be advantageous. To rapidly sequence MERS-CoV, we developed an amplicon-based approach coupled to Oxford Nanopore long read length sequencing. The advantage of this approach is that insertions and deletions can be identified – which are the major drivers of genotype change in coronaviruses. This and a metagenomic approach were evaluated on clinical samples from patients with MERS. The data illustrated that whole genome or near whole genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants including deletion mutants. Whereas, the metagenomic analysis provided information of the background microbiome.

scholarly journals Long‐read whole‐genome sequencing for the genetic diagnosis of dystrophinopathies

2020 ◽  
Vol 7 (10) ◽  
pp. 2041-2046
Author(s):  
Zhiying Xie ◽  
Chengyue Sun ◽  
Siwen Zhang ◽  
Yilin Liu ◽  
Meng Yu ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Diagnosis ◽  
Whole Genome ◽  
Long Read

scholarly journals HLA*PRG:LA – HLA typing from linearly projected graph alignments

2018 ◽  
Author(s):  
Alexander T Dilthey ◽  
Alexander J Mentzer ◽  
Raphael Carapito ◽  
Clare Cutland ◽  
Nezih Cereb ◽  
...  
Keyword(s):  
Type Inference ◽  
Hla Typing ◽  
Supplementary Information ◽  
Whole Genome ◽  
Typical Sample ◽  
Whole Exome ◽  
Oxford Nanopore ◽  
Hla Type ◽  
Long Read ◽  
Genome Assemblies

AbstractSummary:HLA*PRG:LA implements a new graph alignment model for HLA type inference, based on the projection of linear alignments onto a variation graph. It enables accurate HLA type inference from whole-genome (99% accuracy) and whole-exome (93% accuracy) Illumina data; from long-read Oxford Nanopore and Pacific Biosciences data (98% accuracy for whole-genome and targeted data); and from genome assemblies. Computational requirements for a typical sample vary between 0.7 and 14 CPU hours per sample.Availability and Implementation:HLA*PRG:LA is implemented in C++ and Perl and freely available from https://github.com/DiltheyLab/HLA-PRG-LA (GPL v3).Contact:[email protected] informationSupplementary data are available online.

scholarly journals Mobile Colistin Resistance Genetic Determinants of Non-typhoid Salmonella enterica Isolates from Russia

2021 ◽  
Author(s):  
K. V. Kuleshov ◽  
A. S. Pavlova ◽  
E. D. Shedko ◽  
Y. V. Mikhaylova ◽  
G. Margos ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enterica ◽  
Diarrheal Disease ◽  
Whole Genome ◽  
Genetic Determinants ◽  
Colistin Resistance ◽  
Oxford Nanopore ◽  
Susceptible Isolate ◽  
Long Read

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently pose a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carries mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n=586). mcr-1 genes were detected using a previously developed qPCR assay and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological background.

scholarly journals De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

F1000Research ◽  
2018 ◽  
Vol 6 ◽  
pp. 618 ◽  
Author(s):  
Michael Liem ◽  
Hans J. Jansen ◽  
Ron P. Dirks ◽  
Christiaan V. Henkel ◽  
G. Paul H. van Heusden ◽  
...  
Keyword(s):  
De Novo ◽  
Sequence Data ◽  
New Technology ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Short Read ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Before And After ◽  
Long Read

Background: The introduction of the MinION sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. Nanopore sequence data offers great potential for de novo assembly of complex genomes without using other technologies. Furthermore, Nanopore data combined with other sequencing technologies is highly useful for accurate annotation of all genes in the genome. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. We double corrected assemblies from four different assemblers with PILON and assessed sequence correctness before and after PILON correction with a set of 290 Fungi genes using BUSCO. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae (Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. We also compared various technical and software developments for the nanopore sequencing protocol, showing that nanopore-derived assemblies provide the highest contiguity. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

scholarly journals DNA Extraction Method Optimized for Nontuberculous Mycobacteria Long-Read Whole Genome Sequencing

2018 ◽  
Author(s):  
Jennifer M. Bouso ◽  
Paul J. Planet
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Nontuberculous Mycobacteria ◽  
Systemic Disease ◽  
Matrix Material ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies

AbstractNontuberculous mycobacteria (NTM) are a major cause of pulmonary and systemic disease in at-risk populations. Gaps in knowledge about transmission patterns, evolution, and pathogenicity during infection have prompted a recent surge in genomic NTM research. Increased availability and affordability of whole genome sequencing (WGS) techniques, including the advent of Oxford Nanopore Technologies, provide new opportunities to sequence complete NTM genomes at a fraction of the previous cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to their slow growth and recalcitrant cell wall. Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA. Our refined method was compared to 6 other methods with variations in timing of mechanical and enzymatic digestion, quantity of matrix material, and reagents used in extraction and precipitation. We also demonstrate the ability of our optimized protocol to produce sufficient DNA to yield near-complete NTM genome assemblies using Oxford Nanopore Technologies long-read sequencing.

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