Nanopore sequencing analysis of integron gene cassettes in sewages and soils

2022 ◽  
pp. 152766
Author(s):  
Xin-Li An ◽  
Olusegun K. Abass ◽  
Cai-Xia Zhao ◽  
Mei-Rong Xu ◽  
Ting Pan ◽  
...  
Keyword(s):  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Gene Cassettes

scholarly journals Cas9-Assisted Targeting of CHromosome segments (CATCH) for targeted nanopore sequencing and optical genome mapping

2017 ◽  
Author(s):  
Tslil Gabrieli ◽  
Hila Sharim ◽  
Yael Michaeli ◽  
Yuval Ebenstein
Keyword(s):  
Single Molecule ◽  
Genome Mapping ◽  
Single Point ◽  
Read Length ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Whole Genome Analysis ◽  
Long Read

ABSTRACTVariations in the genetic code, from single point mutations to large structural or copy number alterations, influence susceptibility, onset, and progression of genetic diseases and tumor transformation. Next-generation sequencing analysis is unable to reliably capture aberrations larger than the typical sequencing read length of several hundred bases. Long-read, single-molecule sequencing methods such as SMRT and nanopore sequencing can address larger variations, but require costly whole genome analysis. Here we describe a method for isolation and enrichment of a large genomic region of interest for targeted analysis based on Cas9 excision of two sites flanking the target region and isolation of the excised DNA segment by pulsed field gel electrophoresis. The isolated target remains intact and is ideally suited for optical genome mapping and long-read sequencing at high coverage. In addition, analysis is performed directly on native genomic DNA that retains genetic and epigenetic composition without amplification bias. This method enables detection of mutations and structural variants as well as detailed analysis by generation of hybrid scaffolds composed of optical maps and sequencing data at a fraction of the cost of whole genome sequencing.

scholarly journals Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

2015 ◽  
Vol 7 (1) ◽  
Author(s):  
Alexander L. Greninger ◽  
Samia N. Naccache ◽  
Scot Federman ◽  
Guixia Yu ◽  
Placide Mbala ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Viral Pathogens

scholarly journals Genopo: a nanopore sequencing analysis toolkit for portable Android devices

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Hiruna Samarakoon ◽  
Sanoj Punchihewa ◽  
Anjana Senanayake ◽  
Jillian M. Hammond ◽  
Igor Stevanovski ◽  
...  
Keyword(s):  
Computational Analysis ◽  
Smartphone Application ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Android Application ◽  
Sequencing Data ◽  
Human Coronavirus ◽  
Dna And Rna ◽  
Bioinformatics Tools

Abstract The advent of portable nanopore sequencing devices has enabled DNA and RNA sequencing to be performed in the field or the clinic. However, advances in in situ genomics require parallel development of portable, offline solutions for the computational analysis of sequencing data. Here we introduce Genopo, a mobile toolkit for nanopore sequencing analysis. Genopo compacts popular bioinformatics tools to an Android application, enabling fully portable computation. To demonstrate its utility for in situ genome analysis, we use Genopo to determine the complete genome sequence of the human coronavirus SARS-CoV-2 in nine patient isolates sequenced on a nanopore device, with Genopo executing this workflow in less than 30 min per sample on a range of popular smartphones. We further show how Genopo can be used to profile DNA methylation in a human genome sample, illustrating a flexible, efficient architecture that is suitable to run many popular bioinformatics tools and accommodate small or large genomes. As the first ever smartphone application for nanopore sequencing analysis, Genopo enables the genomics community to harness this cheap, ubiquitous computational resource.

scholarly journals Quality control and statistical evaluation of combinatorial DNA libraries using nanopore sequencing

BioTechniques ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 379-383
Author(s):  
Cédric Lood ◽  
Hans Gerstmans ◽  
Yves Briers ◽  
Vera van Noort ◽  
Rob Lavigne
Keyword(s):  
Ad Hoc ◽  
Comprehensive Evaluation ◽  
Combinatorial Libraries ◽  
Initial Step ◽  
Building Blocks ◽  
Small Subset ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Long Reads

Protein engineering and synthetic biology applications increasingly rely on the assembly of modular libraries composed of thousands of different combinations of DNA building blocks. At present, the validation of such libraries is performed by Sanger sequencing analysis on a small subset of clones on an ad hoc basis. Here, we implement a systematic procedure for the comprehensive evaluation of combinatorial libraries, immediately after their creation in vitro, using long reads sequencing technology. After an initial step of nanopore sequencing, we use straightforward bioinformatics tools to tabulate the composition and synteny of the building blocks in each read. We subsequently use exploratory statistics to assess the library and validate its diversity before carrying downstream cloning and screening assays.

scholarly journals Integrons and Gene Cassettes in theEnterobacteriaceae

2001 ◽  
Vol 45 (9) ◽  
pp. 2658-2661 ◽  
Author(s):  
Peter A. White ◽  
Christopher J. McIver ◽  
William D. Rawlinson
Keyword(s):  
Sequencing Analysis ◽  
Degenerate Primers ◽  
Gene Cassettes ◽  
Conserved Regions ◽  
Class 1 ◽  
Urinary Isolates

ABSTRACT Integrons were detected in 59 of 120 (49%) urinary isolates ofEnterobacteriaceae by PCR using degenerate primers targeted to conserved regions of class 1, 2, and 3 integrase genes. PCR sequencing analysis of the cassette arrays revealed a predominance of cassettes that confer resistance to the aminoglycosides and trimethoprim.

scholarly journals Genetic characterization of multidrug resistance in Shigella spp. from Japan

2006 ◽  
Vol 55 (12) ◽  
pp. 1685-1691 ◽  
Author(s):  
Ashraf M. Ahmed ◽  
Kimi Furuta ◽  
Kei Shimomura ◽  
Yoshio Kasama ◽  
Tadashi Shimamoto
Keyword(s):  
Shigella Flexneri ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Shigella Sonnei ◽  
Sequencing Analysis ◽  
Class 1 Integron ◽  
Gene Cassettes ◽  
Class 1 ◽  
Shigella Spp

This study characterized the genetic basis of antimicrobial resistance of a number of Shigella spp. isolated from humans from 2000 to 2004 in Hiroshima prefecture, Japan. A total of 26 isolates of Shigella spp. were included in this study. Antimicrobial susceptibility tests revealed high levels of resistance, especially to ampicillin, streptomycin, trimethoprim, tetracycline, nalidixic acid and ciprofloxacin. PCR and DNA sequencing were used for screening and characterization of antibiotic-resistance determinants. PCR sequencing analysis revealed the presence of only one type of class 1 integron in one isolate of Shigella sonnei. This class 1 integron was 1904 bp and contained two gene cassettes: a probable esterase/lipase (estX) and aadA1, which confers resistance to streptomycin and spectinomycin. Two types of class 2 integron were identified in this study. One was the classic type (2158 bp) and carried the three conserved resistance gene cassettes of the class 2 integron, dfrA1, sat1 and aadA1, which confer resistance to trimethoprim, streptothricin and streptomycin/spectinomycin, respectively. This type was detected in both Shigella sonnei (14 isolates) and Shigella flexneri (five isolates). The other type was shorter (1313 bp) and carried only two gene cassettes, dfrA1 and sat1. This integron was detected in a single isolate of Shigella sonnei. PFGE patterns showed limited diversity within clusters of the same species. Furthermore, an extended-spectrum β-lactamase gene, bla OXA-30, which confers resistance to ampicillin, was characterized in all isolates of Shigella flexneri except the oldest strain, which was isolated in 2000. Southern blot hybridization and conjugation experiments showed that bla OXA-30 was located in the chromosome.

scholarly journals Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

2015 ◽  
Author(s):  
Alex L Greninger ◽  
Samia N Naccache ◽  
Scot Federman ◽  
Guixia Yu ◽  
Placide Mbala ◽  
...  
Keyword(s):  
Real Time ◽  
Ebola Virus ◽  
Hemorrhagic Fever ◽  
Turnaround Time ◽  
Clinical Samples ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Viral Pathogens ◽  
Web Based ◽  
Lower Titer

We report unbiased metagenomic detection of chikungunya virus (CHIKV), Ebola virus (EBOV), and hepatitis C virus (HCV) from four human blood samples by MinION nanopore sequencing coupled to a newly developed, web-based pipeline for real-time bioinformatics analysis on a computational server or laptop(MetaPORE). At titers ranging from 107-108 copies per milliliter, reads to EBOV from two patients with acute hemorrhagic fever and CHIKV from an asymptomatic blood donor were detected within 4 to 10 minutes of data acquisition, while lower titer HCV virus (1x105 copies per milliliter) was detected within 40 minutes. Analysis of mapped nanopore reads alone, despite an average individual error rate of 24% [range 8-49%], permitted identification of the correct viral strain in all 4 isolates, and 90% of the genome of CHIKV was recovered with >98% accuracy. Using nanopore sequencing, metagenomic detection of viral pathogens directly from clinical samples was performed within an unprecedented <6 hours sample-to-answer turnaround time and in a timeframe amenable for actionable clinical and public health diagnostics.

scholarly journals Gross Chromosomal Rearrangements in Kluyveromyces Marxianus Revealed by Illumina and Oxford Nanopore Sequencing

2020 ◽  
Author(s):  
Lin Ding ◽  
Harrison D. Macdonald ◽  
Hamilton O Smith ◽  
Clyde A. Hutchison III ◽  
Chuck Merryman ◽  
...  
Keyword(s):  
Kluyveromyces Marxianus ◽  
Chromosomal Rearrangements ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Illumina Miseq ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Random Integration ◽  
Gross Chromosomal Rearrangements ◽  
Oxford Nanopore

Kluyveromyces marxianus (K. marxianus) is a newly emerging industrially relevant yeast. It is known to possess a highly efficient Non-Homologous End Joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the Gross Chromosomal Rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in Eukaryotes, which could potentially provide insights for cancer research.

scholarly journals Gross Chromosomal Rearrangements in Kluyveromyces marxianus Revealed by Illumina and Oxford Nanopore Sequencing

2020 ◽  
Vol 21 (19) ◽  
pp. 7112
Author(s):  
Lin Ding ◽  
Harrison D. Macdonald ◽  
Hamilton O Smith ◽  
Clyde A. Hutchison III ◽  
Chuck Merryman ◽  
...  
Keyword(s):  
Kluyveromyces Marxianus ◽  
Chromosomal Rearrangements ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Illumina Miseq ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Random Integration ◽  
Gross Chromosomal Rearrangements ◽  
Oxford Nanopore

Kluyveromyces marxianus (K. marxianus) is an increasingly popular industrially relevant yeast. It is known to possess a highly efficient non-homologous end joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus otherwise wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the gross chromosomal rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in eukaryotes, which could potentially provide insights for cancer research.

Molecular Characterization of a Type I Quantitative Factor V Deficiency in a Thrombosis Patient that Is “Pseudo Homozygous” for Activated Protein C Resistance

1997 ◽  
Vol 77 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Joan F Guasch ◽  
Ruud P M Lensen ◽  
Rogier M Bertina
Keyword(s):  
Protein C ◽  
Dna Analysis ◽  
Activated Protein C ◽  
Type I ◽  
Factor V ◽  
Sequencing Analysis ◽  
Apc Resistance ◽  
Quantitative Factor ◽  
Factor V Deficiency ◽  
Fv Leiden

SummaryResistance to activated protein C (APC), which is associated with the FV Leiden mutation in the large majority of the cases, is the most common genetic risk factor for thrombosis. Several laboratory tests have been developed to detect the APC-resistance phenotype. The result of the APC-resistance test (APC-sensitivity ratio, APC-SR) usually correlates well with the FV Leiden genotype, but recently some discrepancies have been reported. Some thrombosis patients that are heterozygous for FV Leiden show an APC-SR usually found only in homozygotes for the defect. Some of those patients proved to be compound heterozygotes for the FV Leiden mutation and for a type I quantitative factor V deficiency. We have investigated a thrombosis patient characterized by an APC-SR that would predict homozygosity for FV Leiden. DNA analysis showed that he was heterozygous for the mutation. Sequencing analysis of genomic DNA revealed that the patient also is heterozygous for a G5509→A substitution in exon 16 of the factor V gene. This mutation interferes with the correct splicing of intron 16 and leads to the presence of a null allele, which corresponds to the “non-FV Leiden” allele. The conjunction of these two defects in the patient apparently leads to the same phenotype as observed in homozygotes for the FV Leiden mutation.

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