Dataset Information

Targeted MinION sequencing of transgenes.

ABSTRACT: The presence of genetically modified organisms (GMO) is commonly assessed using real-time PCR methods targeting the most common transgenic elements found in GMOs. Once the presence of GM material has been established using these screening methods, GMOs are further identified using a battery of real-time PCR methods, each being specific of one GM event and usually targeting the junction of the plant genome and of the transgenic DNA insert. If, using these specific methods, no GMO could be identified, the presence of an unauthorized GMO is suspected. In this context, the aim of this work was to develop a fast and simple method to obtain the sequence of the transgene and of its junction with plant DNA, with the presence of a screening sequence as only prior knowledge. An unauthorized GM petunia, recently found on the French market, was used as template during the development of this new molecular tool. The innovative proposed protocol is based on the circularization of fragmented DNA followed by the amplification of the transgene and of its flanking regions using long-range inverse PCR. Sequencing was performed using the Oxford Nanopore MinION technology and a bioinformatic pipeline was developed.

SUBMITTER: Boutigny AL

PROVIDER: S-EPMC7492250 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Targeted MinION sequencing of transgenes.

Boutigny Anne-Laure AL Fioriti Florent F Rolland Mathieu M

Scientific reports 20200915 1

The presence of genetically modified organisms (GMO) is commonly assessed using real-time PCR methods targeting the most common transgenic elements found in GMOs. Once the presence of GM material has been established using these screening methods, GMOs are further identified using a battery of real-time PCR methods, each being specific of one GM event and usually targeting the junction of the plant genome and of the transgenic DNA insert. If, using these specific methods, no GMO could be identif ...[more]

PMID: 32934250

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Project description:BackgroundAn approach utilizing the long-read capability of the Oxford Nanopore MinION to rapidly sequence bacterial ribosomal operons of complex natural communities was developed. Microbial fingerprinting employs domain-specific forward primers (16S rRNA subunit), reverse primers (23S rRNA subunit), and a high-fidelity Taq polymerase with proofreading capabilities. Amplicons contained both ribosomal subunits for broad-based phylogenetic assignment (~ 3900 bp of sequence), plus the intergenic spacer (ITS) region (~ 300 bp) for potential strain-specific identification.ResultsTo test the approach, bacterial rRNA operons (~ 4200 bp) were amplified from six DNA samples employing a mixture of farm soil and bioreactor DNA in known concentrations. Each DNA sample mixture was barcoded, sequenced in quadruplicate (n = 24), on two separate 6-h runs using the MinION system (R7.3 flow cell; MAP005 and 006 chemistry). From nearly 90,000 MinION reads, roughly 33,000 forward and reverse sequences were obtained. This yielded over 10,000 2D sequences which were analyzed using a simplified data analysis pipeline based on NCBI Blast and assembly with Geneious software. The method could detect over 1000 operational taxonomic units in the sample sets in a quantitative manner. Global sequence coverage for the various rRNA operons ranged from 1 to 1951x. An iterative assembly scheme was developed to reconstruct those rRNA operons with > 35x coverage from a set of 30 operational taxonomic units (OTUs) among the Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, and Gemmatimonadetes. Phylogenetic analysis of the 16S rRNA and 23S rRNA genes from each operon demonstrated similar tree topologies with species/strain-level resolution.ConclusionsThis sequencing method represents a cost-effective way to profile microbial communities. Because the MinION is small, portable, and runs on a laptop, the possibility of microbiota characterization in the field or on robotic platforms becomes realistic.

Dataset Information

Targeted MinION sequencing of transgenes.

Publications

Targeted MinION sequencing of transgenes.

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