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CONTRAILS: A tool for rapid identification of transgene integration sites in complex, repetitive genomes using low-coverage paired-end sequencing.

Lambirth KC, Whaley AM, Schlueter JA, Bost KL, Piller KJ - Genom Data (2015)

Bottom Line: Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR.This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications.Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, United States.

ABSTRACT
Transgenic crops have become a staple in modern agriculture, and are typically characterized using a variety of molecular techniques involving proteomics and metabolomics. Characterization of the transgene insertion site is of great interest, as disruptions, deletions, and genomic location can affect product selection and fitness, and identification of these regions and their integrity is required for regulatory agencies. Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR. This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications. Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution.

No MeSH data available.


Related in: MedlinePlus

Experimental pipeline. Flowchart detailing each major step in the pipeline, from DNA extraction and sequencing to alignment to the reference genome and T-DNA sequence.
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f0005: Experimental pipeline. Flowchart detailing each major step in the pipeline, from DNA extraction and sequencing to alignment to the reference genome and T-DNA sequence.

Mentions: Here, we present and demonstrate CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing): a pipeline using existing bioinformatics tools and paired-end Illumina next-generation genomic sequencing to identify and characterize transgene insertion locations in the highly complex and repetitive genome of the legume Glycine max (Fig. 1). Paired-end reads spanning the T-DNA insertion junction allow for one read to map to the reference genome, and the other to map to the transgene sequence. Using short insert (≤ 500 b.p.) paired-end reads allows the user to narrow the insertion site to a genomic region of 500 b.p. or less, provided assembly is assisted with an established reference genome. In some cases, it is possible for a single read to span both genomic and T-DNA sequences at the transgene insertion junction, giving immediate confirmation of insert location and neighboring sequences at single base resolution. However if this is not achieved, the matched paired-end reads will disclose the location well within conventional PCR amplification range for rapid characterization of the T-DNA junction sites. Using this technique, we have identified and characterized a single T-DNA insert site in a transgenic line expressing recombinant hTG protein [30] to single-base resolution. These results are consistent with previous Southern blot and western blot screens, confirming the findings of the NGS analysis. Using this pipeline in conjunction with event-specific PCR assays, we were able to fully characterize flanking genomic sequences surrounding the T-DNA location.


CONTRAILS: A tool for rapid identification of transgene integration sites in complex, repetitive genomes using low-coverage paired-end sequencing.

Lambirth KC, Whaley AM, Schlueter JA, Bost KL, Piller KJ - Genom Data (2015)

Experimental pipeline. Flowchart detailing each major step in the pipeline, from DNA extraction and sequencing to alignment to the reference genome and T-DNA sequence.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664744&req=5

f0005: Experimental pipeline. Flowchart detailing each major step in the pipeline, from DNA extraction and sequencing to alignment to the reference genome and T-DNA sequence.
Mentions: Here, we present and demonstrate CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing): a pipeline using existing bioinformatics tools and paired-end Illumina next-generation genomic sequencing to identify and characterize transgene insertion locations in the highly complex and repetitive genome of the legume Glycine max (Fig. 1). Paired-end reads spanning the T-DNA insertion junction allow for one read to map to the reference genome, and the other to map to the transgene sequence. Using short insert (≤ 500 b.p.) paired-end reads allows the user to narrow the insertion site to a genomic region of 500 b.p. or less, provided assembly is assisted with an established reference genome. In some cases, it is possible for a single read to span both genomic and T-DNA sequences at the transgene insertion junction, giving immediate confirmation of insert location and neighboring sequences at single base resolution. However if this is not achieved, the matched paired-end reads will disclose the location well within conventional PCR amplification range for rapid characterization of the T-DNA junction sites. Using this technique, we have identified and characterized a single T-DNA insert site in a transgenic line expressing recombinant hTG protein [30] to single-base resolution. These results are consistent with previous Southern blot and western blot screens, confirming the findings of the NGS analysis. Using this pipeline in conjunction with event-specific PCR assays, we were able to fully characterize flanking genomic sequences surrounding the T-DNA location.

Bottom Line: Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR.This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications.Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, United States.

ABSTRACT
Transgenic crops have become a staple in modern agriculture, and are typically characterized using a variety of molecular techniques involving proteomics and metabolomics. Characterization of the transgene insertion site is of great interest, as disruptions, deletions, and genomic location can affect product selection and fitness, and identification of these regions and their integrity is required for regulatory agencies. Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR. This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications. Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution.

No MeSH data available.


Related in: MedlinePlus