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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

Plasmid map of hTG construct. The hTG plasmid map shows all regions included in the transformation plasmid utilized in the Agrobacterium transformation of the original ST77 event. The T-DNA construct contains the soybean β-conglycinin promoter (7S), tobacco etch virus translational enhancer element (TEV), human thyroglobulin gene (hTG), cauliflower mosaic virus terminator element (T35S) followed by the selectable marker cassette comprised of the nopaline synthase promoter (NOS promoter), phosphinothricin acetyltransferase gene (BAR ORF), and nopaline synthase terminator element (NOS Term). The aad A region of the vector confers antibiotic resistance to spectinomycin and streptomycin for selection of Agrobacterium.
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f0010: Plasmid map of hTG construct. The hTG plasmid map shows all regions included in the transformation plasmid utilized in the Agrobacterium transformation of the original ST77 event. The T-DNA construct contains the soybean β-conglycinin promoter (7S), tobacco etch virus translational enhancer element (TEV), human thyroglobulin gene (hTG), cauliflower mosaic virus terminator element (T35S) followed by the selectable marker cassette comprised of the nopaline synthase promoter (NOS promoter), phosphinothricin acetyltransferase gene (BAR ORF), and nopaline synthase terminator element (NOS Term). The aad A region of the vector confers antibiotic resistance to spectinomycin and streptomycin for selection of Agrobacterium.

Mentions: The soybean reference genome sequence version 2.75 was obtained from Phytozome [31] and amended with an extra chromosome scaffold containing the T-DNA sequence located between the left and right border repeat regions (Fig. 2) [30]. Paired sequence reads from the previously described seed genomic DNA sequencing were aligned to the constructed reference using Bowtie (ver. 2.2.1) [32] with parameters –un-conc to specify discordant read output. Default Bowtie search methods were used with zero allowed mismatches to limit ambiguous alignments due to the abundance of highly repetitive and homologous endogenous sequences, and in global mode to not trim read ends to enhance alignment scores.


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)

Plasmid map of hTG construct. The hTG plasmid map shows all regions included in the transformation plasmid utilized in the Agrobacterium transformation of the original ST77 event. The T-DNA construct contains the soybean β-conglycinin promoter (7S), tobacco etch virus translational enhancer element (TEV), human thyroglobulin gene (hTG), cauliflower mosaic virus terminator element (T35S) followed by the selectable marker cassette comprised of the nopaline synthase promoter (NOS promoter), phosphinothricin acetyltransferase gene (BAR ORF), and nopaline synthase terminator element (NOS Term). The aad A region of the vector confers antibiotic resistance to spectinomycin and streptomycin for selection of Agrobacterium.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0010: Plasmid map of hTG construct. The hTG plasmid map shows all regions included in the transformation plasmid utilized in the Agrobacterium transformation of the original ST77 event. The T-DNA construct contains the soybean β-conglycinin promoter (7S), tobacco etch virus translational enhancer element (TEV), human thyroglobulin gene (hTG), cauliflower mosaic virus terminator element (T35S) followed by the selectable marker cassette comprised of the nopaline synthase promoter (NOS promoter), phosphinothricin acetyltransferase gene (BAR ORF), and nopaline synthase terminator element (NOS Term). The aad A region of the vector confers antibiotic resistance to spectinomycin and streptomycin for selection of Agrobacterium.
Mentions: The soybean reference genome sequence version 2.75 was obtained from Phytozome [31] and amended with an extra chromosome scaffold containing the T-DNA sequence located between the left and right border repeat regions (Fig. 2) [30]. Paired sequence reads from the previously described seed genomic DNA sequencing were aligned to the constructed reference using Bowtie (ver. 2.2.1) [32] with parameters –un-conc to specify discordant read output. Default Bowtie search methods were used with zero allowed mismatches to limit ambiguous alignments due to the abundance of highly repetitive and homologous endogenous sequences, and in global mode to not trim read ends to enhance alignment scores.

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