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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

Aligned sequenced PCR products and insert layout. (A) Section of the insert location between two soybean genes. Colored bars represent sequences from the PCR amplicons of the junction sites that aligned to the soybean reference genome on chromosome 3. Purple is the product from primer F1, green from primer R1, yellow from primer F2, and blue from primer R2. 40 bases of genomic DNA have been deleted as a result of the insertion, shown as the uncolored region between the primer products. Start bases for each primer product are shown, as well as their alignment to either the sense or antisense DNA strand. (B) Illustration of the constructed consensus sequence of the T-DNA insert locus, showing the location of the primers used for junction characterization, flanking genomic DNA sequences, and inserted T-DNA elements.
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f0020: Aligned sequenced PCR products and insert layout. (A) Section of the insert location between two soybean genes. Colored bars represent sequences from the PCR amplicons of the junction sites that aligned to the soybean reference genome on chromosome 3. Purple is the product from primer F1, green from primer R1, yellow from primer F2, and blue from primer R2. 40 bases of genomic DNA have been deleted as a result of the insertion, shown as the uncolored region between the primer products. Start bases for each primer product are shown, as well as their alignment to either the sense or antisense DNA strand. (B) Illustration of the constructed consensus sequence of the T-DNA insert locus, showing the location of the primers used for junction characterization, flanking genomic DNA sequences, and inserted T-DNA elements.

Mentions: Junction sites were amplified for both the left and right border sequences generating products of ~ 400 bases and ~ 550 bases respectively. Sequencing results identified products of 373 and 530 bases for the right and left border PCR amplicons, respectively. The primers used for amplification, their attributes and the sequences generated are shown in Fig. 3B. Alignments of these sequences to both the soybean genome reference and the T-DNA sequence identified the insertion site to single-base resolution at base 44,332,733. Furthermore, alignments revealed a 40 base pair deletion at the insertion locus on chromosome 3 as shown in Fig. 4A. This deleted sequence was not part of an existing regulatory region, exon, or gene. In addition, 159 bases were deleted from the 5′ end of the right border region from the T-DNA, but left the 7S promoter intact. From the junction sequencing data, we constructed a consensus sequence of the insert relative to the genome which is illustrated in Fig. 4B.


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)

Aligned sequenced PCR products and insert layout. (A) Section of the insert location between two soybean genes. Colored bars represent sequences from the PCR amplicons of the junction sites that aligned to the soybean reference genome on chromosome 3. Purple is the product from primer F1, green from primer R1, yellow from primer F2, and blue from primer R2. 40 bases of genomic DNA have been deleted as a result of the insertion, shown as the uncolored region between the primer products. Start bases for each primer product are shown, as well as their alignment to either the sense or antisense DNA strand. (B) Illustration of the constructed consensus sequence of the T-DNA insert locus, showing the location of the primers used for junction characterization, flanking genomic DNA sequences, and inserted T-DNA elements.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0020: Aligned sequenced PCR products and insert layout. (A) Section of the insert location between two soybean genes. Colored bars represent sequences from the PCR amplicons of the junction sites that aligned to the soybean reference genome on chromosome 3. Purple is the product from primer F1, green from primer R1, yellow from primer F2, and blue from primer R2. 40 bases of genomic DNA have been deleted as a result of the insertion, shown as the uncolored region between the primer products. Start bases for each primer product are shown, as well as their alignment to either the sense or antisense DNA strand. (B) Illustration of the constructed consensus sequence of the T-DNA insert locus, showing the location of the primers used for junction characterization, flanking genomic DNA sequences, and inserted T-DNA elements.
Mentions: Junction sites were amplified for both the left and right border sequences generating products of ~ 400 bases and ~ 550 bases respectively. Sequencing results identified products of 373 and 530 bases for the right and left border PCR amplicons, respectively. The primers used for amplification, their attributes and the sequences generated are shown in Fig. 3B. Alignments of these sequences to both the soybean genome reference and the T-DNA sequence identified the insertion site to single-base resolution at base 44,332,733. Furthermore, alignments revealed a 40 base pair deletion at the insertion locus on chromosome 3 as shown in Fig. 4A. This deleted sequence was not part of an existing regulatory region, exon, or gene. In addition, 159 bases were deleted from the 5′ end of the right border region from the T-DNA, but left the 7S promoter intact. From the junction sequencing data, we constructed a consensus sequence of the insert relative to the genome which is illustrated in Fig. 4B.

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