Recently, the research group of Xionglei He at the School of Life Sciences, Sun Yat‑sen University, published in The Plant Journal the research article entitled “Genome-wide profiling the integration patterns with T7-PCR” (doi:10.1111/tpj.70403)
In order to address the shortcomings of existing integration–site localization techniques, they developed a method based on in-vitro transcription mediated by the bacteriophage T7 polymerase, designated T7-PCR. By means of RNA conversion and enrichment of the junction fragments of integration sites, the signal-to-noise ratio was substantially improved.
During the study, experiments were conducted on rice (Oryza sativa), soybean (Glycine max) and arabidopsis (Arabidopsis thaliana) under the specific environmental and light-control conditions of the Jiupo artificial climate chambers in the Nine-Plot facility. The authors validated the generality and high efficiency of the method, demonstrating superior performance compared to current NGS (next-generation sequencing) techniques. The method is suitable for high-throughput transgenic screening, and lays the foundation for the development of large-fragment targeted integration tools.
Exogenous gene-introduction technologies are widely applied in agriculture and medicine, and accurately elucidating the integration sites of transgenes is of critical importance for ensuring their efficacy and safety. Existing transgenic insertion mechanisms suffer from random insertion, structural variation or low efficiency; and current tools for integration-site localization have shortcomings including high cost, low signal-to-noise ratio and long processing time.
In this study, the authors developed the T7-PCR method based on T7 in-vitro transcription (T7-IVT), which enriches junction fragments and removes background DNA to optimise the localisation workflow. Validated across multiple species and multiple integration scenarios, T7-PCR was shown to outperform existing methods in both accuracy and efficiency, particularly when applied to complex integration patterns.
Figure 1 Comparison of three NGS-based library construction methods, highlighting key differences in adaptor introduction strategies, workflow design, and the risk of chimeric artefacts.
Through yeast experiments the workflow of T7-PCR was proven effective: the product size distribution matched expectations, and insertion sites could be located with high coverage and accuracy; key parameters were optimised (DNA input amount and IVT time had little effect, whereas PCR cycle number needed to be controlled to avoid false positives).
T7-PCR is applicable in Arabidopsis, rice and soybean, and can accurately identify T-DNA insertion sites as well as structural variations such as reverse-orientation tandem repeats or deletions; the false-negative rate is low, occurring only when there is severe boundary deletion, which can be improved by optimising the position of the T7 promoter.
In comparison with adaptor-ligation PCR (AL-PCR): while the enrichment efficiency of both methods was comparable, T7-PCR exhibited a higher proportion of high-quality reads and longer mapping length; AL-PCR suffers from false positives and false negatives, whereas T7-PCR reduces non-specific amplification and chimera reads, yielding a superior signal-to-noise ratio.
Figure 2 Schematic illustration of the T7-PCR principle and standard workflow.
Figure 3 Mapping of T-DNA–mediated insertions in plants.
In the multimeric integration analysis, T7-PCR demonstrated high accuracy. By converting the flanking genomic sequence into RNA via T7-IVT, enriching the target signal prior to PCR and eliminating background noise, the method dramatically reduced chimeric artefacts. It outperformed traditional PCR enrichment methods and biotin-labeling approaches, and offers a high-efficiency, accurate and cost-effective solution. It can be used to assess the on-target efficiency and off-target frequency of emerging targeted-integration tools, to decipher structural variation near insertion sites, and to facilitate high-throughput screening of transgenic materials and development of targeted-locus integration tools.